U.S. patent application number 16/636900 was filed with the patent office on 2020-11-19 for oligonucleotide compositions and methods thereof.
The applicant listed for this patent is WAVE LIFE SCIENCES LTD.. Invention is credited to Sethumadhavan Divakaramenon, Jean-Cosme Dodart, Naoki Iwamoto, Pachamuthu Kandasamy, Yuanjing Liu, Genliang Lu, Subramanian Marappan, Chandra Vargeese, Jason Jingxin Zhang, Zhong Zhong.
Application Number | 20200362337 16/636900 |
Document ID | / |
Family ID | 1000005061694 |
Filed Date | 2020-11-19 |
United States Patent
Application |
20200362337 |
Kind Code |
A1 |
Dodart; Jean-Cosme ; et
al. |
November 19, 2020 |
OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF
Abstract
Among other things, the present disclosure provides
oligonucleotides, compositions, and methods thereof. Among other
things, the present disclosure encompasses the recognition that
structural elements of oligonucleotides, such as base sequence,
chemical modifications (e.g., modifications of sugar, base, and/or
internucleotidic linkages) or patterns thereof, conjugation with
additional chemical moieties, and/or stereochemistry [e.g.,
stereochemistry of backbone chiral centers (chiral internucleotidic
linkages)], and/or patterns thereof, can have significant impact on
oligonucleotide properties and activities, e.g., knockdown ability,
stability, delivery, etc. In some embodiments, the oligonucleotides
decrease the expression, activity and/or level of a C9orf72 gene,
including but not limited to, one comprising a repeat expansion, or
a gene product thereof. In some embodiments, the present disclosure
provides methods for treatment of diseases using provided
oligonucleotide compositions, for example, in treatment of
C9orf72-related disorders.
Inventors: |
Dodart; Jean-Cosme; (Boston,
MA) ; Liu; Yuanjing; (Arlington, MA) ;
Vargeese; Chandra; (Schwenksville, PA) ; Zhong;
Zhong; (Hingham, MA) ; Iwamoto; Naoki;
(Brighton, MA) ; Zhang; Jason Jingxin; (Walpole,
MA) ; Kandasamy; Pachamuthu; (Belmont, MA) ;
Divakaramenon; Sethumadhavan; (Lexington, MA) ; Lu;
Genliang; (Winchester, MA) ; Marappan;
Subramanian; (Acton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
WAVE LIFE SCIENCES LTD. |
Singapore |
|
SG |
|
|
Family ID: |
1000005061694 |
Appl. No.: |
16/636900 |
Filed: |
August 7, 2018 |
PCT Filed: |
August 7, 2018 |
PCT NO: |
PCT/US18/45653 |
371 Date: |
February 5, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62542778 |
Aug 8, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/11 20130101; C12N 2310/341 20130101; C12N 2310/315
20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A composition comprising an oligonucleotide, wherein the
oligonucleotide comprises at least one modification of a sugar,
base or internucleotidic linkage, and the base sequence of the
oligonucleotide comprises at least 15 contiguous bases of a base
sequence that is identical with or complementary to a base sequence
of a C9orf72 gene or a transcript thereof.
2. The composition of claim 1, wherein the oligonucleotide reduces
level of a repeat expansion-containing C9orf72 transcript when
administered to a system comprising the C9orf72 transcript.
3. The composition of claim 2, wherein the repeat
expansion-containing C9orf72 transcript comprises at least 30, 50,
100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 GGGGCC
repeats.
4. The composition of claim 4, wherein the reduction of level of
the repeat-expansion-containing C9orf72 transcript as measured by
percentage is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 fold of the reduction of level
of the non-repeat-expansion-containing C9orf72 transcript as
measured by percentage.
5. The composition of claim 1, wherein the oligonucleotide
hybridizes a site in C9orf72 exon 1a, intron 1, exon 1b, or exon
2.
6. The composition of any of the preceding claims, wherein the
oligonucleotide comprises at least one internucleotidic linkage
wherein the linkage phosphorus is in the Sp configuration.
7. The composition of claim 6, wherein the oligonucleotide
comprises a core and at least two wings.
8. The composition of claim 7, wherein the pattern of sugar
modifications of the first wing differs from the pattern of sugar
modifications of the second wing.
9. The composition of claim 8, wherein the first wing comprises a
2'-OMe and the second wing does not.
10. The composition of claim 8, wherein the second wing comprises a
2'-MOE and the first wing does not.
11. The composition of claim 8, wherein the first wing comprises a
2'-OMe and the second wing does not and wherein the second wing
comprises a 2'-MOE and the first wing does not.
12. A composition comprising oligonucleotides of a particular
oligonucleotide type characterized by: a) a common base sequence;
b) a common pattern of backbone linkages; c) a common pattern of
backbone chiral centers; which composition is chirally controlled
in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same common base
sequence, for oligonucleotides of the particular oligonucleotide
type; and wherein the oligonucleotide targets C9orf72.
13. An oligonucleotide of or comprising a wing-core-wing structure,
or a wing-core structure, or a core-wing structure, wherein the
core comprises a pattern of backbone chiral centers (linkage
phosphorus) of: (Np)t[(Op/Rp)n(Sp)m]y, wherein: t is 1-50; n is
1-10; m is 1-50; y is 1-10; Np is either Rp or Sp; Sp indicates the
S configuration of a chiral linkage phosphorus of a chiral modified
internucleotidic linkage; Op indicates an achiral linkage
phosphorus of a natural phosphate linkage; and Rp indicates the S
configuration of a chiral linkage phosphorus of a chiral modified
internucleotidic linkage; y is 1-10; each wing independently
comprises one or more nucleobases; and wherein the base sequence of
the oligonucleotide comprises at least 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, or 25 contiguous bases of a base sequence that is
identical with or complementary to a base sequence of a C9orf72
gene or a transcript thereof.
14. The oligonucleotide of claim 13, wherein the oligonucleotide is
of a wing-core-wing structure.
15. The oligonucleotide of any one of claims 13-14, wherein Np is
Sp.
16. The oligonucleotide of any one of claims 13-14, wherein the
pattern comprises at least one Rp.
17. The oligonucleotide of any one of 13-16, wherein the pattern
comprises at least one Op.
18. The oligonucleotide of any one of claims 13-14, wherein the
pattern is (Np)t[(Op)n(Sp)m]y.
19. The oligonucleotide of any one of claims 13-14, wherein the
pattern is (Np)t[(Rp)n(Sp)m]y.
20. The oligonucleotide of any one of claims 13-19, wherein at
least one n is 1.
21. The oligonucleotide of any one of claims 13-19, wherein each n
is 1.
22. The oligonucleotide of any one of claims 13-21, wherein y is
1.
23. The oligonucleotide of any one of claims 13-21, wherein y is
2.
24. The oligonucleotide of any one of claims 13-23, wherein t is
2-20.
25. The oligonucleotide of any one of claims 13-24, wherein at
least one m is 2-20.
26. The oligonucleotide of any one of claims 13-24, wherein at
least one m is 3, 4, 5, 6, 7, 8, 9, or 10.
27. The oligonucleotide of any one of claims 13-26, wherein each m
is independently 2-20.
28. The oligonucleotide of any one of claims 13-27, wherein the two
rings comprise different sugar modifications.
29. The oligonucleotide of any one of claims 13-27, wherein one
wing comprises a sugar modification that is not in the other
wing.
30. The oligonucleotide of any one of claims 13-29, wherein
nucleoside units of the core comprise no 2'-substitutions (two --H
at 2' position).
31. The oligonucleotide of any one of claims 13-30, wherein
nucleoside units of the core comprise no sugar modifications.
32. The oligonucleotide of any one of claims 13-31, wherein each
wing nucleoside unit independently comprises a sugar
modification.
33. The oligonucleotide of any one of claims 13-32, wherein the
base sequence of the oligonucleotide comprises a sequence that is
not identical or complementary to any repeats.
34. The oligonucleotide of any one of claims 13-32, wherein the
base sequence of the oligonucleotide comprises a sequence that is
not identical or complementary to the GGGGCC repeats.
35. The oligonucleotide of any one of claims 13-32, wherein the
base sequence of the oligonucleotide is not identical or
complementary to the GGGGCC repeats.
36. The oligonucleotide of any one of claims 13-35, wherein the
base sequence of the oligonucleotide comprises a sequence targeting
a C9orf72 intro sequence.
37. The oligonucleotide of claim 36, wherein the oligonucleotide
preferentially reduces level of a disease-associated C9orf72
product.
38. The oligonucleotide of claim 37, wherein the product is a
transcript comprising expanded GGGGCC repeats.
39. The oligonucleotide of claim 37, wherein the product is a
transcript comprising at least 30, 50, 100, 200, 300, 400, or 500
GGGGCC repeats.
40. The oligonucleotide of claim 37, wherein the product is an
antisense transcript comprising expanded GGGGCC repeats.
41. The oligonucleotide of claim 37, wherein the product is a
dipeptide repeat protein.
42. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-5987, WV-6408, WV-7117, WV-8009, WV-8010, WV-8011, WV-8012,
WV-8548, WV-8550, WV-9510, WV-11532, WV-12444, WV-12446, WV-12481,
WV-12482, WV-12483, or WV-12484.
43. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-12481, WV-12482, WV-12483, or WV-12484.
44. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-5987 or WV-7117.
45. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-8011.
46. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-8012.
47. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-11532.
48. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-6408.
49. The oligonucleotide of claim 13, wherein the oligonucleotide is
WV-12446.
50. The oligonucleotide of any one of claims 13-49, having a
diastereomeric purity of at least 50%, 60%, 70%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99%.
51. The composition of any one of claims 1-12, wherein the
oligonucleotide is an oligonucleotide of any one of claims
13-50.
52. A pharmaceutical composition comprising an oligonucleotide of
any one of the preceding claims or a pharmaceutically acceptable
salt thereof.
53. An oligonucleotide composition comprising a plurality of
oligonucleotides which have: a) a common base sequence; b) a common
pattern of backbone linkages; c) a common pattern of backbone
chiral centers; which composition is chirally controlled in that
level of the plurality of oligonucleotides in the composition is
not random; and wherein each oligonucleotide of the particular
oligonucleotide type is independently an oligonucleotide of any of
claims 13-50 or a salt thereof.
54. An oligonucleotide composition comprising oligonucleotides of a
particular oligonucleotide type characterized by: a) a common base
sequence; b) a common pattern of backbone linkages; c) a common
pattern of backbone chiral centers; which composition is chirally
controlled in that it is enriched, relative to a substantially
racemic preparation of oligonucleotides having the same common base
sequence, for oligonucleotides of the particular oligonucleotide
type; and wherein each oligonucleotide of the particular
oligonucleotide type is independently an oligonucleotide of any of
claims 13-50 or a salt thereof.
55. A method, comprising administering to a subject suffering from
or susceptible to a condition, disorder, and/or disease related to
C9orf72 expanded repeats an oligonucleotide or composition of any
one of the preceding claims.
56. The method of claim 55, wherein the condition, disorder, and/or
disease is amyotrophic lateral sclerosis (ALS), frontotemporal
dementia (FTD), corticobasal degeneration syndrome (CBD), atypical
Parkinsonian syndrome, olivopontocerebellar degeneration (OPCD), or
Alzheimer's disease.
57. The method of claim 55, wherein the condition, disorder, and/or
disease is amyotrophic lateral sclerosis (ALS).
58. The method of claim 55, wherein the condition, disorder, and/or
disease is frontotemporal dementia (FTD).
59. A method of decreasing the activity, expression and/or level of
a C9orf72 target gene or its gene product in a cell, comprising
introducing into the cell an oligonucleotide or composition of any
of preceding claims.
60. A method for preferential knockdown of a repeat
expansion-containing C9orf72 RNA transcript relative to a
non-repeat expansion-containing C9orf72 RNA transcript in a cell,
comprising contacting a cell comprising the repeat
expansion-containing C9orf72 RNA transcript and the non-repeat
expansion-containing C9orf72 RNA transcript with an oligonucleotide
or composition of any one of the preceding claims, wherein the
oligonucleotide comprises a sequence present in or complementary to
a sequence in the repeat expansion-containing C9orf72 RNA
transcript, wherein the oligonucleotide directs preferential
knockdown of a repeat expansion-containing C9orf72 RNA transcript
relative to a non-repeat expansion-containing C9orf72 RNA
transcript in a cell.
61. A compound, oligonucleotide, composition, or method described
in the specification.
Description
BACKGROUND
[0001] Oligonucleotides targeting the gene C9orf72 (e.g., C9orf72
oligonucleotides) are useful in various applications, e.g.,
therapeutic, diagnostic, and/or research applications, including
but not limited to treatment of various C9orf72-related
disorders.
SUMMARY
[0002] The present disclosure provides oligonucleotides, and
compositions thereof, that can reduce levels of C9orf72 transcripts
(or products thereof). In some embodiments, provided
oligonucleotides and compositions can preferentially reduce levels
of disease-associated transcripts of C9orf72 (or products thereof)
over non-disease-associated transcripts of C9orf72 (see, e.g., FIG.
1). Example C9orf72 transcripts include transcripts from either
strand of the C9orf72 gene and from various starting points. In
some embodiments, at least some C9orf72 transcripts are translated
into proteins; in some embodiments, at least some C9orf72
transcripts are not translated into proteins. In some embodiments,
certain C9orf72 transcripts contain predominantly intronic
sequences.
[0003] A hexanucleotide repeat expansion in C9orf72 (Chromosome 9,
open reading frame 72) is reportedly the most frequent genetic
cause of amyotrophic lateral sclerosis (ALS) and frontotemporal
dementia (FTD). C9orf72 gene variants comprising the repeat
expansion and/or products thereof are also associated with other
C9orf72-related disorders, such as corticobasal degeneration
syndrome (CBD), atypical Parkinsonian syndrome,
olivopontocerebellar degeneration (OPCD), primary lateral sclerosis
(PLS), progressive muscular atrophy (PMA), Huntington's disease
(HD) phenocopy, Alzheimer's disease (AD), bipolar disorder,
schizophrenia, and other non-motor disorders. In some embodiments,
the present disclosure provides compositions and methods related to
oligonucleotides which target a C9orf72 target (e.g., a C9orf72
oligonucleotide) and are capable of knocking down or decreasing
expression, level and/or activity of the C9orf72 target gene and/or
a gene product thereof (a transcript, particularly a repeat
expansion containing transcript, a protein, etc.).
[0004] In some embodiments, an oligonucleotide targets a
pathological or disease-associated C9orf72 mutation or variant
comprising a repeat expansion. In some embodiments, a C9orf72 gene
product is a RNA (e.g., a mRNA, mature RNA or pre-mRNA) transcribed
from a C9orf72 gene, a protein translated from a C9orf72 RNA
transcript (e.g., a dipeptide repeat protein translated from the
hexanucleotide repeat), or a focus (plural: foci) (which reportedly
comprises RNA comprising the repeat expansion bound by RNA-binding
proteins). In some embodiments, a C9orf72 oligonucleotide is
capable of mediating preferential knockdown of a repeat
expansion-containing C9orf72 RNA relative to a non-repeat
expansion-containing C9orf72 RNA (a C9orf72 RNA which does not
contain a repeat expansion). In some embodiments, a C9orf72
oligonucleotide decreases the expression, activity and/or level of
a deleterious C9orf72 gene product (e.g., a RNA comprising a repeat
expansion, a dipeptide repeat protein or a focus) without
decreasing the expression, activity and/or level of a wild-type or
non-deleterious C9orf72 gene product. In some embodiments, a
C9orf72 oligonucleotide decreases the expression, activity and/or
level of a deleterious C9orf72 gene product, but does not decrease
the expression, activity and/or level of a wild-type or
non-deleterious C9orf72 protein enough to eliminate or
significantly suppress a beneficial and/or necessary biological
activity or activities of C9orf72 protein. Beneficial and/or
necessary activities of C9orf72 protein are widely known and
include but not limited to restricting inflammation, preventing
autoimmunity and preventing premature mortality.
[0005] Among other things, the present disclosure encompasses the
recognition that controlling structural elements of C9orf72
oligonucleotides can have a significant impact on oligonucleotide
properties and/or activities, including knockdown of a C9orf72
target gene. In some embodiments, knockdown of a target gene is
mediated by RNase H or steric hindrance affecting translation. In
some embodiments, controlled structural elements of C9orf72
oligonucleotides include but are not limited to: base sequence,
chemical modifications (e.g., modifications of a sugar, base and/or
internucleotidic linkage) or patterns thereof, alterations in
stereochemistry (e.g., stereochemistry of a backbone chiral
internucleotidic linkage) or patterns thereof, wing structure, core
structure, wing-core structure, wing-core-wing structure, or
core-wing structure, and/or conjugation with an additional chemical
moiety (e.g., a carbohydrate moiety, a targeting moiety, etc.). In
some embodiments, the present disclosure provides technologies
(e.g., compounds, methods, etc.) for improving C9orf72
oligonucleotide stability while maintaining or increasing
oligonucleotide activity, including compositions of
improved-stability oligonucleotides. In some embodiments, provided
oligonucleotides target C9orf72 or products thereof. In some
embodiments, a target gene is a C9orf72.
[0006] In some embodiments, the present disclosure encompasses the
recognition that various optional additional chemical moieties,
such as carbohydrate moieties, targeting moieties, etc., when
incorporated into c9orf72 oligonucleotides, can improve one or more
properties. In some embodiments, an additional chemical moiety is
selected from: glucose, GluNAc (N-acetyl amine glucosamine) and
anisamide moieties. These and other moieties are described in more
detail herein, e.g., in Examples 1 and 2. In some embodiments, an
oligonucleotide can comprise two or more additional chemical
moieties, wherein the additional chemical moieties are identical or
non-identical, or are of the same category (e.g., carbohydrate
moiety, sugar moiety, targeting moiety, etc.) or not of the same
category. In some embodiments, certain additional chemical moieties
facilitate delivery of oligonucleotides to desired cells, tissues
and/or organs, including but not limited to particular cells, parts
or portions of the central nervous system (e.g., cerebral cortex,
hippocampus, spinal cord, etc.). In some embodiments, certain
additional chemical moieties facilitate internalization of
oligonucleotides. In some embodiments, certain additional chemical
moieties increase oligonucleotide stability. In some embodiments,
the present disclosure provides technologies for incorporating
various additional chemical moieties into oligonucleotides. In some
embodiments, the present disclosure provides, for example, reagents
and methods, for introducing additional chemical moieties through
internucleotidic linkages, sugars and/or nucleobases (e.g., by
covalent linkage, optionally via a linker, to a site on a sugar, a
nucleobase, or an internucleotidic linkage).
[0007] In some embodiments, the present disclosure demonstrates
that surprisingly high target specificity can be achieved with
oligonucleotides, e.g., C9orf72 oligonucleotides, whose structures
include one or more features as described herein [including, but
not limited to, base sequences disclosed herein (wherein each U can
be optionally and independently substituted by T and vice versa),
and/or chemical modifications and/or stereochemistry and/or
patterns thereof and/or combinations thereof, e.g., examples
illustrated in FIG. 2].
[0008] In some embodiments, the present disclosure demonstrates
that certain provided structural elements, technologies and/or
features are particularly useful for oligonucleotides that knock
down C9orf72. Regardless, however, the teachings of the present
disclosure are not limited to oligonucleotides that participate in
or operate via any particular biochemical mechanism. In some
embodiments, the present disclosure provides oligonucleotides
capable of operating via a mechanism such as double-stranded RNA
interference, single-stranded RNA interference or which acts as an
antisense oligonucleotide which decreases the expression, activity
and/or level of a C9orf72 gene or a gene product thereof via a
RNase H-mediated mechanism or steric hindrance of translation.
[0009] Further, the present disclosure pertains to any C9orf72
oligonucleotide which operates through any mechanism, and which
comprises any sequence, structure or format (or portion thereof)
described herein, wherein the oligonucleotide comprises at least
one non-naturally-occurring modification of a base, sugar or
internucleotidic linkage. In some embodiments, the present
disclosure pertains to any C9orf72 oligonucleotide which comprises
at least one stereocontrolled internucleotidic linkage (including
but not limited to a phosphorothioate linkage in the Sp or Rp
configuration). In some embodiments, the present disclosure
pertains to any C9orf72 oligonucleotide which operates through any
mechanism, and which comprises at least one stereocontrolled
internucleotidic linkage (including but not limited to a
phosphorothioate linkage in the Sp or Rp configuration). In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide which comprises any sequence, structure or format
(or portion thereof) described herein, an optional additional
chemical moiety (including but not limited to a carbohydrate
moiety, and a targeting moiety), stereochemistry or patterns of
stereochemistry, internucleotidic linkage or pattern of
internucleotidic linkages; modification of sugar(s) or pattern of
modifications of sugars; modification of base(s) or patterns of
modifications of bases.
[0010] In some embodiments, a C9orf72 disorder-associated target
allele contains a hexanucleotide repeat expansion in intron 1,
including but not limited to G4C2 or (GGGGCC)ng, wherein ng is 30
or more. In some embodiments, ng is 50 or more. In some
embodiments, ng is 100 or more. In some embodiments, ng is 150 or
more. In some embodiments, ng is 200 or more. In some embodiments,
ng is 300 or more. In some embodiments, ng is 500 or more.
[0011] The C9orf72 G4C2 repeat expansion in intron 1 reportedly
accounts for 1 in 10 ALS cases among European-ancestry populations.
G4C2 repeats are reportedly of only about .about.10% of the
transcripts (e.g., transcripts V3 and V1 of the pathological allele
illustrated in FIG. 1), with gain of function toxicities, at least
partially mediated by the dipeptide repeat proteins and foci
formation by, for example, repeat-expansion containing transcripts
and/or spliced-out repeat-expansion containing introns and/or
antisense transcription of the repeat-expansion containing region
and various nucleic-acid binding proteins. In some embodiments, V1
is reportedly transcribed at very low levels (around 1% of the
total C9orf72 transcript level) and does not contribute
significantly to the levels of transcripts comprising
hexanucleotide repeat expansions. Reportedly, intron nucleic acid
containing repeat expansions can be retained as pre-mRNA, partially
spliced RNA, and/or spliced out introns, and RNA foci comprising
these nucleic acids are associated with RNA binding protein
sequestration. C9orf72 RNA foci are described in, for example, Liu
et al., 2017, Cell Chemical Biology 24, 1-8; Niblock et al. Acta
Neuropathologica Communications (2016) 4:18. Aberrant protein
products comprising dipeptide repeat proteins (DPR proteins) are
reportedly produced from the repeat expansion, with toxicity to
neurons. In some embodiment, the present disclosure provides
oligonucleotides and compositions and methods thereof which target
an intron sequence close to the G4C2 repeats, and can reduce levels
of repeat expansion-containing transcripts, proteins encoded
thereby, and/or related foci. In some embodiment, the present
disclosure provides C9orf72 oligonucleotides and compositions
thereof which target an intron sequence close to the G4C2 repeats,
to specifically knockdown the repeat expansion-containing
transcripts via RNAse-H, with minimal impact on normal C9orf 72
transcripts. In some embodiments, compared to existing data, the
present disclosure demonstrates that provided technologies
targeting an intron sequence (e.g., between the repeats and exon
1b) can effectively and/or preferentially reduce levels of repeat
expansion-containing products.
[0012] Without wishing to be bound by any particular theory, the
present disclosure notes that several possible mechanisms for the
deleterious and disease-associated effects of the repeat expansion
have been proposed in the literature. See for example: Edbauer et
al. 2016 Curr. Opin. Neurobiol. 36: 99-106; Conlon et al. Elife.
2016 Sep. 13; 5. pii: e17820; Xi et al. 2015 Acta Neuropathol. 129:
715-727; Cohen-Hada et al. 2015 Stem Cell Rep. 7: 927-940; and
Burguete et al. eLife 2015; 4:e08881. Among other things, the
present disclosure provides technologies that can reduce or remove
one or more or all deleterious and disease-associated C9orf72
products and/or disease-associated effects.
[0013] Without wishing to be bound by any particular theory, the
present disclosure notes that a possible mechanism of a deleterious
effect of repeat expansion-containing C9orf72 transcripts is the
generation of foci. Reportedly, the repeat expansion results in
retention of intron 1-containing C9orf72 mRNA. The majority of
intron 1-retaining C9orf72 mRNA accumulates in the nucleus where it
is targeted to a specific degradation pathway unable to process
G4C2 RNA repeats. The RNAs subsequently aggregate into foci, which
also comprise RNA-binding proteins, sequestering them from their
normal functions. Niblock Acta Neuropathol Commun. 2016; 4: 18.
Reportedly antisense foci comprising antisense C9orf72 products are
present at a significantly higher frequency in cerebellar Purkinje
neurons and motor neurons, whereas sense foci are present at a
significantly higher frequency in cerebellar granule neurons.
Cooper-Knock et al. Acta Neuropathol (2015) 130:63-75. In some
embodiments, the present disclosure provides technologies for
reducing levels of foci. In some embodiments, provided technologies
reduce levels of or remove antisense foci and/or sense foci in one
or more types of neurons.
[0014] Without wishing to be bound by any particular theory, the
present disclosure notes that another possible mechanism of a
deleterious effect of repeat expansion-containing C9orf72
transcripts is the generation of dipeptide repeat (DPR) proteins. A
small proportion of intron 1-retaining C9orf72 mRNA is exported to
the cytoplasm for RAN (repeat-associated non-AUG translation)
translation in all six reading frames into DPRs. Niblock Acta
Neuropathol Commun. 2016; 4: 18. Cooper-Knock et al. also reported
that inclusions containing sense or antisense derived dipeptide
repeat proteins were present at significantly higher frequency in
cerebellar granule neurons or motor neurons, respectively; and in
motor neurons, which are the primary target of pathology in ALS,
the presence of antisense foci but not sense foci correlated with
mislocalisation of TDP-43, which is a hallmark of ALS
neurodegeneration. In some embodiments, provided technologies
reduce levels of one or more or all of C9orf72 DPR protein
products.
[0015] In some embodiments, gain- and/or loss-of-function
mechanisms lead to neurodegeneration in a C9orf72-related disorder.
See, for example: Mizielinska et al. 2014 Science 345: 1192-94;
Chew et al. 2015 Science 348: 1151-1154; Jiang et al. 2016 Neuron
90: 535-550; and Liu et al. 2016 Neuron 90: 521-534; Gendron et al.
Cold Spring Harb. Perspect. Med. 2017 Jan. 27. pii: a024224;
Haeusler et al. Nat Rev Neurosci. 2016 June; 17(6):383-95; Koppers
et al. Ann. Neurol. 2015; 78:426-438; Todd et al. J. Neurochem.
2016 138 (Suppl. 1) 145-162. In some embodiments, provided
technologies reduce undesired gained functions, and/or restore or
enhance desired functions.
[0016] In some embodiments, provided oligonucleotides and
compositions and methods thereof are useful for treatment of any of
several C9orf72-related disorders, including but not limited to
amyotrophic lateral sclerosis (ALS). In some embodiments, ALS is
MIM: 612069. Amyotrophic lateral sclerosis (ALS) is a reportedly a
fatal neurodegenerative disease characterized clinically by
progressive paralysis leading to death, often from respiratory
failure, typically within two to three years of symptom onset
(Rowland and Shneider, N. Engl. J. Med., 2001, 344, 1688-1700). ALS
reportedly is the third most common neurodegenerative disease in
the Western world (Hirtz et al., Neurology, 2007, 68, 326-337), and
there are currently no effective therapies. Approximately 10% of
cases are familial in nature, whereas the bulk of patients
diagnosed with the disease are classified as sporadic as they
appear to occur randomly throughout the population (Chio et al.,
Neurology, 2008, 70, 533-537). Clinical, genetic, and
epidemiological data reportedly support the hypothesis that ALS and
frontotemporal dementia (FTD) represent an overlapping continuum of
disease, characterized pathologically by the presence of TDP-43
positive inclusions throughout the central nervous system (Lillo
and Hodges, J. Clin. Neurosci., 2009, 16, 1131-1135; Neumann et
al., Science, 2006, 314, 130-133). A number of genes have been
discovered as potentially causative for classical familial ALS, for
example, SOD1, TARDBP, FUS, OPTN, and VCP (Johnson et al., Neuron,
2010, 68, 857-864; Kwiatkowski et al., Science, 2009, 323,
1205-1208; Maruyama et al., Nature, 2010, 465, 223-226; Rosen et
al., Nature, 1993, 362, 59-62; Sreedharan et al., Science, 2008,
319, 1668-1672; Vance et al., Brain, 2009, 129, 868-876). Linkage
analysis of kindreds involving multiple cases of ALS, FTD, and
ALS-FTD had reportedly suggested that there was an important locus
for the disease on the short arm of chromosome 9, identified as
C9orf72 (Boxer et al., J. Neurol. Neurosurg. Psychiatry, 2011, 82,
196-203; Morita et al., Neurology, 2006, 66, 839-844; Pearson et
al. J. Neurol., 2011, 258, 647-655; Vance et al., Brain, 2006, 129,
868-876). This mutation had been found to be the most common
genetic cause of ALS and FTD. In some embodiments, ALS-FTD causing
mutation is a large hexanucleotide (e.g., GGGGCC or G.sub.4C.sub.2)
repeat expansion in the first intron of the C9orf72 gene on
chromosome 9 (Renton et al., Neuron, 2011, 72, 257-268;
DeJesus-Hernandez et al., Neuron, 2011, 72, 245-256). A founder
haplotype, covering the C9orf72 gene, is present in the majority of
cases linked to this region (Renton et al., Neuron, 2011, 72,
257-268). This locus on chromosome 9p21 accounts for nearly half of
familial ALS and nearly one-quarter of all ALS cases in a cohort of
405 Finnish patients (Laaksovirta et al, Lancet Neurol., 2010, 9,
978-985). The incidence of ALS is reportedly 1:50,000. Familial ALS
reportedly represents 5-10% of all ALS cases; C9orf72 mutations
reportedly can be the most common cause of ALS (40-50%). ALS is
reportedly associated with degeneration of both upper and lower
motor neurons in the motor cortex of the brain, the brain stem, and
the spinal cord. Symptoms of ALS reportedly include: muscle
weakness and/or muscle atrophy, trouble swallowing or breathing,
cramping, stiffness. Respiratory failure is reportedly the main
cause of death. In some embodiments, provided technologies reduces
severity and/or removes one or more of symptoms related to ALS or
other C9orf72 related conditions, disorders and/or diseases.
[0017] In some embodiments, provided oligonucleotides and
compositions and methods thereof are useful for treatment of any of
several C9orf72-related disorders, including but not limited to
frontotemporal dementia (FTD). In some embodiments, FTD is referred
to as frontotemporal lobar degeneration or FTLD, MIM: 600274.
Frontotemporal dementia, reportedly the second most common form of
presenile dementia, is reportedly associated with focal atrophy of
the frontal or temporal lobes. Boxer et al. 2005 Alzheimer Dis.
Assoc. Disord. 19 (Suppl 1):S3-S6. FTD shares extensive clinical,
pathological, and molecular overlap with amyotrophic lateral
sclerosis. As reported by Gijselinck, Cold Spring Harb. Perspect.
Med. 2017 Jan. 27. pii: a026757, there are reportedly families and
individual patients in which both diseases occur (ALS-FTD)
(Lomen-Hoerth et al. 2002 Neurology 59:1077-1079), and TDP-43
inclusions (Arai et al. 2006 Biochem. Biophys. Res. Comm. 351:
602-611; Neumann et al. 2006 Science 314: 130-133) in ALS and FTLD
patients can be indistinguishable (Tsuji et al. 2012 Brain 135:
3380-3391), despite the pathological distribution being different
for ALS and FTLD patients. There is reportedly evidence that common
disease pathways may be involved in ALS and FTLD because their
clinical and pathological hallmarks overlap; hence, the pure forms
of these diseases are considered the two extremes of one disease
continuum (Lillo and Hodges 2009 J. Clin. Neurosci. 16: 1131-1135).
Genetic studies reportedly identified mutations in the same genes
in FTLD and ALS--for example, TBK1, TARDBP, FUS, VCP (Neumann et
al. 2006; Kovacs et al. 2009 Mov. Disord. 24: 1843-1847; Johnson et
al. 2010 Neuron 68: 857-864; Van Langenhove et al. 2010 Neurology
74: 366-371; Cirulli et al. 2015 Science 347: 1436-1441;
Freischmidt et al. 2015 Nat. Neurosci. 18: 631-636; Pottier et al.
2015 Acta Neuropathol. 130: 77-92). Genetic evidence for a common
disease pathomechanism was reportedly provided by the
identification of the repeat expansion mutations in C9orf72 in
patients with ALS, FTLD, and ALS-FTD (Gijselinck et al. 2010 Arch.
Neurol. 67: 606-616; De Jesus-Hernandez et al. 2011 Neuron 72:
245-256; Renton et al. 2011 Neuron 72: 257-268).
[0018] In some embodiments, a C9orf72 target is a specific allele
(e.g., one with a repeat expansion) and level, expression and/or
activity of one or more products (e.g., RNA and/or protein products
such as dipeptide repeat proteins or DPRs) are intended to be
altered. In many embodiments, a C9orf72 target allele is one whose
presence and/or expression is associated (e.g., correlated) with
presence, incidence, and/or severity, of one or more diseases
and/or conditions, including but not limited to ALS and FTD or
other C9orf72-related disorders, or a symptom thereof.
Alternatively or additionally, in some embodiments, a C9orf72
target allele is one for which alteration of expression, level
and/or activity of one or more gene products correlates with
improvement (e.g., delay of onset, reduction of severity,
responsiveness to other therapy, etc.) in one or more aspects of a
disease and/or condition, including but not limited to ALS and FTD
or other C9orf72-related disorders.
[0019] In some embodiments, a neurological disease is characterized
by neuronal hyperexcitability. In some embodiments, a 50% reduction
in C9orf72 activity, due to and/or in the presence of the (GGGGCC)
expansion, reportedly increases neurotransmission through the
glutamate receptors NMDA, AMPA, and kainite. In addition, glutamate
receptors reportedly accumulate on neurons. The increased
neurotransmission and accumulation of glutamate receptors
reportedly leads to glutamate-induced excitotoxicity due to the
neuronal hyperexcitability. Inhibiting glutamate receptors would
reportedly treat the neuronal hyperexcitability. Clearance of
dipeptide repeat proteins generated from the expansion reportedly
is impaired, enhancing their neurotoxicity. C9orf72 reportedly
promotes early endosomal trafficking through activation of RAB5,
which requires phosphatidylinositol 3-phosphase (PI3P). PIKFYVE
converts PI3P to phosphatidylinositol (3,5)-bisphosphate
(PI(3,5)P2). Inhibiting PIKFYVE reportedly would compensate for
altered RAB5 levels by increasing PI3P levels to enable early
endosomal maturation, which would ultimately lead to the clearance
of dipeptide repeat proteins. Neurons reportedly also use endosomal
trafficking to regulate sodium and potassium ion channel
localization. Inhibiting PIKFYVE reportedly may also treat neuronal
hyperexcitability. In some embodiments, provided technologies
reduce neuronal hyperexcitability. In some embodiments, provided
technologies may be administered as part of the same treatment
regime as an inhibitor of PIKFYVE.
[0020] In some embodiments, the present disclosure provides an
oligonucleotide composition comprising a first plurality of
oligonucleotides which share: [0021] 1) a common base sequence;
[0022] 2) a common pattern of backbone linkages; and [0023] 3) a
common pattern of backbone chiral centers, which composition is a
substantially pure preparation of a single oligonucleotide in that
a non-random or controlled level of the oligonucleotides in the
composition have the common base sequence and length, the common
pattern of backbone linkages, and the common pattern of backbone
chiral centers.
[0024] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides capable of directing C9orf72 knockdown, wherein
oligonucleotides are of a particular oligonucleotide type
characterized by: [0025] 1) a common base sequence and length;
[0026] 2) a common pattern of backbone linkages; and [0027] 3) a
common pattern of backbone chiral centers; which composition is
chirally controlled in that it is enriched, relative to a
substantially racemic preparation of oligonucleotides having the
same base sequence and length, for oligonucleotides of the
particular oligonucleotide type.
[0028] In some embodiments, a provided oligonucleotide (which can
target C9orf72 or target a target other than C9orf72) comprises one
or more blocks. In some embodiments, a block comprises one or more
consecutive nucleosides, and/or nucleotides, and/or sugars, or
bases, and/or internucleotidic linkages. In some embodiments, a
provided oligonucleotide comprises three or more blocks, wherein
the blocks on either end are not identical and the oligonucleotide
is thus asymmetric. In some embodiments, a block is a wing or a
core.
[0029] In some embodiments, a c9orf72 oligonucleotide comprises at
least one wing and at least one core, wherein a wing differs
structurally from a core in that a wing comprises a structure
[e.g., stereochemistry, additional chemical moiety, or chemical
modification at a sugar, base or internucleotidic linkage (or
pattern thereof)] different than the core, or vice versa. In some
embodiments, a provided oligonucleotide comprises a wing-core-wing
structure. In some embodiments, a provided oligonucleotide
comprises a wing-core, core-wing, or wing-core-wing structure,
wherein one wing differs in structure [e.g., stereochemistry,
additional chemical moiety, or chemical modification at a sugar,
base or internucleotidic linkage (or pattern thereof)] from the
other wing and the core (for example, an asymmetrical
oligonucleotide). In some embodiments, an oligonucleotide has or
comprises a wing-core, core-wing, or wing-core-wing structure, and
a block is a wing or core. In some embodiments, a core is also
referenced to as a gap.
[0030] In general, properties of oligonucleotide compositions as
described herein can be assessed using any appropriate assay.
[0031] Those of skill in the art will be aware of and/or will
readily be able to develop appropriate assays for particular
oligonucleotide compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1 describes example C9orf72 transcripts. V3, V2 and V1
transcripts produced from a healthy and a pathological C9orf72
allele are illustrated, wherein the pathological allele contains a
hexanucleotide repeat expansion [horizontal bar, indicated by
(GGGGCC).sub.30+]. The downward-pointing arrow indicates the
position of some example C9orf72 oligonucleotides targeting intron
1.
[0033] FIG. 2 presents certain provided formats of oligonucleotides
as examples.
[0034] FIGS. 3A and 3B present certain provided C9orf72
oligonucleotides as examples. Structural details of these
oligonucleotides are further described in, for example, Table
1A.
[0035] FIG. 4 presents example data demonstrating that provided
C9orf72 oligonucleotides can provide preferential knockdown of
repeat expansion-containing C9orf72 transcripts relative to total
C9orf72 transcripts (including non-repeat expansion-containing
C9orf72 transcripts). FIG. 4A shows knockdown of repeating
expansion-containing transcripts by administration of WV-3662 and
WV-3536 (which represent the base sequence of SEQ ID NO: 0553 of
WO2015054676, and SEQ ID NO: 0057 of WO2016168592, respectively),
and WV-6408, normalized to controls. FIG. 4B shows knockdown of
total C9orf72 transcripts by administration of WV-3662, WV-3536,
and WV-6408. In FIGS. 4A and 4B, concentrations of oligonucleotides
used were: 0.1, 0.3, 1, 3, and 10 .mu.M from left to right. FIG. 4C
shows knockdown of repeating expansion-containing transcripts
provided by control oligonucleotides WV-2376 and WV-3542, and
example oligonucleotides WV-3688, WV-6408, WV-7658, WV-7659,
WV-8010, and WV-8011. Concentrations were 1 (left column) and 10
.mu.M (right column). FIG. 4D shows knockdown of total transcripts
by administration of control oligonucleotides WV-2376 and WV-3542.
Concentrations were 1 (left column) and 10 .mu.M (right
column).
[0036] FIG. 5 presents example data demonstrating in vivo potency
of provided C9orf72 oligonucleotides in the C9-BAC mouse spinal
cord. WV-2376 is a negative control oligonucleotide. Present data
were those of WV-6408, WV-8009, WV-8010, WV-8011, and WV-8012. FIG.
5A shows knockdown of total transcripts (including repeat
expansion-containing and non-repeat expansion-containing
transcripts). FIG. 5B shows knockdown of V3 (repeat
expansion-containing) transcripts. FIG. 5C shows knockdown of
Intron/AS transcripts (with probes targeting a region 3' to the
repeat transcript expansion, the detected area includes both sense
and antisense transcripts of the intronic region). PBS, phosphate
buffered saline (negative control).
[0037] FIG. 6 presents example data demonstrating the in vivo
potency of some C9orf72 oligonucleotides in the C9-BAC mouse
cortex. WV-2376 is a negative control oligonucleotide which does
not target C9orf72; presented data were those of: WV-6408, WV-8009,
WV-8010, WV-8011, and WV-8012. FIG. 6A shows knockdown of total
transcripts (including repeat expansion-containing and non-repeat
expansion-containing transcripts). FIG. 6B shows knockdown of V3
(repeat expansion-containing) transcripts. FIG. 6C shows knockdown
of Intron/AS transcripts (with probes targeting a region 3' to the
repeat transcript expansion, the detected area includes both sense
and antisense transcripts of the intronic region).
[0038] FIGS. 7A to 7D present example data on the activity of
provided Malat1 oligonucleotides conjugated to various chemical
moieties, for example, sulfonamide or anisamide. FIG. 7A shows
example data of Malat1 oligonucleotides in knocking down Malat1 in
spinal cord; FIG. 7B shows example distribution data of various
Malat1 oligonucleotides (ASO or antisense oligonucleotides) in
spinal cord; FIG. 7C shows the knockdown of Malat1 in cortex; and
FIG. 7D shows the distribution of the test oligonucleotides in
cortex. Presented data were those of: WV-3174, WV-7558, WV-7559,
and WV-7560, administered ICV, 1.times.50 .mu.g.
[0039] FIGS. 8A to H show the effect of certain provided C9orf72
oligonucleotides on C9orf72 transcripts in C9-BAC mice. C9orf72
oligonucleotides tested were: WV-6408, WV-8009, WV-8010, WV-8011,
and WV-8012. Negative controls were PBS (phosphate-buffered saline)
and WV-2376, which does not target C9orf72. Transcripts were
analyzed from the cerebral cortex (FIGS. 8A to D) and spinal cord
(FIGS. 8E to H). Transcripts analyzed were: All transcripts (FIGS.
8A and E); V3 (FIGS. 8B and F); V3 (exon 1a) (FIGS. 8C and G); and
Intron1/AS (FIGS. 8D and H). The data in FIG. 9 and FIG. 10 are
from the same in-vivo mouse study.
[0040] FIGS. 9A and 9B show example distribution data of C9orf72
oligonucleotides in spinal cord (FIG. 9A) and cerebral cortex (FIG.
9B) of C9-BAC mice. C9orf72 oligonucleotides tested were: WV-6408,
WV-8009, WV-8010, WV-8011, and WV-8012. Negative controls were PBS
(phosphate-buffered saline) and WV-2376, which does not target
C9orf72.
[0041] FIG. 10 shows example data of C9orf72 oligonucleotides on
the level of polyGP (a dipeptide repeat protein) in the hippocampus
of C9-BAC mice. C9orf72 oligonucleotides tested were: WV-6408,
WV-8009, WV-8010, WV-8011, and WV-8012. Negative controls were PBS
(phosphate-buffered saline) and WV-2376, which does not target
C9orf72.
[0042] FIG. 11A shows an example hybridization ELISA assay for
measuring oligonucleotide levels, e.g., in tissues and fluids,
including but not limited to animal biopsies. FIG. 11B shows
example chemistry for binding a primary amine-labeled capture probe
to an amino-reactive solid support, such as a plate comprising
maleic anhydride.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
Definitions
[0043] As used herein, the following definitions shall apply unless
otherwise indicated. For purposes of this disclosure, the chemical
elements are identified in accordance with the Periodic Table of
the Elements, CAS version, Handbook of Chemistry and Physics, 75th
Ed. Additionally, general principles of organic chemistry are
described in "Organic Chemistry", Thomas Sorrell, University
Science Books, Sausalito: 1999, and "March's Advanced Organic
Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley
& Sons, New York: 2001.
[0044] Aliphatic: As used herein, "aliphatic" means a
straight-chain (i.e., unbranched) or branched, substituted or
unsubstituted hydrocarbon chain that is completely saturated or
that contains one or more units of unsaturation, or a substituted
or unsubstituted monocyclic, bicyclic, or polycyclic hydrocarbon
ring that is completely saturated or that contains one or more
units of unsaturation (but not aromatic), or combinations thereof.
In some embodiments, aliphatic groups contain 1-50 aliphatic carbon
atoms. In some embodiments, aliphatic groups contain 1-20 aliphatic
carbon atoms. In other embodiments, aliphatic groups contain 1-10
aliphatic carbon atoms. In other embodiments, aliphatic groups
contain 1-9 aliphatic carbon atoms. In other embodiments, aliphatic
groups contain 1-8 aliphatic carbon atoms. In other embodiments,
aliphatic groups contain 1-7 aliphatic carbon atoms. In other
embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms.
In still other embodiments, aliphatic groups contain 1-5 aliphatic
carbon atoms, and in yet other embodiments, aliphatic groups
contain 1, 2, 3, or 4 aliphatic carbon atoms. Suitable aliphatic
groups include, but are not limited to, linear or branched,
substituted or unsubstituted alkyl, alkenyl, alkynyl groups and
hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or
(cycloalkyl)alkenyl.
[0045] Alkenyl: As used herein, the term "alkenyl" refers to an
alkyl group, as defined herein, having one or more double
bonds.
[0046] Alkyl: As used herein, the term "alkyl" is given its
ordinary meaning in the art and may include saturated aliphatic
groups, including straight-chain alkyl groups, branched-chain alkyl
groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. In some
embodiments, an alkyl has 1-100 carbon atoms. In certain
embodiments, a straight chain or branched chain alkyl has about
1-20 carbon atoms in its backbone (e.g., C.sub.1-C.sub.2 for
straight chain, C.sub.2-C.sub.20 for branched chain), and
alternatively, about 1-10. In some embodiments, cycloalkyl rings
have from about 3-10 carbon atoms in their ring structure where
such rings are monocyclic, bicyclic, or polycyclic, and
alternatively about 5, 6 or 7 carbons in the ring structure. In
some embodiments, an alkyl group may be a lower alkyl group,
wherein a lower alkyl group comprises 1-4 carbon atoms (e.g.,
C.sub.1-C.sub.4 for straight chain lower alkyls).
[0047] Alkynyl: As used herein, the term "alkynyl" refers to an
alkyl group, as defined herein, having one or more triple
bonds.
[0048] Animal: As used herein, the term "animal" refers to any
member of the animal kingdom. In some embodiments, "animal" refers
to humans, at any stage of development. In some embodiments,
"animal" refers to non-human animals, at any stage of development.
In certain embodiments, the non-human animal is a mammal (e.g., a
rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep,
cattle, a primate and/or a pig). In some embodiments, animals
include, but are not limited to, mammals, birds, reptiles,
amphibians, fish and/or worms. In some embodiments, an animal may
be a transgenic animal, a genetically-engineered animal and/or a
clone.
[0049] Approximately: As used herein, the terms "approximately" or
"about" in reference to a number are generally taken to include
numbers that fall within a range of 5%, 10%, 15%, or 20% in either
direction (greater than or less than) of the number unless
otherwise stated or otherwise evident from the context (except
where such number would be less than 0% or exceed 100% of a
possible value). In some embodiments, use of the term "about" in
reference to dosages means .+-.5 mg/kg/day.
[0050] Aryl: The term "aryl", as used herein, used alone or as part
of a larger moiety as in "aralkyl," "aralkoxy," or "aryloxyalkyl,"
refers to monocyclic, bicyclic or polycyclic ring systems having a
total of five to thirty ring members, wherein at least one ring in
the system is aromatic. In some embodiments, an aryl group is a
monocyclic, bicyclic or polycyclic ring system having a total of
five to fourteen ring members, wherein at least one ring in the
system is aromatic, and wherein each ring in the system contains 3
to 7 ring members. In some embodiments, an aryl group is a biaryl
group. The term "aryl" may be used interchangeably with the term
"aryl ring." In certain embodiments of the present disclosure,
"aryl" refers to an aromatic ring system which includes, but not
limited to, phenyl, biphenyl, naphthyl, binaphthyl, anthracyl and
the like, which may bear one or more substituents. Also included
within the scope of the term "aryl," as it is used herein, is a
group in which an aromatic ring is fused to one or more
non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,
phenanthridinyl, or tetrahydronaphthyl, and the like.
[0051] Comparable: The term "comparable" is used herein to describe
two (or more) sets of conditions or circumstances that are
sufficiently similar to one another to permit comparison of results
obtained or phenomena observed. In some embodiments, comparable
sets of conditions or circumstances are characterized by a
plurality of substantially identical features and one or a small
number of varied features. Those of ordinary skill in the art will
appreciate that sets of conditions are comparable to one another
when characterized by a sufficient number and type of substantially
identical features to warrant a reasonable conclusion that
differences in results obtained or phenomena observed under the
different sets of conditions or circumstances are caused by or
indicative of the variation in those features that are varied.
[0052] Cycloaliphatic: The term "cycloaliphatic," "carbocycle,"
"carbocyclyl," "carbocyclic radical," and "carbocyclic ring," are
used interchangeably, and as used herein, refer to saturated or
partially unsaturated, but non-aromatic, cyclic aliphatic
monocyclic, bicyclic, or polycyclic ring systems, as described
herein, having, unless otherwise specified, from 3 to 30 ring
members. Cycloaliphatic groups include, without limitation,
cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,
cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl,
norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, a
cycloaliphatic group has 3-6 carbons. In some embodiments, a
cycloaliphatic group is saturated and is cycloalkyl. The term
"cycloaliphatic" may also include aliphatic rings that are fused to
one or more aromatic or nonaromatic rings, such as
decahydronaphthyl or tetrahydronaphthyl. In some embodiments, a
cycloaliphatic group is bicyclic. In some embodiments, a
cycloaliphatic group is tricyclic. In some embodiments, a
cycloaliphatic group is polycyclic. In some embodiments,
"cycloaliphatic" refers to C.sub.3-C.sub.6 monocyclic hydrocarbon,
or C.sub.8-C.sub.10 bicyclic or polycyclic hydrocarbon, that is
completely saturated or that contains one or more units of
unsaturation, but which is not aromatic, that has a single point of
attachment to the rest of the molecule, or a C.sub.9-C.sub.16
polycyclic hydrocarbon that is completely saturated or that
contains one or more units of unsaturation, but which is not
aromatic, that has a single point of attachment to the rest of the
molecule.
[0053] Dosing regimen: As used herein, a "dosing regimen" or
"therapeutic regimen" refers to a set of unit doses (typically more
than one) that are administered individually to a subject,
typically separated by periods of time. In some embodiments, a
given therapeutic agent has a recommended dosing regimen, which may
involve one or more doses. In some embodiments, a dosing regimen
comprises a plurality of doses each of which are separated from one
another by a time period of the same length; in some embodiments, a
dosing regime comprises a plurality of doses and at least two
different time periods separating individual doses. In some
embodiments, all doses within a dosing regimen are of the same unit
dose amount. In some embodiments, different doses within a dosing
regimen are of different amounts. In some embodiments, a dosing
regimen comprises a first dose in a first dose amount, followed by
one or more additional doses in a second dose amount different from
the first dose amount. In some embodiments, a dosing regimen
comprises a first dose in a first dose amount, followed by one or
more additional doses in a second dose amount same as the first
dose amount.
[0054] Heteroaliphatic: The term "heteroaliphatic", as used herein,
is given its ordinary meaning in the art and refers to aliphatic
groups as described herein in which one or more carbon atoms are
independently replaced with one or more heteroatoms (e.g., oxygen,
nitrogen, sulfur, silicon, phosphorus, and the like). In some
embodiments, one or more units selected from C, CH, CH.sub.2, and
CH.sub.3 are independently replaced by one or more heteroatoms
(including oxidized and/or substituted form thereof). In some
embodiments, a heteroaliphatic group is heteroalkyl. In some
embodiments, a heteroaliphatic group is heteroalkenyl.
[0055] Heteroalkyl: The term "heteroalkyl", as used herein, is
given its ordinary meaning in the art and refers to alkyl groups as
described herein in which one or more carbon atoms are
independently replaced with one or more heteroatoms (e.g., oxygen,
nitrogen, sulfur, silicon, phosphorus, and the like). Examples of
heteroalkyl groups include, but are not limited to, alkoxy,
poly(ethylene glycol)-, alkyl-substituted amino, tetrahydrofuranyl,
piperidinyl, morpholinyl, etc.
[0056] Heteroaryl: The terms "heteroaryl" and "heteroar-", as used
herein, used alone or as part of a larger moiety, e.g.,
"heteroaralkyl," or "heteroaralkoxy," refer to monocyclic, bicyclic
or polycyclic ring systems having a total of five to thirty ring
members, wherein at least one ring in the system is aromatic and at
least one aromatic ring atom is a heteroatom. In some embodiments,
a heteroaryl group is a group having 5 to 10 ring atoms (i.e.,
monocyclic, bicyclic or polycyclic), in some embodiments 5, 6, 9,
or 10 ring atoms. In some embodiments, a heteroaryl group has 6,
10, or 14.pi. electrons shared in a cyclic array; and having, in
addition to carbon atoms, from one to five heteroatoms. Heteroaryl
groups include, without limitation, thienyl, furanyl, pyrrolyl,
imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl,
oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,
pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,
naphthyridinyl, and pteridinyl. In some embodiments, a heteroaryl
is a heterobiaryl group, such as bipyridyl and the like. The terms
"heteroaryl" and "heteroar-", as used herein, also include groups
in which a heteroaromatic ring is fused to one or more aryl,
cycloaliphatic, or heterocyclyl rings, where the radical or point
of attachment is on the heteroaromatic ring. Non-limiting examples
include indolyl, isoindolyl, benzothienyl, benzofuranyl,
dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl,
isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl,
4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl,
phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and
pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be
monocyclic, bicyclic or polycyclic. The term "heteroaryl" may be
used interchangeably with the terms "heteroaryl ring," "heteroaryl
group," or "heteroaromatic," any of which terms include rings that
are optionally substituted. The term "heteroaralkyl" refers to an
alkyl group substituted by a heteroaryl group, wherein the alkyl
and heteroaryl portions independently are optionally
substituted.
[0057] Heteroatom: The term "heteroatom", as used herein, means an
atom that is not carbon or hydrogen. In some embodiments, a
heteroatom is boron, oxygen, sulfur, nitrogen, phosphorus, or
silicon (including any oxidized form of nitrogen, sulfur,
phosphorus, or silicon; the quaternized form of any basic nitrogen
or a substitutable nitrogen of a heterocyclic ring (for example, N
as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR (as
in N-substituted pyrrolidinyl); etc.).
[0058] Heterocycle: As used herein, the terms "heterocycle,"
"heterocyclyl," "heterocyclic radical," and "heterocyclic ring", as
used herein, are used interchangeably and refer to a monocyclic,
bicyclic or polycyclic ring moiety (e.g., 3-30 membered) that is
saturated or partially unsaturated and has one or more heteroatom
ring atoms. In some embodiments, a heterocyclyl group is a stable
5- to 7-membered monocyclic or 7- to 10-membered bicyclic
heterocyclic moiety that is either saturated or partially
unsaturated, and having, in addition to carbon atoms, one or more,
preferably one to four, heteroatoms, as defined above. When used in
reference to a ring atom of a heterocycle, the term "nitrogen"
includes substituted nitrogen. As an example, in a saturated or
partially unsaturated ring having 0-3 heteroatoms selected from
oxygen, sulfur and nitrogen, the nitrogen may be N (as in
3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or .sup.+NR (as
in N-substituted pyrrolidinyl). A heterocyclic ring can be attached
to its pendant group at any heteroatom or carbon atom that results
in a stable structure and any of the ring atoms can be optionally
substituted. Examples of such saturated or partially unsaturated
heterocyclic radicals include, without limitation,
tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl,
pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,
decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl,
dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and
quinuclidinyl. The terms "heterocycle," "heterocyclyl,"
"heterocyclyl ring," "heterocyclic group," "heterocyclic moiety,"
and "heterocyclic radical," are used interchangeably herein, and
also include groups in which a heterocyclyl ring is fused to one or
more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl,
3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl. A
heterocyclyl group may be monocyclic, bicyclic or polycyclic. The
term "heterocyclylalkyl" refers to an alkyl group substituted by a
heterocyclyl, wherein the alkyl and heterocyclyl portions
independently are optionally substituted.
[0059] In vitro: As used herein, the term "in vitro" refers to
events that occur in an artificial environment, e.g., in a test
tube or reaction vessel, in cell culture, etc., rather than within
an organism (e.g., animal, plant and/or microbe).
[0060] In vivo: As used herein, the term "in vivo" refers to events
that occur within an organism (e.g., animal, plant and/or
microbe).
[0061] Optionally Substituted: As described herein, compounds,
e.g., oligonucleotides, of the disclosure may contain optionally
substituted and/or substituted moieties. In general, the term
"substituted," whether preceded by the term "optionally" or not,
means that one or more hydrogens of the designated moiety are
replaced with a suitable substituent. Unless otherwise indicated,
an "optionally substituted" group may have a suitable substituent
at each substitutable position of the group, and when more than one
position in any given structure may be substituted with more than
one substituent selected from a specified group, the substituent
may be either the same or different at every position. In some
embodiments, an optionally substituted group is unsubstituted.
Combinations of substituents envisioned by this disclosure are
preferably those that result in the formation of stable or
chemically feasible compounds. The term "stable," as used herein,
refers to compounds that are not substantially altered when
subjected to conditions to allow for their production, detection,
and, in certain embodiments, their recovery, purification, and use
for one or more of the purposes disclosed herein.
[0062] Suitable monovalent substituents on a substitutable atom,
e.g., a suitable carbon atom, are independently halogen;
--(CH.sub.2).sub.0-4R.sup..largecircle.;
--(CH.sub.2).sub.0-4OR.sup..largecircle.; --O(CH.sub.2).sub.0-4R,
--O--(CH.sub.2).sub.0-4C(O)OR.sup..largecircle.;
--(CH.sub.2).sub.0-4CH(OR.sup..largecircle.).sub.2;
--(CH.sub.2).sub.0-4Ph, which may be substituted with
R.sup..largecircle.; --(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1Ph
which may be substituted with R.sup..largecircle.; --CH.dbd.CHPh,
which may be substituted with R.sup..largecircle.;
--(CH.sub.2).sub.0-4O(CH.sub.2).sub.0-1-pyridyl which may be
substituted with R.sup..largecircle.; --NO.sub.2; --CN; --N.sub.3;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.).sub.2;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)R.sup..largecircle.;
--N(R.sup..largecircle.)C(S)R.sup..largecircle.;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)NR.sup..largecircle..sub.2;
--N(R.sup..largecircle.)C(S)NR.sup..largecircle..sub.2;
--(CH.sub.2).sub.0-4N(R.sup..largecircle.)C(O)OR.sup..largecircle.;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)R.sup..largecircle.;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)NR.sup..largecircle..su-
b.2;
--N(R.sup..largecircle.)N(R.sup..largecircle.)C(O)OR.sup..largecircle-
.; --(CH.sub.2).sub.0-4C(O)R.sup..largecircle.;
--C(S)R.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)OR.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)SR.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)OSiR.sup..largecircle..sub.3;
--(CH.sub.2).sub.0-4OC(O)R.sup..largecircle.;
--OC(O)(CH.sub.2).sub.0-4SR, --SC(S)SR.sup..largecircle.;
--(CH.sub.2).sub.0-4SC(O)R.sup..largecircle.;
--(CH.sub.2).sub.0-4C(O)NR.sup..largecircle..sub.2;
--C(S)NR.sup..largecircle..sub.2; --C(S)SR.sup..largecircle.;
--SC(S)SR.sup..largecircle.,
--(CH.sub.2).sub.0-4OC(O)NR.sup..largecircle..sub.2;
--C(O)N(OR.sup..largecircle.)R.sup..largecircle.;
--C(O)C(O)R.sup..largecircle.;
--C(O)CH.sub.2C(O)R.sup..largecircle.;
--C(NOR.sup..largecircle.)R.sup..largecircle.;
--(CH.sub.2).sub.0-4SSR.sup..largecircle.;
--(CH.sub.2).sub.0-4S(O).sub.2R.sup..largecircle.;
--(CH.sub.2).sub.0-4S(O).sub.2OR.sup..largecircle.;
--(CH.sub.2).sub.0-4OS(O).sub.2R.sup..largecircle.;
--S(O).sub.2NR.sup..largecircle..sub.2;
--(CH.sub.2).sub.0-4S(O)R.sup..largecircle.;
--N(R.sup..largecircle.)S(O).sub.2NR.sup..largecircle..sub.2;
--N(R.sup..largecircle.)S(O).sub.2R.sup..largecircle.;
--N(OR.sup..largecircle.)R.sup..largecircle.;
--C(NH)NR.sup..largecircle..sub.2; --Si(R.sup..largecircle.).sub.3;
--OSi(R.sup..largecircle.).sub.3; --B(R.sup..largecircle.).sub.2;
--OB(R.sup..largecircle.).sub.2; --OB(OR.sup..largecircle.).sub.2;
--P(R.sup..largecircle.).sub.2; --P(OR.sup..largecircle.).sub.2;
--OP(R.sup..largecircle.).sub.2; --OP(OR.sup..largecircle.).sub.2;
--P(O)(R.sup..largecircle.).sub.2;
--P(O)(OR.sup..largecircle.).sub.2;
--OP(O)(R.sup..largecircle.).sub.2;
--OP(O)(OR.sup..largecircle.).sub.2;
--OP(O)(OR.sup..largecircle.)(SR.sup..largecircle.);
--SP(O)(R.sup..largecircle.).sub.2;
--SP(O)(OR.sup..largecircle.).sub.2;
--N(R.sup..largecircle.)P(O)(R.sup..largecircle.).sub.2;
--N(R.sup..largecircle.)P(O)(OR.sup..largecircle.).sub.2;
--P(R.sup..largecircle.).sub.2[B(R.sup..largecircle.).sub.3];
--P(OR.sup..largecircle.).sub.2[B(R.sup..largecircle.).sub.3];
--OP(R.sup..largecircle.).sub.2[B(R.sup..largecircle.).sub.3];
--OP(OR.sup..largecircle.).sub.2[B(R.sup..largecircle.).sub.3];
--(C.sub.1-4 straight or branched
alkylene)O--N(R.sup..largecircle.).sub.2; or --(C.sub.1-4 straight
or branched alkylene)C(O)O--N(R.sup..largecircle.).sub.2, wherein
each R.sup..largecircle. may be substituted as defined below and is
independently hydrogen, C.sub.1-20 aliphatic, C.sub.1-20
heteroaliphatic having 1-5 heteroatoms independently selected from
nitrogen, oxygen, sulfur, silicon and phosphorus,
--CH.sub.2--(C.sub.6-14 aryl), --O(CH.sub.2).sub.0-1(C.sub.6-14
aryl), --CH.sub.2-(5-14 membered heteroaryl ring), a 5-20 membered,
monocyclic, bicyclic, or polycyclic, saturated, partially
unsaturated or aryl ring having 0-5 heteroatoms independently
selected from nitrogen, oxygen, sulfur, silicon and phosphorus, or,
notwithstanding the definition above, two independent occurrences
of R.sup..largecircle., taken together with their intervening
atom(s), form a 5-20 membered, monocyclic, bicyclic, or polycyclic,
saturated, partially unsaturated or aryl ring having 0-5
heteroatoms independently selected from nitrogen, oxygen, sulfur,
silicon and phosphorus, which may be substituted as defined
below.
[0063] Suitable monovalent substituents on R.sup..largecircle. (or
the ring formed by taking two independent occurrences of
R.sup..largecircle. together with their intervening atoms), are
independently halogen, --(CH.sub.2).sub.0-2R.sup..circle-solid.,
-(haloR.sup..circle-solid.), --(CH.sub.2).sub.0-2OH,
--(CH.sub.2).sub.0-2OR.sup..circle-solid.,
--(CH.sub.2).sub.0-2CH(OR.sup..circle-solid.).sub.2;
--O(haloR.sup..circle-solid.), --CN, --N.sub.3,
--(CH.sub.2).sub.0-2C(O)R.sup..circle-solid.,
--(CH.sub.2).sub.0-2C(O)OH,
--(CH.sub.2).sub.0-2C(O)OR.sup..circle-solid.,
--(CH.sub.2).sub.0-2SR.sup..circle-solid., --(CH.sub.2).sub.0-2SH,
--(CH.sub.2).sub.0-2NH.sub.2,
--(CH.sub.2).sub.0-2NHR.sup..circle-solid.,
--(CH.sub.2).sub.0-2NR.sup..circle-solid..sub.2, --NO.sub.2,
--SiR.sup..circle-solid..sub.3, --OSiR.sup..circle-solid..sub.3,
--C(O)SR.sup..circle-solid., --(C.sub.1-4 straight or branched
alkylene)C(O)OR.sup..circle-solid., or --SSR.sup..circle-solid.
wherein each R.sup..circle-solid. is unsubstituted or where
preceded by "halo" is substituted only with one or more halogens,
and is independently selected from C.sub.1-4 aliphatic,
--CH.sub.2Ph, --O(CH.sub.2).sub.0-1Ph, and a 5-6-membered
saturated, partially unsaturated, or aryl ring having 0-4
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. Suitable divalent substituents on a saturated carbon atom
of R.sup..largecircle. include .dbd.O and .dbd.S.
[0064] Suitable divalent substituents, e.g., on a suitable carbon
atom, are independently the following: .dbd.O, .dbd.S,
.dbd.NNR*.sub.2, .dbd.NNHC(O)R*, .dbd.NNHC(O)OR*,
.dbd.NNHS(O).sub.2R*, .dbd.NR*, .dbd.NOR*,
--O(C(R*.sub.2)).sub.2-3O--, or --S(C(R*.sub.2)).sub.2-3S--,
wherein each independent occurrence of R* is selected from
hydrogen, C.sub.1-6 aliphatic which may be substituted as defined
below, and an unsubstituted 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. Suitable divalent
substituents that are bound to vicinal substitutable carbons of an
"optionally substituted" group include: --O(CR*.sub.2).sub.2-3O--,
wherein each independent occurrence of R* is selected from
hydrogen, C.sub.1-6 aliphatic which may be substituted as defined
below, and an unsubstituted 5-6-membered saturated, partially
unsaturated, or aryl ring having 0-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0065] Suitable substituents on the aliphatic group of R* are
independently halogen, --R.sup..circle-solid.,
-(haloR.sup..circle-solid.), --OH, --OR.sup..circle-solid.,
--O(haloR.sup..circle-solid.), --CN, --C(O)OH,
--C(O)OR.sup..circle-solid., --NH.sub.2, --NHR.sup..circle-solid.,
--NR.sup..circle-solid..sub.2, or --NO.sub.2, wherein each
R.sup..circle-solid. is unsubstituted or where preceded by "halo"
is substituted only with one or more halogens, and is independently
C.sub.1-4 aliphatic, --CH.sub.2Ph, --O(CH.sub.2).sub.0-1Ph, or a
5-6-membered saturated, partially unsaturated, or aryl ring having
0-4 heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0066] Oral: The phrases "oral administration" and "administered
orally" as used herein have their art-understood meaning referring
to administration by mouth of a compound or composition.
[0067] Parenteral: The phrases "parenteral administration" and
"administered parenterally" as used herein have their
art-understood meaning referring to modes of administration other
than enteral and topical administration, usually by injection, and
include, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal, and intrasternal injection and
infusion.
[0068] Partially unsaturated: As used herein, the term "partially
unsaturated" refers to a ring moiety that includes at least one
double or triple bond. The term "partially unsaturated" is intended
to encompass rings having multiple sites of unsaturation, but is
not intended to include aryl or heteroaryl moieties, as herein
defined.
[0069] Pharmaceutical composition: As used herein, the term
"pharmaceutical composition" refers to an active agent, formulated
together with one or more pharmaceutically acceptable carriers. In
some embodiments, an active agent is present in unit dose amount
appropriate for administration in a therapeutic regimen that shows
a statistically significant probability of achieving a
predetermined therapeutic effect when administered to a relevant
population. In some embodiments, pharmaceutical compositions may be
specially formulated for administration in solid or liquid form,
including those adapted for the following: oral administration, for
example, drenches (aqueous or non-aqueous solutions or
suspensions), tablets, e.g., those targeted for buccal, sublingual,
and systemic absorption, boluses, powders, granules, pastes for
application to the tongue; parenteral administration, for example,
by subcutaneous, intramuscular, intravenous or epidural injection
as, for example, a sterile solution or suspension, or
sustained-release formulation; topical application, for example, as
a cream, ointment, or a controlled-release patch or spray applied
to the skin, lungs, or oral cavity; intravaginally or
intrarectally, for example, as a pessary, cream, or foam;
sublingually; ocularly; transdermally; or nasally, pulmonary, and
to other mucosal surfaces.
[0070] Pharmaceutically acceptable: As used herein, the phrase
"pharmaceutically acceptable" refers to those compounds, materials,
compositions and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0071] Pharmaceutically acceptable carrier: As used herein, the
term "pharmaceutically acceptable carrier" means a
pharmaceutically-acceptable material, composition or vehicle, such
as a liquid or solid filler, diluent, excipient, or solvent
encapsulating material, involved in carrying or transporting the
subject compound from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. Some examples of
materials which can serve as pharmaceutically-acceptable carriers
include: sugars, such as lactose, glucose and sucrose; starches,
such as corn starch and potato starch; cellulose, and its
derivatives, such as sodium carboxymethyl cellulose, ethyl
cellulose and cellulose acetate; powdered tragacanth; malt;
gelatin; talc; excipients, such as cocoa butter and suppository
waxes; oils, such as peanut oil, cottonseed oil, safflower oil,
sesame oil, olive oil, corn oil and soybean oil; glycols, such as
propylene glycol; polyols, such as glycerin, sorbitol, mannitol and
polyethylene glycol; esters, such as ethyl oleate and ethyl
laurate; agar; buffering agents, such as magnesium hydroxide and
aluminum hydroxide; alginic acid; pyrogen-free water; isotonic
saline; Ringer's solution; ethyl alcohol; pH buffered solutions;
polyesters, polycarbonates and/or polyanhydrides; and other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0072] Pharmaceutically acceptable salt: The term "pharmaceutically
acceptable salt", as used herein, refers to salts of such compounds
that are appropriate for use in pharmaceutical contexts, i.e.,
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge, et al. describes pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19
(1977). In some embodiments, pharmaceutically acceptable salt
include, but are not limited to, nontoxic acid addition salts,
which are salts of an amino group formed with inorganic acids such
as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric
acid and perchloric acid or with organic acids such as acetic acid,
maleic acid, tartaric acid, citric acid, succinic acid or malonic
acid or by using other methods used in the art such as ion
exchange. In some embodiments, pharmaceutically acceptable salts
include, but are not limited to, adipate, alginate, ascorbate,
aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,
camphorate, camphorsulfonate, citrate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptonate, glycerophosphate, gluconate, hemisulfate,
heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate,
nitrate, oleate, oxalate, palmitate, pamoate, pectinate,
persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like. In
some embodiments, a provided compound comprises one or more acidic
groups, e.g., an oligonucleotide, and a pharmaceutically acceptable
salt is an alkali, alkaline earth metal, or ammonium (e.g., an
ammonium salt of N(R).sub.3, wherein each R is independently
defined and described in the present disclosure) salt.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. In some
embodiments, a pharmaceutically acceptable salt is a sodium salt.
In some embodiments, a pharmaceutically acceptable salt is a
potassium salt. In some embodiments, a pharmaceutically acceptable
salt is a calcium salt. In some embodiments, pharmaceutically
acceptable salts include, when appropriate, nontoxic ammonium,
quaternary ammonium, and amine cations formed using counterions
such as halide, hydroxide, carboxylate, sulfate, phosphate,
nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl
sulfonate. In some embodiments, a provided compound comprises more
than one acid groups, for example, a provided oligonucleotide may
comprise two or more acidic groups (e.g., in natural phosphate
linkages and/or modified internucleotidic linkages). In some
embodiments, a pharmaceutically acceptable salt, or generally a
salt, of such a compound comprises two or more cations, which can
be the same or different. In some embodiments, in a
pharmaceutically acceptable salt (or generally, a salt), all
ionizable hydrogen in the acidic groups are replaced with cations.
In some embodiments, a pharmaceutically acceptable salt is a sodium
salt of a provided oligonucleotide. In some embodiments, a
pharmaceutically acceptable salt is a sodium salt of a provided
oligonucleotide, wherein each acidic phosphate group exists as a
salt form (all sodium salt).
[0073] Protecting group: The term "protecting group," as used
herein, is well known in the art and includes those described in
detail in Protecting Groups in Organic Synthesis, T. W. Greene and
P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, the
entirety of which is incorporated herein by reference. Also
included are those protecting groups specially adapted for
nucleoside and nucleotide chemistry described in Current Protocols
in Nucleic Acid Chemistry, edited by Serge L. Beaucage et al. June
2012, the entirety of Chapter 2 is incorporated herein by
reference. Suitable amino-protecting groups include methyl
carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc),
9-(2-sulfo)fluorenylmethyl carbamate,
9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),
1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethylcarbamate(Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl
carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc), cinnamyl
carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl
carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate,
benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, phenothiazinyl-(10)-carbonyl
derivative, N'-p-toluenesulfonylaminocarbonyl derivative,
N'-phenylaminothiocarbonyl derivative, t-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxycarbonylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, 2,4,6-trimethylbenzyl carbamate, formamide, acetamide,
chloroacetamide, trichloroacetamide, trifluoroacetamide,
phenylacetamide, 3-phenylpropanamide, picolinamide,
3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,
p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,
acetoacetamide, (N'-dithiobenzyloxycarbonylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,
4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide
(Dts), N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,
N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),
5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one,
5-substituted 1,3-dibenzyl-1,3,5-triazacyclohexan-2-one,
1-substituted 3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N'-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine,
N-copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine,
amine N-oxide, diphenylphosphinamide (Dpp),
dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt),
dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl
phosphoramidate, benzenesulfenamide, o-nitrobenzenesulfenamide
(Nps), 2,4-dinitrobenzenesulfenamide,
pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,
triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), (3-trimethylsilylethanesulfonamide (SES),
9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide,
trifluoromethylsulfonamide,andphenacylsulfonamide.
[0074] Suitably protected carboxylic acids further include, but are
not limited to, silyl-, alkyl-, alkenyl-, aryl-, and
arylalkyl-protected carboxylic acids. Examples of suitable silyl
groups include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triisopropylsilyl, and the like. Examples of
suitable alkyl groups include methyl, benzyl, p-methoxybenzyl,
3,4-dimethoxybenzyl, trityl, t-butyl, tetrahydropyran-2-yl.
Examples of suitable alkenyl groups include allyl. Examples of
suitable aryl groups include optionally substituted phenyl,
biphenyl, or naphthyl. Examples of suitable arylalkyl groups
include optionally substituted benzyl (e.g., p-methoxybenzyl (MPM),
3,4-dimethoxybenzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl), and 2- and 4-picolyl.
[0075] Suitable hydroxyl protecting groups include methyl,
methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,
(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),
p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),
guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),
siloxymethyl, 2-methoxyethoxymethyl (MEM),
2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,
2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),
3-bromotetrahydropyranyl, tetrahydrothiopyranyl,
1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP),
4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl
S,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl
(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS),
diethylisopropylsilyl(DEIPS), dimethylthexylsilyl,
t-butyldimethylsilyl(TBDMS), t-butyldiphenylsilyl (TBDPS),
tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,
diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS),
formate, benzoylformate, acetate, chloroacetate, dichloroacetate,
trichloroacetate, trifluoroacetate, methoxyacetate,
triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,
3-phenylpropionate, 4-oxopentanoate(levulinate),
4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,
adamantoate, crotonate, 4-methoxycrotonate, benzoate,
p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl
carbonate, 9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl
carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc),
2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl
carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),
alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl
carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate,
alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl
carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl
carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-1-napththyl
carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,
4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,
2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,
4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxycarbonyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts). For protecting 1,2- or 1,3-diols, the protecting groups
include methylene acetal, ethylidene acetal, 1-t-butylethylidene
ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene
acetal, 2,2,2-trichloroethylidene acetal, acetonide,
cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene
ketal, benzylidene acetal, p-methoxybenzylidene acetal,
2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal,
2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene
acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho
ester, 1-ethoxyethylidine ortho ester, 1,2-dimethoxyethylidene
ortho ester, .alpha.-methoxybenzylidene ortho ester,
1-(N,N-dimethylamino)ethylidene derivative,
.alpha.-(N,N'-dimethylamino)benzylidene derivative,
2-oxacyclopentylidene ortho ester, di-t-butylsilylene group (DTBS),
1,3-(1,1,3,3-tetraisopropyldisiloxanylidene) derivative (TIPDS),
tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cyclic
carbonates, cyclic boronates, ethyl boronate, and phenyl
boronate.
[0076] In some embodiments, a hydroxyl protecting group is acetyl,
t-butyl, tbutoxymethyl, methoxymethyl, tetrahydropyranyl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl,
p-chlorophenyl, 2,4-dinitrophenyl, benzyl, benzoyl,
p-phenylbenzoyl, 2,6-dichlorobenzyl, diphenylmethyl, p-nitrobenzyl,
triphenylmethyl (trityl), 4,4'-dimethoxytrityl, trimethylsilyl,
triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,
triphenylsilyl, triisopropylsilyl, benzoylformate, chloroacetyl,
trichloroacetyl, trifiuoroacetyl, pivaloyl, 9-fluorenylmethyl
carbonate, mesylate, tosylate, triflate, trityl, monomethoxytrityl
(MMTr), 4,4'-dimethoxytrityl, (DMTr) and 4,4',4''-trimethoxytrityl
(TMTr), 2-cyanoethyl (CE or Cne), 2-(trimethylsilyl)ethyl (TSE),
2-(2-nitrophenyl)ethyl, 2-(4-cyanophenyl)ethyl
2-(4-nitrophenyl)ethyl (NPE), 2-(4-nitrophenylsulfonyl)ethyl,
3,5-dichlorophenyl, 2,4-dimethylphenyl, 2-nitrophenyl,
4-nitrophenyl, 2,4,6-trimethylphenyl, 2-(2-nitrophenyl)ethyl,
butylthiocarbonyl, 4,4',4''-tris(benzoyloxy)trityl,
diphenylcarbamoyl, levulinyl, 2-(dibromomethyl)benzoyl (Dbmb),
2-(isopropylthiomethoxymethyl)benzoyl (Ptmt), 9-phenylxanthen-9-yl
(pixyl) or 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In some
embodiments, each of the hydroxyl protecting groups is,
independently selected from acetyl, benzyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl and 4,4'-dimethoxytrityl. In some embodiments,
the hydroxyl protecting group is selected from the group consisting
of trityl, monomethoxytrityl and 4,4'-dimethoxytrityl group. In
some embodiments, a phosphorous linkage protecting group is a group
attached to the phosphorous linkage (e.g., an internucleotidic
linkage) throughout oligonucleotide synthesis. In some embodiments,
a protecting group is attached to a sulfur atom of an
phosphorothioate group. In some embodiments, a protecting group is
attached to an oxygen atom of an internucleotide phosphorothioate
linkage. In some embodiments, a protecting group is attached to an
oxygen atom of the internucleotide phosphate linkage. In some
embodiments a protecting group is 2-cyanoethyl (CE or Cne),
2-trimethylsilylethyl, 2-nitroethyl, 2-sulfonylethyl, methyl,
benzyl, o-nitrobenzyl, 2-(p-nitrophenyl)ethyl (NPE or Npe),
2-phenylethyl, 3-(N-tert-butylcarboxamido)-1-propyl, 4-oxopentyl,
4-methylthio-1-butyl, 2-cyano-1,1-dimethylethyl,
4-N-methylaminobutyl, 3-(2-pyridyl)-1-propyl,
2-[N-methyl-N-(2-pyridyl)]aminoethyl, 2-(N-formyl,
N-methyl)aminoethyl, or
4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl.
[0077] Sample: A "sample" as used herein is a specific organism or
material obtained therefrom. In some embodiments, a sample is a
biological sample obtained or derived from a source of interest, as
described herein. In some embodiments, a source of interest
comprises an organism, such as an animal or human. In some
embodiments, a biological sample comprises biological tissue or
fluid. In some embodiments, a biological sample is or comprises
bone marrow; blood; blood cells; ascites; tissue or fine needle
biopsy samples; cell-containing body fluids; free floating nucleic
acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal
fluid; pleural fluid; feces; lymph; gynecological fluids; skin
swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages
such as a ductal lavages or broncheoalveolar lavages; aspirates;
scrapings; bone marrow specimens; tissue biopsy specimens; surgical
specimens; feces, other body fluids, secretions and/or excretions;
and/or cells therefrom, etc. In some embodiments, a biological
sample is or comprises cells obtained from an individual. In some
embodiments, a sample is a "primary sample" obtained directly from
a source of interest by any appropriate means. For example, in some
embodiments, a primary biological sample is obtained by methods
selected from the group consisting of biopsy (e.g., fine needle
aspiration or tissue biopsy), surgery, collection of body fluid
(e.g., blood, lymph, feces etc.), etc. In some embodiments, as will
be clear from context, the term "sample" refers to a preparation
that is obtained by processing (e.g., by removing one or more
components of and/or by adding one or more agents to) a primary
sample. For example, filtering using a semi-permeable membrane.
Such a "processed sample" may comprise, for example nucleic acids
or proteins extracted from a sample or obtained by subjecting a
primary sample to techniques such as amplification or reverse
transcription of mRNA, isolation and/or purification of certain
components, etc. In some embodiments, a sample is an organism. In
some embodiments, a sample is a plant. In some embodiments, a
sample is an animal. In some embodiments, a sample is a human. In
some embodiments, a sample is an organism other than a human.
[0078] Subject: As used herein, the term "subject" or "test
subject" refers to any organism to which a provided compound or
composition is administered in accordance with the present
disclosure e.g., for experimental, diagnostic, prophylactic and/or
therapeutic purposes. Typical subjects include animals (e.g.,
mammals such as mice, rats, rabbits, non-human primates, and
humans; insects; worms; etc.) and plants. In some embodiments, a
subject may be suffering from and/or susceptible to a disease,
disorder and/or condition.
[0079] Substantially: As used herein, the term "substantially"
refers to the qualitative condition of exhibiting total or
near-total extent or degree of a characteristic or property of
interest. A base sequence which is substantially complementary to a
second sequence is not identical to the second sequence, but is
mostly or nearly identical to the second sequence. In addition, one
of ordinary skill in the biological arts will understand that
biological and chemical phenomena rarely, if ever, go to completion
and/or proceed to completeness or achieve or avoid an absolute
result. The term "substantially" is therefore used herein to
capture the potential lack of completeness inherent in many
biological and/or chemical phenomena.
[0080] Suffering from: An individual who is "suffering from" a
disease, disorder and/or condition has been diagnosed with and/or
displays one or more symptoms of a disease, disorder and/or
condition.
[0081] Susceptible to: An individual who is "susceptible to" a
disease, disorder and/or condition is one who has a higher risk of
developing the disease, disorder and/or condition than does a
member of the general public. In some embodiments, an individual
who is susceptible to a disease, disorder and/or condition is
predisposed to have that disease, disorder and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder and/or condition may not have been diagnosed with the
disease, disorder and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder and/or
condition may exhibit symptoms of the disease, disorder and/or
condition. In some embodiments, an individual who is susceptible to
a disease, disorder and/or condition may not exhibit symptoms of
the disease, disorder and/or condition. In some embodiments, an
individual who is susceptible to a disease, disorder, and/or
condition will develop the disease, disorder, and/or condition. In
some embodiments, an individual who is susceptible to a disease,
disorder, and/or condition will not develop the disease, disorder,
and/or condition.
[0082] Systemic: The phrases "systemic administration,"
"administered systemically," "peripheral administration," and
"administered peripherally" as used herein have their
art-understood meaning referring to administration of a compound or
composition such that it enters the recipient's system.
[0083] Therapeutic agent: As used herein, the phrase "therapeutic
agent" refers to any agent that, when administered to a subject,
has a therapeutic effect and/or elicits a desired biological and/or
pharmacological effect. In some embodiments, a therapeutic agent is
any substance that can be used to alleviate, ameliorate, relieve,
inhibit, prevent, delay onset of, reduce severity of, and/or reduce
incidence of one or more symptoms or features of a disease,
disorder, and/or condition.
[0084] Therapeutically effective amount: As used herein, the term
"therapeutically effective amount" means an amount of a substance
(e.g., a therapeutic agent, composition, and/or formulation) that
elicits a desired biological response when administered as part of
a therapeutic regimen. In some embodiments, a therapeutically
effective amount of a substance is an amount that is sufficient,
when administered to a subject suffering from or susceptible to a
disease, disorder, and/or condition, to treat, diagnose, prevent,
and/or delay the onset of the disease, disorder, and/or condition.
As will be appreciated by those of ordinary skill in this art, the
effective amount of a substance may vary depending on such factors
as the desired biological endpoint, the substance to be delivered,
the target cell or tissue, etc. For example, the effective amount
of compound in a formulation to treat a disease, disorder, and/or
condition is the amount that alleviates, ameliorates, relieves,
inhibits, prevents, delays onset of, reduces severity of and/or
reduces incidence of one or more symptoms or features of the
disease, disorder, and/or condition. In some embodiments, a
therapeutically effective amount is administered in a single dose;
in some embodiments, multiple unit doses are required to deliver a
therapeutically effective amount.
[0085] Treat: As used herein, the term "treat," "treatment," or
"treating" refers to any method used to partially or completely
alleviate, ameliorate, relieve, inhibit, prevent, delay onset of,
reduce severity of, and/or reduce incidence of one or more symptoms
or features of a disease, disorder, and/or condition. Treatment may
be administered to a subject who does not exhibit signs of a
disease, disorder, and/or condition. In some embodiments, treatment
may be administered to a subject who exhibits only early signs of
the disease, disorder, and/or condition, for example for the
purpose of decreasing the risk of developing pathology associated
with the disease, disorder, and/or condition.
[0086] Unsaturated: The term "unsaturated," as used herein, means
that a moiety has one or more units of unsaturation.
[0087] Unit dose: The expression "unit dose" as used herein refers
to an amount administered as a single dose and/or in a physically
discrete unit of a pharmaceutical composition. In many embodiments,
a unit dose contains a predetermined quantity of an active agent.
In some embodiments, a unit dose contains an entire single dose of
the agent. In some embodiments, more than one unit dose is
administered to achieve a total single dose. In some embodiments,
administration of multiple unit doses is required, or expected to
be required, in order to achieve an intended effect. A unit dose
may be, for example, a volume of liquid (e.g., an acceptable
carrier) containing a predetermined quantity of one or more
therapeutic agents, a predetermined amount of one or more
therapeutic agents in solid form, a sustained release formulation
or drug delivery device containing a predetermined amount of one or
more therapeutic agents, etc. It will be appreciated that a unit
dose may be present in a formulation that includes any of a variety
of components in addition to the therapeutic agent(s). For example,
acceptable carriers (e.g., pharmaceutically acceptable carriers),
diluents, stabilizers, buffers, preservatives, etc., may be
included as described infra. It will be appreciated by those
skilled in the art, in many embodiments, a total appropriate daily
dosage of a particular therapeutic agent may comprise a portion, or
a plurality, of unit doses, and may be decided, for example, by the
attending physician within the scope of sound medical judgment. In
some embodiments, the specific effective dose level for any
particular subject or organism may depend upon a variety of factors
including the disorder being treated and the severity of the
disorder; activity of specific active compound employed; specific
composition employed; age, body weight, general health, sex and
diet of the subject; time of administration, and rate of excretion
of the specific active compound employed; duration of the
treatment; drugs and/or additional therapies used in combination or
coincidental with specific compound(s) employed, and like factors
well known in the medical arts.
[0088] Wild-type: As used herein, the term "wild-type" has its
art-understood meaning that refers to an entity having a structure
and/or activity as found in nature in a "normal" (as contrasted
with mutant, diseased, altered, etc) state or context. Those of
ordinary skill in the art will appreciate that wild type genes and
polypeptides often exist in multiple different forms (e.g.,
alleles).
[0089] Nucleic acid: The term "nucleic acid", as used herein,
includes any nucleotides and polymers thereof. The term
"polynucleotide", as used herein, refers to a polymeric form of
nucleotides of any length, either ribonucleotides (RNA) or
deoxyribonucleotides (DNA). These terms refer to the primary
structure of the molecules and, thus, include double- and
single-stranded DNA, and double- and single-stranded RNA. These
terms include, as equivalents, analogs of either RNA or DNA made
from modified nucleotides and/or modified polynucleotides, such as,
though not limited to, methylated, protected and/or capped
nucleotides or polynucleotides. The terms encompass poly- or
oligo-ribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides
(DNA); RNA or DNA derived from N-glycosides or C-glycosides of
nucleobases and/or modified nucleobases; nucleic acids derived from
sugars and/or modified sugars; and nucleic acids derived from
phosphate bridges and/or modified internucleotide linkages. The
term encompasses nucleic acids containing any combinations of
nucleobases, modified nucleobases, sugars, modified sugars,
phosphate bridges or modified internucleotidic linkages. Examples
include, and are not limited to, nucleic acids containing ribose
moieties, nucleic acids containing deoxy-ribose moieties, nucleic
acids containing both ribose and deoxyribose moieties, nucleic
acids containing ribose and modified ribose moieties. Unless
otherwise specified, the prefix poly--refers to a nucleic acid
containing 2 to about 10,000 nucleotide monomer units and wherein
the prefix oligo--refers to a nucleic acid containing 2 to about
200 nucleotide monomer units.
[0090] Nucleotide: The term "nucleotide" as used herein refers to a
monomeric unit of a polynucleotide that consists of a nucleobase, a
sugar, and one or more internucleotidic linkages. The naturally
occurring bases (guanine, (G), adenine, (A), cytosine, (C),
thymine, (T), and uracil (U)) are derivatives of purine or
pyrimidine, though it should be understood that naturally and
non-naturally occurring base analogs are also included. The
naturally occurring sugar is the pentose (five-carbon sugar)
deoxyribose (which forms DNA) or ribose (which forms RNA), though
it should be understood that naturally and non-naturally occurring
sugar analogs are also included. Nucleotides are linked via
internucleotidic linkages to form nucleic acids, or
polynucleotides. Many internucleotidic linkages are known in the
art (such as, though not limited to, phosphate, phosphorothioates,
boranophosphates and the like). Artificial nucleic acids include
PNAs (peptide nucleic acids), phosphotriesters, phosphorothionates,
H-phosphonates, phosphoramidates, boranophosphates,
methylphosphonates, phosphonoacetates, thiophosphonoacetates and
other variants of the phosphate backbone of native nucleic acids,
such as those described herein. In some embodiments, a natural
nucleotide comprises a naturally occurring base, sugar and
internucleotidic linkage. As used herein, the term "nucleotide"
also encompasses structural analogs used in lieu of natural or
naturally-occurring nucleotides, such as modified nucleotides and
nucleotide analogs.
[0091] Modified nucleotide: The term "modified nucleotide" includes
any chemical moiety which differs structurally from a natural
nucleotide but is capable of performing at least one function of a
natural nucleotide. In some embodiments, a modified nucleotide
comprises a modification at a sugar, base and/or internucleotidic
linkage. In some embodiments, a modified nucleotide comprises a
modified sugar, modified nucleobase and/or modified
internucleotidic linkage. In some embodiments, a modified
nucleotide is capable of at least one function of a nucleotide,
e.g., forming a subunit in a polymer capable of base-pairing to a
nucleic acid comprising an at least complementary sequence of
bases.
[0092] Analog: The term "analog" includes any chemical moiety which
differs structurally from a reference chemical moiety or class of
moieties, but which is capable of performing at least one function
of such a reference chemical moiety or class of moieties. As
non-limiting examples, a nucleotide analog differs structurally
from a nucleotide but performs at least one function of a
nucleotide; a nucleobase analog differs structurally from a
nucleobase but performs at least one function of a nucleobase;
etc.
[0093] Nucleoside: The term "nucleoside" refers to a moiety wherein
a nucleobase or a modified nucleobase is covalently bound to a
sugar or a modified sugar.
[0094] Modified nucleoside: The term "modified nucleoside" refers
to a moiety derived from or chemically similar to a natural
nucleoside, but which comprises a chemical modification which
differentiates it from a natural nucleoside. Non-limiting examples
of modified nucleosides include those which comprise a modification
at the base and/or the sugar. Non-limiting examples of modified
nucleosides include those with a 2' modification at a sugar.
Non-limiting examples of modified nucleosides also include abasic
nucleosides (which lack a nucleobase). In some embodiments, a
modified nucleoside is capable of at least one function of a
nucleoside, e.g., forming a moiety in a polymer capable of
base-pairing to a nucleic acid comprising an at least complementary
sequence of bases.
[0095] Nucleoside analog: The term "nucleoside analog" refers to a
chemical moiety which is chemically distinct from a natural
nucleoside, but which is capable of performing at least one
function of a nucleoside. In some embodiments, a nucleoside analog
comprises an analog of a sugar and/or an analog of a nucleobase. In
some embodiments, a modified nucleoside is capable of at least one
function of a nucleoside, e.g., forming a moiety in a polymer
capable of base-pairing to a nucleic acid comprising a
complementary sequence of bases.
[0096] Sugar: The term "sugar" refers to a monosaccharide or
polysaccharide in closed and/or open form. In some embodiments,
sugars are monosaccharides. In some embodiments, sugars are
polysaccharides. Sugars include, but are not limited to, ribose,
deoxyribose, pentofuranose, pentopyranose, and hexopyranose
moieties. As used herein, the term "sugar" also encompasses
structural analogs used in lieu of conventional sugar molecules,
such as glycol, polymer of which forms the backbone of the nucleic
acid analog, glycol nucleic acid ("GNA"), etc. As used herein, the
term "sugar" also encompasses structural analogs used in lieu of
natural or naturally-occurring nucleotides, such as modified sugars
and nucleotide sugars.
[0097] Modified sugar: The term "modified sugar" refers to a moiety
that can replace a sugar. A modified sugar mimics the spatial
arrangement, electronic properties, or some other physicochemical
property of a sugar.
[0098] Nucleobase: The term "nucleobase" refers to the parts of
nucleic acids that are involved in the hydrogen-bonding that binds
one nucleic acid strand to another complementary strand in a
sequence specific manner. The most common naturally-occurring
nucleobases are adenine (A), guanine (G), uracil (U), cytosine (C),
and thymine (T). In some embodiments, the naturally-occurring
nucleobases are modified adenine, guanine, uracil, cytosine, or
thymine. In some embodiments, the naturally-occurring nucleobases
are methylated adenine, guanine, uracil, cytosine, or thymine. In
some embodiments, a nucleobase is a "modified nucleobase," e.g., a
nucleobase other than adenine (A), guanine (G), uracil (U),
cytosine (C), and thymine (T). In some embodiments, the modified
nucleobases are methylated adenine, guanine, uracil, cytosine, or
thymine. In some embodiments, the modified nucleobase mimics the
spatial arrangement, electronic properties, or some other
physicochemical property of the nucleobase and retains the property
of hydrogen-bonding that binds one nucleic acid strand to another
in a sequence specific manner. In some embodiments, a modified
nucleobase can pair with all of the five naturally occurring bases
(uracil, thymine, adenine, cytosine, or guanine) without
substantially affecting the melting behavior, recognition by
intracellular enzymes or activity of the oligonucleotide duplex. As
used herein, the term "nucleobase" also encompasses structural
analogs used in lieu of natural or naturally-occurring nucleotides,
such as modified nucleobases and nucleobase analogs.
[0099] Modified nucleobase: The terms "modified nucleobase",
"modified base" and the like refer to a chemical moiety which is
chemically distinct from a nucleobase, but which is capable of
performing at least one function of a nucleobase. In some
embodiments, a modified nucleobase is a nucleobase which comprises
a modification. In some embodiments, a modified nucleobase is
capable of at least one function of a nucleobase, e.g., forming a
moiety in a polymer capable of base-pairing to a nucleic acid
comprising an at least complementary sequence of bases.
[0100] Blocking group: The term "blocking group" refers to a group
that masks the reactivity of a functional group. The functional
group can be subsequently unmasked by removal of the blocking
group. In some embodiments, a blocking group is a protecting
group.
[0101] Moiety: The term "moiety" refers to a specific segment or
functional group of a molecule. Chemical moieties are often
recognized chemical entities embedded in or appended to a
molecule.
[0102] Solid support: The term "solid support" refers to any
support which enables synthesis of nucleic acids. In some
embodiments, the term refers to a glass or a polymer, that is
insoluble in the media employed in the reaction steps performed to
synthesize nucleic acids, and is derivatized to comprise reactive
groups. In some embodiments, the solid support is Highly
Cross-linked Polystyrene (HCP) or Controlled Pore Glass (CPG). In
some embodiments, the solid support is Controlled Pore Glass (CPG).
In some embodiments, the solid support is hybrid support of
Controlled Pore Glass (CPG) and Highly Cross-linked Polystyrene
(HCP).
[0103] Homology: "Homology" or "identity" or "similarity" refers to
sequence similarity between two nucleic acid molecules. Homology
and identity can each be determined by comparing a position in each
sequence which can be aligned for purposes of comparison. When an
equivalent position in the compared sequences is occupied by the
same base, then the molecules are identical at that position; when
the equivalent site occupied by the same or a similar nucleic acid
residue (e.g., similar in steric and/or electronic nature), then
the molecules can be referred to as homologous (similar) at that
position. Expression as a percentage of homology/similarity or
identity refers to a function of the number of identical or similar
nucleic acids at positions shared by the compared sequences. A
sequence which is "unrelated" or "non-homologous" shares less than
40% identity, less than 35% identity, less than 30% identity, or
less than 25% identity with a sequence described herein. In
comparing two sequences, the absence of residues (amino acids or
nucleic acids) or presence of extra residues also decreases the
identity and homology/similarity.
[0104] In some embodiments, the term "homology" describes a
mathematically based comparison of sequence similarities which is
used to identify genes with similar functions or motifs. The
nucleic acid sequences described herein can be used as a "query
sequence" to perform a search against public databases, for
example, to identify other family members, related sequences or
homologs. In some embodiments, such searches can be performed using
the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.
(1990) J. Mol. Biol. 215:403-10. In some embodiments, BLAST
nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to nucleic acid molecules of the disclosure. In some embodiments,
to obtain gapped alignments for comparison purposes, Gapped BLAST
can be utilized as described in Altschul et al., (1997) Nucleic
Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and BLAST) can be used (See www.ncbi.nlm.nih.gov).
[0105] Identity: As used herein, "identity" means the percentage of
identical nucleotide residues at corresponding positions in two or
more sequences when the sequences are aligned to maximize sequence
matching, i.e., taking into account gaps and insertions. Identity
can be readily calculated by known methods, including but not
limited to those known in the art, including but not limited to
those cited in WO2017/192679.
[0106] Oligonucleotide: The term "oligonucleotide" refers to a
polymer or oligomer of nucleotides, and may contain any combination
of natural and non-natural nucleobases, sugars, and
internucleotidic linkages.
[0107] Oligonucleotides can be single-stranded or double-stranded.
A single-stranded oligonucleotide can have double-stranded regions
(formed by two portions of the single-stranded oligonucleotide) and
a double-stranded oligonucleotide, which comprises two
oligonucleotide chains, can have single-stranded regions for
example, at regions where the two oligonucleotide chains are not
complementary to each other. Example oligonucleotides include, but
are not limited to structural genes, genes including control and
termination regions, self-replicating systems such as viral or
plasmid DNA, single-stranded and double-stranded RNAi agents and
other RNA interference reagents (RNAi agents or iRNA agents),
shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA
mimics, supermirs, aptamers, antimirs, antagomirs, UI adaptors,
triplex-forming oligonucleotides, G-quadruplex oligonucleotides,
RNA activators, immuno-stimulatory oligonucleotides, and decoy
oligonucleotides.
[0108] Internucleotidic linkage: As used herein, the phrase
"internucleotidic linkage" refers generally to a linkage linking
nucleoside units of an oligonucleotide or a nucleic acid. In some
embodiments, an internucleotidic linkage is a phosphodiester
linkage, as found in naturally occurring DNA and RNA molecules
(natural phosphate linkage). In some embodiments, an
internucleotidic linkage includes a modified internucleotidic
linkage. In some embodiments, an internucleotidic linkage is a
"modified internucleotidic linkage" wherein each oxygen atom of the
phosphodiester linkage is optionally and independently replaced by
an organic or inorganic moiety. In some embodiments, such an
organic or inorganic moiety is selected from but not limited to
.dbd.S, .dbd.Se, .dbd.NR', --SR', --SeR', --N(R').sub.2,
B(R').sub.3, --S--, --Se--, and --N(R')--, wherein each R' is
independently as defined and described in the present disclosure.
In some embodiments, an internucleotidic linkage is a
phosphotriester linkage, phosphorothioate diester linkage
##STR00001##
or modified phosphorothioate triester linkage. In some embodiments,
an internucleotidic linkage is one of, e.g., PNA (peptide nucleic
acid) or PMO (phosphorodiamidate Morpholino oligomer) linkage. It
is understood by a person of ordinary skill in the art that an
internucleotidic linkage may exist as an anion or cation at a given
pH due to the existence of acid or base moieties in the
linkage.
[0109] Non-limiting examples of modified internucleotidic linkages
are modified internucleotidic linkages designated s, s1, s2, s3,
s4, s5, s6, s7, s8, s9, s10, s11, s12, s13, s14, s15, s16, s17 and
s18 as described in WO 2017/210647.
[0110] For instance, (Rp, Sp)-ATsCs1GA has 1) a phosphorothioate
internucleotidic linkage
##STR00002##
[0111] between T and C; and 2) a phosphorothioate triester
internucleotidic linkage having the structure of
##STR00003##
between C and G. Unless otherwise specified, the Rp/Sp designations
preceding an oligonucleotide sequence describe the configurations
of chiral linkage phosphorus atoms in the internucleotidic linkages
sequentially from 5' to 3' of the oligonucleotide sequence. For
instance, in (Rp, Sp)-ATsCs1GA, the phosphorus in the "s" linkage
between T and C has Rp configuration and the phosphorus in "s1"
linkage between C and G has Sp configuration. In some embodiments,
"All-(Rp)" or "All-(Sp)" is used to indicate that all chiral
linkage phosphorus atoms in oligonucleotide have the same Rp or Sp
configuration, respectively.
[0112] Oligonucleotide type: As used herein, the phrase
"oligonucleotide type" is used to define an oligonucleotide that
has a particular base sequence, pattern of backbone linkages (i.e.,
pattern of internucleotidic linkage types, for example, phosphate,
phosphorothioate, etc.), pattern of backbone chiral centers (i.e.
pattern of linkage phosphorus stereochemistry (Rp/Sp)), and pattern
of backbone phosphorus modifications (e.g., pattern of "-XLR.sup.1"
groups in formula I). In some embodiments, oligonucleotides of a
common designated "type" are structurally identical to one
another.
[0113] One of skill in the art will appreciate that synthetic
methods of the present disclosure provide for a degree of control
during the synthesis of an oligonucleotide strand such that each
nucleotide unit of the oligonucleotide strand can be designed
and/or selected in advance to have a particular stereochemistry at
the linkage phosphorus and/or a particular modification at the
linkage phosphorus, and/or a particular base, and/or a particular
sugar. In some embodiments, an oligonucleotide strand is designed
and/or selected in advance to have a particular combination of
stereocenters at the linkage phosphorus. In some embodiments, an
oligonucleotide strand is designed and/or determined to have a
particular combination of modifications at the linkage phosphorus.
In some embodiments, an oligonucleotide strand is designed and/or
selected to have a particular combination of bases. In some
embodiments, an oligonucleotide strand is designed and/or selected
to have a particular combination of one or more of the above
structural characteristics. In some embodiments, the present
disclosure provides compositions comprising or consisting of a
plurality of oligonucleotide molecules (e.g., chirally controlled
oligonucleotide compositions). In some embodiments, all such
molecules are of the same type (i.e., are structurally identical to
one another). In many embodiments, however, provided compositions
comprise a plurality of oligonucleotides of different types,
typically in pre-determined relative amounts.
[0114] Chiral control: As used herein, "chiral control" refers to
control of the stereochemical designation of a chiral linkage
phosphorus in a chiral internucleotidic linkage within an
oligonucleotide. In some embodiments, a control is achieved through
a chiral element that is absent from the sugar and base moieties of
an oligonucleotide, for example, in some embodiments, a control is
achieved through use of one or more chiral auxiliaries during
oligonucleotide preparation as exemplified in the present
disclosure, which chiral auxiliaries often are part of chiral
phosphoramidites used during oligonucleotide preparation. In
contrast to chiral control, a person having ordinary skill in the
art appreciates that conventional oligonucleotide synthesis which
does not use chiral auxiliaries cannot control stereochemistry at a
chiral internucleotidic linkage if such conventional
oligonucleotide synthesis is used to form the chiral
internucleotidic linkage. In some embodiments, the stereochemical
designation of each chiral linkage phosphorus in a chiral
internucleotidic linkage within an oligonucleotide is
controlled.
[0115] Chirally controlled oligonucleotide composition: The terms
"chirally controlled oligonucleotide composition", "chirally
controlled nucleic acid composition", and the like, as used herein,
refers to a composition that comprises a plurality of
oligonucleotides (or nucleic acids) which share 1) a common base
sequence, 2) a common pattern of backbone linkages, and 3) a common
pattern of backbone phosphorus modifications, wherein the plurality
of oligonucleotides (or nucleic acids) share the same
stereochemistry at one or more chiral internucleotidic linkages
(chirally controlled internucleotidic linkages, whose chiral
linkage phosphorus is Rp or Sp in the composition, not a random Rp
and Sp mixture as non-chirally controlled internucleotidic
linkage). Level of the plurality of oligonucleotides (or nucleic
acids) in a chirally controlled oligonucleotide composition is
pre-determined/controlled (e.g., through chirally controlled
oligonucleotide preparation to stereoselectively form one or more
chiral internucleotidic linkages). In some embodiments, about
1%-100%, (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%,
40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%,
50%-90%, or about 5% 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a
chirally controlled oligonucleotide composition are
oligonucleotides of the plurality. In some embodiments, about
1%-100%, (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%,
40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%,
50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least
5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99%) of all oligonucleotides in a
chirally controlled oligonucleotide composition that share the
common base sequence, the common pattern of backbone linkages, and
the common pattern of backbone phosphorus modifications are
oligonucleotides of the plurality. In some embodiments, a
predetermined level is be about 1%-100%, (e.g., about 5%-100%,
10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%,
70%-100%, 80-100%, 90-100%, 95-100%, 50%-90%, or about 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%) of all oligonucleotides in a composition, or of all
oligonucleotides in a composition that share a common base sequence
(e.g., of a plurality of oligonucleotide or an oligonucleotide
type), or of all oligonucleotides in a composition that share a
common base sequence, a common pattern of backbone linkages, and a
common pattern of backbone phosphorus modifications are
oligonucleotides of the plurality, or of all oligonucleotides in a
composition that share a common base sequence, a common patter of
base modifications, a common pattern of sugar modifications, a
common pattern of internucleotidic linkage types, and/or a common
pattern of internucleotidic linkage modifications. In some
embodiments, the plurality of oligonucleotides share the same
stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20,
10-15, 10-20, 10-25, 10-30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20)
chiral internucleotidic linkages. In some embodiments, the
plurality of oligonucleotides share the same stereochemistry at
about 1%-100% (e.g., about 5%-100%, 10%-100%, 20%-100%, 30%-100%,
40%-100%, 50%-100%, 60%-100%, 70%-100%, 80-100%, 90-100%, 95-100%,
50%-90%, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, or 99%) of chiral internucleotidic
linkages. In some embodiments, each chiral internucleotidic linkage
is a chiral controlled internucleotidic linkage, and the
composition is a completely chirally controlled oligonucleotide
composition. In some embodiments, not all chiral internucleotidic
linkages are chiral controlled internucleotidic linkages, and the
composition is a partially chirally controlled oligonucleotide
composition. In some embodiments, a chirally controlled
oligonucleotide composition comprises non-random or controlled
levels of individual oligonucleotide or nucleic acids types. For
instance, in some embodiments a chirally controlled oligonucleotide
composition comprises one oligonucleotide type. In some
embodiments, a chirally controlled oligonucleotide composition
comprises more than one oligonucleotide type. In some embodiments,
a chirally controlled oligonucleotide composition comprises
multiple oligonucleotide types. In some embodiments, a chirally
controlled oligonucleotide composition is a composition of
oligonucleotides of a oligonucleotide type, which composition
comprises a non-random or controlled level of a plurality of
oligonucleotides of the oligonucleotide type.
[0116] Chirally pure: as used herein, the phrase "chirally pure" is
used to describe an oligonucleotide or compositions thereof, in
which all are nearly all (the rest are impurities) of the
oligonucleotide molecules exist in a single diastereomeric form
with respect to the linkage phosphorus atoms.
[0117] Predetermined: By predetermined (or pre-determined) is meant
deliberately selected or non-random or controlled, for example as
opposed to randomly occurring, random, or achieved without control.
Those of ordinary skill in the art, reading the present
specification, will appreciate that the present disclosure provides
technologies that permit selection of particular chemistry and/or
stereochemistry features to be incorporated into oligonucleotide
compositions, and further permits controlled preparation of
oligonucleotide compositions having such chemistry and/or
stereochemistry features. Such provided compositions are
"predetermined" as described herein. Compositions that may contain
certain oligonucleotides because they happen to have been generated
through a process that are not controlled to intentionally generate
the particular chemistry and/or stereochemistry features are not
"predetermined" compositions. In some embodiments, a predetermined
composition is one that can be intentionally reproduced (e.g.,
through repetition of a controlled process). In some embodiments, a
predetermined level of a plurality of oligonucleotides in a
composition means that the absolute amount, and/or the relative
amount (ratio, percentage, etc.) of the plurality of
oligonucleotides in the composition is controlled. In some
embodiments, a predetermined level of a plurality of
oligonucleotides in a composition is achieved through chirally
controlled oligonucleotide preparation.
[0118] Linkage phosphorus: as defined herein, the phrase "linkage
phosphorus" is used to indicate that the particular phosphorus atom
being referred to is the phosphorus atom present in the
internucleotidic linkage, which phosphorus atom corresponds to the
phosphorus atom of a phosphodiester internucleotidic linkage as
occurs in naturally occurring DNA and RNA. In some embodiments, a
linkage phosphorus atom is in a modified internucleotidic linkage,
wherein each oxygen atom of a phosphodiester linkage is optionally
and independently replaced by an organic or inorganic moiety. In
some embodiments, a linkage phosphorus atom is the P of Formula I.
In some embodiments, a linkage phosphorus atom is chiral. In some
embodiments, a linkage phosphorus atom is achiral.
[0119] P-modification: as used herein, the term "P-modification"
refers to any modification at the linkage phosphorus other than a
stereochemical modification. In some embodiments, a P-modification
comprises addition, substitution, or removal of a pendant moiety
covalently attached to a linkage phosphorus. In some embodiments,
the "P-modification" is --X-L-R.sup.1 wherein each of X, L and R'
is independently as defined and described in the present
disclosure.
[0120] Blockmer: the term "blockmer," as used herein, refers to an
oligonucleotide strand whose pattern of structural features
characterizing each individual nucleotide unit is characterized by
the presence of at least two consecutive nucleotide units sharing a
common structural feature at the internucleotidic phosphorus
linkage. By common structural feature is meant common
stereochemistry at the linkage phosphorus or a common modification
at the linkage phosphorus. In some embodiments, the at least two
consecutive nucleotide units sharing a common structure feature at
the internucleotidic phosphorus linkage are referred to as a
"block". In some embodiments, a provided oligonucleotide is a
blockmer.
[0121] In some embodiments, a blockmer is a "stereoblockmer," e.g.,
at least two consecutive nucleotide units have the same
stereochemistry at the linkage phosphorus. Such at least two
consecutive nucleotide units form a "stereoblock."
[0122] In some embodiments, a blockmer is a "P-modification
blockmer," e.g., at least two consecutive nucleotide units have the
same modification at the linkage phosphorus. Such at least two
consecutive nucleotide units form a "P-modification block". For
instance, (Rp, Sp)-ATsCsGA is a P-modification blockmer because at
least two consecutive nucleotide units, the Ts and the Cs, have the
same P-modification (i.e., both are a phosphorothioate diester). In
the same oligonucleotide of (Rp, Sp)-ATsCsGA, TsCs forms a block,
and it is a P-modification block.
[0123] In some embodiments, a blockmer is a "linkage blockmer,"
e.g., at least two consecutive nucleotide units have identical
stereochemistry and identical modifications at the linkage
phosphorus. At least two consecutive nucleotide units form a
"linkage block". For instance, (Rp, Rp)-ATsCsGA is a linkage
blockmer because at least two consecutive nucleotide units, the Ts
and the Cs, have the same stereochemistry (both Rp) and
P-modification (both phosphorothioate). In the same oligonucleotide
of (Rp, Rp)-ATsCsGA, TsCs forms a block, and it is a linkage
block.
[0124] In some embodiments, a blockmer comprises one or more blocks
independently selected from a stereoblock, a P-modification block
and a linkage block. In some embodiments, a blockmer is a
stereoblockmer with respect to one block, and/or a P-modification
blockmer with respect to another block, and/or a linkage blockmer
with respect to yet another block.
[0125] Altmer: the term "altmer," as used herein, refers to an
oligonucleotide strand whose pattern of structural features
characterizing each individual nucleotide unit is characterized in
that no two consecutive nucleotide units of the oligonucleotide
strand share a particular structural feature at the
internucleotidic phosphorus linkage. In some embodiments, an altmer
is designed such that it comprises a repeating pattern. In some
embodiments, an altmer is designed such that it does not comprise a
repeating pattern. In some embodiments, a provided oligonucleotide
is a altmer.
[0126] In some embodiments, an altmer is a "stereoaltmer," e.g., no
two consecutive nucleotide units have the same stereochemistry at
the linkage phosphorus.
[0127] In some embodiments, an altmer is a "P-modification altmer"
e.g., no two consecutive nucleotide units have the same
modification at the linkage phosphorus. For instance,
All-(Sp)-CAs1GsT, in which each linkage phosphorus has a different
P-modification than the others.
[0128] In some embodiments, an altmer is a "linkage altmer," e.g.,
no two consecutive nucleotide units have identical stereochemistry
or identical modifications at the linkage phosphorus.
[0129] Unimer: the term "unimer," as used herein, refers to an
oligonucleotide strand whose pattern of structural features
characterizing each individual nucleotide unit is such that all
nucleotide units within the strand share at least one common
structural feature at the internucleotidic phosphorus linkage. By
common structural feature is meant common stereochemistry at the
linkage phosphorus or a common modification at the linkage
phosphorus. In some embodiments, a provided oligonucleotide is a
unimer.
[0130] In some embodiments, a unimer is a "stereounimer," e.g., all
nucleotide units have the same stereochemistry at the linkage
phosphorus.
[0131] In some embodiments, a unimer is a "P-modification unimer",
e.g., all nucleotide units have the same modification at the
linkage phosphorus.
[0132] In some embodiments, a unimer is a "linkage unimer," e.g.,
all nucleotide units have the same stereochemistry and the same
modifications at the linkage phosphorus.
[0133] Gapmer: as used herein, the term "gapmer" refers to an
oligonucleotide strand characterized in that at least one
internucleotidic phosphorus linkage of the oligonucleotide strand
is a phosphate diester linkage, for example such as those found in
naturally occurring DNA or RNA. In some embodiments, more than one
internucleotidic phosphorus linkage of the oligonucleotide strand
is a phosphate diester linkage such as those found in naturally
occurring DNA or RNA. In some embodiments, a provided
oligonucleotide is a gapmer.
[0134] Skipmer: as used herein, the term "skipmer" refers to a type
of gapmer in which every other internucleotidic phosphorus linkage
of the oligonucleotide strand is a phosphate diester linkage, for
example such as those found in naturally occurring DNA or RNA, and
every other internucleotidic phosphorus linkage of the
oligonucleotide strand is a modified internucleotidic linkage. In
some embodiments, a provided oligonucleotide is a skipmer.
[0135] For purposes of this disclosure, the chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87,
inside cover.
[0136] The methods and structures described herein relating to
compounds and compositions of the disclosure also apply to the
pharmaceutically acceptable acid or base addition salts and all
stereoisomeric forms of these compounds and compositions.
Description of Certain Embodiments
[0137] Oligonucleotides provide useful molecular tools in a wide
variety of applications. For example, oligonucleotides (e.g.,
oligonucleotides which target C9orf72) are useful in therapeutic,
diagnostic, and research applications, including the treatment of a
variety of conditions, disorders, and diseases. The use of
naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is
limited, for example, by their susceptibility to endo- and
exo-nucleases. As such, various synthetic counterparts have been
developed to circumvent these shortcomings. These include synthetic
oligonucleotides that contain chemical modifications, e.g., base
modifications, sugar modifications, backbone modifications, etc.,
which, among other things, render these molecules less susceptible
to degradation and improve other properties of oligonucleotides.
From a structural point of view, modifications to internucleotidic
linkages can introduce chirality, and certain properties of
oligonucleotides may be affected by configurations of phosphorus
atoms that form the backbone of oligonucleotides. For example, in
vitro studies have shown that properties of antisense
oligonucleotides, such as binding affinity, sequence specific
binding to complementary RNA, stability to nucleases, are affected
by, inter alia, chirality of backbone phosphorus atoms. Various
modifications are efficacious for C9orf72 oligonucleotides.
Oligonucleotides and Compositions
[0138] In some embodiments, the present disclosure provides an
oligonucleotide comprising a region of consecutive nucleotidic
units:
(Nu.sup.M)t[(Nu.sup.O)n(Nu.sup.M)m]y
wherein: [0139] each Nu.sup.M is independently a nucleotidic unit
comprising a modified internucleotidic linkage; [0140] each
Nu.sup.O is independently a nucleotidic unit comprising a natural
phosphate linkage; [0141] each of t, n, and m is independently
1-20; and [0142] y is 1-10.
[0143] In some embodiments, as demonstrated in the present
disclosure, such oligonucleotides provide improved properties,
e.g., improved stability, and/or activities.
[0144] In some embodiments, y is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In some embodiments, y is 1. In some embodiments, y is 2. In some
embodiments, y is 3. In some embodiments, y is 4. In some
embodiments, y is 5. In some embodiments, y is 6. In some
embodiments, y is 7. In some embodiments, y is 8. In some
embodiments, y is 9. In some embodiments, y is 10.
[0145] As defined herein, each Nu.sup.M independently comprises a
modified internucleotidic linkage. In some embodiments, a modified
internucleotidic linkage is a chiral internucleotidic linkage. In
some embodiments, a modified internucleotidic linkage is of formula
I or a salt form thereof. In some embodiments, a modified
internucleotidic linkage is chiral and is of formula I or a salt
form thereof. In some embodiments, a modified internucleotidic
linkage is a phosphorothioate diester linkage. In some embodiments,
a modified internucleotidic linkage is chiral and is chirally
controlled. In some embodiments, each modified internucleotidic
linkage is chirally controlled. In some embodiments,
internucleotidic linkage of Nu.sup.M is a chirally controlled
phosphorothioate diester linkage. In some embodiments, Nu.sup.M of
a provided oligonucleotides comprises different types of modified
internucleotidic linkages. In some embodiments, Nu.sup.M of a
provided oligonucleotides comprises chiral internucleotidic
linkages having linkage phosphorus atoms of different
configuration. In some embodiments, Nu.sup.M of a provided
oligonucleotides comprises different types of modified
internucleotidic linkages. In some embodiments, Nu.sup.M of a
provided oligonucleotides comprises chiral internucleotidic
linkages having linkage phosphorus atoms of different
configuration. In some embodiments, at least one chiral
internucleotidic linkage of Nu.sup.M is Sp at its linkage
phosphorus. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9,
or 10 Nu.sup.M each independently comprise a chiral
internucleotidic linkage of Sp at its linkage phosphorus. In some
embodiments, each chiral internucleotidic linkage of Nu.sup.M is Sp
at its linkage phosphorus. In some embodiments, at least one chiral
internucleotidic linkage of Nu.sup.M is Rp at its linkage
phosphorus. In some embodiments, at least one chiral
internucleotidic linkage of Nu.sup.M is Rp at its linkage
phosphorus, and at least one chiral internucleotidic linkage of
Nu.sup.M is Sp at its linkage phosphorus. Additional nucleotidic
unit comprising modified internucleotidic linkages suitable for
Nu.sup.M are known in the art and/or described in the present
disclosure and can be utilized in accordance with the present
disclosure.
[0146] As defined herein, each Nu.sup.O is independently a
nucleotidic unit comprising a natural phosphate linkage. In some
embodiments, at least one Nu.sup.O is a nucleotidic unit comprising
a natural phosphate linkage, wherein the natural phosphate linkage
is bonded to a 5'-nucleotidic unit and a carbon atom of the sugar
unit of the nucleotidic unit, wherein the carbon atom is bonded to
less than two hydrogen atoms. In some embodiments, each Nu.sup.O is
independently a nucleotidic unit comprising a natural phosphate
linkage, wherein the natural phosphate linkage is bonded to a
5'-nucleotidic unit and a carbon atom of the sugar unit of the
nucleotidic unit, wherein the carbon atom is bonded to less than
two hydrogen atoms. In some embodiments, at least one Nu.sup.O
comprises a structure of --C(R.sup.5s).sub.2--, which structure is
directly boned to the natural phosphate linkage of Nu.sup.O and a
ring moiety of the sugar unit of Nu.sup.O. In some embodiments,
each Nu.sup.O independently comprises a structure of
--C(R.sup.5s).sub.2--, which structure is directly boned to the
natural phosphate linkage of Nu.sup.O and a ring moiety of the
sugar unit of Nu.sup.O.
[0147] In some embodiments, each Nu.sup.O independently has the
structure of formula N-I:
##STR00004##
or a salt form thereof, wherein: [0148] BA is an optionally
substituted group selected from C.sub.1-30 cycloaliphatic,
C.sub.6-30 aryl, C.sub.5-30 heteroaryl having 1-10 heteroatoms,
C.sub.3-30 heterocyclyl having 1-10 heteroatoms, a natural
nucleobase moiety, and a modified nucleobase moiety; [0149] L.sup.O
is a natural phosphate linkage; [0150] L.sup.s is
--C(R.sup.5s).sub.2--, or L; [0151] each R.sup.5s and R.sup.s is
independently --F, --Cl, --Br, --I, --CN, --N.sub.3, --NO,
--NO.sub.2, -L-R', -L-OR', -L-SR', -L-N(R').sub.2, --O-L-OR',
--O-L-SR', or --O-L-N(R').sub.2; [0152] each L is independently a
covalent bond, or a bivalent, optionally substituted, linear or
branched group selected from a C.sub.1-30 aliphatic group and a
C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus,
boron and silicon, wherein one or more methylene units are
optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0153] Ring A
is an optionally substituted 3-20 membered monocyclic, bicyclic or
polycyclic ring having 0-10 heteroatoms; [0154] s is 0-20; [0155]
each R' is independently --R, --C(O)R, --C(O)OR, or --S(O).sub.2R;
and [0156] each R is independently --H, or an optionally
substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30
heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl,
C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having
1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms,
and 3-30 membered heterocyclyl having 1-10 heteroatoms, or [0157]
two R groups are optionally and independently taken together to
form a covalent bond, or. [0158] two or more R groups on the same
atom are optionally and independently taken together with the atom
to form an optionally substituted, 3-30 membered monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-10
heteroatoms; or [0159] two or more R groups on two or more atoms
are optionally and independently taken together with their
intervening atoms to form an optionally substituted, 3-30 membered
monocyclic, bicyclic or polycyclic ring having, in addition to the
intervening atoms, 0-10 heteroatoms.
[0160] In some embodiments,
##STR00005##
has the structure of
##STR00006##
wherein each of R.sup.1s, R.sup.2s, R.sup.3s, and R.sup.4s is
independently R.sup.s and as described in the present disclosure.
In some embodiments,
##STR00007##
has the structure of
##STR00008##
wherein each of R.sup.1s, R.sup.2s, R.sup.3s, and R.sup.4s is
independently as described in the present disclosure. In some
embodiments,
##STR00009##
has the structure of
##STR00010##
wherein each of R.sup.1s, R.sup.2s, R.sup.3s, and R.sup.4s is
independently as described in the present disclosure.
[0161] In some embodiments, L.sup.s is --C(R.sup.5s).sub.2--. In
some embodiments, one R.sup.5s is --H and L.sup.s is
--CHR.sup.5s--. In some embodiments, each R.sup.5s is independently
R. In some embodiments, In some embodiments, --C(R.sup.5s).sub.2--
is --C(R).sub.2--. In some embodiments, one R.sup.5s is --H and
--C(R.sup.5s).sub.2-- is --CHR--. In some embodiments, R is not
hydrogen. In some embodiments, R is optionally substituted
C.sub.1-6 aliphatic. In some embodiments, R is optionally
substituted C.sub.1-6 alkyl. In some embodiments, R is substituted.
In some embodiments, R is unsubstituted. In some embodiments, R is
methyl. Additional example R groups are widely described in the
present disclosure. In some embodiments, the C of
--C(R.sup.5s).sub.2-- is chiral and is R. In some embodiments, the
C of --C(R.sup.5s).sub.2-- is chiral and is S. In some embodiments,
--C(R.sup.5s).sub.2-- is --(R)--CHMe-. In some embodiments,
--C(R.sup.5s).sub.2-- is --(S)--CHMe-.
[0162] In some embodiments, a region of consecutive nucleotidic
units comprises a pattern of backbone chiral centers (linkage
phosphorus) of (Np)t[(Op)n(Sp)m]y, wherein each variable is
independently as described in the present disclosure. In some
embodiments, a region of consecutive nucleotidic units comprises a
pattern of backbone chiral centers (linkage phosphorus) of
(Sp)t[(Op)n(Sp)m]y, wherein each variable is independently as
described in the present disclosure.
[0163] In some embodiments, the present disclosure provides
oligonucleotides that comprise one or two wings and a core, and
comprise or are of a wing-core-wing, a core-wing, or a wing-core
structure. In some embodiments, provided oligonucleotides comprise
or are of a wing-core-wing structure. In some embodiments, provided
oligonucleotides comprise or are of a core-wing structure. In some
embodiments, provided oligonucleotides comprise or are of a
wing-core structure. In some embodiments, a core of is a region of
consecutive nucleotidic unit as described in the present
disclosure. In some embodiments, each wing independently comprises
one or more nucleobases as described in the present disclosure.
[0164] In some embodiments, a wing-core-wing motif is described as
"X-Y-Z", where "X" represents the length of the 5' wing, "Y"
represents the length of the core, and "Z" represents the length of
the 3' wing. In some embodiments, the core is positioned
immediately adjacent to each of the 5' wing and the 3' wing. In
some embodiments, X and Z are the same or different lengths and/or
have the same or different modifications or patterns of
modifications. In a preferred embodiment, Y is between 8 and 15
nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides.
In some embodiments, an oligonucleotide described herein has or
comprises a wing-core-wing structure of, for example 5-10-5,
5-10-4, 4-10-4, 4-10-3, 3-10-3, 2-10-2, 5-9-5, 5-9-4, 4-9-5, 5-8-5,
5-8-4, 4-8-5, 5-7-5, 4-7-5, 5-7-4, or 4-7-4. In some embodiments,
an oligonucleotide described herein has or comprises a wing-core or
core-wing structure of, for example 5-10, 8-4, 4-12, 12-4, 3-14,
16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8. In some
embodiments, a wing or a core is a block, and a wing-core,
core-wing, or wing-core-wing structure is a blockmer comprising two
or three blocks.
[0165] In some embodiments, an oligonucleotide has a
wing-core-wing-structure, wherein the length (in bases) of the
first wing is represented by X, the length of the core is
represented by Y and the length of the second wing is represented
by Z, wherein X--Y--Z is any of: 1-5-1, 1-6-1, 1-7-1, 1-8-1, 1-9-1,
1-10-1, 1-11-1, 1-12-1, 1-13-1, 1-14-1, 1-15-1, 1-16-1, 1-17-1,
1-18-1, 1-19-1, 1-20-1, 1-5-2, 1-6-2, 1-7-2, 1-8-2, 1-9-2, 1-10-2,
1-11-2, 1-12-2, 1-13-2, 1-14-2, 1-15-2, 1-16-2, 1-17-2, 1-18-2,
1-19-2, 1-20-2, 1-5-3, 1-6-3, 1-7-3, 1-8-3, 1-9-3, 1-10-3, 1-11-3,
1-12-3, 1-13-3, 1-14-3, 1-15-3, 1-16-3, 1-17-3, 1-18-3, 1-19-3,
1-20-3, 1-5-4, 1-6-4, 1-7-4, 1-8-4, 1-9-4, 1-10-4, 1-11-4, 1-12-4,
1-13-4, 1-14-4, 1-15-4, 1-16-4, 1-17-4, 1-18-4, 1-19-4, 1-20-4,
1-5-5, 1-6-5, 1-7-5, 1-8-5, 1-9-5, 1-10-5, 1-11-5, 1-12-5, 1-13-5,
1-14-5, 1-15-5, 1-16-5, 1-17-5, 1-18-5, 1-19-5, 1-20-5, 2-5-1,
2-6-1, 2-7-1, 2-8-1, 2-9-1, 2-10-1, 2-12-1, 2-12-1, 2-13-1, 2-14-1,
2-15-1, 2-16-1, 2-17-1, 2-18-1, 2-19-1, 2-20-1, 2-5-2, 2-6-2,
2-7-2, 2-8-2, 2-9-2, 2-10-2, 2-12-2, 2-12-2, 2-13-2, 2-14-2,
2-15-2, 2-16-2, 2-17-2, 2-18-2, 2-19-2, 2-20-2, 2-5-3, 2-6-3,
2-7-3, 2-8-3, 2-9-3, 2-10-3, 2-12-3, 2-12-3, 2-13-3, 2-14-3,
2-15-3, 2-16-3, 2-17-3, 2-18-3, 2-19-3, 2-20-3, 2-5-4, 2-6-4,
2-7-4, 2-8-4, 2-9-4, 2-10-4, 2-12-4, 2-12-4, 2-13-4, 2-14-4,
2-15-4, 2-16-4, 2-17-4, 2-18-4, 2-19-4, 2-20-4, 2-5-5, 2-6-5,
2-7-5, 2-8-5, 2-9-5, 2-10-5, 2-12-5, 2-12-5, 2-13-5, 2-14-5,
2-15-5, 2-16-5, 2-17-5, 2-18-5, 2-19-5, 2-20-5, 3-5-1, 3-6-1,
3-7-1, 3-8-1, 3-9-1, 3-10-1, 3-13-1, 3-14-1, 3-13-1, 3-14-1,
3-15-1, 3-16-1, 3-17-1, 3-18-1, 3-19-1, 3-20-1, 3-5-2, 3-6-2,
3-7-2, 3-8-2, 3-9-2, 3-10-2, 3-13-2, 3-14-2, 3-13-2, 3-14-2,
3-15-2, 3-16-2, 3-17-2, 3-18-2, 3-19-2, 3-20-2, 3-5-3, 3-6-3,
3-7-3, 3-8-3, 3-9-3, 3-10-3, 3-13-3, 3-14-3, 3-13-3, 3-14-3,
3-15-3, 3-16-3, 3-17-3, 3-18-3, 3-19-3, 3-20-3, 3-5-4, 3-6-4,
3-7-4, 3-8-4, 3-9-4, 3-10-4, 3-13-4, 3-14-4, 3-13-4, 3-14-4,
3-15-4, 3-16-4, 3-17-4, 3-18-4, 3-19-4, 3-20-4, 3-5-5, 3-6-5,
3-7-5, 3-8-5, 3-9-5, 3-10-5, 3-13-5, 3-14-5, 3-13-5, 3-14-5,
3-15-5, 3-16-5, 3-17-5, 3-18-5, 3-19-5, 3-20-5, 4-5-1, 4-6-1,
4-7-1, 4-8-1, 4-9-1, 4-10-1, 4-14-1, 4-14-1, 4-13-1, 4-14-1,
4-15-1, 4-16-1, 4-17-1, 4-18-1, 4-19-1, 4-20-1, 4-5-2, 4-6-2,
4-7-2, 4-8-2, 4-9-2, 4-10-2, 4-14-2, 4-14-2, 4-13-2, 4-14-2,
4-15-2, 4-16-2, 4-17-2, 4-18-2, 4-19-2, 4-20-2, 4-5-3, 4-6-3,
4-7-3, 4-8-3, 4-9-3, 4-10-3, 4-14-3, 4-14-3, 4-13-3, 4-14-3,
4-15-3, 4-16-3, 4-17-3, 4-18-3, 4-19-3, 4-20-3, 4-5-4, 4-6-4,
4-7-4, 4-8-4, 4-9-4, 4-10-4, 4-14-4, 4-14-4, 4-13-4, 4-14-4,
4-15-4, 4-16-4, 4-17-4, 4-18-4, 4-19-4, 4-20-4, 4-5-5, 4-6-5,
4-7-5, 4-8-5, 4-9-5, 4-10-5, 4-14-5, 4-14-5, 4-13-5, 4-14-5,
4-15-5, 4-16-5, 4-17-5, 4-18-5, 4-19-5, 4-20-5, 5-5-1, 5-6-1,
5-7-1, 5-8-1, 5-9-1, 5-10-1, 5-15-1, 5-12-1, 5-13-1, 5-14-1,
5-15-1, 5-16-1, 5-17-1, 5-18-1, 5-19-1, 5-20-1, 5-5-2, 5-6-2,
5-7-2, 5-8-2, 5-9-2, 5-10-2, 5-15-2, 5-12-2, 5-13-2, 5-14-2,
5-15-2, 5-16-2, 5-17-2, 5-18-2, 5-19-2, 5-20-2, 5-5-3, 5-6-3,
5-7-3, 5-8-3, 5-9-3, 5-10-3, 5-15-3, 5-12-3, 5-13-3, 5-14-3,
5-15-3, 5-16-3, 5-17-3, 5-18-3, 5-19-3, 5-20-3, 5-5-4, 5-6-4,
5-7-4, 5-8-4, 5-9-4, 5-10-4, 5-15-4, 5-12-4, 5-13-4, 5-14-4,
5-15-4, 5-16-4, 5-17-4, 5-18-4, 5-19-4, 5-20-4, 5-5-5, 5-6-5,
5-7-5, 5-8-5, 5-9-5, 5-10-5, 5-15-5, 5-12-5, 5-13-5, 5-14-5,
5-15-5, 5-16-5, 5-17-5, 5-18-5, 5-19-5, 5-20-5, 1-5-6, 1-6-6,
1-7-6, 1-8-6, 1-9-6, 1-10-6, 1-11-6, 1-12-6, 1-13-6, 1-14-6,
1-15-6, 1-16-6, 1-17-6, 1-18-6, 1-19-6, 1-20-6, 2-5-6, 2-6-6,
2-7-6, 2-8-6, 2-9-6, 2-10-6, 2-11-6, 2-12-6, 2-13-6, 2-14-6,
2-15-6, 2-16-6, 2-17-6, 2-18-6, 2-19-6, 2-20-6, 3-5-6, 3-6-6,
3-7-6, 3-8-6, 3-9-6, 3-10-6, 3-11-6, 3-12-6, 3-13-6, 3-14-6,
3-15-6, 3-16-6, 3-17-6, 3-18-6, 3-19-6, 3-20-6, 4- 5-6, 4-6-6,
4-7-6, 4-8-6, 4-9-6, 4-10-6, 4-11-6, 4-12-6, 4-13-6, 4-14-6,
4-15-6, 4-16-6, 4-17-6, 4-18-6, 4-19-6, 4-20-6, 5-5-6, 5-6-6,
5-7-6, 5-8-6, 5-9-6, 5-10-6, 5-11-6, 5-12-6, 5-13-6, 5-14-6,
5-15-6, 5-16- 6, 5-17-6, 5-18-6, 5-19-6, 5-20-6, 6-5-6, 6-6-6,
6-7-6, 6-8-6, 6-9-6, 6-10-6, 6-11-6, 6-12-6, 6-13-6, 6-14- 6,
6-15-6, 6-16-6, 6-17-6, 6-18-6, 6-19-6, 6-20-6, 7-5-6, 7-6-6,
7-7-6, 7-8-6, 7-9-6, 7-10-6, 7-11-6, 7-12- 6, 7-13-6, 7-14-6,
7-15-6, 7-16-6, 7-17-6, 7-18-6, 7-19-6, 7-20-6, 1-5-7, 1-6-7,
1-7-7, 1-8-7, 1-9-7, 1-10- 7, 1-11-7, 1-12-7, 1-13-7, 1-14-7,
1-15-7, 1-16-7, 1-17-7, 1-18-7, 1-19-7, 1-20-7, 2-5-7, 2-6-7,
2-7-7, 2-8-7, 2-9-7, 2-10-7, 2-11-7, 2-12-7, 2-13-7, 2-14-7,
2-15-7, 2-16-7, 2-17-7, 2-18-7, 2-19-7, 2-20-7, 3-5-7, 3-6-7,
3-7-7, 3-8-7, 3-9-7, 3-10-7, 3-11-7, 3-12-7, 3-13-7, 3-14-7,
3-15-7, 3-16-7, 3-17-7, 3-18-7, 3-19-7, 3-20-7, 4-5-7, 4-6-7,
4-7-7, 4-8-7, 4-9-7, 4-10-7, 4-11-7, 4-12-7, 4-13-7, 4-14-7,
4-15-7, 4-16-7, 4-17-7, 4-18-7, 4-19-7, 4-20-7, 5-5-7, 5-6-7,
5-7-7, 5-8-7, 5-9-7, 5-10-7, 5-11-7, 5-12-7, 5-13-7, 5-14-7,
5-15-7, 5-16-7, 5-17-7, 5-18-7, 5-19-7, 5-20-7, 6-5-7, 6-6-7,
6-7-7, 6-8-7, 6-9-7, 6-10-7, 6-11-7, 6-12-7, 6-13-7, 6-14-7,
6-15-7, 6-16-7, 6-17-7, 6-18-7, 6-19-7, 6-20-7, 7-5-7, 7-6-7,
7-7-7, 7-8-7, 7-9-7, 7-10-7, 7-11-7, 7-12-7, 7-13-7, 7-14-7,
7-15-7, 7-16-7, 7-17-7, 7-18-7, 7-19-7, or 7-20-7.
[0166] In some embodiments, the present disclosure provides an
oligonucleotide comprising or of a wing-core-wing, core-wing or
wing-core structure, wherein: [0167] the core comprises a pattern
of backbone chiral centers (linkage phosphorus) of:
[0167] (Np)t[(Op/Rp)n(Sp)m]y,
wherein: [0168] Np is either Rp or Sp; [0169] Sp indicates the S
configuration of a chiral linkage phosphorus of a chiral modified
internucleotidic linkage; [0170] Op indicates an achiral linkage
phosphorus of a natural phosphate linkage; and [0171] Rp indicates
the S configuration of a chiral linkage phosphorus of a chiral
modified internucleotidic linkage; and [0172] each wing
independently comprises one or more nucleobases.
[0173] In some embodiments, the present disclosure provides an
oligonucleotide comprising or of a wing-core-wing, core-wing or
wing-core structure, wherein: [0174] the core is or comprises a
region of consecutive nucleotidic units
(Nu.sup.M)t[(Nu.sup.O)n(Nu.sup.M)m]y, which region of consecutive
nucleotidic units has a pattern of backbone chiral centers (linkage
phosphorus) of (Np)t[(Op)n(Sp)m]y, [0175] wherein each variable is
independently as described in the present disclosure.
[0176] In some embodiments, (Np)t[(Op/Rp)n(Sp)m]y comprises at
least one Op. In some embodiments, (Np)t[(Op/Rp)n(Sp)m]y comprises
at least one Rp. In some embodiments, (Np)t[(Op/Rp)n(Sp)m]y is
(Np)t[(Op)n(Sp)m]y. In some embodiments, (Np)t[(Op/Rp)n(Sp)m]y is
(Np)t[(Rp)n(Sp)m]y.
[0177] In some embodiments, a wing comprises one or more sugar
modifications. In some embodiments, the two wings of a
wing-core-wing structure comprise different sugar modifications. In
some embodiments, sugar modifications provide improved stability
compared to absence of sugar modifications.
[0178] In some embodiments, certain sugar modifications, e.g.,
2'-MOE, provides more stability under otherwise identical
conditions than 2'-OMe. In some embodiments, a wing comprises
2'-MOE modifications. In some embodiments, each nucleoside unit of
a wing comprising a pyrimidine base (e.g., C, U, T, etc.) comprises
a 2'-MOE modification. In some embodiments, each sugar unit of a
wing comprises a 2'-MOE modification. In some embodiments, each
nucleoside unit of a wing comprising a purine base (e.g., A, G,
etc.) comprises no 2'-MOE modification (e.g., 2'-OMe, no
2'-modification, etc.). In some embodiments, each nucleoside unit
of a wing comprising a purine base comprises a 2'-OMe modification.
In some embodiments, each internucleotidic linkage at the
3'-position of a sugar unit comprising a 2'-MOE modification is a
natural phosphate linkage. In some embodiments, each
internucleotidic linkage at the 3'-position of a sugar unit
comprising a 2'-MOE modification is a natural phosphate linkage,
except that if the wing is a 5'-wing to the core, the first
internucleotidic linkage of the wing is a modified internucleotidic
linkage, e.g., a phosphorothioate diester linkage, and the
internucleotidic linkage linking the 3'-end nucleoside unit of the
wing and the 5'-end nucleoside unit of the core is a modified
internucleotidic linkage, e.g., a phosphorothioate diester linkage;
and if the wing is a 3'-wing to the core, the last internucleotidic
linkage of the wing is a modified internucleotidic linkage, e.g., a
phosphorothioate diester linkage, and the internucleotidic linkage
linking the 3'-end nucleoside unit of the core and the 5'-end
nucleoside unit of the wing is a modified internucleotidic linkage,
e.g., a phosphorothioate diester linkage (e.g., see WV-7127,
WV-7128, etc.). In some embodiments, such a wing is a 5'-wing. In
some embodiments, such a wing is a 3'-wing.
[0179] In some embodiments, a wing comprises no 2'-MOE
modifications. In some embodiments, a wing comprises 2'-OMe
modifications. In some embodiments, each nucleoside unit of a wing
independently comprises a 2'-OMe modifications. Among other things,
the present disclosure encompasses the recognition that
oligonucleotides with 2'-OMe modifications are less stable than
comparable oligonucleotides with 2'-MOE modifications under certain
conditions. In some embodiments, modified non-natural
internucleotidic linkages, such as phosphorothioate diester
linkages, in some instances particularly Sp phosphorothioate
diester linkages, can be utilized to improve properties, e.g.,
stability, of oligonucleotides. In some embodiments, a wing
comprises no 2'-MOE modifications, and each internucleotidic
linkage between nucleoside units of the wing is a modified
internucleotidic linkage. In some embodiments, a wing comprises no
2'-MOE modifications, each nucleoside unit of the wing comprise a
2'-OMe modification, and each internucleotidic linkage between
nucleoside units of the wing is a modified internucleotidic
linkage. In some embodiments, a modified internucleotidic linkage
is a phosphorothioate diester lineage. In some embodiments, a
modified internucleotidic linkage is a chirally controlled
internucleotidic linkage. In some embodiments, a modified
internucleotidic linkage is a chirally controlled internucleotidic
linkage wherein the linkage phosphorus is of Sp configuration. In
some embodiments, a modified internucleotidic linkage is a chirally
controlled internucleotidic linkage wherein the linkage phosphorus
is of Rp configuration. In some embodiments, a modified
internucleotidic linkage is a Sp phosphorothioate diester linkage.
In some embodiments, a modified internucleotidic linkage is a Rp
phosphorothioate diester linkage. In some embodiments, such a wing
is a 5'-wing. In some embodiments, such a wing is a 3'-wing.
[0180] Among other things, the present disclosure encompasses the
recognition that 2'-modifications and/or modified internucleotidic
linkages can be utilized either individually or in combination to
fine-tune properties, e.g., stability, and/or activities of
oligonucleotides.
[0181] In some embodiments, a wing comprises one or more natural
phosphate linkages. In some embodiments, a wing comprises one or
more consecutive natural phosphate linkages. In some embodiments, a
wing comprises one or more natural phosphate linkages and one or
more modified internucleotidic linkages. In some embodiments, a
modified internucleotidic linkage is a phosphorothioate diester
linkage. In some embodiments, a modified internucleotidic linkage
is a Sp phosphorothioate diester linkage.
[0182] In some embodiments, a wing comprises no natural phosphate
linkages, and each internucleotidic linkage of the wing is
independently a modified internucleotidic linkage. In some
embodiments, a modified internucleotidic linkage is chiral and
chirally controlled. In some embodiments, a modified
internucleotidic linkage is a phosphorothioate diester linkage. In
some embodiments, a modified internucleotidic linkage is a Sp
phosphorothioate diester linkage.
[0183] In some embodiments, for an oligonucleotide comprising or is
a wing-core-wing structure, the two wings are different in that
they contain different levels and/or types of chemical
modifications, backbone chiral center stereochemistry, and/or
patterns thereof. In some embodiments, the two wings are different
in that they contain different levels and/or types of sugar
modifications, and/or internucleotidic linkages, and/or
internucleotidic linkage stereochemistry, and/or patterns thereof.
For example, in some embodiments, one wing comprises 2'-OR
modifications wherein R is optionally substituted C.sub.1-6 alkyl
(e.g., 2-MOE), while the other wing comprises no such
modifications, or lower level (e.g., by number and/or percentage)
of such modifications; additionally and alternatively, one wing
comprises natural phosphate linkages while the other wing comprises
no natural phosphate linkages or lower level (e.g., by number
and/or percentage) of natural phosphate linkages; additionally and
alternatively, one wing may comprise a certain type of modified
internucleotidic linkages (e.g., phosphorothioate diester
internucleotidic linkage) while the other wing comprises no natural
phosphate linkages or lower level (e.g., by number and/or
percentage) of the type of modified internucleotidic linkages;
additionally and alternatively, one wing may comprise chiral
modified internucleotidic linkages comprising linkage phosphorus
atoms of a particular configuration (e.g., Rp or Sp), while the
other wing comprises no or lower level of chiral modified
internucleotidic linkages comprising linkage phosphorus atoms of
the particular configuration; alternatively or additionally, each
wing may comprise a different pattern of sugar modification,
internucleotidic linkages, and/or backbone chiral centers. In some
embodiments, one wing comprises one or more natural phosphate
linkages and one or more 2'-OR modifications wherein R is not --H
or -Me, and the other wing comprises no natural phosphate linkages
and no 2'-OR modifications wherein R is not --H or -Me. In some
embodiments, one wing comprises one or more natural phosphate
linkages and one or more 2'-MOE modifications, and each
internucleotidic linkage in the other wing is a phosphorothioate
linkage and each sugar unit of the other wing comprises a 2'-OMe
modification. In some embodiments, one wing comprises one or more
natural phosphate linkages and one or more 2'-MOE modifications,
and each internucleotidic linkage in the other wing is a Sp
phosphorothioate linkage and each sugar unit of the other wing
comprises a 2'-OMe modification.
[0184] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein one wing comprises a 2'-OMe and
the other wing comprises a bicyclic sugar. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein one
wing comprises a 2'-OMe and the other wing comprises a bicyclic
sugar, and the majority of the sugars in the core comprise a
2'-deoxy.
[0185] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and the majority of the sugars in the other
wing are a bicyclic sugar. In some embodiments, an oligonucleotide
comprises a wing-core-wing structure, wherein the majority of the
sugars in one wing comprise a 2'-OMe and the majority of the sugars
in the other wing are a bicyclic sugar, and the majority of the
sugars in the core comprise a 2'-deoxy.
[0186] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and, in the other wing, at least one sugar
is a bicyclic sugar and at least one sugar comprises a 2'-OMe. In
some embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-OMe and, in the other wing, at least one sugar is a bicyclic
sugar and at least one sugar comprises a 2'-OMe, and the majority
of the sugars in the core comprise a 2'-deoxy.
[0187] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are a bicyclic sugar and, in the other wing, at least one
sugar is a bicyclic sugar and at least one sugar comprises a
2'-OMe. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are a bicyclic sugar and, in the other wing, at least one
sugar is a bicyclic sugar and at least one sugar comprises a
2'-OMe, and the majority of the sugars in the core comprise a
2'-deoxy.
[0188] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and, in the other wing, at least two sugars
are a bicyclic sugar and at least two sugars comprise a 2'-OMe. In
some embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-OMe and, in the other wing, at least two sugars are a bicyclic
sugar and at least two sugars comprise a 2'-OMe, and the majority
of the sugars in the core comprise a 2'-deoxy.
[0189] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are a bicyclic sugar and, in the other wing, at least two
sugars are a bicyclic sugar and at least two sugars comprise a
2'-OMe. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are a bicyclic sugar and, in the other wing, at least two
sugars are a bicyclic sugar and at least two sugars comprise a
2'-OMe, and the majority of the sugars in the core comprise a
2'-deoxy.
[0190] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a 2'-OMe and each sugar in the other wing comprises a bicyclic
sugar. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a 2'-OMe and each sugar in the other wing comprises a bicyclic
sugar, and the majority of the sugars in the core comprise a
2'-deoxy.
[0191] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a bicyclic sugar, each sugar in the other wing comprises a 2'-OMe,
and each sugar in the core comprises a 2'-deoxy.
[0192] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein one wing comprises a bicyclic
sugar and the other wing comprises a 2'-MOE. In some embodiments,
an oligonucleotide comprises a wing-core-wing structure, wherein
one wing comprises a bicyclic sugar and the other wing comprises a
2'-MOE, and the majority of the sugars in the core comprise a
2'-deoxy.
[0193] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a bicyclic sugar and the majority of the sugars in
the other wing comprise a 2'-MOE. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein the
majority of the sugars in one wing comprise a bicyclic sugar and
the majority of the sugars in the other wing comprise a 2'-MOE, and
the majority of the sugars in the core comprise a 2'-deoxy.
[0194] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a bicyclic sugar and, in the other wing, at least one
sugar comprises a 2'-MOE and at least one sugar are a bicyclic
sugar. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a bicyclic sugar and, in the other wing, at least one
sugar comprises a 2'-MOE and at least one sugar are a bicyclic
sugar, and the majority of the sugars in the core comprise a
2'-deoxy.
[0195] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-MOE and, in the other wing, at least one sugar
comprises a 2'-MOE and at least one sugar are a bicyclic sugar. In
some embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-MOE and, in the other wing, at least one sugar comprises a
2'-MOE and at least one sugar are a bicyclic sugar, and the
majority of the sugars in the core comprise a 2'-deoxy.
[0196] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a bicyclic sugar and, in the other wing, at least two
sugars comprise a 2'-MOE and at least two sugars is a bicyclic
sugar. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a bicyclic sugar and, in the other wing, at least two
sugars comprise a 2'-MOE and at least two sugars is a bicyclic
sugar, and the majority of the sugars in the core comprise a
2'-deoxy.
[0197] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-MOE and, in the other wing, at least two sugars
comprise a 2'-MOE and at least two sugars is a bicyclic sugar. In
some embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-MOE and, in the other wing, at least two sugars comprise a
2'-MOE and at least two sugars is a bicyclic sugar, and the
majority of the sugars in the core comprise a 2'-deoxy.
[0198] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing are a
bicyclic sugar and each sugar in the other wing comprises a 2'-MOE.
In some embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein each sugar in one wing are a bicyclic sugar and
each sugar in the other wing comprises a 2'-MOE, and the majority
of the sugars in the core comprise a 2'-deoxy.
[0199] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a 2'-MOE, each sugar in the other wing are a bicyclic sugar, and
each sugar in the core comprises a 2'-deoxy.
[0200] In some embodiments, a bicyclic sugar is a LNA, a cEt or
BNA.
[0201] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein one wing comprises a 2'-OMe and
the other wing comprises 2'-F. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein one
wing comprises a 2'-OMe and the other wing comprises 2'-F, and the
majority of the sugars in the core comprise a 2'-deoxy.
[0202] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and the majority of the sugars in the other
wing are 2'-F. In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and the majority of the sugars in the other
wing are 2'-F, and the majority of the sugars in the core comprise
a 2'-deoxy.
[0203] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and, in the other wing, at least one sugar
is 2'-F and at least one sugar comprises a 2'-OMe. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-OMe and, in the other wing, at least one sugar is 2'-F and at
least one sugar comprises a 2'-OMe, and the majority of the sugars
in the core comprise a 2'-deoxy.
[0204] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are 2'-F and, in the other wing, at least one sugar is 2'-F
and at least one sugar comprises a 2'-OMe. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein the
majority of the sugars in one wing are 2'-F and, in the other wing,
at least one sugar is 2'-F and at least one sugar comprises a
2'-OMe, and the majority of the sugars in the core comprise a
2'-deoxy.
[0205] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-OMe and, in the other wing, at least two sugars
are 2'-F and at least two sugars comprise a 2'-OMe. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-OMe and, in the other wing, at least two sugars are 2'-F and
at least two sugars comprise a 2'-OMe, and the majority of the
sugars in the core comprise a 2'-deoxy.
[0206] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing are 2'-F and, in the other wing, at least two sugars are 2'-F
and at least two sugars comprise a 2'-OMe. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein the
majority of the sugars in one wing are 2'-F and, in the other wing,
at least two sugars are 2'-F and at least two sugars comprise a
2'-OMe, and the majority of the sugars in the core comprise a
2'-deoxy.
[0207] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a 2'-OMe and each sugar in the other wing comprises 2'-F. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein each sugar in one wing comprises a 2'-OMe and
each sugar in the other wing comprises 2'-F, and the majority of
the sugars in the core comprise a 2'-deoxy.
[0208] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
2'-F, each sugar in the other wing comprises a 2'-OMe, and each
sugar in the core comprises a 2'-deoxy.
[0209] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein one wing comprises 2'-F and the
other wing comprises a 2'-MOE. In some embodiments, an
oligonucleotide comprises a wing-core-wing structure, wherein one
wing comprises 2'-F and the other wing comprises a 2'-MOE, and the
majority of the sugars in the core comprise a 2'-deoxy.
[0210] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise 2'-F and the majority of the sugars in the other wing
comprise a 2'-MOE. In some embodiments, an oligonucleotide
comprises a wing-core-wing structure, wherein the majority of the
sugars in one wing comprise 2'-F and the majority of the sugars in
the other wing comprise a 2'-MOE, and the majority of the sugars in
the core comprise a 2'-deoxy.
[0211] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise 2'-F and, in the other wing, at least one sugar
comprises a 2'-MOE and at least one sugar are 2'-F. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
2'-F and, in the other wing, at least one sugar comprises a 2'-MOE
and at least one sugar are 2'-F, and the majority of the sugars in
the core comprise a 2'-deoxy.
[0212] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-MOE and, in the other wing, at least one sugar
comprises a 2'-MOE and at least one sugar are 2'-F. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-MOE and, in the other wing, at least one sugar comprises a
2'-MOE and at least one sugar are 2'-F, and the majority of the
sugars in the core comprise a 2'-deoxy.
[0213] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise 2'-F and, in the other wing, at least two sugars
comprise a 2'-MOE and at least two sugars is 2'-F. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
2'-F and, in the other wing, at least two sugars comprise a 2'-MOE
and at least two sugars is 2'-F, and the majority of the sugars in
the core comprise a 2'-deoxy.
[0214] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein the majority of the sugars in one
wing comprise a 2'-MOE and, in the other wing, at least two sugars
comprise a 2'-MOE and at least two sugars is 2'-F. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein the majority of the sugars in one wing comprise
a 2'-MOE and, in the other wing, at least two sugars comprise a
2'-MOE and at least two sugars is 2'-F, and the majority of the
sugars in the core comprise a 2'-deoxy.
[0215] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing is 2'-F
and each sugar in the other wing comprises a 2'-MOE. In some
embodiments, an oligonucleotide comprises a wing-core-wing
structure, wherein each sugar in one wing is 2'-F and each sugar in
the other wing comprises a 2'-MOE, and the majority of the sugars
in the core comprise a 2'-deoxy.
[0216] In some embodiments, an oligonucleotide comprises a
wing-core-wing structure, wherein each sugar in one wing comprises
a 2'-MOE, each sugar in the other wing are 2'-F, and each sugar in
the core comprises a 2'-deoxy.
[0217] In some embodiments, a C9orf72 oligonucleotides has a
wing-core-wing structure and has an asymmetrical format. In some
embodiments of a C9orf72 oligonucleotide having an asymmetrical
format, one wing differs from another. In some embodiments of a
C9orf72 oligonucleotide having an asymmetrical format, one wing
differs from another in the sugar modifications or pattern thereof,
or the backbone internucleotidic linkages or pattern thereof, or
the backbone chiral centers or pattern thereof. In some embodiments
of an oligonucleotide having an asymmetrical format, the core
comprises 1 or more 2'-deoxy sugars. In some embodiments of an
oligonucleotide having an asymmetrical format, the core comprises 5
or more consecutive 2'-deoxy sugars. In some embodiments of an
oligonucleotide having an asymmetrical format, the core comprises
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive 2'-deoxy
sugars. Some non-limiting examples of C9orf72 oligonucleotides
having an asymmetrical format are shown herein. In some embodiments
of a C9orf72 oligonucleotide having an asymmetrical format, a first
wing and a second wing independently has a pattern of
2'-modifications of sugars which is or comprises F, FF, FFF, FFFF,
FFFFF, FMMMF, FMMMF, LMMMm, m, M, mm, MM, mmm, mMm, MMm, MMM, mmm,
mmmm, mMMm, MMMm, MMMM, mmmm, mmmmm, MMMMM, mMMMm, MMMMM, mmmmm, or
any pattern of 2'-modifications of any wing of any oligonucleotide
described herein, wherein the pattern of 2'-modifications of the
first and second wing are different, and wherein m=2'-OMe;
M=2'-MOE; F=2'-F; and L=LNA. In some embodiments of an
oligonucleotide having an asymmetrical format, a first wing and a
second wing independently has a pattern of internucleotidic
linkages which is or comprises PS, PO, PS-PS, PS-PO, PO-PS, PO-PO,
PO-PS-PS, PS-PO-PO-PO-PS, PS-PO-PO-PS, PS-PS-PS-PS, PS-PS-PS-PS-PS,
PS-Xn-Xn-Xn-PS, or any pattern of internucleotidic linkages of any
wing of any oligonucleotide described herein, wherein the pattern
of internucleotidic linkages of the first and second wing are
different, and wherein PS=Phosphorothioate; PO=phosphodiester;
Xn=any neutral internucleotidic linkage. In some embodiments of an
oligonucleotide having an asymmetrical format, a first wing and a
second wing independently has a pattern of stereochemistry of
internucleotidic linkages which is or comprises PO, SR, Sp, Rp,
Sp-PO, Rp-PO, PO-Sp, PO-Rp, PO-PO-PO, Sp-PO-PO, Rp-PO-PO,
Rp-PO-PO-PO-Rp, Rp-PO-PO-Rp-Rp, Rp-PO-Rp-PO-Rp, Rp-Rp-PO-PO-Rp,
Sp-PO-PO-PO-Sp, Sp-Sp-Sp-Sp, Sp-Sp-Sp-Sp, Sp-Sp-Sp-Sp-Sp,
Sp-Xn-Xn-Xn-Sp, SR-PO-PO-PO-SR, SR-SR-SR-SR, SR-SR-SR-SR-SR,
SR-Xn-Xn-Xn-SR, or any pattern of stereochemistry of
internucleotidic linkages of any wing of any oligonucleotide
described herein, wherein the pattern of stereochemistry of
internucleotidic linkages of the first and second wing are
different, and wherein SR=internucleotidic linkage which is
stereorandom (e.g., not chirally controlled); PO=phosphodiester
(which lacks a chiral center); Sp=internucleotidic linkage in the
Sp configuration; Rp=internucleotidic linkage in the Rp
configuration; Xn=a neutral internucleotidic linkage, which can be
independently stereocontrolled (in the Rp or Sp configuration) or
stereorandom. In some embodiments of an oligonucleotide having an
asymmetrical format, the first wing is the 5' wing (the wing closer
to the 5'-end of the oligonucleotide) and the second wing is the
3'-wing (the wing closer to the 3'-end of the oligonucleotide). In
some embodiments of an oligonucleotide having an asymmetrical
format, the first wing is the 3' wing (the wing closer to the
3'-end of the oligonucleotide) and the second wing is the 5'-wing
(the wing closer to the 5'-end of the oligonucleotide). In some
embodiments, the first and second wing are the same or different
lengths.
[0218] In some embodiments, an oligonucleotide having an
asymmetrical structure (e.g., wherein one wing differs chemically
from another wing) has an improved biological activity compared to
an oligonucleotide having the same base sequence but a different
structure (e.g., a symmetric structure wherein both wings have the
same pattern of chemical modifications; or a different asymmetrical
structure). In some embodiments, improved biological activity
includes improved decrease of the expression, activity, and/or
level or a gene or gene product. In some embodiments, improved
biological activity is improved delivery to a cellular nucleus. In
some embodiments, improved biological activity is improved delivery
to a cellular nucleus and one wing in an oligonucleotide having an
asymmetrical structure comprises a 2'-F or two or more 2'-F. In
some embodiments, improved biological activity is improved delivery
to a cellular nucleus and one wing in an oligonucleotide having an
asymmetrical structure comprises a 2'-MOE or two or more 2'-MOE. In
some embodiments, improved biological activity is improved delivery
to a cellular nucleus and one wing in an oligonucleotide having an
asymmetrical structure comprises a 2'-OMe or two or more 2'-OMe. In
some embodiments, improved biological activity is improved delivery
to a cellular nucleus and one wing in an oligonucleotide having an
asymmetrical structure comprises a bicyclic sugar or two or more
bicyclic sugars.
[0219] In some embodiments, a core comprises no 2'-substitution,
and each sugar unit is a natural sugar unit found in natural
unmodified DNA. In some embodiments, a core comprises one or more
2'-halogen modification. In some embodiments, a core comprises one
or more 2'-F modification. In some embodiments, no less than 70%,
80%, 90% or 100% of internucleotidic linkages in a core is a
modified internucleotidic linkage. In some embodiments, no less
than 70%, 80%, or 90% of internucleotidic linkages in a core is
independently a modified internucleotidic linkage of Sp
configuration, and the core also contains 1, 2, 3, 4, or 5
internucleotidic linkages selected from modified internucleotidic
linkages of Rp configuration and natural phosphate linkages. In
some embodiments, the core also contains 1 or 2 internucleotidic
linkages selected from modified internucleotidic linkages of Rp
configuration and natural phosphate linkages. In some embodiments,
the core also contains 1 and no more than 1 internucleotidic
linkage selected from a modified internucleotidic linkage of Rp
configuration and a natural phosphate linkage, and the rest
internucleotidic linkages are independently modified
internucleotidic linkages of Sp configuration. In some embodiments,
the core also contains 2 and no more than 2 internucleotidic
linkage each independently selected from a modified
internucleotidic linkage of Rp configuration and a natural
phosphate linkage, and the rest internucleotidic linkages are
independently modified internucleotidic linkages of Sp
configuration. In some embodiments, the core also contains 1 and no
more than 1 natural phosphate linkage, and the rest
internucleotidic linkages are independently modified
internucleotidic linkages of Sp configuration. In some embodiments,
the core also contains 2 and no more than 2 natural phosphate
linkages, and the rest internucleotidic linkages are independently
modified internucleotidic linkages of Sp configuration. In some
embodiments, the core also contains 1 and no more than 1 modified
internucleotidic linkage of Rp configuration, and the rest
internucleotidic linkages are independently modified
internucleotidic linkages of Sp configuration. In some embodiments,
the core also contains 2 and no more than 2 modified
internucleotidic linkages of Rp configuration, and the rest
internucleotidic linkages are independently modified
internucleotidic linkages of Sp configuration. In some embodiments,
the two natural phosphate linkages, or the two modified
internucleotidic linkages of Rp configuration, are separated by two
or more modified internucleotidic linkages of Sp configuration. In
some embodiments, a modified internucleotidic linkage is of formula
I. In some embodiments, a modified internucleotidic linkage is a
phosphorothioate diester linkage.
[0220] Core and wings can be of various lengths. In some
embodiments, a core comprises no less than 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases. In some
embodiments, a wing comprises no less than 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 nucleobases. In some embodiments, a wing comprises no more
than 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleobases. In some
embodiments, for a wing-core-wing structure, both wings are of the
same length, for example, of 5 nucleobases. In some embodiments,
the two wings are of different lengths. In some embodiments, a core
is no less than 40%, 45%, 50%, 60%, 70%, 80%, or 90% of total
oligonucleotide length as measured by percentage of nucleoside
units within the core. In some embodiments, a core is no less than
50% of total oligonucleotide length.
[0221] In some embodiments, the present disclosure provides
oligonucleotides comprising additional chemistry moieties,
optionally connected to the oligonucleotide moiety through a
linker. In some embodiments, the present disclosure provides
oligonucleotides comprising (R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-,
wherein: [0222] each R.sup.D is independently a chemical moiety;
[0223] each of L.sup.M1, L.sup.M2, and L.sup.M3 is independently a
covalent bond, or a bivalent or multivalent, optionally
substituted, linear or branched group selected from a C.sub.1-30
aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10
heteroatoms, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0224] each
Cy.sup.L is independently an optionally substituted tetravalent
group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20
aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms,
and a 3-20 membered heterocyclyl ring; and b is 1-1000.
[0225] In some embodiments, each of L.sup.M1 L.sup.M2, and L.sup.M3
is independently a covalent bond, or a bivalent or multivalent,
optionally substituted, linear or branched group selected from a
C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group
having 1-5 heteroatoms, wherein one or more methylene units are
optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L.
[0226] In some embodiments, L.sup.M1 comprises one or more
--N(R')-- and one or more --C(O)--. In some embodiments, a linker
or L.sup.M1 is or comprises
##STR00011##
wherein n.sup.L is 1-8. In some embodiments, a linker or
-L.sup.M1-L.sup.M2-L.sup.M3- is
##STR00012##
or a salt form thereof, wherein n is 1-8. In some embodiments, a
linker or -L.sup.M1-L.sup.M2-L.sup.M3- is
##STR00013##
or a salt form thereof, wherein: [0227] n.sup.L is 18 [0228] each
amino group independently connects to a moiety; and [0229] the P
atom connects to the 5'-OH of the oligonucleotide. In some
embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00014##
[0229] In some embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00015##
In some embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00016##
In some embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00017##
In some embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00018##
In some embodiments, the moiety and the linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises
##STR00019##
In some embodiments, the moiety and the linker, or
(e)b-L.sup.M1-L.sup.M2-L.sup.M3- is or comprises
##STR00020##
In some embodiments, the linker, or L.sup.M1, is or comprises
##STR00021##
In some embodiments, the moiety and linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3-, is or comprises:
##STR00022##
In some embodiments, the moiety and linker, or
(R.sup.D)b-L.sup.M1-L.sup.M2-L.sup.M3- is or comprises:
##STR00023##
[0230] In some embodiments, n.sup.L is 1-8. In some embodiments,
n.sup.L is 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, n.sup.L
is 1. In some embodiments, n.sup.L is 2. In some embodiments,
n.sup.L is 3. In some embodiments, n.sup.L is 4. In some
embodiments, n.sup.L is 5. In some embodiments, n.sup.L is 6. In
some embodiments, n.sup.L is 7. In some embodiments, n.sup.L8.
[0231] In some embodiments, L.sup.M2 is a covalent bond, or a
bivalent, optionally substituted, linear or branched group selected
from a C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic
group having 1-5 heteroatoms, wherein one or more methylene units
are optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L. In some
embodiments, L.sup.M2 is a covalent bond, or a bivalent, optionally
substituted, linear or branched group selected from a C.sub.1-10
aliphatic group and a C.sub.1-10 heteroaliphatic group having 1-5
heteroatoms, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, or --P(O)(R')--. In some embodiments, L.sup.M2 is a
covalent bond, or a bivalent, optionally substituted, linear or
branched C.sub.1-10 aliphatic wherein one or more methylene units
are optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--N(R')--, or --C(O)--. In some embodiments, L.sup.M2 is
--NH--(CH.sub.2).sub.6--, wherein --NH-- is bonded to L.sup.M1.
[0232] In some embodiments, L.sup.M3 is --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')--,
--OP(O)(SR')--, --OP(O)(R')--, --OP(O)(NR')--, --OP(S)(OR')--,
--OP(S)(SR')--, --OP(S)(R')--, --OP(S)(NR')--, --OP(R')--,
--OP(OR')--, --OP(SR')--, --OP(NR')--, or --OP(OR')[B(R').sub.3]--.
In some embodiments, L.sup.M3 is --OP(O)(OR')--, or --OP(O)(SR')--,
wherein --O-- is bonded to L.sup.M2. In some embodiments, the P
atom is connected to a sugar unit, a nucleobase unit, or an
internucleotidic linkage. In some embodiments, the P atom is
connected to a --OH group through formation of a P--O bond. In some
embodiments, the P atom is connected to the 5'-OH group through
formation of a P--O bond.
[0233] In some embodiments, L.sup.M1 is a covalent bond. In some
embodiments, L.sup.M2 is a covalent bond. In some embodiments,
L.sup.M3 is a covalent bond. In some embodiments, L.sup.M1 is
L.sup.M2 as described in the present disclosure. In some
embodiments, L.sup.M1 is L.sup.M3 as described in the present
disclosure. In some embodiments, L.sup.M2 is L.sup.M1 as described
in the present disclosure. In some embodiments, L.sup.M2 is
L.sup.M3 as described in the present disclosure. In some
embodiments, L.sup.M3 is L.sup.M1 as described in the present
disclosure. In some embodiments, L.sup.M3 is L.sup.M2 as described
in the present disclosure. In some embodiments, L.sup.M1 is
L.sup.M1 as described in the present disclosure. In some
embodiments, L.sup.M1 is L.sup.2 as described in the present
disclosure. In some embodiments, L.sup.M is L.sup.M3 as described
in the present disclosure. In some embodiments, L.sup.M1 is
-L.sup.M2, wherein each of L.sup.M1 and L.sup.M2 is independently
as described in the present disclosure. In some embodiments,
L.sup.M1 is L.sup.M1-L.sup.M3, wherein each of L.sup.M1 and
L.sup.M3 is independently as described in the present disclosure.
In some embodiments, L.sup.M is L.sup.M2-L.sup.M3, wherein each of
L.sup.M2 and L.sup.M3 is independently as described in the present
disclosure. In some embodiments, L.sup.M is
L.sup.M1-L.sup.M2-L.sup.M3, wherein each of L.sup.M1, L.sup.M2 and
L.sup.M3 is independently as described in the present
disclosure.
[0234] In some embodiments, each R.sup.D is independently a
chemical moiety as described in the present disclosure. In some
embodiments, R.sup.D is targeting moiety. In some embodiments,
R.sup.D is or comprises a carbohydrate moiety. In some embodiments,
R is or comprises a lipid moiety. In some embodiments, R.sup.D is
or comprises a ligand moiety for, e.g., cell receptors such as a
sigma receptor, an asialoglycoprotein receptor, etc. In some
embodiments, a ligand moiety is or comprises an anisamide moiety,
which may be a ligand moiety for a sigma receptor. In some
embodiments, a ligand moiety is or comprises a GalNAc moiety, which
may be a ligand moiety for an asialoglycoprotein receptor. In some
embodiments, R.sup.D is selected from optionally substituted
phenyl,
##STR00024##
wherein n' is 0 or 1, and each other variable is independently as
described in the present disclosure. In some embodiments, R.sup.s
is F. In some embodiments, R.sup.s is OMe. In some embodiments,
R.sup.s is OH. In some embodiments, R.sup.s is NHAc. In some
embodiments, R.sup.s is NHCOCF.sub.3. In some embodiments, R' is H.
In some embodiments, R is H. In some embodiments, R.sup.2s is NHAc,
and R.sup.5s is OH. In some embodiments, R.sup.2s is p-anisoyl, and
R.sup.5s is OH. In some embodiments, R.sup.2s is NHAc and R.sup.5s
is p-anisoyl. In some embodiments, R.sup.2s is OH, and R.sup.5s is
p-anisoyl. In some embodiments, R.sup.D is selected from
##STR00025## ##STR00026## ##STR00027##
Further embodiments of R.sup.D includes additional chemical moiety
embodiments, e.g., those described in Example, Example 2, etc.
[0235] In some embodiments, n' is 1. In some embodiments, n' is
0.
[0236] In some embodiments, n'' is 1. In some embodiments, n'' is
2.
[0237] In some embodiments, the present disclosure provides a
provided compound, e.g., an oligonucleotide of a provided
composition, having the structure of formula O-I:
##STR00028##
or a salt thereof, wherein: [0238] R.sup.E is a 5'-end group;
[0239] each BA is independently an optionally substituted group
selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl,
C.sub.5-30 heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus, boron and
silicon, a natural nucleobase moiety, and a modified nucleobase
moiety; [0240] each R.sup.s is independently --F, --Cl, --Br, --I,
--CN, --N.sub.3, --NO, --NO.sub.2, -L-R', -L-OR', -L-SR',
-L-N(R').sub.2, --O-L-OR', --O-L-SR', or --O-L-N(R').sub.2; [0241]
s is 0-20; [0242] L.sup.s is --C(R.sup.5s).sub.2--, or L; [0243]
each L is independently a covalent bond, or a bivalent, optionally
substituted, linear or branched group selected from a C.sub.1-30
aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus, boron and silicon, wherein one or more methylene units
are optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0244] each
Cy.sup.L is independently an optionally substituted tetravalent
group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20
aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and a 3-20 membered heterocyclyl ring having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus, boron and silicon; [0245] each Ring A is independently
an optionally substituted 3-20 membered monocyclic, bicyclic or
polycyclic ring having 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon; [0246] each
L.sup.P is independently an internucleotidic linkage; [0247] z is
1-1000; [0248] L.sup.3E is L or -L-L-; [0249] R.sup.3E is --R',
-L-R', --OR', or a solid support; [0250] each R' is independently
--R, --C(O)R, --C(O)OR, or --S(O).sub.2R; [0251] each R is
independently --H, or an optionally substituted group selected from
C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic,
C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, or [0252] two R groups are optionally and
independently taken together to form a covalent bond, or: [0253]
two or more R groups on the same atom are optionally and
independently taken together with the atom to form an optionally
substituted, 3-30 membered monocyclic, bicyclic or polycyclic ring
having, in addition to the atom, 0-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
[0254] two or more R groups on two or more atoms are optionally and
independently taken together with their intervening atoms to form
an optionally substituted, 3-30 membered monocyclic, bicyclic or
polycyclic ring having, in addition to the intervening atoms, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0255] In some embodiments, each L.sup.P independently has the
structure of formula I:
##STR00029##
or a salt form thereof, wherein: [0256] P.sup.L is P(.dbd.W), P, or
P--B(R').sub.3; [0257] W is O, S or Se; [0258] R.sup.1 is -L-R,
halogen, --CN, --NO.sub.2, --Si(R').sub.3, --OR', --SR', or
--N(R').sub.2; [0259] each of X, Y and Z is independently --O--,
--S--, --N(-L-R.sup.1)--, or L; [0260] each R' is independently
--R, --C(O)R, --C(O)OR, or --S(O).sub.2R; [0261] each L is
independently a covalent bond, or a bivalent, optionally
substituted, linear or branched group selected from a C.sub.1-30
aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus, boron and silicon, wherein one or more methylene units
are optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0262] each R
is independently --H, or an optionally substituted group selected
from C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic,
C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, or [0263] two R groups are optionally and
independently taken together to form a covalent bond, or. [0264]
two or more R groups on the same atom are optionally and
independently taken together with the atom to form an optionally
substituted, 3-30 membered monocyclic, bicyclic or polycyclic ring
having, in addition to the atom, 0-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
[0265] two or more R groups on two or more atoms are optionally and
independently taken together with their intervening atoms to form
an optionally substituted, 3-30 membered monocyclic, bicyclic or
polycyclic ring having, in addition to the intervening atoms, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0266] In some embodiments, each L.sup.P independently has the
structure of formula I, and R.sup.E is --C(R.sup.5s).sub.3,
-L-P.sup.DB, --C(R.sup.5s).sub.2OH, -L-R.sup.5s, or
-L-P.sup.5s-L-R.sup.5, or a salt form thereof, wherein each
variable is independently as described in the present
disclosure.
[0267] In some embodiments, each L.sup.P independently has the
structure of formula I, and R.sup.E is --C(R.sup.5s).sub.3,
-L-P.sup.DB, --C(R.sup.5s).sub.2OH, -L-R.sup.5s, or
-L-P.sup.5s-L-R.sup.5, or a salt form thereof, wherein each
variable is independently as described in the present
disclosure.
[0268] In some embodiments, R.sup.E is --C(R.sup.5s).sub.3,
--C(R.sup.5s).sub.2OH, or -L-R.sup.5s; [0269] each BA is
independently an optionally substituted group selected from
C.sub.5-30 heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, and
C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus, boron and
silicon; [0270] each Ring A is independently an optionally
substituted 3-20 membered monocyclic, bicyclic or polycyclic ring
having 0-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon; and [0271] each L.sup.P
independently has the structure of formula I, wherein each variable
is independently as described in the present disclosure.
[0272] In some embodiments, R.sup.E is --C(R.sup.5s).sub.3,
--C(R.sup.5s).sub.2OH, or -L-R.sup.5s [0273] each BA is
independently an optionally substituted C.sub.5-30 heteroaryl
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein the heteroaryl
comprises one or more heteroatoms selected from oxygen and
nitrogen; [0274] each Ring A is independently an optionally
substituted 5-10 membered monocyclic or bicyclic saturated ring
having 0-5 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein the ring
comprises at least one oxygen atom; and [0275] each L.sup.P
independently has the structure of formula I, wherein each variable
is independently as described in the present disclosure.
[0276] In some embodiments, R.sup.E is --C(R.sup.5s).sub.3,
--C(R.sup.5s).sub.2OH, or -L-R.sup.5s; [0277] each BA is
independently an optionally substituted or protected nucleobase
selected from adenine, cytosine, guanosine, thymine, and uracil;
[0278] each Ring A is independently an optionally substituted 5-7
membered monocyclic or bicyclic saturated ring having one or more
oxygen atoms; and [0279] each L.sup.P independently has the
structure of formula I, wherein each variable is independently as
described in the present disclosure.
[0280] In some embodiments, R.sup.E is a 5'-end group as described
herein. In some embodiments, R.sup.E is --C(R.sup.5s).sub.3,
-L-P.sup.DB, --C(R.sup.5s).sub.2OH, -L-R.sup.5s, or
-L-P.sup.5s-L-R.sup.s, or a salt form thereof, wherein each
variable is independently as described in the present disclosure.
In some embodiments, R.sup.E is --CH.sub.2OH. In some embodiments,
R.sup.E is --CH.sub.2OP(O)(OR).sub.2 or a salt form thereof,
wherein each R is independently as described in the present
disclosure. In some embodiments, R.sup.E is
--CH.sub.2OP(O)(OH).sub.2 or a salt form thereof. In some
embodiments, R.sup.E is --CH.sub.2OP(O)(OR)(SR) or a salt form
thereof, wherein each R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
--CH.sub.2OP(O)(SH)(OH) or a salt form thereof. In some
embodiments, R.sup.E is (E)-CH.dbd.CHP(O)(OR).sub.2 or a salt form
thereof, wherein each R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
(E)-CH.dbd.CHP(O)(OH).sub.2.
[0281] In some embodiments, R.sup.E is --CH.sub.2OH. In some
embodiments, R.sup.E is --CH.sub.2OP(O)(R).sub.2 or a salt form
thereof, wherein each R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
--CH.sub.2P(O)(OR).sub.2 or a salt form thereof, wherein each R is
independently as described in the present disclosure. In some
embodiments, R.sup.E is --CH.sub.2OP(O)(OH).sub.2 or a salt form
thereof. In some embodiments, R.sup.E is --CH.sub.2OP(O)(OR)(SR) or
a salt form thereof, wherein each R is independently as described
in the present disclosure. In some embodiments, R.sup.E is
--CH.sub.2OP(O)(SH)(OH) or a salt form thereof. In some
embodiments, R.sup.E is (E)-CH.dbd.CHP(O)(OR).sub.2 or a salt form
thereof, wherein each R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
(E)-CH.dbd.CHP(O)(OH).sub.2.
[0282] In some embodiments, R.sup.E is --CH(R.sup.5s)--OH, wherein
R.sup.5s is as described in the present disclosure. In some
embodiments, R.sup.E is --CH(R.sup.5s)--OP(O)(R).sub.2 or a salt
form thereof, wherein each R.sup.5s and R is independently as
described in the present disclosure. In some embodiments, R.sup.E
is --CH(R.sup.5s)--OP(O)(OR).sub.2 or a salt form thereof, wherein
each R.sup.5s and R is independently as described in the present
disclosure. In some embodiments, R.sup.E is
--CH(R.sup.5s)--OP(O)(OH).sub.2 or a salt form thereof. In some
embodiments, R.sup.E is --CH(R.sup.5s)--OP(O)(OR)(SR) or a salt
form thereof. In some embodiments, R.sup.E is
--CH(R)--OP(O)(OH)(SH) or a salt form thereof. In some embodiments,
R.sup.E is --(R)--CH(R.sup.5s)--OH, wherein R.sup.5s is as
described in the present disclosure. In some embodiments, R.sup.E
is --(R)--CH(R.sup.5s)--OP(O)(R).sub.2 or a salt form thereof,
wherein each R.sup.5s and R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
--(R)--CH(R.sup.5s)--OP(O)(OR).sub.2 or a salt form thereof,
wherein each R.sup.5s and R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
--(R)--CH(R.sup.5s)--OP(O)(OH).sub.2 or a salt form thereof. In
some embodiments, R.sup.E is --(R)--CH(R.sup.5s)--OP(O)(OR)(SR) or
a salt form thereof. In some embodiments, R.sup.E is
--(R)--CH(R.sup.5s)--OP(O)(OH)(SH) or a salt form thereof. In some
embodiments, R.sup.E is --(S)--CH(R.sup.5s)--OH, wherein R.sup.5s
is as described in the present disclosure. In some embodiments,
R.sup.E is --(S)--CH(R.sup.5s)--OP(O)(R).sub.2 or a salt form
thereof, wherein each R.sup.5s and R is independently as described
in the present disclosure. In some embodiments, R.sup.E is
--(S)--CH(R.sup.5s)--OP(O)(OR).sub.2 or a salt form thereof,
wherein each R.sup.5s and R is independently as described in the
present disclosure. In some embodiments, R.sup.E is
--(S)--CH(R.sup.5s)--OP(O)(OH).sub.2 or a salt form thereof. In
some embodiments, R.sup.E is --(S)--CH(R.sup.5s)--OP(O)(OR)(SR) or
a salt form thereof. In some embodiments, R.sup.E is
--(S)--CH(R.sup.5s)--OP(O)(OH)(SH) or a salt form thereof. In some
embodiments, R.sup.5s is optionally substituted C.sub.1, C.sub.2,
C.sub.3, or C.sub.4 aliphatic. In some embodiments, R.sup.5s is
C.sub.1, C.sub.2, C.sub.3, or C.sub.4 aliphatic or haloaliphatic.
In some embodiments, R.sup.5s is optionally substituted --CH.sub.3.
In some embodiments, R.sup.5s is --CH.sub.3.
[0283] In some embodiments, BA is an optionally substituted group
selected from C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl,
C.sub.5-30 heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a
natural nucleobase moiety, and a modified nucleobase moiety. In
some embodiments, BA is an optionally substituted group selected
from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.3-30 heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a
natural nucleobase moiety, and a modified nucleobase moiety. In
some embodiments, BA is an optionally substituted group selected
from C.sub.5-30 heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, a
natural nucleobase moiety, and a modified nucleobase moiety. In
some embodiments, BA is optionally substituted C.sub.5-30
heteroaryl having 1-10 heteroatoms independently selected from
oxygen, nitrogen, and sulfur. In some embodiments, BA is optionally
substituted natural nucleobases and tautomers thereof. In some
embodiments, BA is protected natural nucleobases and tautomers
thereof. Various nucleobase protecting groups for oligonucleotide
synthesis are known and can be utilized in accordance with the
present disclosure. In some embodiments, BA is an optionally
substituted nucleobase selected from adenine, cytosine, guanosine,
thymine, and uracil, and tautomers thereof. In some embodiments, BA
is an optionally protected nucleobase selected from adenine,
cytosine, guanosine, thymine, and uracil, and tautomers
thereof.
[0284] In some embodiments, BA is optionally substituted C.sub.3-30
cycloaliphatic. In some embodiments, BA is optionally substituted
C.sub.6-30 aryl. In some embodiments, BA is optionally substituted
C.sub.3-30 heterocyclyl. In some embodiments, BA is optionally
substituted C.sub.5-30 heteroaryl. In some embodiments, BA is an
optionally substituted natural base moiety. In some embodiments, BA
is an optionally substituted modified base moiety. BA is an
optionally substituted group selected from C.sub.3-30
cycloaliphatic, C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, and
C.sub.5-30 heteroaryl. In some embodiments, BA is an optionally
substituted group selected from C.sub.3-30 cycloaliphatic,
C.sub.6-30 aryl, C.sub.3-30 heterocyclyl, C.sub.5-30 heteroaryl,
and a natural nucleobase moiety.
[0285] In some embodiments, BA is connected through an aromatic
ring. In some embodiments, BA is connected through a heteroatom. In
some embodiments, BA is connected through a ring heteroatom of an
aromatic ring. In some embodiments, BA is connected through a ring
nitrogen atom of an aromatic ring.
[0286] In some embodiments, BA is a natural nucleobase moiety. In
some embodiments, BA is an optionally substituted natural
nucleobase moiety. In some embodiments, BA is a substituted natural
nucleobase moiety. In some embodiments, BA is natural nucleobase A,
T, C, U, or G. In some embodiments, BA is an optionally substituted
group selected from natural nucleobases A, T, C, U, and G.
[0287] In some embodiments, BA is an optionally substituted group,
which group is formed by removing a --H from
##STR00030##
or a tautomer thereof. In some embodiments, BA is an optionally
substituted group, which group is formed by removing a --H from
##STR00031##
In some embodiments, BA is an optionally substituted group which
group is selected from
##STR00032##
and tautomeric forms thereof. In some embodiments, BA is an
optionally substituted group which group is selected from
##STR00033##
In some embodiments, BA is an optionally substituted group, which
group is formed by removing a --H from
##STR00034##
and tautomers thereof. In some embodiments, BA is an optionally
substituted group, which group is formed by removing a --H from
##STR00035##
In some embodiments, BA is an optionally substituted group which
group is selected from
##STR00036##
and tautomeric forms thereof. In some embodiments, BA is an
optionally substituted group which group is selected from
##STR00037##
In some embodiments, BA is optionally substituted
##STR00038##
A or a tautomeric form thereof. In some embodiments, BA is
optionally substituted
##STR00039##
In some embodiments, BA is optionally substituted
##STR00040##
or a tautomeric form thereof. In some embodiments, BA is optionally
substituted
##STR00041##
In some embodiments, BA is optionally substituted
##STR00042##
or a tautomeric form thereof. In some embodiments, BA is optionally
substituted
##STR00043##
In some embodiments, BA is optionally substituted
##STR00044##
or a tautomeric form thereof. In some embodiments, BA is optionally
substituted
##STR00045##
In some embodiments, BA is optionally substituted
##STR00046##
or a tautomeric form thereof. In some embodiments, BA is optionally
substituted
##STR00047##
In some embodiments, BA is
##STR00048##
In some embodiments, BA is
##STR00049##
In some embodiments, BA is
##STR00050##
In some embodiments, BA is
##STR00051##
In some embodiments, BA is
##STR00052##
In some embodiments, BA of the 5'-end nucleoside unit of a provided
oligonucleotide, e.g., an oligonucleotide of formula VIII, is an
optionally substituted group, which group is formed by removing a
--H from
##STR00053##
In some embodiments, BA of the 5'-end nucleoside unit is an
optionally substituted group which group is selected from
##STR00054##
In some embodiments, BA of the 5'-end nucleoside unit is an
optionally substituted group, which group is formed by removing a
--H from
##STR00055##
In some embodiments, BA of the 5'-end nucleoside unit is an
optionally substituted group which group is selected from
##STR00056##
In some embodiments, BA of the 5'-end nucleoside unit is optionally
substituted
##STR00057##
In some embodiments, BA of the 5'-end nucleoside unit is optionally
substituted
##STR00058##
In some embodiments, BA of the 5'-end nucleoside unit is optionally
substituted
##STR00059##
In some embodiments, BA of the 5'-end nucleoside unit is optionally
substituted
##STR00060##
In some embodiments, BA of the 5'-end nucleoside unit is optionally
substituted
##STR00061##
In some embodiments, BA of the 5'-end nucleoside unit is
##STR00062##
In some embodiments, BA of the 5'-end nucleoside unit is
##STR00063##
In some embodiments, BA of the 5'-end nucleoside unit is
##STR00064##
In some embodiments, BA of the 5'-end nucleoside unit is
##STR00065##
In some embodiments, BA of the 5'-end nucleoside unit is
##STR00066##
[0288] In some embodiments, BA is H
##STR00067##
In some embodiments, BA is
##STR00068##
In some embodiments, BA is
##STR00069##
In some embodiments, BA is
##STR00070##
In some embodiments, BA is
##STR00071##
In some embodiments, BA is
##STR00072##
In some embodiments, BA is
##STR00073##
In some embodiments, BA is
##STR00074##
In some embodiments, BA is
##STR00075##
In some embodiments, BA is
##STR00076##
In some embodiments, a protection group is --Ac. In some
embodiments, a protection group is -Bz. In some embodiments, a
protection group is -iBu for nucleobase.
[0289] In some embodiments, BA is an optionally substituted purine
base residue. In some embodiments, BA is a protected purine base
residue. In some embodiments, BA is an optionally substituted
adenine residue. In some embodiments, BA is a protected adenine
residue. In some embodiments, BA is an optionally substituted
guanine residue. In some embodiments, BA is a protected guanine
residue. In some embodiments, BA is an optionally substituted
cytosine residue. In some embodiments, BA is a protected cytosine
residue. In some embodiments, BA is an optionally substituted
thymine residue. In some embodiments, BA is a protected thymine
residue. In some embodiments, BA is an optionally substituted
uracil residue. In some embodiments, BA is a protected uracil
residue. In some embodiments, BA is an optionally substituted
5-methylcytosine residue. In some embodiments, BA is a protected
5-methylcytosine residue.
[0290] In some embodiments, BA is a protected base residue as used
in oligonucleotide preparation. In some embodiments, BA is a base
residue illustrated in US 2011/0294124, US 2015/0211006, US
2015/0197540, and WO 2015/107425, each of which is incorporated
herein by reference.
[0291] In some embodiments, BA is a modified nucleobase illustrated
in WO 2017/192679.
[0292] In some embodiments, each R.sup.s is independently --H,
halogen, --CN, --N.sub.3, --NO, --NO.sub.2, -L.sup.s-R',
-L.sup.s-Si(R).sub.3, -L.sup.s-OR', -L.sup.s-SR',
-L.sup.s-N(R').sub.2, --O-L.sup.s-R', --O-L.sup.s-Si(R).sub.3,
--O-L.sup.s-OR', --O-L.sup.s-SR', or --O-L.sup.s-N(R').sub.2 as
described in the present disclosure. In some embodiments, R.sup.s
is --H. In some embodiments, R.sup.s is not --H.
[0293] In some embodiments, R.sup.s is R', wherein R is as
described in the present disclosure. In some embodiments, R.sup.s
is R, wherein R is as described in the present disclosure. In some
embodiments, R.sup.s is optionally substituted C.sub.1-30
heteroaliphatic. In some embodiments, R.sup.s comprises one or more
silicon atoms. In some embodiments, R.sup.s is
--CH.sub.2Si(Ph).sub.2CH.sub.3.
[0294] In some embodiments, R.sup.s is -L.sup.s-R'. In some
embodiments, R.sup.s is -L.sup.s-R' wherein -L.sup.s-- is a
bivalent, optionally substituted C.sub.1-30 heteroaliphatic group.
In some embodiments, R.sup.s is --CH.sub.2Si(Ph).sub.2CH.sub.3.
[0295] In some embodiments, R.sup.s is --F. In some embodiments,
R.sup.s is --Cl. In some embodiments, R.sup.s is --Br. In some
embodiments, R.sup.s is --I. In some embodiments, R.sup.s is --CN.
In some embodiments, R.sup.s is --N.sub.3. In some embodiments,
R.sup.s is --NO. In some embodiments, R.sup.s is --NO.sub.2. In
some embodiments, R.sup.s is -L.sup.s--Si(R).sub.3. In some
embodiments, R.sup.s is --Si(R).sub.3. In some embodiments, R is
-L.sup.s-R'. In some embodiments, R.sup.s is --R'. In some
embodiments, R.sup.s is -L.sup.s-OR'. In some embodiments, R.sup.s
is --OR'. In some embodiments, R.sup.s is -L.sup.s-SR'. In some
embodiments, R.sup.s is --SR'. In some embodiments, R.sup.s is
-L-N(R').sub.2. In some embodiments, R.sup.s is --N(R').sub.2. In
some embodiments, R.sup.s is --O-L.sup.s-R'. In some embodiments,
R.sup.s is --O-L.sup.s--Si(R).sub.3. In some embodiments, R.sup.s
is --O-L-OR'. In some embodiments, R.sup.s is --O-L.sup.s-SR'. In
some embodiments, R is --O-L.sup.s-N(R').sub.2. In some
embodiments, R.sup.s is a 2'-modification as described in the
present disclosure. In some embodiments, R.sup.s is --OR, wherein R
is as described in the present disclosure. In some embodiments,
R.sup.s is --OR, wherein R is optionally substituted C.sub.1-6
aliphatic. In some embodiments, R.sup.s is --OMe. In some
embodiments, R.sup.s is --OCH.sub.2CH.sub.2OMe.
[0296] In some embodiments, s is 0-20. In some embodiments, s is
1-20. In some embodiments, s is 1-5. In some embodiments, s is 1.
In some embodiments, s is 2. In some embodiments, s is 3. In some
embodiments, s is 4. In some embodiments, s is 5. In some
embodiments, s is 6. In some embodiments, s is 7. In some
embodiments, s is 8. In some embodiments, s is 9. In some
embodiments, s is 10. In some embodiments, s is 11. In some
embodiments, s is 12. In some embodiments, s is 13. In some
embodiments, s is 14. In some embodiments, s is 15. In some
embodiments, s is 16. In some embodiments, s is 17. In some
embodiments, s is 18. In some embodiments, s is 19. In some
embodiments, s is 20.
[0297] In some embodiments, L.sup.s is L, wherein L is as described
in the present disclosure. In some embodiments, L is a bivalent
optionally substituted methylene group.
[0298] As described herein, each L is independently a covalent
bond, or a bivalent, optionally substituted, linear or branched
group selected from a C.sub.1-30 aliphatic group and a C.sub.1-30
heteroaliphatic group having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus, boron and
silicon, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L.
[0299] In some embodiments, L is a covalent bond, or a bivalent,
optionally substituted, linear or branched group selected from a
C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein one or more
methylene units are optionally and independently replaced by an
optionally substituted group selected from C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--, and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L. In some
embodiments, L is a covalent bond, or a bivalent, optionally
substituted, linear or branched C.sub.1-30 aliphatic group, wherein
one or more methylene units are optionally and independently
replaced by an optionally substituted group selected from C.sub.1-6
alkylene, C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--,
--O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --C(O)S--, --C(O)O--,
--P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--,
--P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--,
--P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--, and one or
more carbon atoms are optionally and independently replaced with
Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent,
optionally substituted, linear or branched C.sub.1-30
heteroaliphatic group having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
wherein one or more methylene units are optionally and
independently replaced by an optionally substituted group selected
from C.sub.1-6 alkylene, C.sub.1-6 alkenylene, --C.ident.C--,
--C(R').sub.2--, --O--, --S--, --S--S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)O--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--C(O)S--, --C(O)O--, --P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--,
--P(O)(NR')--, --P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--,
--P(S)(NR')--, --P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--, and one or
more carbon atoms are optionally and independently replaced with
Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent,
optionally substituted, linear or branched group selected from a
C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein one or more
methylene units are optionally and independently replaced by an
optionally substituted group selected from C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, or --C(O)O--, and one or more
carbon atoms are optionally and independently replaced with
Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent,
optionally substituted, linear or branched group selected from a
C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group
having 1-5 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein one or more
methylene units are optionally and independently replaced by an
optionally substituted group selected from C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, and --C(O)O--, and one or more
carbon atoms are optionally and independently replaced with
Cy.sup.L. In some embodiments, L is a covalent bond, or a bivalent,
optionally substituted, linear or branched group selected from a
C.sub.1-10 aliphatic group and a C.sub.1-10 heteroaliphatic group
having 1-5 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon, wherein one or more
methylene units are optionally and independently replaced by an
optionally substituted group selected from --C(R').sub.2--, --O--,
--S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --C(O)S--, and --C(O)O--.
[0300] In some embodiments, L is a covalent bond. In some
embodiments, L is optionally substituted bivalent C.sub.1-30
aliphatic. In some embodiments, L is optionally substituted
bivalent C.sub.1-30 heteroaliphatic having 1-10 heteroatoms
independently selected from boron, oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0301] In some embodiments, aliphatic moieties, e.g. those of L, R,
etc., either monovalent or bivalent or multivalent, and can contain
any number of carbon atoms (before any optional substitution)
within its range, e.g., C.sub.1, C.sub.2, C.sub.3, C.sub.4,
C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11,
C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17,
C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23,
C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29,
C.sub.30, etc. In some embodiments, heteroaliphatic moieties, e.g.
those of L, R, etc., either monovalent or bivalent or multivalent,
and can contain any number of carbon atoms (before any optional
substitution) within its range, e.g., C.sub.1, C.sub.2, C.sub.3,
C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,
C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22,
C.sub.23, C.sub.24, C.sub.25, C.sub.26, C.sub.27, C.sub.28,
C.sub.29, C.sub.30, etc.
[0302] In some embodiments, one or more methylene unit is
optionally and independently substituted with --O--, --S--,
--N(R')--, --C(O)--, --S(O)--, --S(O).sub.2--, --P(O)(OR')--,
--P(O)(SR')--, --P(S)(OR')--, or --P(S)(OR')--. In some
embodiments, a methylene unit is replaced with --O--. In some
embodiments, a methylene unit is replaced with --S--. In some
embodiments, a methylene unit is replaced with --N(R')--. In some
embodiments, a methylene unit is replaced with --C(O)--. In some
embodiments, a methylene unit is replaced with --S(O)--. In some
embodiments, a methylene unit is replaced with --S(O).sub.2--. In
some embodiments, a methylene unit is replaced with --P(O)(OR')--.
In some embodiments, a methylene unit is replaced with
--P(O)(SR')--. In some embodiments, a methylene unit is replaced
with --P(O)(R')--. In some embodiments, a methylene unit is
replaced with --P(O)(NR')--. In some embodiments, a methylene unit
is replaced with --P(S)(OR')--. In some embodiments, a methylene
unit is replaced with --P(S)(SR')--. In some embodiments, a
methylene unit is replaced with --P(S)(R')--. In some embodiments,
a methylene unit is replaced with --P(S)(NR')--. In some
embodiments, a methylene unit is replaced with --P(R')--. In some
embodiments, a methylene unit is replaced with --P(OR')--. In some
embodiments, a methylene unit is replaced with --P(SR')--. In some
embodiments, a methylene unit is replaced with --P(NR')--. In some
embodiments, a methylene unit is replaced with
--P(OR')[B(R').sub.3]--. In some embodiments, one or more methylene
unit is optionally and independently substituted with --O--, --S--,
--N(R')--, --C(O)--, --S(O)--, --S(O).sub.2--, --P(O)(OR')--,
--P(O)(SR')--, --P(S)(OR')--, or --P(S)(OR')--. In some
embodiments, a methylene unit is replaced with --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--, each of which may independently be an
internucleotidic linkage.
[0303] In some embodiments, L, e.g., when connected to R, is
--CH.sub.2--. In some embodiments, L is --C(R).sub.2--, wherein at
least one R is not hydrogen. In some embodiments, L is --CHR--. In
some embodiments, R is hydrogen. In some embodiments, L is --CHR--,
wherein R is not hydrogen. In some embodiments, C of --CHR-- is
chiral. In some embodiments, L is --(R)--CHR--, wherein C of
--CHR-- is chiral. In some embodiments, L is --(S)--CHR--, wherein
C of --CHR-- is chiral. In some embodiments, R is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R is
optionally substituted C.sub.1-6 alkyl. In some embodiments, R is
optionally substituted C.sub.1-5 aliphatic. In some embodiments, R
is optionally substituted C.sub.1-5 alkyl. In some embodiments, R
is optionally substituted C.sub.1-4 aliphatic. In some embodiments,
R is optionally substituted C.sub.1-4 alkyl. In some embodiments, R
is optionally substituted C.sub.1-3 aliphatic. In some embodiments,
R is optionally substituted C.sub.1-3 alkyl. In some embodiments, R
is optionally substituted C.sub.2 aliphatic. In some embodiments, R
is optionally substituted methyl. In some embodiments, R is
C.sub.1-6 aliphatic. In some embodiments, R is C.sub.1-6 alkyl. In
some embodiments, R is C.sub.15 aliphatic. In some embodiments, R
is C.sub.1-5 alkyl. In some embodiments, R is C.sub.1-4 aliphatic.
In some embodiments, R is C.sub.1-4 alkyl. In some embodiments, R
is C.sub.1-3 aliphatic. In some embodiments, R is C.sub.1-3 alkyl.
In some embodiments, R is C.sub.2 aliphatic. In some embodiments, R
is methyl. In some embodiments, R is C.sub.1-6 haloaliphatic. In
some embodiments, R is C.sub.1-6 haloalkyl. In some embodiments, R
is C.sub.15 haloaliphatic. In some embodiments, R is C.sub.1-5
haloalkyl. In some embodiments, R is C.sub.1-4 haloaliphatic. In
some embodiments, R is C.sub.1-4 haloalkyl. In some embodiments, R
is C.sub.1-3 haloaliphatic. In some embodiments, R is C.sub.1-3
haloalkyl. In some embodiments, R is C.sub.2 haloaliphatic. In some
embodiments, R is methyl substituted with one or more halogen. In
some embodiments, R is --CF.sub.3. In some embodiments, L is
optionally substituted --CH.dbd.CH--. In some embodiments, L is
optionally substituted (E)-CH.dbd.CH--. In some embodiments, L is
optionally substituted (Z)--CH.dbd.CH--. In some embodiments, L is
--C.ident.C--.
[0304] In some embodiments, L comprises at least one phosphorus
atom. In some embodiments, at least one methylene unit of L is
replaced with --P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--,
--P(O)(NR')--, --P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--,
--P(S)(NR')--, --P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--.
[0305] In some embodiments, Cy.sup.L is an optionally substituted
tetravalent group selected from a C.sub.3-20 cycloaliphatic ring, a
C.sub.6-20 aryl ring, a 5-20 membered heteroaryl ring having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon, and a 3-20 membered heterocyclyl ring
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus, boron and silicon.
[0306] In some embodiments, Cy.sup.L is monocyclic. In some
embodiments, Cy.sup.L is bicyclic. In some embodiments, Cy.sup.L is
polycyclic.
[0307] In some embodiments, Cy.sup.L is saturated. In some
embodiments, Cy.sup.L is partially unsaturated. In some
embodiments, Cy.sup.L is aromatic. In some embodiments, Cy.sup.L is
or comprises a saturated ring moiety. In some embodiments, Cy.sup.L
is or comprises a partially unsaturated ring moiety. In some
embodiments, Cy.sup.L is or comprises an aromatic ring moiety.
[0308] In some embodiments, Cy.sup.L is an optionally substituted
C.sub.3-20 cycloaliphatic ring as described in the present
disclosure (for example, those described for R but tetravalent). In
some embodiments, a ring is an optionally substituted saturated
C.sub.3-20 cycloaliphatic ring. In some embodiments, a ring is an
optionally substituted partially unsaturated C.sub.3-20
cycloaliphatic ring. A cycloaliphatic ring can be of various sizes
as described in the present disclosure. In some embodiments, a ring
is 3, 4, 5, 6, 7, 8, 9, or 10-membered. In some embodiments, a ring
is 3-membered. In some embodiments, a ring is 4-membered. In some
embodiments, a ring is 5-membered. In some embodiments, a ring is
6-membered. In some embodiments, a ring is 7-membered. In some
embodiments, a ring is 8-membered. In some embodiments, a ring is
9-membered. In some embodiments, a ring is 10-membered. In some
embodiments, a ring is an optionally substituted cyclopropyl
moiety. In some embodiments, a ring is an optionally substituted
cyclobutyl moiety. In some embodiments, a ring is an optionally
substituted cyclopentyl moiety. In some embodiments, a ring is an
optionally substituted cyclohexyl moiety. In some embodiments, a
ring is an optionally substituted cycloheptyl moiety. In some
embodiments, a ring is an optionally substituted cyclooctanyl
moiety. In some embodiments, a cycloaliphatic ring is a cycloalkyl
ring. In some embodiments, a cycloaliphatic ring is monocyclic. In
some embodiments, a cycloaliphatic ring is bicyclic. In some
embodiments, a cycloaliphatic ring is polycyclic. In some
embodiments, a ring is a cycloaliphatic moiety as described in the
present disclosure for R with more valences.
[0309] In some embodiments, Cy.sup.L is an optionally substituted
6-20 membered aryl ring. In some embodiments, a ring is an
optionally substituted tetravalent phenyl moiety. In some
embodiments, a ring is a tetravalent phenyl moiety. In some
embodiments, a ring is an optionally substituted naphthalene
moiety. A ring can be of different size as described in the present
disclosure. In some embodiments, an aryl ring is 6-membered. In
some embodiments, an aryl ring is 10-membered. In some embodiments,
an aryl ring is 14-membered. In some embodiments, an aryl ring is
monocyclic. In some embodiments, an aryl ring is bicyclic. In some
embodiments, an aryl ring is polycyclic. In some embodiments, a
ring is an aryl moiety as described in the present disclosure for R
with more valences.
[0310] In some embodiments, Cy.sup.L is an optionally substituted
5-20 membered heteroaryl ring having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In
some embodiments, Cy.sup.L is an optionally substituted 5-20
membered heteroaryl ring having 1-10 heteroatoms independently
selected from oxygen, nitrogen, and sulfur. In some embodiments, as
described in the present disclosure, heteroaryl rings can be of
various sizes and contain various numbers and/or types of
heteroatoms. In some embodiments, a heteroaryl ring contains no
more than one heteroatom. In some embodiments, a heteroaryl ring
contains more than one heteroatom. In some embodiments, a
heteroaryl ring contains no more than one type of heteroatom. In
some embodiments, a heteroaryl ring contains more than one type of
heteroatoms. In some embodiments, a heteroaryl ring is 5-membered.
In some embodiments, a heteroaryl ring is 6-membered. In some
embodiments, a heteroaryl ring is 8-membered. In some embodiments,
a heteroaryl ring is 9-membered. In some embodiments, a heteroaryl
ring is 10-membered. In some embodiments, a heteroaryl ring is
monocyclic. In some embodiments, a heteroaryl ring is bicyclic. In
some embodiments, a heteroaryl ring is polycyclic. In some
embodiments, a heteroaryl ring is a nucleobase moiety, e.g., A, T,
C, G, U, etc. In some embodiments, a ring is a heteroaryl moiety as
described in the present disclosure for R with more valences.
[0311] In some embodiments, Cy.sup.L is a 3-20 membered
heterocyclyl ring having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, Cy.sup.L is a 3-20 membered heterocyclyl ring having
1-10 heteroatoms independently selected from oxygen, nitrogen, and
sulfur. In some embodiments, a heterocyclyl ring is saturated. In
some embodiments, a heterocyclyl ring is partially unsaturated. A
heterocyclyl ring can be of various sizes as described in the
present disclosure. In some embodiments, a ring is 3, 4, 5, 6, 7,
8, 9, or 10-membered. In some embodiments, a ring is 3-membered. In
some embodiments, a ring is 4-membered. In some embodiments, a ring
is 5-membered. In some embodiments, a ring is 6-membered. In some
embodiments, a ring is 7-membered. In some embodiments, a ring is
8-membered. In some embodiments, a ring is 9-membered. In some
embodiments, a ring is 10-membered. Heterocyclyl rings can contain
various numbers and/or types of heteroatoms. In some embodiments, a
heterocyclyl ring contains no more than one heteroatom. In some
embodiments, a heterocyclyl ring contains more than one heteroatom.
In some embodiments, a heterocyclyl ring contains no more than one
type of heteroatom. In some embodiments, a heterocyclyl ring
contains more than one type of heteroatoms. In some embodiments, a
heterocyclyl ring is monocyclic. In some embodiments, a
heterocyclyl ring is bicyclic. In some embodiments, a heterocyclyl
ring is polycyclic. In some embodiments, a ring is a heterocyclyl
moiety as described in the present disclosure for R with more
valences.
[0312] As readily appreciated by a person having ordinary skill in
the art, many suitable ring moieties are extensively described in
and can be used in accordance with the present disclosure, for
example, those described for R (which may have more valences for
Cy.sup.L).
[0313] In some embodiments, Cy.sup.L is a sugar moiety in a nucleic
acid. In some embodiments, Cy.sup.L is an optionally substituted
furanose moiety. In some embodiments, Cy.sup.L is a pyranose
moiety. In some embodiments, Cy.sup.L is an optionally substituted
furanose moiety found in DNA. In some embodiments, Cy.sup.L is an
optionally substituted furanose moiety found in RNA. In some
embodiments, Cy.sup.L is an optionally substituted
2'-deoxyribofuranose moiety. In some embodiments, Cy.sup.L is an
optionally substituted ribofuranose moiety. In some embodiments,
substitutions provide sugar modifications as described in the
present disclosure. In some embodiments, an optionally substituted
2'-deoxyribofuranose moiety and/or an optionally substituted
ribofuranose moiety comprise substitution at a 2'-position. In some
embodiments, a 2'-position is a 2'-modification as described in the
present disclosure. In some embodiments, a 2'-modification is --F.
In some embodiments, a 2'-modification is --OR, wherein R is as
described in the present disclosure. In some embodiments, R is not
hydrogen. In some embodiments, Cy.sup.L is a modified sugar moiety,
such as a sugar moiety in LNA. In some embodiments, Cy.sup.L is a
modified sugar moiety, such as a sugar moiety in ENA. In some
embodiments, Cy.sup.L is a terminal sugar moiety of an
oligonucleotide, connecting an internucleotidic linkage and a
nucleobase. In some embodiments, Cy.sup.L is a terminal sugar
moiety of an oligonucleotide, for example, when that terminus is
connected to a solid support optionally through a linker. In some
embodiments, Cy.sup.L is a sugar moiety connecting two
internucleotidic linkages and a nucleobase. Example sugars and
sugar moieties are extensively described in the present
disclosure.
[0314] In some embodiments, Cy.sup.L is a nucleobase moiety. In
some embodiments, a nucleobase is a natural nucleobase, such as A,
T, C, G, U, etc. In some embodiments, a nucleobase is a modified
nucleobase. In some embodiments, Cy.sup.L is optionally substituted
nucleobase moiety selected from A, T, C, G, U, and 5mC. Example
nucleobases and nucleobase moieties are extensively described in
the present disclosure.
[0315] In some embodiments, two Cy.sup.L moieties are bonded to
each other, wherein one Cy.sup.L is a sugar moiety and the other is
a nucleobase moiety. In some embodiments, such a sugar moiety and
nucleobase moiety forms a nucleoside moiety. In some embodiments, a
nucleoside moiety is natural. In some embodiments, a nucleoside
moiety is modified. In some embodiments, Cy.sup.L is an optionally
substituted natural nucleoside moiety selected from adenosine,
5-methyluridine, cytidine, guanosine, uridine, 5-methylcytidine,
2'-deoxyadenosine, thymidine, 2'-deoxycytidine, 2'-deoxyguanosine,
2'-deoxyuridine, and 5-methyl-2'-deoxycytidine. Example nucleosides
and nucleosides moieties are extensive described in the present
disclosure.
[0316] In some embodiments, for example in L.sup.s, Cy.sup.L is an
optionally substituted nucleoside moiety bonded to an
internucleotidic linkage, for example, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, --OP(OR')[B(R').sub.3]O--,
etc., which may form an optionally substituted nucleotidic unit.
Example nucleotides and nucleosides moieties are extensive
described in the present disclosure.
[0317] In some embodiments, each Ring A is independently an
optionally substituted 3-20 membered monocyclic, bicyclic or
polycyclic ring having 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, Ring A is an optionally substituted ring, which ring
is as described in the present disclosure. In some embodiments, a
ring is
##STR00077##
In some embodiments, a ring is
##STR00078##
In some embodiments, Ring A is or comprises a ring of a sugar
moiety. In some embodiments, Ring A is or comprises a ring of a
modified sugar moiety.
[0318] In some embodiments, a sugar unit is of the structure
##STR00079##
wherein each variable is independently as described in the present
disclosure. In some embodiments, a nucleoside unit is of the
structure
##STR00080##
wherein each variable is independently as described in the present
disclosure. In some embodiments, a nucleotide unit, e.g., Nu.sup.M,
Nu.sup.O, etc., is of the structure
##STR00081##
wherein each variable is independently as described in the present
disclosure. In some embodiments, for Nu.sup.O, L.sup.P is a natural
phosphate linkage, and L.sup.s is --C(R.sup.5s).sub.2-- as
described in the present disclosure.
[0319] In some embodiments, L.sup.s is --C(R.sup.5s).sub.2--
and
##STR00082##
is as described in the present disclosure. In some embodiments,
##STR00083##
BA is connected at C1, and each of R.sup.1s, R.sup.2s, R.sup.3s,
R.sup.4s and R.sup.5s is independently as described in the present
disclosure. In some embodiments,
##STR00084##
wherein R.sup.2s is as described in the present disclosure. In some
embodiments,
##STR00085##
wherein R.sup.2s is not --OH. In some embodiments,
##STR00086##
wherein R.sup.2s and R.sup.4s are R, and the two R groups are taken
together with their intervening atoms to form an optionally
substituted ring. In some embodiments,
##STR00087##
or Ring A, is optionally substituted
##STR00088##
In some embodiments,
##STR00089##
or Ring A, is
##STR00090##
[0320] In some embodiments,
##STR00091##
or Ring A, is
##STR00092##
[0322] In some embodiments, each of R.sup.1s, R.sup.2s, R.sup.3s,
R.sup.4s, and R.sup.5s is independently R.sup.s, wherein R.sup.s is
as described in the present disclosure.
[0323] In some embodiments, R.sup.1s is R.sup.s wherein R.sup.s is
as described in the present disclosure. In some embodiments,
R.sup.1s is at 1'-position (BA is at 1'-position). In some
embodiments, R.sup.1s is --H. In some embodiments, R.sup.1s is --F.
In some embodiments, R.sup.1s is --Cl. In some embodiments,
R.sup.1s is --Br. In some embodiments, R.sup.1s is --I. In some
embodiments, R.sup.1s is --CN. In some embodiments, R.sup.1s is
--N.sub.3. In some embodiments, R.sup.1s is --NO. In some
embodiments, R.sup.1s is --NO.sub.2. In some embodiments, R.sup.1s
is -L-R'. In some embodiments, R.sup.1s is --R'. In some
embodiments, R.sup.1s is -L-OR'. In some embodiments, R.sup.1s is
--OR'. In some embodiments, R.sup.1s is -L-SR'. In some
embodiments, R.sup.1s is --SR'. In some embodiments, R.sup.1s is
L-L-N(R').sub.2. In some embodiments, R.sup.1s is --N(R').sub.2. In
some embodiments, R.sup.1s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.1s is --OMe. In some embodiments, R.sup.1s
is -MOE. In some embodiments, R.sup.1s is hydrogen. In some
embodiments, R.sup.s at one 1'-position is hydrogen, and R.sup.s at
the other 1'-position is not hydrogen as described herein. In some
embodiments, R.sup.s at both 1'-positions are hydrogen. In some
embodiments, R.sup.s at one 1'-position is hydrogen, and the other
1'-position is connected to an internucleotidic linkage. In some
embodiments, R.sup.1s is --F. In some embodiments, R.sup.1s is
--Cl. In some embodiments, R.sup.1s is --Br. In some embodiments,
R.sup.1s is --I. In some embodiments, R.sup.1s is --CN. In some
embodiments, R.sup.1s is --N.sub.3. In some embodiments, R.sup.1s
is --NO. In some embodiments, R.sup.1s is --NO.sub.2. In some
embodiments, R.sup.1s is -L-R'. In some embodiments, R.sup.1s is
--R'. In some embodiments, R.sup.1s is -L-OR'. In some embodiments,
R.sup.1s is --OR'. In some embodiments, R.sup.1s is -L-SR'. In some
embodiments, R.sup.1s is --SR'. In some embodiments, R.sup.1s is
-L-N(R').sub.2. In some embodiments, R.sup.1s is --N(R').sub.2. In
some embodiments, R.sup.1s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.1s is --OH. In some embodiments, R.sup.1s
is --OMe. In some embodiments, R.sup.1s is -MOE. In some
embodiments, R.sup.1s is hydrogen. In some embodiments, one
R.sup.1s at a 1'-position is hydrogen, and the other R.sup.1s at
the other 1'-position is not hydrogen as described herein. In some
embodiments, R.sup.1s at both 1'-positions are hydrogen. In some
embodiments, R.sup.1s is --O-L.sup.s-OR'. In some embodiments,
R.sup.1s is --O-L.sup.s-OR', wherein L.sup.s is optionally
substituted C.sub.1-6 alkylene, and R' is optionally substituted
C.sub.1-6 aliphatic. In some embodiments, R.sup.1s is
--O-(optionally substituted C.sub.1-6 alkylene)-OR'. In some
embodiments, R.sup.1s is --O-(optionally substituted C.sub.1-6
alkylene)-OR', wherein R' is optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.1s is --OCH.sub.2CH.sub.2OMe.
[0324] In some embodiments, R.sup.2s is R.sup.s wherein R.sup.s is
as described in the present disclosure. In some embodiments, if
there are two R.sup.2s at the 2'-position, one R.sup.2s is --H and
the other is not. In some embodiments, R.sup.2s is at 2'-position
(BA is at 1'-position). In some embodiments, R.sup.2s is --H. In
some embodiments, R.sup.2s is --F. In some embodiments, R.sup.2s is
--Cl. In some embodiments, R.sup.2s is --Br. In some embodiments,
R.sup.2s is --I. In some embodiments, R.sup.2s is --CN. In some
embodiments, R.sup.2s is --N.sub.3. In some embodiments, R.sup.2s
is --NO. In some embodiments, R.sup.2s is --NO.sub.2. In some
embodiments, R.sup.2s is -L-R'. In some embodiments, R.sup.2s is
--R'. In some embodiments, R.sup.2s is -L-OR'. In some embodiments,
R.sup.2s is --OR'. In some embodiments, R.sup.2s is -L-SR'. In some
embodiments, R.sup.2s is --SR'. In some embodiments, R.sup.2s is
L-L-N(R').sub.2. In some embodiments, R.sup.2s is --N(R').sub.2. In
some embodiments, R.sup.2s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.2s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.2s is --OMe. In some embodiments, R.sup.2s
is -MOE. In some embodiments, R.sup.2s is hydrogen. In some
embodiments, R.sup.s at one 2'-position is hydrogen, and R.sup.s at
the other 2'-position is not hydrogen as described herein. In some
embodiments, R.sup.s at both 2'-positions are hydrogen. In some
embodiments, R.sup.s at one 2'-position is hydrogen, and the other
2'-position is connected to an internucleotidic linkage. In some
embodiments, R.sup.2s is --F. In some embodiments, R.sup.2s is
--Cl. In some embodiments, R.sup.2s is --Br. In some embodiments,
R.sup.2s is --I. In some embodiments, R.sup.2s is --CN. In some
embodiments, R.sup.2s is --N.sub.3. In some embodiments, R.sup.2s
is --NO. In some embodiments, R.sup.2s is --NO.sub.2. In some
embodiments, R.sup.2s is -L-R'. In some embodiments, R.sup.2s is
--R'. In some embodiments, R.sup.2s is -L-OR'. In some embodiments,
R.sup.2s is --OR'. In some embodiments, R.sup.2s is -L-SR'. In some
embodiments, R.sup.2s is --SR'. In some embodiments, R.sup.2s is
-L-N(R').sub.2. In some embodiments, R.sup.2s is --N(R').sub.2. In
some embodiments, R.sup.2s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.2s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.2s is --OH. In some embodiments, R.sup.2s
is --OMe. In some embodiments, R.sup.2s is -MOE. In some
embodiments, R.sup.2s is hydrogen. In some embodiments, one
R.sup.2s at a 2'-position is hydrogen, and the other R.sup.2s at
the other 2'-position is not hydrogen as described herein. In some
embodiments, R.sup.2s at both 2'-positions are hydrogen. In some
embodiments, R.sup.2s is --O-L-OR'. In some embodiments, R.sup.2s
is --O-L-OR', wherein L.sup.s is optionally substituted C.sub.1-6
alkylene, and R' is optionally substituted C.sub.1-6 aliphatic. In
some embodiments, R.sup.2s is --O-(optionally substituted C.sub.1-6
alkylene)-OR'. In some embodiments, R.sup.2s is --O-(optionally
substituted C.sub.1-6 alkylene)-OR', wherein R' is optionally
substituted C.sub.1-6 alkyl. In some embodiments, R.sup.2s is
--OCH.sub.2CH.sub.2OMe.
[0325] In some embodiments, R.sup.3s is R.sup.s wherein R.sup.s is
as described in the present disclosure. In some embodiments,
R.sup.3s is at 3'-position (BA is at 1'-position). In some
embodiments, R.sup.3s is --H. In some embodiments, R.sup.3 is --F.
In some embodiments, R.sup.3s is --Cl. In some embodiments,
R.sup.3s is --Br. In some embodiments, R.sup.3s is --I. In some
embodiments, R.sup.3s is --CN. In some embodiments, R.sup.3s is
--N.sub.3. In some embodiments, R.sup.3s is --NO. In some
embodiments, R.sup.3s is --NO.sub.2. In some embodiments, R.sup.3s
is -L-R'. In some embodiments, R.sup.s is --R'. In some
embodiments, R.sup.3s is -L-OR'. In some embodiments, R.sup.3s is
--OR'. In some embodiments, R.sup.3s is -L-SR'. In some
embodiments, R.sup.3s is --SR'. In some embodiments, R.sup.3s is
-L-N(R').sub.2. In some embodiments, R.sup.3s is --N(R').sub.2. In
some embodiments, R.sup.3s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.3s is --OMe. In some embodiments, R.sup.3s
is -MOE. In some embodiments, R.sup.3s is hydrogen. In some
embodiments, R.sup.s at one 3'-position is hydrogen, and R.sup.s at
the other 3'-position is not hydrogen as described herein. In some
embodiments, R.sup.s at both 3'-positions are hydrogen. In some
embodiments, R.sup.s at one 3'-position is hydrogen, and the other
3'-position is connected to an internucleotidic linkage. In some
embodiments, R.sup.s is --F. In some embodiments, R.sup.3s is --Cl.
In some embodiments, R.sup.3s is --Br. In some embodiments,
R.sup.3s is --I. In some embodiments, R.sup.3s is --CN. In some
embodiments, R.sup.3s is --N.sub.3. In some embodiments, R.sup.3s
is --NO. In some embodiments, R.sup.3s is --NO.sub.2. In some
embodiments, R.sup.3s is -L-R'. In some embodiments, R.sup.3s is
--R'. In some embodiments, R.sup.3s is -L-OR'. In some embodiments,
R.sup.3s is --OR'. In some embodiments, R.sup.s is -L-SR'. In some
embodiments, R.sup.3s is --SR'. In some embodiments, R.sup.3s is
L-L-N(R').sub.2. In some embodiments, R.sup.3s is --N(R').sub.2. In
some embodiments, R.sup.3s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.3s is --OH. In some embodiments, R.sup.3s
is --OMe. In some embodiments, R.sup.3s is -MOE. In some
embodiments, R.sup.3s is hydrogen.
[0326] In some embodiments, R.sup.4s is R.sup.s wherein R.sup.s is
as described in the present disclosure. In some embodiments,
R.sup.4s is at 4'-position (BA is at 1'-position). In some
embodiments, R.sup.4s is --H. In some embodiments, R.sup.4s is --F.
In some embodiments, R.sup.4s is --Cl. In some embodiments,
R.sup.4s is --Br. In some embodiments, R.sup.4s is --I. In some
embodiments, R.sup.4s is --CN. In some embodiments, R.sup.4s is
--N.sub.3. In some embodiments, R.sup.4s is --NO. In some
embodiments, R.sup.4s is --NO.sub.2. In some embodiments, R.sup.4s
is -L-R'. In some embodiments, R.sup.4s is --R'. In some
embodiments, R.sup.4s is -L-OR'. In some embodiments, R.sup.4s is
--OR'. In some embodiments, R.sup.4s is -L-SR'. In some
embodiments, R.sup.4s is --SR'. In some embodiments, R.sup.4s is
-L-N(R').sub.2. In some embodiments, R.sup.4s is --N(R').sub.2. In
some embodiments, R.sup.4s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.4s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.4s is --OMe. In some embodiments, R.sup.4s
is -MOE. In some embodiments, R.sup.4s is hydrogen. In some
embodiments, R.sup.s at one 4'-position is hydrogen, and R.sup.s at
the other 4'-position is not hydrogen as described herein. In some
embodiments, R.sup.s at both 4'-positions are hydrogen. In some
embodiments, R.sup.s at one 4'-position is hydrogen, and the other
4'-position is connected to an internucleotidic linkage. In some
embodiments, R.sup.4 is --F. In some embodiments, R.sup.4s is --Cl.
In some embodiments, R.sup.4s is --Br. In some embodiments,
R.sup.4s is --I. In some embodiments, R.sup.4s is --CN. In some
embodiments, R.sup.4s is --N.sub.3. In some embodiments, R.sup.4s
is --NO. In some embodiments, R.sup.4s is --NO.sub.2. In some
embodiments, R.sup.4s is -L-R'. In some embodiments, R.sup.4s is
--R'. In some embodiments, R.sup.4s is -L-OR'. In some embodiments,
R.sup.4s is --OR'. In some embodiments, R.sup.4s is -L-SR'. In some
embodiments, R.sup.4s is --SR'. In some embodiments, R.sup.4s is
L-L-N(R').sub.2. In some embodiments, R.sup.4s is --N(R').sub.2. In
some embodiments, R.sup.4s is --OR', wherein R' is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.4s is
--OR', wherein R' is optionally substituted C.sub.1-6 alkyl. In
some embodiments, R.sup.4s is --OH. In some embodiments, R.sup.4s
is --OMe. In some embodiments, R.sup.4s is -MOE. In some
embodiments, R.sup.4s is hydrogen.
[0327] In some embodiments, R.sup.5s is R.sup.s wherein R.sup.s is
as described in the present disclosure. In some embodiments,
R.sup.5s is R' wherein R' is as described in the present
disclosure. In some embodiments, R.sup.5s is --H. In some
embodiments, two or more R.sup.5s are connected to the same carbon
atom, and at least one is not --H. In some embodiments, R.sup.5s is
not --H. In some embodiments, R.sup.5s is --F. In some embodiments,
R.sup.5s is --Cl. In some embodiments, R.sup.5s is --Br. In some
embodiments, R.sup.5s is --I. In some embodiments, R.sup.5s is
--CN. In some embodiments, R.sup.5s is --N.sub.3. In some
embodiments, R.sup.5s is --NO. In some embodiments, R.sup.5s is
--NO.sub.2. In some embodiments, R.sup.5s is -L-R'. In some
embodiments, R.sup.5s is --R'. In some embodiments, R.sup.5s is
-L-OR'. In some embodiments, R.sup.5s is --OR'. In some
embodiments, R.sup.5s is -L-SR'. In some embodiments, R.sup.5s is
--SR'. In some embodiments, R.sup.5s is L-L-N(R').sub.2. In some
embodiments, R.sup.5s is --N(R').sub.2. In some embodiments,
R.sup.5s is --OR', wherein R' is optionally substituted C.sub.1-6
aliphatic. In some embodiments, R.sup.5s is --OR', wherein R' is
optionally substituted C.sub.1-6 alkyl. In some embodiments,
R.sup.5s is --OH. In some embodiments, R.sup.5s is --OMe. In some
embodiments, R.sup.5s is -MOE. In some embodiments, R.sup.5s is
hydrogen.
[0328] In some embodiments, R.sup.5s is optionally substituted
C.sub.1-6 aliphatic as described in the present disclosure, e.g.,
C.sub.1-6 aliphatic embodiments described for R or other variables.
In some embodiments, R.sup.5s is optionally substituted C.sub.1-6
alkyl. In some embodiments, R.sup.5s is methyl. In some
embodiments, R.sup.5s is ethyl.
[0329] In some embodiments, R.sup.5s is a protected hydroxyl group
suitable for oligonucleotide synthesis. In some embodiments,
R.sup.5s is --OR', wherein R' is optionally substituted C.sub.1-6
aliphatic. In some embodiments, R.sup.5s is DMTrO-. Example
protecting groups are widely known for use in accordance with the
present disclosure. For additional examples, see Greene, T. W.;
Wuts, P. G. M. Protective Groups in Organic Synthesis, 2nd ed.;
Wiley: New York, 1991, and WO/2011/005761, WO/2013/012758,
WO/2014/012081, WO/2015/107425, WO/2010/064146, WO/2014/010250,
WO/2011/108682, WO/2012/039448, and WO/2012/073857, protecting
groups of each of which are hereby incorporated by reference.
[0330] In some embodiments, two or more of R.sup.1s, R.sup.2s,
R.sup.3s, R.sup.4s, and R.sup.5s are R and can be taken together
with intervening atom(s) to form a ring as described in the present
disclosure. In some embodiments, R.sup.2s and R.sup.4s are R taken
together to form a ring, and a sugar moiety can be a bicyclic sugar
moiety, e.g., a LNA sugar moiety.
[0331] In some embodiments, L.sup.s is --C(R.sup.5s).sub.2--,
wherein each R.sup.5s is independently as described in the present
disclosure. In some embodiments, one of R.sup.5s is H and the other
is not H. In some embodiments, none of R.sup.5s is H. In some
embodiments, L is --CHR'--, wherein each R.sup.5s is independently
as described in the present disclosure. In some embodiments,
--C(R.sup.5s).sub.2-- is 5'-C, optionally substituted, of a sugar
moiety. In some embodiments, the C of --C(R.sup.5s).sub.2-- is
connected to linkage phosphorus and a sugar wing moiety. In some
embodiments, the C of --C(R.sup.5s).sub.2-- is of R configuration.
In some embodiments, the C of --C(R.sup.5s).sub.2-- is of S
configuration. As described in the present disclosure, in some
embodiments, R.sup.5s is optionally substituted C.sub.1-6
aliphatic; in some embodiments, R.sup.5s is methyl.
[0332] In some embodiments, provided compounds comprise one or more
bivalent or multivalent optionally substituted rings, e.g., Ring A,
Cy.sup.L, those formed by two or more R groups (R and (combinations
of) variables that can be R) taken together, etc. In some
embodiments, a ring is a cycloaliphatic, aryl, heteroaryl, or
heterocyclyl group as described for R but bivalent or multivalent.
As appreciated by those skilled in the art, ring moieties described
for one variable, e.g., Ring A, can also be applicable to other
variables, e.g., Cy.sup.L, if requirements of the other variables,
e.g., number of heteroatoms, valence, etc., are satisfied. Example
rings are extensively described in the present disclosure.
[0333] In some embodiments, a ring, e.g., in Ring A, R, etc. which
is optionally substituted, is a 3-20 membered monocyclic, bicyclic
or polycyclic ring having 0-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0334] In some embodiments, a ring can be of any size within its
range, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20-membered.
[0335] In some embodiments, a ring is monocyclic. In some
embodiments, a ring is saturated and monocyclic. In some
embodiments, a ring is monocyclic and partially saturated. In some
embodiments, a ring is monocyclic and aromatic.
[0336] In some embodiments, a ring is bicyclic. In some
embodiments, a ring is polycyclic. In some embodiments, a bicyclic
or polycyclic ring comprises two or more monocyclic ring moieties,
each of which can be saturated, partially saturated, or aromatic,
and each which can contain no or 1-10 heteroatoms. In some
embodiments, a bicyclic or polycyclic ring comprises a saturated
monocyclic ring. In some embodiments, a bicyclic or polycyclic ring
comprises a saturated monocyclic ring containing no heteroatoms. In
some embodiments, a bicyclic or polycyclic ring comprises a
saturated monocyclic ring comprising one or more heteroatoms. In
some embodiments, a bicyclic or polycyclic ring comprises a
partially saturated monocyclic ring. In some embodiments, a
bicyclic or polycyclic ring comprises a partially saturated
monocyclic ring containing no heteroatoms. In some embodiments, a
bicyclic or polycyclic ring comprises a partially saturated
monocyclic ring comprising one or more heteroatoms. In some
embodiments, a bicyclic or polycyclic ring comprises an aromatic
monocyclic ring. In some embodiments, a bicyclic or polycyclic ring
comprises an aromatic monocyclic ring containing no heteroatoms. In
some embodiments, a bicyclic or polycyclic ring comprises an
aromatic monocyclic ring comprising one or more heteroatoms. In
some embodiments, a bicyclic or polycyclic ring comprises a
saturated ring and a partially saturated ring, each of which
independently contains one or more heteroatoms. In some
embodiments, a bicyclic ring comprises a saturated ring and a
partially saturated ring, each of which independently comprises no,
or one or more heteroatoms. In some embodiments, a bicyclic ring
comprises an aromatic ring and a partially saturated ring, each of
which independently comprises no, or one or more heteroatoms. In
some embodiments, a polycyclic ring comprises a saturated ring and
a partially saturated ring, each of which independently comprises
no, or one or more heteroatoms. In some embodiments, a polycyclic
ring comprises an aromatic ring and a partially saturated ring,
each of which independently comprises no, or one or more
heteroatoms. In some embodiments, a polycyclic ring comprises an
aromatic ring and a saturated ring, each of which independently
comprises no, or one or more heteroatoms. In some embodiments, a
polycyclic ring comprises an aromatic ring, a saturated ring, and a
partially saturated ring, each of which independently comprises no,
or one or more heteroatoms. In some embodiments, a ring comprises
at least one heteroatom. In some embodiments, a ring comprises at
least one nitrogen atom. In some embodiments, a ring comprises at
least one oxygen atom. In some embodiments, a ring comprises at
least one sulfur atom.
[0337] As appreciated by those skilled in the art in accordance
with the present disclosure, a ring is typically optionally
substituted. In some embodiments, a ring is unsubstituted. In some
embodiments, a ring is substituted. In some embodiments, a ring is
substituted on one or more of its carbon atoms. In some
embodiments, a ring is substituted on one or more of its
heteroatoms. In some embodiments, a ring is substituted on one or
more of its carbon atoms, and one or more of its heteroatoms. In
some embodiments, two or more substituents can be located on the
same ring atom. In some embodiments, all available ring atoms are
substituted. In some embodiments, not all available ring atoms are
substituted. In some embodiments, in provided structures where
rings are indicated to be connected to other structures (e.g., Ring
A in
##STR00093##
"optionally substituted" is to mean that, besides those structures
already connected, remaining substitutable ring positions, if any,
are optionally substituted.
[0338] In some embodiments, a ring is a bivalent or multivalent
C.sub.3-30 cycloaliphatic ring. In some embodiments, a ring is a
bivalent or multivalent C.sub.3-20 cycloaliphatic ring. In some
embodiments, a ring is a bivalent or multivalent C.sub.3-10
cycloaliphatic ring. In some embodiments, a ring is a bivalent or
multivalent 3-30 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 3-7 membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 3-membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 4-membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 5-membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 6-membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent 7-membered saturated or partially unsaturated
carbocyclic ring. In some embodiments, a ring is a bivalent or
multivalent cyclohexyl ring. In some embodiments, a ring is a
bivalent or multivalent cyclopentyl ring. In some embodiments, a
ring is a bivalent or multivalent cyclobutyl ring. In some
embodiments, a ring is a bivalent or multivalent cyclopropyl
ring.
[0339] In some embodiments, a ring is a bivalent or multivalent
C.sub.6-30 aryl ring. In some embodiments, a ring is a bivalent or
multivalent phenyl ring.
[0340] In some embodiments, a ring is a bivalent or multivalent
8-10 membered bicyclic saturated, partially unsaturated or aryl
ring. In some embodiments, a ring is a bivalent or multivalent 8-10
membered bicyclic saturated ring. In some embodiments, a ring is a
bivalent or multivalent 8-10 membered bicyclic partially
unsaturated ring. In some embodiments, a ring is a bivalent or
multivalent 8-10 membered bicyclic aryl ring. In some embodiments,
a ring is a bivalent or multivalent naphthyl ring.
[0341] In some embodiments, a ring is a bivalent or multivalent
5-30 membered heteroaryl ring having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In
some embodiments, a ring is a bivalent or multivalent 5-30 membered
heteroaryl ring having 1-10 heteroatoms independently selected from
oxygen, nitrogen, and sulfur. In some embodiments, a ring is a
bivalent or multivalent 5-30 membered heteroaryl ring having 1-5
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. In some embodiments, a ring is a bivalent
or multivalent 5-30 membered heteroaryl ring having 1-5 heteroatoms
independently selected from oxygen, nitrogen, and sulfur.
[0342] In some embodiments, a ring is a bivalent or multivalent 5-6
membered monocyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, a ring is a bivalent or multivalent 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, sulfur, and oxygen.
[0343] In some embodiments, a ring is a bivalent or multivalent
5-membered monocyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, a ring is a bivalent or multivalent 6-membered
monocyclic heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0344] In certain embodiments, a ring is a bivalent or multivalent
8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, a ring is a bivalent or multivalent 5,6-fused
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, and sulfur. In some embodiments, a ring is a
bivalent or multivalent 5,6-fused heteroaryl ring having 1-5
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, a ring is a bivalent or multivalent
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0345] In some embodiments, a ring is a bivalent or multivalent
3-30 membered heterocyclic ring having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon. In some embodiments, a ring is a bivalent or
multivalent 3-7 membered saturated or partially unsaturated
heterocyclic ring having 1-3 heteroatoms independently selected
from nitrogen, oxygen, and sulfur. In certain embodiments, a ring
is a bivalent or multivalent 5-7 membered partially unsaturated
monocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a
bivalent or multivalent 5-6 membered partially unsaturated
monocyclic ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, and sulfur. In certain embodiments, a ring is a
bivalent or multivalent 5-membered partially unsaturated monocyclic
ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In certain embodiments, a ring is a bivalent or
multivalent 6-membered partially unsaturated monocyclic ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, a ring is a bivalent or multivalent
7-membered partially unsaturated monocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, a ring is a bivalent or multivalent
3-membered heterocyclic ring having one heteroatom selected from
nitrogen, oxygen or sulfur. In some embodiments, a ring is a
bivalent or multivalent 4-membered heterocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, a ring is a bivalent or multivalent
5-membered heterocyclic ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, a
ring is a bivalent or multivalent 6-membered heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, a ring is a bivalent or
multivalent 7-membered heterocyclic ring having 1-3 heteroatoms
independently selected from nitrogen, oxygen, and sulfur.
[0346] In some embodiments, a ring is a bivalent or multivalent
7-10 membered bicyclic saturated or partially unsaturated
heterocyclic ring having 1-5 heteroatoms independently selected
from nitrogen, oxygen, and sulfur. In some embodiments, a ring is a
bivalent or multivalent 8-10 membered bicyclic heteroaryl ring
having 1-5 heteroatoms independently selected from nitrogen,
oxygen, and sulfur.
[0347] In some embodiments, a ring is a bivalent or multivalent
5,6-fused heteroaryl ring having 1-5 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In certain embodiments,
a ring is a bivalent or multivalent 6,6-fused heteroaryl ring
having 1-5 heteroatoms independently selected from nitrogen,
oxygen, and sulfur.
[0348] In some embodiments, a ring formed by two or more groups
taken together, which is typically optionally substituted, is a
monocyclic saturated 5-7 membered ring having no additional
heteroatoms in addition to intervening heteroatoms, if any. In some
embodiments, a ring formed by two or more groups taken together is
a monocyclic saturated 5-membered ring having no additional
heteroatoms in addition to intervening heteroatoms, if any. In some
embodiments, a ring formed by two or more groups taken together is
a monocyclic saturated 6-membered ring having no additional
heteroatoms in addition to intervening heteroatoms, if any. In some
embodiments, a ring formed by two or more groups taken together is
a monocyclic saturated 7-membered ring having no additional
heteroatoms in addition to intervening heteroatoms, if any.
[0349] In some embodiments, a ring formed by two or more groups
taken together is a bicyclic, saturated, partially unsaturated, or
aryl 5-30 membered ring having, in addition to the intervening
heteroatoms, if any, 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, a ring formed by two or more groups taken together is
a bicyclic, saturated, partially unsaturated, or aryl 5-30 membered
ring having, in addition to the intervening heteroatoms, if any,
0-10 heteroatoms independently selected from oxygen, nitrogen, and
sulfur. In some embodiments, a ring formed by two or more groups
taken together is a bicyclic and saturated 8-10 membered bicyclic
ring having no additional heteroatoms in addition to intervening
heteroatoms, if any. In some embodiments, a ring formed by two or
more groups taken together is a bicyclic and saturated 8-membered
bicyclic ring having no additional heteroatoms in addition to
intervening heteroatoms, if any. In some embodiments, a ring formed
by two or more groups taken together is a bicyclic and saturated
9-membered bicyclic ring having no additional heteroatoms in
addition to intervening heteroatoms, if any. In some embodiments, a
ring formed by two or more groups taken together is a bicyclic and
saturated 10-membered bicyclic ring having no additional
heteroatoms in addition to intervening heteroatoms, if any. In some
embodiments, a ring formed by two or more groups taken together is
bicyclic and comprises a 5-membered ring fused to a 5-membered
ring. In some embodiments, a ring formed by two or more groups
taken together is bicyclic and comprises a 5-membered ring fused to
a 6-membered ring. In some embodiments, the 5-membered ring
comprises one or more intervening nitrogen, phosphorus and oxygen
atoms as ring atoms. In some embodiments, a ring formed by two or
more groups taken together comprises a ring system having the
backbone structure of
##STR00094##
[0350] In some embodiments, a ring formed by two or more groups
taken together is a polycyclic, saturated, partially unsaturated,
or aryl 3-30 membered ring having, in addition to the intervening
heteroatoms, if any, 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, a ring formed by two or more groups taken together is
a polycyclic, saturated, partially unsaturated, or aryl 3-30
membered ring having, in addition to the intervening heteroatoms,
if any, 0-10 heteroatoms independently selected from oxygen,
nitrogen, and sulfur.
[0351] In some embodiments, a ring formed by two or more groups
taken together is monocyclic, bicyclic or polycyclic and comprises
a 5-10 membered monocyclic ring whose ring atoms comprise one or
more intervening nitrogen, phosphorus and/or oxygen atoms. In some
embodiments, a ring formed by two or more groups taken together is
monocyclic, bicyclic or polycyclic and comprises a 5-9 membered
monocyclic ring whose ring atoms comprise one or more intervening
nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a
ring formed by two or more groups taken together is monocyclic,
bicyclic or polycyclic and comprises a 5-8 membered monocyclic ring
whose ring atoms comprise one or more intervening nitrogen,
phosphorus and/or oxygen atoms. In some embodiments, a ring formed
by two or more groups taken together is monocyclic, bicyclic or
polycyclic and comprises a 5-7 membered monocyclic ring whose ring
atoms comprise one or more intervening nitrogen, phosphorus and/or
oxygen atoms. In some embodiments, a ring formed by two or more
groups taken together is monocyclic, bicyclic or polycyclic and
comprises a 5-6 membered monocyclic ring whose ring atoms comprise
one or more intervening nitrogen, phosphorus and/or oxygen
atoms.
[0352] In some embodiments, a ring formed by two or more groups
taken together is monocyclic, bicyclic or polycyclic and comprises
a 5-membered monocyclic ring whose ring atoms comprise one or more
intervening nitrogen, phosphorus and/or oxygen atoms. In some
embodiments, a ring formed by two or more groups taken together is
monocyclic, bicyclic or polycyclic and comprises a 6-membered
monocyclic ring whose ring atoms comprise one or more intervening
nitrogen, phosphorus and/or oxygen atoms. In some embodiments, a
ring formed by two or more groups taken together is monocyclic,
bicyclic or polycyclic and comprises a 7-membered monocyclic ring
whose ring atoms comprise one or more intervening nitrogen,
phosphorus and/or oxygen atoms. In some embodiments, a ring formed
by two or more groups taken together is monocyclic, bicyclic or
polycyclic and comprises a 8-membered monocyclic ring whose ring
atoms comprise one or more intervening nitrogen, phosphorus and/or
oxygen atoms. In some embodiments, a ring formed by two or more
groups taken together is monocyclic, bicyclic or polycyclic and
comprises a 9-membered monocyclic ring whose ring atoms comprise
one or more intervening nitrogen, phosphorus and/or oxygen atoms.
In some embodiments, a ring formed by two or more groups taken
together is monocyclic, bicyclic or polycyclic and comprises a
10-membered monocyclic ring whose ring atoms comprise one or more
intervening nitrogen, phosphorus and/or oxygen atoms.
[0353] In some embodiments, a ring formed by two or more groups
taken together is monocyclic, bicyclic or polycyclic and comprises
a 5-membered ring whose ring atoms consist of carbon atoms and the
intervening nitrogen, phosphorus and oxygen atoms. In some
embodiments, a ring formed by two or more groups taken together is
monocyclic, bicyclic or polycyclic and comprises a 6-membered ring
whose ring atoms consist of carbon atoms and the intervening
nitrogen, phosphorus and oxygen atoms. In some embodiments, a ring
formed by two or more groups taken together is monocyclic, bicyclic
or polycyclic and comprises a 7-membered ring whose ring atoms
consist of carbon atoms and the intervening nitrogen, phosphorus
and oxygen atoms. In some embodiments, a ring formed by two or more
groups taken together is monocyclic, bicyclic or polycyclic and
comprises a 8-membered ring whose ring atoms consist of carbon
atoms and the intervening nitrogen, phosphorus and oxygen atoms. In
some embodiments, a ring formed by two or more groups taken
together is monocyclic, bicyclic or polycyclic and comprises a
9-membered ring whose ring atoms consist of carbon atoms and the
intervening nitrogen, phosphorus and oxygen atoms. In some
embodiments, a ring formed by two or more groups taken together is
monocyclic, bicyclic or polycyclic and comprises a 10-membered ring
whose ring atoms consist of carbon atoms and the intervening
nitrogen, phosphorus and oxygen atoms.
[0354] In some embodiments, rings described herein are
unsubstituted. In some embodiments, rings described herein are
substituted. In some embodiments, substituents are selected from
those described in example compounds provided in the present
disclosure.
[0355] As described herein, each L.sup.P is independently an
internucleotidic linkage as described in the present disclosure,
e.g., a natural phosphate linkage, a phosphorothioate diester
linkage, a modified internucleotidic linkage, a chiral
internucleotidic linkage, etc., In some embodiments, each L.sup.P
is independently a linkage having the structure of formula IIn some
embodiments, L.sup.3E is -L.sup.s- or -L.sup.s-L.sup.s-. In some
embodiments, L.sup.3E is -L.sup.s-. In some embodiments, L.sup.3E
is -L.sup.s-L.sup.s-. In some embodiments, L.sup.3E is a covalent
bond. In some embodiments, L.sup.3E is a linker used in
oligonucleotide synthesis. In some embodiments, L.sup.3E is a
linker used in solid phase oligonucleotide synthesis. Various types
of linkers are known and can be utilized in accordance with the
present disclosure. In some embodiments, a linker is a succinate
linker (--O--C(O)--CH.sub.2--CH.sub.2--C(O)--). In some
embodiments, a linker is an oxalyl linker (--O--C(O)--C(O)--). In
some embodiments, L.sup.3E is a succinyl-piperidine linker (SP)
linker. In some embodiments, L.sup.3E is a succinyl linker. In some
embodiments, L.sup.3E is a Q-linker.
[0356] In some embodiments, R.sup.3E is --R', -L.sup.s-R', --OR',
or a solid support. In some embodiments, R.sup.3E is --R'. In some
embodiments, R.sup.3E is -L.sup.s-R'. In some embodiments, R.sup.3E
is --OR'. In some embodiments, R.sup.3E is a solid support. In some
embodiments, R.sup.3E is --H. In some embodiments, -L-R.sup.3E is
--H. In some embodiments, R.sup.3E is --OH. In some embodiments,
-L-R.sup.3E is --OH. In some embodiments, R.sup.3E is optionally
substituted C.sub.1-6 aliphatic. In some embodiments, R.sup.3E is
optionally substituted C.sub.1-6 alkyl. In some embodiments,
R.sup.3E is --OR'. In some embodiments, R.sup.3E is --OH. In some
embodiments, R.sup.3E is --OR', wherein R' is not hydrogen. In some
embodiments, R.sup.3E is --OR', wherein R' is optionally
substituted C.sub.1-6 alkyl.
In some embodiments, R.sup.3E is a 3'-end cap (e.g., those used in
RNAi technologies).
[0357] In some embodiments, R.sup.3E is a solid support. In some
embodiments, R.sup.3E is a solid support for oligonucleotide
synthesis. Various types of solid support are known and can be
utilized in accordance with the present disclosure. In some
embodiments, a solid support is HCP. In some embodiments, a solid
support is CPG.
[0358] In some embodiments, R' is --R, --C(O)R, --C(O)OR, or
--S(O).sub.2R, wherein R is as described in the present disclosure.
In some embodiments, R' is R, wherein R is as described in the
present disclosure. In some embodiments, R' is --C(O)R, wherein R
is as described in the present disclosure. In some embodiments, R'
is --C(O)OR, wherein R is as described in the present disclosure.
In some embodiments, R' is --S(O).sub.2R, wherein R is as described
in the present disclosure. In some embodiments, R' is hydrogen. In
some embodiments, R' is not hydrogen. In some embodiments, R' is R,
wherein R is optionally substituted C.sub.1-20 aliphatic as
described in the present disclosure. In some embodiments, R' is R,
wherein R is optionally substituted C.sub.1-20 heteroaliphatic as
described in the present disclosure. In some embodiments, R' is R,
wherein R is optionally substituted C.sub.6-20 aryl as described in
the present disclosure. In some embodiments, R' is R, wherein R is
optionally substituted C.sub.6-20 arylaliphatic as described in the
present disclosure. In some embodiments, R' is R, wherein R is
optionally substituted C.sub.6-20 arylheteroaliphatic as described
in the present disclosure. In some embodiments, R' is R, wherein R
is optionally substituted 5-20 membered heteroaryl as described in
the present disclosure. In some embodiments, R' is R, wherein R is
optionally substituted 3-20 membered heterocyclyl as described in
the present disclosure. In some embodiments, two or more R' are R,
and are optionally and independently taken together to form an
optionally substituted ring as described in the present
disclosure.
[0359] In some embodiments, each R is independently --H, or an
optionally substituted group selected from C.sub.1-30 aliphatic,
C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30
membered heteroaryl having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30
membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
[0360] two R groups are optionally and independently taken together
to form a covalent bond, or: [0361] two or more R groups on the
same atom are optionally and independently taken together with the
atom to form an optionally substituted, 3-30 membered, monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon; or [0362] two or more R groups on two or
more atoms are optionally and independently taken together with
their intervening atoms to form an optionally substituted, 3-30
membered, monocyclic, bicyclic or polycyclic ring having, in
addition to the intervening atoms, 0-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0363] In some embodiments, each R is independently --H, or an
optionally substituted group selected from C.sub.1-30 aliphatic,
C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30
membered heteroaryl having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30
membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
[0364] two R groups are optionally and independently taken together
to form a covalent bond, or. [0365] two or more R groups on the
same atom are optionally and independently taken together with the
atom to form an optionally substituted, 3-30 membered, monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. [0366] two or more R groups on two or more
atoms are optionally and independently taken together with their
intervening atoms to form an optionally substituted, 3-30 membered,
monocyclic, bicyclic or polycyclic ring having, in addition to the
intervening atoms, 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon.
[0367] In some embodiments, each R is independently --H, or an
optionally substituted group selected from C.sub.1-20 aliphatic,
C.sub.1-20 heteroaliphatic having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.6-20 aryl, C.sub.6-20 arylaliphatic, C.sub.6-20
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20
membered heteroaryl having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20
membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
[0368] two R groups are optionally and independently taken together
to form a covalent bond, or: [0369] two or more R groups on the
same atom are optionally and independently taken together with the
atom to form an optionally substituted, 3-20 membered monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. [0370] two or more R groups on two or more
atoms are optionally and independently taken together with their
intervening atoms to form an optionally substituted, 3-20 membered
monocyclic, bicyclic or polycyclic ring having, in addition to the
intervening atoms, 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon.
[0371] In some embodiments, each R is independently --H, or an
optionally substituted group selected from C.sub.1-30 aliphatic,
C.sub.1-30 heteroaliphatic having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.6-30 aryl, C.sub.6-30 arylaliphatic, C.sub.6-30
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-30
membered heteroaryl having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-30
membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0372] In some embodiments, each R is independently --H, or an
optionally substituted group selected from C.sub.1-20 aliphatic,
C.sub.1-20 heteroaliphatic having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
C.sub.6-20 aryl, C.sub.6-20 arylaliphatic, C.sub.6-20
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, 5-20
membered heteroaryl having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon, and 3-20
membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon.
[0373] In some embodiments, R is hydrogen. In some embodiments, R
is not hydrogen. In some embodiments, R is an optionally
substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30
heteroaliphatic having 1-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon, C.sub.6-30 aryl,
a 5-30 membered heteroaryl ring having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and a 3-30 membered heterocyclic ring having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0374] In some embodiments, R is hydrogen or an optionally
substituted group selected from C.sub.1-20 aliphatic, phenyl, a 3-7
membered saturated or partially unsaturated carbocyclic ring, an
8-10 membered bicyclic saturated, partially unsaturated or aryl
ring, a 5-6 membered monocyclic heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, and
sulfur, a 4-7 membered saturated or partially unsaturated
heterocyclic ring having 1-3 heteroatoms independently selected
from nitrogen, oxygen, and sulfur, a 7-10 membered bicyclic
saturated or partially unsaturated heterocyclic ring having 1-5
heteroatoms independently selected from nitrogen, oxygen, and
sulfur, or an 8-10 membered bicyclic heteroaryl ring having 1-5
heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0375] In some embodiments, R is optionally substituted C.sub.1-30
aliphatic. In some embodiments, R is optionally substituted
C.sub.1-20 aliphatic. In some embodiments, R is optionally
substituted C.sub.1-15 aliphatic. In some embodiments, R is
optionally substituted C.sub.1-10 aliphatic. In some embodiments, R
is optionally substituted C.sub.1-6 aliphatic. In some embodiments,
R is optionally substituted C.sub.1-6 alkyl. In some embodiments, R
is optionally substituted hexyl, pentyl, butyl, propyl, ethyl or
methyl. In some embodiments, R is optionally substituted hexyl. In
some embodiments, R is optionally substituted pentyl. In some
embodiments, R is optionally substituted butyl. In some
embodiments, R is optionally substituted propyl. In some
embodiments, R is optionally substituted ethyl. In some
embodiments, R is optionally substituted methyl. In some
embodiments, R is hexyl. In some embodiments, R is pentyl. In some
embodiments, R is butyl. In some embodiments, R is propyl. In some
embodiments, R is ethyl. In some embodiments, R is methyl. In some
embodiments, R is isopropyl. In some embodiments, R is n-propyl. In
some embodiments, R is tert-butyl. In some embodiments, R is
sec-butyl. In some embodiments, R is n-butyl. In some embodiments,
R is --(CH.sub.2).sub.2CN.
[0376] In some embodiments, R is optionally substituted C.sub.3-30
cycloaliphatic. In some embodiments, R is optionally substituted
C.sub.3-20 cycloaliphatic. In some embodiments, R is optionally
substituted C.sub.3-10 cycloaliphatic. In some embodiments, R is
optionally substituted cyclohexyl. In some embodiments, R is
cyclohexyl. In some embodiments, R is optionally substituted
cyclopentyl. In some embodiments, R is cyclopentyl. In some
embodiments, R is optionally substituted cyclobutyl. In some
embodiments, R is cyclobutyl. In some embodiments, R is optionally
substituted cyclopropyl. In some embodiments, R is cyclopropyl.
[0377] In some embodiments, R is an optionally substituted 3-30
membered saturated or partially unsaturated carbocyclic ring. In
some embodiments, R is an optionally substituted 3-7 membered
saturated or partially unsaturated carbocyclic ring. In some
embodiments, R is an optionally substituted 3-membered saturated or
partially unsaturated carbocyclic ring. In some embodiments, R is
an optionally substituted 4-membered saturated or partially
unsaturated carbocyclic ring. In some embodiments, R is an
optionally substituted 5-membered saturated or partially
unsaturated carbocyclic ring. In some embodiments, R is an
optionally substituted 6-membered saturated or partially
unsaturated carbocyclic ring. In some embodiments, R is an
optionally substituted 7-membered saturated or partially
unsaturated carbocyclic ring. In some embodiments, R is optionally
substituted cycloheptyl. In some embodiments, R is cycloheptyl. In
some embodiments, R is optionally substituted cyclohexyl. In some
embodiments, R is cyclohexyl. In some embodiments, R is optionally
substituted cyclopentyl. In some embodiments, R is cyclopentyl. In
some embodiments, R is optionally substituted cyclobutyl. In some
embodiments, R is cyclobutyl. In some embodiments, R is optionally
substituted cyclopropyl. In some embodiments, R is cyclopropyl.
[0378] In some embodiments, when R is or comprises a ring
structure, e.g., cycloaliphatic, cycloheteroaliphatic, aryl,
heteroaryl, etc., the ring structure can be monocyclic, bicyclic or
polycyclic. In some embodiments, R is or comprises a monocyclic
structure. In some embodiments, R is or comprises a bicyclic
structure. In some embodiments, R is or comprises a polycyclic
structure.
[0379] In some embodiments, R is optionally substituted C.sub.1-30
heteroaliphatic having 1-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, R is optionally substituted C.sub.1-20 heteroaliphatic
having 1-10 heteroatoms. In some embodiments, R is optionally
substituted C.sub.1-20 heteroaliphatic having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus or
silicon, optionally including one or more oxidized forms of
nitrogen, sulfur, phosphorus or selenium. In some embodiments, R is
optionally substituted C.sub.1-30 heteroaliphatic comprising 1-10
groups independently selected from
##STR00095##
--N.dbd., .ident.N, --S--, --S(O)--, --S(O).sub.2--, --O--,
.dbd.O,
##STR00096##
[0381] In some embodiments, R is optionally substituted C.sub.6-30
aryl. In some embodiments, R is optionally substituted phenyl. In
some embodiments, R is phenyl. In some embodiments, R is
substituted phenyl.
[0382] In some embodiments, R is an optionally substituted 8-10
membered bicyclic saturated, partially unsaturated or aryl ring. In
some embodiments, R is an optionally substituted 8-10 membered
bicyclic saturated ring. In some embodiments, R is an optionally
substituted 8-10 membered bicyclic partially unsaturated ring. In
some embodiments, R is an optionally substituted 8-10 membered
bicyclic aryl ring. In some embodiments, R is optionally
substituted naphthyl.
[0383] In some embodiments, R is optionally substituted 5-30
membered heteroaryl ring having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In
some embodiments, R is optionally substituted 5-30 membered
heteroaryl ring having 1-10 heteroatoms independently selected from
oxygen, nitrogen, and sulfur. In some embodiments, R is optionally
substituted 5-30 membered heteroaryl ring having 1-5 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon. In some embodiments, R is optionally substituted 5-30
membered heteroaryl ring having 1-5 heteroatoms independently
selected from oxygen, nitrogen, and sulfur.
[0384] In some embodiments, R is an optionally substituted 5-6
membered monocyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is a substituted 5-6 membered monocyclic heteroaryl
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6
membered monocyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is an optionally substituted 5-6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, sulfur, and oxygen. In some embodiments, R is a
substituted 5-6 membered monocyclic heteroaryl ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is an unsubstituted 5-6 membered
monocyclic heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, sulfur, and oxygen.
[0385] In some embodiments, R is an optionally substituted
5-membered monocyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen or sulfur. In some
embodiments, R is an optionally substituted 6-membered monocyclic
heteroaryl ring having 1-4 heteroatoms independently selected from
nitrogen, oxygen, and sulfur.
[0386] In some embodiments, R is an optionally substituted
5-membered monocyclic heteroaryl ring having one heteroatom
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is selected from optionally substituted pyrrolyl, furanyl, or
thienyl.
[0387] In some embodiments, R is an optionally substituted
5-membered heteroaryl ring having two heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In certain embodiments,
R is an optionally substituted 5-membered heteroaryl ring having
one nitrogen atom, and an additional heteroatom selected from
sulfur or oxygen. Example R groups include but are not limited to
optionally substituted pyrazolyl, imidazolyl, thiazolyl,
isothiazolyl, oxazolyl or isoxazolyl.
[0388] In some embodiments, R is an optionally substituted
5-membered heteroaryl ring having three heteroatoms independently
selected from nitrogen, oxygen, and sulfur. Example R groups
include but are not limited to optionally substituted triazolyl,
oxadiazolyl or thiadiazolyl.
[0389] In some embodiments, R is an optionally substituted
5-membered heteroaryl ring having four heteroatoms independently
selected from nitrogen, oxygen, and sulfur. Example R groups
include but are not limited to optionally substituted tetrazolyl,
oxatriazolyl and thiatriazolyl.
[0390] In some embodiments, R is an optionally substituted
6-membered heteroaryl ring having 1-4 nitrogen atoms. In some
embodiments, R is an optionally substituted 6-membered heteroaryl
ring having 1-3 nitrogen atoms. In other embodiments, R is an
optionally substituted 6-membered heteroaryl ring having 1-2
nitrogen atoms. In some embodiments, R is an optionally substituted
6-membered heteroaryl ring having four nitrogen atoms. In some
embodiments, R is an optionally substituted 6-membered heteroaryl
ring having three nitrogen atoms. In some embodiments, R is an
optionally substituted 6-membered heteroaryl ring having two
nitrogen atoms. In certain embodiments, R is an optionally
substituted 6-membered heteroaryl ring having one nitrogen atom.
Example R groups include but are not limited to optionally
substituted pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,
triazinyl, or tetrazinyl.
[0391] In certain embodiments, R is an optionally substituted 8-10
membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In other embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In
certain embodiments, R is an optionally substituted 5,6-fused
heteroaryl ring having 1 heteroatom independently selected from
nitrogen, oxygen, and sulfur. In some embodiments, R is an
optionally substituted indolyl. In some embodiments, R is an
optionally substituted azabicyclo[3.2.1]octanyl. In certain
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having 2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted azaindolyl. In some embodiments, R is an optionally
substituted benzimidazolyl. In some embodiments, R is an optionally
substituted benzothiazolyl. In some embodiments, R is an optionally
substituted benzoxazolyl. In some embodiments, R is an optionally
substituted indazolyl. In certain embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having 3 heteroatoms
independently selected from nitrogen, oxygen, and sulfur.
[0392] In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having 1-5 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having two
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having three heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having four
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having five heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0393] In certain embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having one heteroatom independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted indolyl. In some embodiments, R is
optionally substituted benzofuranyl. In some embodiments, R is
optionally substituted benzo[b]thienyl. In certain embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having two
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted
azaindolyl. In some embodiments, R is optionally substituted
benzimidazolyl. In some embodiments, R is optionally substituted
benzothiazolyl. In some embodiments, R is optionally substituted
benzoxazolyl. In some embodiments, R is an optionally substituted
indazolyl. In certain embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having three heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted oxazolopyridiyl, thiazolopyridinyl or
imidazopyridinyl. In certain embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having four heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is optionally substituted purinyl,
oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl,
thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl,
thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments,
R is an optionally substituted 5,6-fused heteroaryl ring having
five heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0394] In some embodiments, R is optionally substituted
1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl,
4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl,
thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl,
pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some
embodiments, R is optionally substituted dihydropyrroloimidazolyl,
1H-furoimidazolyl, 1H-thienoimidazolyl, furooxazolyl,
furoisoxazolyl, 4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl,
thienooxazolyl, thienoisoxazolyl, 4H-pyrrolothiazolyl,
furothiazolyl, thienothiazolyl, 1H-imidazoimidazolyl,
imidazooxazolylorimidazo[5,1-b]thiazolyl.
[0395] In certain embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 6,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In other embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having 1 heteroatom independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted quinolinyl. In some embodiments, R is
an optionally substituted isoquinolinyl. In some embodiments, R is
an optionally substituted 6,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted
quinazoline or a quinoxaline.
[0396] In some embodiments, R is 3-30 membered heterocyclic ring
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon. In some embodiments, R is
3-30 membered heterocyclic ring having 1-10 heteroatoms
independently selected from oxygen, nitrogen, and sulfur. In some
embodiments, R is 3-30 membered heterocyclic ring having 1-5
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. In some embodiments, R is 3-30 membered
heterocyclic ring having 1-5 heteroatoms independently selected
from oxygen, nitrogen, and sulfur.
[0397] In some embodiments, R is an optionally substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is a substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In certain embodiments, R is an optionally
substituted 5-7 membered partially unsaturated monocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In certain embodiments, R is an optionally
substituted 5-6 membered partially unsaturated monocyclic ring
having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In certain embodiments, R is an optionally
substituted 5-membered partially unsaturated monocyclic ring having
1-3 heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, R is an optionally substituted
6-membered partially unsaturated monocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, R is an optionally substituted
7-membered partially unsaturated monocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted 3-membered
heterocyclic ring having one heteroatom selected from nitrogen,
oxygen or sulfur. In some embodiments, R is optionally substituted
4-membered heterocyclic ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted 5-membered heterocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted 6-membered
heterocyclic ring having 1-3 heteroatoms independently selected
from nitrogen, oxygen, and sulfur. In some embodiments, R is
optionally substituted 7-membered heterocyclic ring having 1-3
heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0398] In some embodiments, R is an optionally substituted
3-membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted 4-membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, and sulfur. In some embodiments, R is an
optionally substituted 5-membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 6-membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 7-membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur.
[0399] In some embodiments, R is an optionally substituted
4-membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted 4-membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
and sulfur. In some embodiments, R is an optionally substituted
4-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom. In some embodiments, R is an optionally
substituted 4-membered partially unsaturated heterocyclic ring
having no more than 1 heteroatom, wherein the heteroatom is
nitrogen. In some embodiments, R is an optionally substituted
4-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom, wherein the heteroatom is oxygen. In some
embodiments, R is an optionally substituted 4-membered partially
unsaturated heterocyclic ring having no more than 1 heteroatom,
wherein the heteroatom is sulfur. In some embodiments, R is an
optionally substituted 4-membered partially unsaturated
heterocyclic ring having 2 oxygen atoms. In some embodiments, R is
an optionally substituted 4-membered partially unsaturated
heterocyclic ring having 2 nitrogen atoms. In some embodiments, R
is an optionally substituted 4-membered saturated or partially
unsaturated heterocyclic ring having 1-2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 4-membered partially unsaturated
heterocyclic ring having 2 heteroatoms independently selected from
nitrogen, oxygen, and sulfur. In some embodiments, R is an
optionally substituted 4-membered partially unsaturated
heterocyclic ring having no more than 1 heteroatom. In some
embodiments, R is an optionally substituted 4-membered partially
unsaturated heterocyclic ring having no more than 1 heteroatom,
wherein the heteroatom is nitrogen. In some embodiments, R is an
optionally substituted 4-membered partially unsaturated
heterocyclic ring having no more than 1 heteroatom, wherein the
heteroatom is oxygen. In some embodiments, R is an optionally
substituted 4-membered partially unsaturated heterocyclic ring
having no more than 1 heteroatom, wherein the heteroatom is sulfur.
In some embodiments, R is an optionally substituted 4-membered
partially unsaturated heterocyclic ring having 2 oxygen atoms. In
some embodiments, R is an optionally substituted 4-membered
partially unsaturated heterocyclic ring having 2 nitrogen
atoms.
[0400] In some embodiments, R is an optionally substituted
5-membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted 5-membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
and sulfur. In some embodiments, R is an optionally substituted
5-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom. In some embodiments, R is an optionally
substituted 5-membered partially unsaturated heterocyclic ring
having no more than 1 heteroatom, wherein the heteroatom is
nitrogen. In some embodiments, R is an optionally substituted
5-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom, wherein the heteroatom is oxygen. In some
embodiments, R is an optionally substituted 5-membered partially
unsaturated heterocyclic ring having no more than 1 heteroatom,
wherein the heteroatom is sulfur. In some embodiments, R is an
optionally substituted 5-membered partially unsaturated
heterocyclic ring having 2 oxygen atoms. In some embodiments, R is
an optionally substituted 5-membered partially unsaturated
heterocyclic ring having 2 nitrogen atoms.
[0401] In some embodiments, R is an optionally substituted
6-membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted 6-membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
and sulfur. In some embodiments, R is an optionally substituted
6-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom. In some embodiments, R is an optionally
substituted 6-membered partially unsaturated heterocyclic ring
having no more than 1 heteroatom, wherein the heteroatom is
nitrogen. In some embodiments, R is an optionally substituted
6-membered partially unsaturated heterocyclic ring having no more
than 1 heteroatom, wherein the heteroatom is oxygen. In some
embodiments, R is an optionally substituted 6-membered partially
unsaturated heterocyclic ring having no more than 1 heteroatom,
wherein the heteroatom is sulfur. In some embodiments, R is an
optionally substituted 6-membered partially unsaturated
heterocyclic ring having 2 oxygen atoms. In some embodiments, R is
an optionally substituted 6-membered partially unsaturated
heterocyclic ring having 2 nitrogen atoms.
[0402] In certain embodiments, R is a 3-7 membered saturated or
partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In
certain embodiments, R is optionally substituted oxiranyl,
oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, oxepaneyl,
aziridineyl, azetidineyl, pyrrolidinyl, piperidinyl, azepanyl,
thiiranyl, thietanyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,
thiepanyl, dioxolanyl, oxathiolanyl, oxazolidinyl, imidazolidinyl,
thiazolidinyl, dithiolanyl, dioxanyl, morpholinyl, oxathianyl,
piperazinyl, thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl,
oxathiepanyl, dithiepanyl, diazepanyl, dihydrofuranonyl,
tetrahydropyranonyl, oxepanonyl, pyrolidinonyl, piperidinonyl,
azepanonyl, dihydrothiophenonyl, tetrahydrothiopyranonyl,
thiepanonyl, oxazolidinonyl, oxazinanonyl, oxazepanonyl,
dioxolanonyl, dioxanonyl, dioxepanonyl, oxathiolinonyl,
oxathianonyl, oxathiepanonyl, thiazolidinonyl, thiazinanonyl,
thiazepanonyl, imidazolidinonyl, tetrahydropyrimidinonyl,
diazepanonyl, imidazolidinedionyl, oxazolidinedionyl,
thiazolidinedionyl, dioxolanedionyl, oxathiolanedionyl,
piperazinedionyl, morpholinedionyl, thiomorpholinedionyl,
tetrahydropyranyl, tetrahydrofuranyl, morpholinyl, thiomorpholinyl,
piperidinyl, piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or
tetrahydrothiopyranyl.
[0403] In certain embodiments, R is an optionally substituted 5-6
membered partially unsaturated monocyclic ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, R is an optionally substituted
tetrahydropyridinyl, dihydrothiazolyl, dihydrooxazolyl, or
oxazolinyl group.
[0404] In some embodiments, R is an optionally substituted 7-10
membered bicyclic saturated or partially unsaturated heterocyclic
ring having 1-5 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is optionally
substituted indolinyl. In some embodiments, R is optionally
substituted isoindolinyl. In some embodiments, R is optionally
substituted 1, 2, 3, 4-tetrahydroquinolinyl. In some embodiments, R
is optionally substituted 1, 2, 3, 4-tetrahydroisoquinolinyl. In
some embodiments, R is an optionally substituted
azabicyclo[3.2.1]octanyl.
[0405] In some embodiments, R is an optionally substituted 8-10
membered bicyclic heteroaryl ring having 1-5 heteroatoms
independently selected from nitrogen, oxygen, and sulfur.
[0406] In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having 1-5 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having 1-4
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having 1-3 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having two
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted
1,4-dihydropyrrolo[3,2-b]pyrrolyl, 4H-furo[3,2-b]pyrrolyl,
4H-thieno[3,2-b]pyrrolyl, furo[3,2-b]furanyl, thieno[3,2-b]furanyl,
thieno[3,2-b]thienyl, 1H-pyrrolo[1,2-a]imidazolyl,
pyrrolo[2,1-b]oxazolyl or pyrrolo[2,1-b]thiazolyl. In some
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having three heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is optionally
substituted dihydropyrroloimidazolyl, 1H-furoimidazolyl,
1H-thienoimidazolyl, furooxazolyl, furoisoxazolyl,
4H-pyrrolooxazolyl, 4H-pyrroloisoxazolyl, thienooxazolyl,
thienoisoxazolyl, 4H-pyrrolothiazolyl, furothiazolyl,
thienothiazolyl, 1H-imidazoimidazolyl, imidazooxazolyl or
imidazo[5,1-b]thiazolyl. In some embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having four heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having five heteroatoms independently selected from nitrogen,
oxygen, and sulfur.
[0407] In some embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having 1-5 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In other embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In certain embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having one heteroatom independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted indolyl. In some embodiments, R is
optionally substituted benzofuranyl. In some embodiments, R is
optionally substituted benzo[b]thienyl. In certain embodiments, R
is an optionally substituted 5,6-fused heteroaryl ring having two
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted
azaindolyl. In some embodiments, R is optionally substituted
benzimidazolyl. In some embodiments, R is optionally substituted
benzothiazolyl. In some embodiments, R is optionally substituted
benzoxazolyl. In some embodiments, R is an optionally substituted
indazolyl. In certain embodiments, R is an optionally substituted
5,6-fused heteroaryl ring having three heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted oxazolopyridiyl, thiazolopyridinyl or
imidazopyridinyl. In certain embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having four heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is optionally substituted purinyl,
oxazolopyrimidinyl, thiazolopyrimidinyl, oxazolopyrazinyl,
thiazolopyrazinyl, imidazopyrazinyl, oxazolopyridazinyl,
thiazolopyridazinyl or imidazopyridazinyl. In certain embodiments,
R is an optionally substituted 5,6-fused heteroaryl ring having
five heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0408] In certain embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having 1-5 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 6,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In other embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having one heteroatom selected from
nitrogen, oxygen, and sulfur. In some embodiments, R is optionally
substituted quinolinyl. In some embodiments, R is optionally
substituted isoquinolinyl. In some embodiments, R is an optionally
substituted 6,6-fused heteroaryl ring having two heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is optionally substituted quinazolinyl,
phthalazinyl, quinoxalinyl or naphthyridinyl. In some embodiments,
R is an optionally substituted 6,6-fused heteroaryl ring having
three heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is optionally substituted
pyridopyrimidinyl, pyridopyridazinyl, pyridopyrazinyl, or
benzotriazinyl. In some embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having four heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is optionally substituted pyridotriazinyl, pteridinyl,
pyrazinopyrazinyl, pyrazinopyridazinyl, pyridazinopyridazinyl,
pyrimidopyridazinyl or pyrimidopyrimidinyl. In some embodiments, R
is an optionally substituted 6,6-fused heteroaryl ring having five
heteroatoms independently selected from nitrogen, oxygen, and
sulfur.
[0409] In some embodiments, R is optionally substituted C.sub.6-30
arylaliphatic. In some embodiments, R is optionally substituted
C.sub.6-20 arylaliphatic. In some embodiments, R is optionally
substituted C.sub.6-10 arylaliphatic. In some embodiments, an aryl
moiety of the arylaliphatic has 6, 10, or 14 aryl carbon atoms. In
some embodiments, an aryl moiety of the arylaliphatic has 6 aryl
carbon atoms. In some embodiments, an aryl moiety of the
arylaliphatic has 10 aryl carbon atoms. In some embodiments, an
aryl moiety of the arylaliphatic has 14 aryl carbon atoms. In some
embodiments, an aryl moiety is optionally substituted phenyl.
[0410] In some embodiments, R is optionally substituted C.sub.6-30
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, R is optionally substituted C.sub.6-30
arylheteroaliphatic having 1-10 heteroatoms independently selected
from oxygen, nitrogen, and sulfur. In some embodiments, R is
optionally substituted C.sub.6-20 arylheteroaliphatic having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. In some embodiments, R is optionally
substituted C.sub.6-20 arylheteroaliphatic having 1-10 heteroatoms
independently selected from oxygen, nitrogen, and sulfur. In some
embodiments, R is optionally substituted C.sub.6-10
arylheteroaliphatic having 1-5 heteroatoms independently selected
from oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, R is optionally substituted C.sub.6-10
arylheteroaliphatic having 1-5 heteroatoms independently selected
from oxygen, nitrogen, and sulfur.
[0411] In some embodiments, two R groups are optionally and
independently taken together to form a covalent bond. In some
embodiments, --C.dbd.O is formed. In some embodiments,
--C.ident.C-- is formed. In some embodiments, --C.ident.C-- is
formed.
[0412] In some embodiments, two or more R groups on the same atom
are optionally and independently taken together with the atom to
form an optionally substituted, 3-30 membered, monocyclic, bicyclic
or polycyclic ring having, in addition to the atom, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. In some embodiments, two or more R groups
on the same atom are optionally and independently taken together
with the atom to form an optionally substituted, 3-20 membered
monocyclic, bicyclic or polycyclic ring having, in addition to the
atom, 0-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon. In some embodiments, two
or more R groups on the same atom are optionally and independently
taken together with the atom to form an optionally substituted,
3-10 membered monocyclic, bicyclic or polycyclic ring having, in
addition to the atom, 0-5 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, two or more R groups on the same atom are optionally
and independently taken together with the atom to form an
optionally substituted, 3-6 membered monocyclic, bicyclic or
polycyclic ring having, in addition to the atom, 0-3 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon. In some embodiments, two or more R groups on the same
atom are optionally and independently taken together with the atom
to form an optionally substituted, 3-5 membered monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-3
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0413] In some embodiments, two or more R groups on two or more
atoms are optionally and independently taken together with their
intervening atoms to form an optionally substituted, 3-30 membered,
monocyclic, bicyclic or polycyclic ring having, in addition to the
intervening atoms, 0-10 heteroatoms independently selected from
oxygen, nitrogen, sulfur, phosphorus and silicon. In some
embodiments, two or more R groups on two or more atoms are
optionally and independently taken together with their intervening
atoms to form an optionally substituted, 3-20 membered monocyclic,
bicyclic or polycyclic ring having, in addition to the intervening
atoms, 0-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon. In some embodiments, two
or more R groups on two or more atoms are optionally and
independently taken together with their intervening atoms to form
an optionally substituted, 3-10 membered monocyclic, bicyclic or
polycyclic ring having, in addition to the intervening atoms, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon. In some embodiments, two or more R groups
on two or more atoms are optionally and independently taken
together with their intervening atoms to form an optionally
substituted, 3-10 membered monocyclic, bicyclic or polycyclic ring
having, in addition to the intervening atoms, 0-5 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon. In some embodiments, two or more R groups on two or
more atoms are optionally and independently taken together with
their intervening atoms to form an optionally substituted, 3-6
membered monocyclic, bicyclic or polycyclic ring having, in
addition to the intervening atoms, 0-3 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon. In
some embodiments, two or more R groups on two or more atoms are
optionally and independently taken together with their intervening
atoms to form an optionally substituted, 3-5 membered monocyclic,
bicyclic or polycyclic ring having, in addition to the intervening
atoms, 0-3 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus and silicon.
[0414] In some embodiments, heteroatoms in R groups, or in the
structures formed by two or more R groups taken together, are
selected from oxygen, nitrogen, and sulfur. In some embodiments, a
formed ring is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20-membered. In some embodiments, a formed ring is
saturated. In some embodiments, a formed ring is partially
saturated. In some embodiments, a formed ring is aromatic. In some
embodiments, a formed ring comprises a saturated, partially
saturated, or aromatic ring moiety. In some embodiments, a formed
ring comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 aromatic ring atoms. In some embodiments, a formed
contains no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 aromatic ring atoms. In some embodiments,
aromatic ring atoms are selected from carbon, nitrogen, oxygen and
sulfur.
[0415] In some embodiments, a ring formed by two or more R groups
(or two or more groups selected from R and variables that can be R)
taken together is a C.sub.3-30 cycloaliphatic, C.sub.6-30 aryl,
5-30 membered heteroaryl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon, or
3-30 membered heterocyclyl having 1-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon,
ring as described for R, but bivalent or multivalent.
[0416] In some embodiments, P.sup.L is P(.dbd.W). In some
embodiments, P.sup.L is P. In some embodiments, P.sup.L is
P.fwdarw.B(R').sub.3. In some embodiments, P of P.sup.L is chiral.
In some embodiments, P of P.sup.L s Rp. In some embodiments, P of
P.sup.L is Sp. In some embodiments, a linkage of formula I is a
phosphate linkage or a salt form thereof. In some embodiments, a
linkage of formula I is a phosphorothioate linkage or a salt form
thereof. In some embodiments, P.sup.L is P*(.dbd.W), wherein P* is
a chiral linkage phosphorus. In some embodiments, P.sup.L is
P*(.dbd.O), wherein P* is a chiral linkage phosphorus.
[0417] In some embodiments, W is O. In some embodiments, W is S. In
some embodiments, W is Se.
[0418] Each of X, Y, Z and R.sup.1 is independently as described in
the present disclosure, for example, as described, in some
embodiments, R.sup.1 is H. In some embodiments, --X-L-R.sup.1 is
--X--R.sup.1. In some embodiments, --X-L-R.sup.1 is --X--H. In some
embodiments, Y and Z are O, and X is S. In some embodiments, Y and
Z are O and X is O. Additional embodiments of each of the variables
are independently described in the present disclosure.
[0419] In some embodiments, a provided oligonucleotide has the
structure of formula O-I. In some embodiments, an oligonucleotide
of formula O-I comprise chemical modifications (e.g., sugar
modification, base modifications, modified internucleotidic
linkages, etc., and patterns thereof), stereochemistry (of 5'-C,
chiral phosphorus, etc., and patterns thereof), base sequences,
etc., as described in the present disclosure. In some embodiments,
a provided oligonucleotide of formula O-I is one selected from in
Table 1A, Table 17, etc.
[0420] In some embodiments, the present disclosure provides
multimers of oligonucleotides. In some embodiments, at least one of
the monomer is a C9orf72 oligonucleotide. In some embodiments, a
multimer is a multimer of the same oligonucleotides. In some
embodiments, a multimer is a multimer of structurally different
oligonucleotides. In some embodiments, each oligonucleotide of a
multimer performs its functions independently through its own
pathways, e.g., RNA interference (RNAi), RNase H dependent, etc. In
some embodiments, provided oligonucleotides exist in an oligomeric
or polymeric form, in which one or more oligonucleotide moieties
are linked together by linkers, e.g., L, L.sup.M, etc., through
nucleobases, sugars, and/or internucleotidic linkages of the
oligonucleotide moieties. For example, in some embodiments, a
provided multimer compound has the structure of
(A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, wherein each variable is
independently as described in the present disclosure.
[0421] In some embodiments, a provided compound, e.g., an
oligonucleotide of a provided composition, has the structure
of:
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D,
(A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, or
(A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b,
or a salt thereof, wherein:
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D,
(A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, or
(A.sup.c)-L.sup.M-(R.sup.D).sub.b,
or a salt thereof, wherein: each A.sup.c is independently an
oligonucleotide moiety (e.g., [H].sub.a-A.sup.c or
[H].sub.b-A.sup.c is an oligonucleotide); a is 1-1000; b is 1-1000;
L.sup.M is a multivalent linker; and each R.sup.D is independently
a chemical moiety.
[0422] In some embodiments, a provided compound, e.g., an
oligonucleotide of a provided composition, have the structure
of:
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D,
(A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, or
(A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b,
or a salt thereof, wherein: each A.sup.c is independently an
oligonucleotide moiety (e.g., [H].sub.a-A.sup.c or
[H].sub.b-A.sup.c is an oligonucleotide); a is 1-1000; b is 1-1000;
each R.sup.D is independently R.sup.LD, R.sup.CD or R.sup.TD;
[0423] R.sup.CD is an optionally substituted, linear or branched
group selected from a C.sub.1-100 aliphatic group and a C.sub.1-100
heteroaliphatic group having 1-30 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus, boron and
silicon, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0424]
R.sup.LD is an optionally substituted, linear or branched group
selected from a C.sub.1-100 aliphatic group wherein one or more
methylene units are optionally and independently replaced with
C.sub.1-6 alkylene, C.sub.1-6 alkenylene, --C.ident.C--,
--C(R').sub.2--, --O--, --S--, --S--S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)O--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--C(O)S--, --C(O)O--, --P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--,
--P(O)(NR')--, --P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--,
--P(S)(NR')--, --P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--; and one or
more carbon atoms are optionally and independently replaced with
Cy.sup.L; [0425] R.sup.TD is a targeting moiety; [0426] each
L.sup.M is independently a covalent bond, or a bivalent or
multivalent, optionally substituted, linear or branched group
selected from a C.sub.1-100 aliphatic group and a C.sub.1-100
heteroaliphatic group having 1-30 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus, boron and
silicon, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L; [0427] each
Cy.sup.L is independently an optionally substituted tetravalent
group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20
aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and a 3-20 membered heterocyclyl ring having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus, boron and silicon; [0428] each R' is independently --R,
--C(O)R, --C(O)OR, or --S(O).sub.2R; and [0429] each R is
independently --H, or an optionally substituted group selected from
C.sub.1-30 aliphatic, C.sub.1-30 heteroaliphatic having 1-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon, C.sub.6-30 aryl, C.sub.6-30 arylaliphatic,
C.sub.6-30 arylheteroaliphatic having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, 5-30 membered heteroaryl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, and 3-30 membered heterocyclyl having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus
and silicon, or [0430] two R groups are optionally and
independently taken together to form a covalent bond, or. [0431]
two or more R groups on the same atom are optionally and
independently taken together with the atom to form an optionally
substituted, 3-30 membered monocyclic, bicyclic or polycyclic ring
having, in addition to the atom, 0-10 heteroatoms independently
selected from oxygen, nitrogen, sulfur, phosphorus and silicon; or
[0432] two or more R groups on two or more atoms are optionally and
independently taken together with their intervening atoms to form
an optionally substituted, 3-30 membered monocyclic, bicyclic or
polycyclic ring having, in addition to the intervening atoms, 0-10
heteroatoms independently selected from oxygen, nitrogen, sulfur,
phosphorus and silicon.
[0433] In some embodiments,
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D, or
(A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b is a conjugate of a
provided oligonucleotide with one or more chemical moieties, e.g.,
targeting moieties, carbohydrate moieties, lipid moieties, etc.
[0434] In some embodiments, (R.sup.D).sub.b-L.sup.M- is
(R.sup.D).sub.b-L.sup.M1-L.sup.M2 as described in the present
disclosure.
[0435] In some embodiments, [H].sub.a-A.sup.c or [H].sub.b-A.sup.c
is an oligonucleotide as described in the present disclosure. In
some embodiments, [H].sub.a-A.sup.c or [H].sub.b-A.sup.c is of
formula O-I.
[0436] In some embodiments, R.sup.D is an additional chemical
moiety as described in the present disclosure. In some embodiments,
R.sup.D is a targeting moiety as described in the present
disclosure. In some embodiments, R.sup.D is R.sup.TD, which is a
targeting moiety as described in the present disclosure (e.g.,
targeting moiety described as embodiment for R.sup.D as targeting
moiety). In some embodiments, In some embodiments, R is R.sup.CD,
wherein R.sup.CD is as described in the present disclosure. In some
embodiments, R.sup.CD comprises one or more carbohydrate moieties.
In some embodiments, R.sup.D is R.sup.LD. In some embodiments,
R.sup.LD is a lipid moiety as described in the present
disclosure.
[0437] In some embodiments, a is 1-100. In some embodiments, a is
1-50. In some embodiments, a is 1-40. In some embodiments, a is
1-30. In some embodiments, a is 1-20. In some embodiments, a is
1-15. In some embodiments, a is 1-10. In some embodiments, a is
1-9. In some embodiments, a is 1-8. In some embodiments, a is 1-7.
In some embodiments, a is 1-6. In some embodiments, a is 1-5. In
some embodiments, a is 1-4. In some embodiments, a is 1-3. In some
embodiments, a is 1-2. In some embodiments, a is 1. In some
embodiments, a is 2. In some embodiments, a is 3. In some
embodiments, a is 4. In some embodiments, a is 5. In some
embodiments, a is 6. In some embodiments, a is 7. In some
embodiments, a is 8. In some embodiments, a is 9. In some
embodiments, a is 10. In some embodiments, a is more than 10.
[0438] In some embodiments, b is 1-100. In some embodiments, b is
1-50. In some embodiments, b is 1-40. In some embodiments, b is
1-30. In some embodiments, b is 1-20. In some embodiments, b is
1-15. In some embodiments, b is 1-10. In some embodiments, b is
1-9. In some embodiments, b is 1-8. In some embodiments, b is 1-7.
In some embodiments, b is 1-6. In some embodiments, b is 1-5. In
some embodiments, b is 1-4. In some embodiments, b is 1-3. In some
embodiments, b is 1-2. In some embodiments, b is 1. In some
embodiments, b is 2. In some embodiments, b is 3. In some
embodiments, b is 4. In some embodiments, b is 5. In some
embodiments, b is 6. In some embodiments, b is 7. In some
embodiments, b is 8. In some embodiments, b is 9. In some
embodiments, b is 10. In some embodiments, b is 1. In some
embodiments, b is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more.
[0439] In some embodiments, z is 1-1000. In some embodiments, z+1
is an oligonucleotide length as described in the present
disclosure. In some embodiments, z is no less than 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments,
z is no less than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In some
embodiments, z is no more than 50, 60, 70, 80, 90, 100, 150, or
200. In some embodiments, z is 5-50, 10-50, 14-50, 14-45, 14-40,
14-35, 14-30, 14-25, 14-100, 14-150, 14-200, 14-250, 14-300, 15-50,
15-45, 15-40, 15-35, 15-30, 15-25, 15-100, 15-150, 15-200, 15-250,
15-300, 16-50, 16-45, 16-40, 16-35, 16-30, 16-25, 16-100, 16-150,
16-200, 16-250, 16-300, 17-50, 17-45, 17-40, 17-35, 17-30, 17-25,
17-100, 17-150, 17-200, 17-250, 17-300, 18-50, 18-45, 18-40, 18-35,
18-30, 18-25, 18-100, 18-150, 18-200, 18-250, 18-300, 19-50, 19-45,
19-40, 19-35, 19-30, 19-25, 19-100, 19-150, 19-200, 19-250, or
19-300. In some embodiments, z is 10. In some embodiments, z is 11.
In some embodiments, z is 12. In some embodiments, z is 13. In some
embodiments, z is 14. In some embodiments, z is 15. In some
embodiments, z is 16. In some embodiments, z is 17. In some
embodiments, z is 18. In some embodiments, z is 19. In some
embodiments, z is 20. In some embodiments, z is 21. In some
embodiments, z is 22. In some embodiments, z is 23. In some
embodiments, z is 24. In some embodiments, z is 25. In some
embodiments, z is 26. In some embodiments, z is 27. In some
embodiments, z is 28. In some embodiments, z is 29. In some
embodiments, z is 30. In some embodiments, z is 31. In some
embodiments, z is 32. In some embodiments, z is 33. In some
embodiments, z is 34.
[0440] In some embodiments, L.sup.M1 is
-L.sup.M1-L.sup.M2-L.sup.M3- as described in the present
disclosure. In some embodiments, L.sup.M is L.sup.M1 as described
in the present disclosure. In some embodiments, L.sup.M is L.sup.M2
as described in the present disclosure. In some embodiments,
L.sup.M is L.sup.M3 as described in the present disclosure.
[0441] In some embodiments, at least one L.sup.M is directly bound
to a sugar unit of a provided oligonucleotide. In some embodiments,
a L.sup.M directly binds to a sugar unit incorporates a lipid
moiety into an oligonucleotide. In some embodiments, a L.sup.M
directly binds to a sugar unit incorporates a carbohydrate moiety
into an oligonucleotide. In some embodiments, a L.sup.M directly
binds to a sugar unit incorporates a R.sup.LD group into an
oligonucleotide. In some embodiments, a L.sup.M directly binds to a
sugar unit incorporates a R.sup.CD group into an oligonucleotide.
In some embodiments, L.sup.M1 is directed bound through 5'-OH of an
oligonucleotide chain. In some embodiments, L.sup.M1 is directed
bound through 3'-OH of an oligonucleotide chain.
[0442] In some embodiments, at least one L.sup.M is directly bound
to an internucleotidic linkage unit of a provided oligonucleotide.
In some embodiments, a L.sup.M directly binds to an
internucleotidic linkage unit incorporates a lipid moiety into an
oligonucleotide. In some embodiments, a L.sup.M directly binds to
an internucleotidic linkage unit incorporates a carbohydrate moiety
into an oligonucleotide. In some embodiments, a L.sup.M directly
binds to an internucleotidic linkage unit incorporates a R.sup.D
group into an oligonucleotide. In some embodiments, a L.sup.M
directly binds to an internucleotidic linkage unit incorporates a
R.sup.CD group into an oligonucleotide.
[0443] In some embodiments, at least one L.sup.M is directly bound
to a nucleobase unit of a provided oligonucleotide. In some
embodiments, a L.sup.M directly binds to a nucleobase unit
incorporates a lipid moiety into an oligonucleotide. In some
embodiments, a L.sup.M directly binds to a nucleobase unit
incorporates a carbohydrate moiety into an oligonucleotide. In some
embodiments, a L.sup.M directly binds to a nucleobase unit
incorporates a R.sup.LD group into an oligonucleotide. In some
embodiments, a L.sup.M directly binds to a nucleobase unit
incorporates a R.sup.CD group into an oligonucleotide.
[0444] In some embodiments, L.sup.M is bivalent. In some
embodiments, L.sup.M is multivalent. In some embodiments, L.sup.M
is
##STR00097##
wherein L.sup.M is directly bond to a nucleobase, for example, as
in:
##STR00098##
In some embodiments, L.sup.M is
##STR00099##
In some embodiments, L.sup.M is
##STR00100##
In some embodiments, L.sup.M is
##STR00101##
In some embodiments, L.sup.M is
##STR00102##
[0445] In some embodiments, R.sup.LD is optionally substituted
C.sub.10, C.sub.15, C.sub.16, C.sub.17, C.sub.18, C.sub.19,
C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, or C.sub.25 to
C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, C.sub.25,
C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30, C.sub.35,
C.sub.40, C.sub.45, C.sub.50, C.sub.60, C.sub.70, or C.sub.80
aliphatic. In some embodiments, R.sup.LD is optionally substituted
C.sub.10-80 aliphatic. In some embodiments, R.sup.LD is optionally
substituted C.sub.2-80 aliphatic. In some embodiments, R.sup.LD is
optionally substituted C.sub.10-70 aliphatic. In some embodiments,
R.sup.LD is optionally substituted C.sub.20-70 aliphatic. In some
embodiments, R.sup.LD is optionally substituted C.sub.10-60
aliphatic. In some embodiments, R.sup.LD is optionally substituted
C.sub.20-60 aliphatic. In some embodiments, R.sup.LD is optionally
substituted C.sub.10-50 aliphatic. In some embodiments, R.sup.LD is
optionally substituted C.sub.20-50 aliphatic. In some embodiments,
R.sup.LD is optionally substituted C.sub.10-40 aliphatic. In some
embodiments, R.sup.LD is optionally substituted C.sub.20-40
aliphatic. In some embodiments, R.sup.LD is optionally substituted
C.sub.10-30 aliphatic. In some embodiments, R.sup.LD is optionally
substituted C.sub.20-30 aliphatic. In some embodiments, R.sup.LD is
unsubstituted C.sub.10, C.sub.15, C.sub.16, C.sub.17, C.sub.18,
C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24, or
C.sub.25 to C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24,
C.sub.25, C.sub.26, C.sub.27, C.sub.28, C.sub.29, C.sub.30,
C.sub.35, C.sub.40, C.sub.45, C.sub.50, C.sub.60, C.sub.70, or
C.sub.80 aliphatic. In some embodiments, R.sup.LD is unsubstituted
C.sub.10-80 aliphatic. In some embodiments, R.sup.LD is
unsubstituted C.sub.20-80 aliphatic. In some embodiments, R.sup.LD
is unsubstituted C.sub.10-70 aliphatic. In some embodiments,
R.sup.LD is unsubstituted C.sub.20-70 aliphatic. In some
embodiments, R.sup.LD is unsubstituted C.sub.10-60 aliphatic. In
some embodiments, R.sup.LD is unsubstituted C.sub.20-60 aliphatic.
In some embodiments, R.sup.LD is unsubstituted C.sub.10-50
aliphatic. In some embodiments, R.sup.LD is unsubstituted
C.sub.20-50 aliphatic. In some embodiments, R.sup.LD is
unsubstituted C.sub.10-40 aliphatic. In some embodiments, R.sup.LD
is unsubstituted C.sub.20-40 aliphatic. In some embodiments,
R.sup.LD is unsubstituted C.sub.10-30 aliphatic. In some
embodiments, R.sup.LD is unsubstituted C.sub.20-30 aliphatic.
[0446] In some embodiments, R.sup.LD is not hydrogen. In some
embodiments, R.sup.LD is a lipid moiety. In some embodiments,
R.sup.LD is a targeting moiety. In some embodiments, R.sup.LD is a
targeting moiety comprising a carbohydrate moiety. In some
embodiments, R.sup.LD is a GalNAc moiety.
[0447] In some embodiments, R.sup.TD is R.sup.LD, wherein R.sup.LD
is independently as described in the present disclosure. In some
embodiments, R.sup.T is R.sup.CD, wherein R.sup.CD is independently
as described in the present disclosure.
[0448] In some embodiments, R.sup.CD is an optionally substituted,
linear or branched group selected from a C.sub.1-30 aliphatic group
and a C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus,
boron and silicon, wherein one or more methylene units are
optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
optionally and independently replaced with Cy.sup.L. In some
embodiments, R.sup.CD is an optionally substituted, linear or
branched group selected from a C.sub.1-30 aliphatic group and a
C.sub.1-30 heteroaliphatic group having 1-10 heteroatoms
independently selected from oxygen, nitrogen, sulfur, phosphorus,
boron and silicon, wherein one or more methylene units are
optionally and independently replaced with C.sub.1-6 alkylene,
C.sub.1-6 alkenylene, --C.ident.C--, --C(R').sub.2--, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)O--, --S(O)--, --S(O).sub.2--,
--S(O).sub.2N(R')--, --C(O)S--, --C(O)O--, --P(O)(OR')--,
--P(O)(SR')--, --P(O)(R')--, --P(O)(NR')--, --P(S)(OR')--,
--P(S)(SR')--, --P(S)(R')--, --P(S)(NR')--, --P(R')--, --P(OR')--,
--P(SR')--, --P(NR')--, --P(OR')[B(R').sub.3]--, --OP(O)(OR')O--,
--OP(O)(SR')O--, --OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--,
--OP(SR')O--, --OP(NR')O--, --OP(R')O--, or
--OP(OR')[B(R').sub.3]O--; and one or more carbon atoms are
independently replaced with a monosaccharide, disaccharide or
polysaccharide moiety. In some embodiments, R.sup.CD is an
optionally substituted, linear or branched group selected from a
C.sub.1-30 aliphatic group and a C.sub.1-30 heteroaliphatic group
having 1-10 heteroatoms independently selected from oxygen,
nitrogen, sulfur, phosphorus, boron and silicon, wherein one or
more methylene units are optionally and independently replaced with
C.sub.1-6 alkylene, C.sub.1-6 alkenylene, --C.ident.C--,
--C(R').sub.2--, --O--, --S--, --S--S--, --N(R')--, --C(O)--,
--C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)O--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--C(O)S--, --C(O)O--, --P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--,
--P(O)(NR')--, --P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--,
--P(S)(NR')--, --P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--; and one or
more carbon atoms are independently replaced with a GalNac
moiety.
[0449] In some embodiments, the present disclosure provides salts
of oligonucleotides, and pharmaceutical compositions thereof. In
some embodiments, a salt is a pharmaceutically acceptable salt. In
some embodiments, each hydrogen ion that may be donated to a base
(e.g., under conditions of an aqueous solution, a pharmaceutical
composition, etc.) is replaced by a non-H.sup.+ cation. For
example, in some embodiments, a pharmaceutically acceptable salt of
an oligonucleotide is an all-metal ion salt, wherein each hydrogen
ion (for example, of --OH, --SH, etc.) of each internucleotidic
linkage (e.g., a natural phosphate linkage, a phosphorothioate
diester linkage, etc.) is replaced by a metal ion. In some
embodiments, a provided salt is an all-sodium salt. In some
embodiments, a provided pharmaceutically acceptable salt is an
all-sodium salt. In some embodiments, a provided salt is an
all-sodium salt, wherein each internucleotidic linkage which is a
natural phosphate linkage (acid form --O--P(O)(OH)--O--), if any,
exists as its sodium salt form (--O--P(O)(ONa)--O--), and each
internucleotidic linkage which is a phosphorothioate diester
linkage (acid form --O--P(O)(SH)--O--), if any, exists as its
sodium salt form (--O--P(O)(SNa)--O--).
[0450] In some embodiments, a provided compound, e.g., a provided
oligonucleotide, has a purity of 60%-100%. In some embodiments, a
purity is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, a purity
is at least 60%. In some embodiments, a purity is at least 70%. In
some embodiments, a purity is at least 80%. In some embodiments, a
purity is at least 85%. In some embodiments, a purity is at least
90%. In some embodiments, a purity is at least 91%. In some
embodiments, a purity is at least 92%. In some embodiments, a
purity is at least 93%. In some embodiments, a purity is at least
94%. In some embodiments, a purity is at least 95%. In some
embodiments, a purity is at least 96%. In some embodiments, a
purity is at least 97%. In some embodiments, a purity is at least
98%. In some embodiments, a purity is at least 99%. In some
embodiments, a purity is at least 99.5%.
[0451] In some embodiments, a provided compound, e.g., a provided
oligonucleotide, has a diastereomeric purity of 60%-100%. In some
embodiments, a diastereomeric purity is at least 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
In some embodiments, a chiral element, e.g., a chiral center
(carbon, phosphorus, etc.) of a provided compound, e.g. a provided
oligonucleotide, has a diastereomeric purity of 60%-100%. In some
embodiments, a chiral element, e.g., a chiral center (carbon,
phosphorus, etc.) has a diastereomeric purity of at least 60%, 65%,
70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99%. In some embodiments, a diastereomeric purity is at least 60%.
In some embodiments, a diastereomeric purity is at least 70%. In
some embodiments, a diastereomeric purity is at least 80%. In some
embodiments, a diastereomeric purity is at least 85%. In some
embodiments, a diastereomeric purity is at least 90%. In some
embodiments, a diastereomeric purity is at least 91%. In some
embodiments, a diastereomeric purity is at least 92%. In some
embodiments, a diastereomeric purity is at least 93%. In some
embodiments, a diastereomeric purity is at least 94%. In some
embodiments, a diastereomeric purity is at least 95%. In some
embodiments, a diastereomeric purity is at least 96%. In some
embodiments, a diastereomeric purity is at least 97%. In some
embodiments, a diastereomeric purity is at least 98%. In some
embodiments, a diastereomeric purity is at least 99%. In some
embodiments, a diastereomeric purity is at least 99.5%.
[0452] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or
more chiral elements of a provided compound each independently have
a diastereomeric purity as described herein. In some embodiments,
at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or more chiral carbon centers of
a provided compound each independently have a diastereomeric purity
as described herein. In some embodiments, at least 1, 2, 3, 4, 5,
6, 7, 8, 9 or more chiral phosphorus centers of a provided compound
each independently have a diastereomeric purity as described
herein.
[0453] In some embodiments, at least 5%-100% of all chiral elements
of a provided compound each independently have a diastereomeric
purity as described herein. In some embodiments, at least 5%, 10%,
15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or 100% of all chiral elements of a provided
compound each independently have a diastereomeric purity as
described herein. In some embodiments, at least 5%-100% of all
chiral phosphorus centers of a provided compound each independently
have a diastereomeric purity as described herein. In some
embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of all chiral
phosphorus centers of a provided compound each independently have a
diastereomeric purity as described herein.
[0454] In some embodiments, each chiral element independently has a
diastereomeric purity as described herein. In some embodiments,
each chiral center independently has a diastereomeric purity as
described herein. In some embodiments, each chiral carbon center
independently has a diastereomeric purity as described herein. In
some embodiments, each chiral phosphorus center independently has a
diastereomeric purity as described herein.
[0455] In some embodiments, a provided compound, e.g.,
oligonucleotide and/or compositions thereof, can modulate
activities and/or functions of a C9orf72 target. In some
embodiments, a C9orf72 target gene is a gene with respect to which
expression and/or activity of one or more C9orf72 gene products
(e.g., RNA and/or protein products) are intended to be altered. In
many embodiments, a C9orf72 target gene is intended to be
inhibited. Thus, when a C9orf72 oligonucleotide as described herein
acts on a particular C9orf72 target gene, presence and/or activity
of one or more gene products of that C9orf72 gene are altered when
the oligonucleotide is present as compared with when it is
absent.
[0456] In some embodiments, a C9orf72 target is a specific allele
(e.g., a pathological allele) with respect to which expression
and/or activity of one or more products (e.g., RNA and/or protein
products) are intended to be altered. In many embodiments, a
C9orf72 target allele is one whose presence and/or expression is
associated (e.g., correlated) with presence, incidence, and/or
severity, of one or more diseases and/or conditions, e.g., a
C9orf72-related disorder. Alternatively or additionally, in some
embodiments, a C9orf72 target allele is one for which alteration of
level and/or activity of one or more gene products correlates with
improvement (e.g., delay of onset, reduction of severity,
responsiveness to other therapy, etc) in one or more aspects of a
disease and/or condition. In some such embodiments, C9orf72
oligonucleotides and methods thereof as described herein may
preferentially or specifically target the pathological allele
relative to the non-pathological allele, e.g., one or more
less-associated/unassociated allele(s). In some embodiments, a
pathological allele of C9orf72 comprises a repeat expansion, e.g.,
a hexanucleotide repeat expansion (HRE), e.g., a hexanucleotide
repeat expansion of greater than about 30 and up to 500 or 1000 or
more.
[0457] In some embodiments, a C9orf72 target sequence is a sequence
to which an oligonucleotide as described herein binds. In many
embodiments, a C9orf72 target sequence is identical to, or is an
exact complement of, a sequence of a provided oligonucleotide, or
of consecutive residues therein (e.g., a provided oligonucleotide
includes a target-binding sequence that is identical to, or an
exact complement of, a C9orf72 target sequence). In some
embodiments, a small number of differences/mismatches is tolerated
between (a relevant portion of) an oligonucleotide and its target
sequence. In many embodiments, a C9orf72 target sequence is present
within a C9orf72 target gene. In many embodiments, a C9orf72 target
sequence is present within a transcript (e.g., an mRNA and/or a
pre-mRNA) produced from a C9orf72 target gene. In some embodiments,
a C9orf72 target sequence includes one or more allelic sites (i.e.,
positions within a C9orf72 target gene at which allelic variation
occurs). In some such embodiments, a provided oligonucleotide binds
to one allele preferentially or specifically relative to one or
more other alleles.
[0458] In some embodiments, C9orf72 (chromosome 9 open reading
frame 72) is a gene or its gene product, also designated as
C90RF72, C9, ALSFTD, FTDALS, FTDALS1, DENNL72; External IDs: MGI:
1920455 HomoloGene: 10137 GeneCards: C9orf72. C9orf72 is also
informally designated C9. C9orf72 Orthologs: Species: Human Entrez:
203228; Ensembl: ENSG00000147894; UniProt: Q96LT7; RefSeq (mRNA):
NM_145005 NM_001256054 NM_018325; RefSeq (protein): NP_001242983
NP_060795 NP_659442; Location (UCSC): Chr 9: 27.55-27.57 Mb;
Species: Mouse Entrez: 73205; Ensembl: ENSMUSG00000028300; UniProt:
Q6DFW0; RefSeq (mRNA): NM_001081343; RefSeq (protein): NP_00107481;
Location (UCSC): Chr 4: 35.19-35.23 Mb. Nucleotides which encode
C9orf72 include, without limitation, GENBANK Accession No.
NM_001256054.1; GENBANK Accession No. NT_008413.18; GENBANK
Accession No. BQ068108.1; GENBANK Accession No. NM_018325.3;
GENBANK Accession No. DN993522.1; GENBANKAccession No. NM_145005.5;
GENBANK Accession No. DB079375.1; GENBANK Accession No. BU194591.1;
Sequence Identifier 4141_014_A 5; Sequence Identifier 4008_73_A;
and GENBANKAccession No. NT_008413.18. C9orf72 reportedly is a 481
amino acid protein with a molecular mass of 54328 Da, which may
undergo post-translational modifications of ubiquitination and
phosphorylation. The expression levels of C9orf72 reportedly may be
highest in the central nervous system and the protein localizes in
the cytoplasm of neurons as well as in presynaptic terminals.
C9orf72 reportedly plays a role in endosomal and lysosomal
trafficking regulation and has been shown to interact with RAB
proteins that are involved in autophagy and endocytic transport.
C9orf72 reportedly activates RAB5, a GTPase that mediates early
endosomal trafficking. Mutations in C9orf72 reportedly have been
associated with ALS and FTD. DeJesus-Hernandez et al. 2011 Neuron
72: 245-256; Renton et al. 2011 Neuron 72: 257-268; and Itzcovich
et al. 2016. Neurobiol. Aging. Volume 40, Pages 192.e13-192.e15. A
hexanucleotide repeat expansion (e.g., (GGGGCC)n) in C9orf72
reportedly may be present in subjects suffering from a neurological
disease, such as a C9orf72-related disorder.
[0459] In some embodiments, C9orf72 is not capitalized and is
rendered as c9orf72.
[0460] In some embodiments, a C9orf72 oligonucleotide can comprise
any of various linkers, additional moieties (including but not
limited to targeting moieties), and/or be chirally controlled
and/or have any of various bases sequences and/or chemical
structures or formats as described herein.
[0461] Various linker, carbohydrate moieties and targeting
moieties, including many known in the art, can be utilized in
accordance with the present disclosure. In some embodiments, a
carbohydrate moiety is a targeting moiety. In some embodiments, a
targeting moiety is a carbohydrate moiety.
[0462] In some embodiments, the present disclosure provides
oligonucleotides and oligonucleotide compositions that are chirally
controlled. For instance, in some embodiments, a provided
composition contains non-random or controlled levels of one or more
individual oligonucleotide types, wherein an oligonucleotide type
is defined by: 1) base sequence; 2) pattern of backbone linkages;
3) pattern of backbone chiral centers; and 4) pattern of backbone
P-modifications. In some embodiments, a particular oligonucleotide
type may be defined by 1A) base identity; 1B) pattern of base
modification; 1C) pattern of sugar modification; 2) pattern of
backbone linkages; 3) pattern of backbone chiral centers; and 4)
pattern of backbone P-modifications. In some embodiments,
oligonucleotides of the same oligonucleotide type are identical. In
some embodiments, the present disclosure provides chirally
controlled C9orf72 oligonucleotide compositions of
oligonucleotides, wherein the composition comprises a non-random or
controlled level of a plurality of oligonucleotides, wherein
oligonucleotides of the plurality share a common base sequence, and
comprise the same configuration of linkage phosphorus at at least
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 chiral internucleotidic linkages
(chirally controlled internucleotidic linkages). In some
embodiments, oligonucleotides of a predetermined level and/or a
provided plurality, e.g., those of formula O-I,
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D,
(c).sub.a-L.sup.M-(A.sup.c).sub.b, or
(A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b, comprise 1-30 chirally
controlled internucleotidic linkages. In some embodiments, provided
C9orf72 oligonucleotides comprise 2-30 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides comprise 5-30 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides comprise
10-30 chirally controlled internucleotidic linkages. In some
embodiments, provided oligonucleotides comprise 1 chirally
controlled internucleotidic linkage. In some embodiments, provided
oligonucleotides comprise 2 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides comprise 3
chirally controlled internucleotidic linkages. In some embodiments,
provided oligonucleotides comprise 4 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides comprise 5 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides comprise 6
chirally controlled internucleotidic linkages. In some embodiments,
provided oligonucleotides comprise 7 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides comprise 8 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides comprise 9
chirally controlled internucleotidic linkages. In some embodiments,
provided oligonucleotides comprise 10 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides comprise 11 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides comprise
12 chirally controlled internucleotidic linkages. In some
embodiments, provided oligonucleotides comprise 13 chirally
controlled internucleotidic linkages. In some embodiments, provided
oligonucleotides comprise 14 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides have 15
chirally controlled internucleotidic linkages. In some embodiments,
provided oligonucleotides have 16 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides have 17 chirally controlled internucleotidic
linkages. In some embodiments, provided oligonucleotides have 18
chirally controlled internucleotidic linkages. In some embodiments,
provided oligonucleotides have 19 chirally controlled
internucleotidic linkages. In some embodiments, provided
oligonucleotides have 20 chirally controlled internucleotidic
linkages. In some embodiments, about 1-100% of all internucleotidic
linkages are chirally controlled internucleotidic linkages. In some
embodiments, a percentage is about 5%-100%. In some embodiments, a
percentage is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 965, 96%, 98%, or
99%. In some embodiments, a percentage is about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 965, 96%, 98%, or 99%.
[0463] In some embodiments, a provided oligonucleotide is a unimer.
In some embodiments, a provided oligonucleotide is a P-modification
unimer. In some embodiments, a provided oligonucleotide is a
stereounimer. In some embodiments, a provided oligonucleotide is a
stereounimer of configuration Rp. In some embodiments, a provided
oligonucleotide is a stereounimer of configuration Sp.
[0464] In some embodiments, a provided oligonucleotide is an
altmer. In some embodiments, a provided oligonucleotide is a
P-modification altmer. In some embodiments, a provided
oligonucleotide is a stereoaltmer.
[0465] In some embodiments, a provided oligonucleotide is a
blockmer. In some embodiments, a provided oligonucleotide is a
P-modification blockmer. In some embodiments, a provided
oligonucleotide is a stereoblockmer.
[0466] In some embodiments, a provided oligonucleotide is a
gapmer.
[0467] In some embodiments, a provided oligonucleotide is a
skipmer.
[0468] In some embodiments, a provided oligonucleotide is a
hemimer. In some embodiments, a hemimer is an oligonucleotide
wherein the 5'-end or the 3'-end region has a sequence that
possesses a structure feature that the rest of the oligonucleotide
does not have. In some embodiments, the 5'-end or the 3'-end region
has or comprises 2 to 20 nucleotides. In some embodiments, a
structural feature is a base modification. In some embodiments, a
structural feature is a sugar modification. In some embodiments, a
structural feature is a P-modification. In some embodiments, a
structural feature is stereochemistry of the chiral
internucleotidic linkage. In some embodiments, a structural feature
is or comprises a base modification, a sugar modification, a
P-modification, or stereochemistry of the chiral internucleotidic
linkage, or combinations thereof. In some embodiments, a hemimer is
an oligonucleotide in which each sugar moiety of the 5'-end region
shares a common modification. In some embodiments, a hemimer is an
oligonucleotide in which each sugar moiety of the 3'-end region
shares a common modification. In some embodiments, a common sugar
modification of the 5' or 3'-end region is not shared by any other
sugar moieties in the oligonucleotide. In some embodiments, an
example hemimer is an oligonucleotide comprising a sequence of
substituted or unsubstituted 2'-O-alkyl sugar modified nucleosides,
bicyclic sugar modified nucleosides, .beta.-D-ribonucleosides or
.beta.-D-deoxyribonucleosides (for example 2'-MOE modified
nucleosides, and LNA.TM. or ENA.TM. bicyclic sugar modified
nucleosides) at one terminus region and a sequence of nucleosides
with a different sugar moiety (such as a substituted or
unsubstituted 2'-O-alkyl sugar modified nucleosides, bicyclic sugar
modified nucleosides or natural ones) at the other terminus region.
In some embodiments, a provided oligonucleotide is a combination of
one or more of unimer, altmer, blockmer, gapmer, hemimer and
skipmer. In some embodiments, a provided oligonucleotide is a
combination of one or more of unimer, altmer, blockmer, gapmer, and
skipmer. For instance, in some embodiments, a provided
oligonucleotide is both an altmer and a gapmer. In some
embodiments, a provided nucleotide is both a gapmer and a skipmer.
One of skill in the chemical and synthetic arts will recognize that
numerous other combinations of patterns are available and are
limited only by the commercial availability and/or synthetic
accessibility of constituent parts required to synthesize a
provided oligonucleotide in accordance with methods of the present
disclosure. In some embodiments, a hemimer structure provides
advantageous benefits. In some embodiments, provided
oligonucleotides are 5'-hemimers that comprises modified sugar
moieties in a 5'-end sequence. In some embodiments, provided
oligonucleotides are 5'-hemimers that comprises modified 2'-sugar
moieties in a 5'-end sequence.
[0469] In some embodiments, a provided oligonucleotide comprises
one or more optionally substituted nucleotides. In some
embodiments, a provided oligonucleotide comprises one or more
modified nucleotides. In some embodiments, a provided
oligonucleotide comprises one or more optionally substituted
nucleosides. In some embodiments, a provided oligonucleotide
comprises one or more modified nucleosides. In some embodiments, a
provided oligonucleotide comprises one or more optionally
substituted LNAs.
[0470] In some embodiments, a provided oligonucleotide comprises
one or more optionally substituted nucleobases. In some
embodiments, a provided oligonucleotide comprises one or more
optionally substituted natural nucleobases. In some embodiments, a
provided oligonucleotide comprises one or more optionally
substituted modified nucleobases. In some embodiments, a provided
oligonucleotide comprises one or more 5-methylcytidine;
5-hydroxymethylcytidine, 5-formylcytosine, or 5-carboxylcytosine.
In some embodiments, a provided oligonucleotide comprises one or
more 5-methylcytidine.
[0471] In some embodiments, each nucleobase of a provided
oligonucleotide, e.g., one of formula O-I,
A.sup.c-[-L.sup.M-(R.sup.D).sub.a].sub.b,
[(A.sup.c).sub.a-L.sup.M].sub.b-R.sup.D,
(A.sup.c).sub.a-L.sup.M-(A.sup.c).sub.b, or
(A.sup.c).sub.a-L.sup.M-(R.sup.D).sub.b, is independently an
optionally substituted or protected nucleobase of adenine,
cytosine, guanosine, thymine, or uracil. In some embodiments, each
BA is independently an optionally substituted or protected
nucleobase of adenine, cytosine, guanosine, thymine, or uracil. As
appreciated by those skilled in the art, various protected
nucleobases, including those widely known in the art, for example,
those used in oligonucleotide preparation (e.g., protected
nucleobases of WO/2010/064146, WO/2011/005761, WO/2013/012758,
WO/2014/010250, US2013/0178612, WO/2014/012081, WO/2015/107425,
WO2017/015555, and WO2017/062862, protected nucleobases of each of
which are incorporated herein by reference), and can be utilized in
accordance with the present disclosure.
[0472] In some embodiments, a provided oligonucleotide comprises
one or more optionally substituted sugars. In some embodiments, a
provided oligonucleotide comprises one or more optionally
substituted sugars found in naturally occurring DNA and RNA. In
some embodiments, a provided oligonucleotide comprises one or more
optionally substituted ribose or deoxyribose. In some embodiments,
a provided oligonucleotide comprises one or more optionally
substituted ribose or deoxyribose, wherein one or more hydroxyl
groups of the ribose or deoxyribose moiety is optionally and
independently replaced by halogen, R', --N(R').sub.2, --OR', or
--SR', wherein each R' is independently described in the present
disclosure. In some embodiments, a provided oligonucleotide
comprises one or more optionally substituted deoxyribose, wherein
the 2' position of the deoxyribose is optionally and independently
substituted with halogen, R', --N(R').sub.2, --OR', or --SR',
wherein each R' is independently described in the present
disclosure. In some embodiments, a provided oligonucleotide
comprises one or more optionally substituted deoxyribose, wherein
the 2' position of the deoxyribose is optionally and independently
substituted with halogen. In some embodiments, a provided
oligonucleotide comprises one or more optionally substituted
deoxyribose, wherein the 2' position of the deoxyribose is
optionally and independently substituted with one or more --F.
halogen. In some embodiments, a provided oligonucleotide comprises
one or more optionally substituted deoxyribose, wherein the 2'
position of the deoxyribose is optionally and independently
substituted with --OR', wherein each R' is independently described
in the present disclosure. In some embodiments, a provided
oligonucleotide comprises one or more optionally substituted
deoxyribose, wherein the 2' position of the deoxyribose is
optionally and independently substituted with --OR', wherein each
R' is independently an optionally substituted C.sub.1-C.sub.6
aliphatic. In some embodiments, a provided oligonucleotide
comprises one or more optionally substituted deoxyribose, wherein
the 2' position of the deoxyribose is optionally and independently
substituted with --OR', wherein each R' is independently an
optionally substituted C.sub.1-C.sub.6 alkyl. In some embodiments,
a provided oligonucleotide comprises one or more optionally
substituted deoxyribose, wherein the 2' position of the deoxyribose
is optionally and independently substituted with --OMe. In some
embodiments, a provided oligonucleotide comprises one or more
optionally substituted deoxyribose, wherein the 2' position of the
deoxyribose is optionally and independently substituted with --O--
methoxyethyl.
[0473] In some embodiments, a provided oligonucleotide is
single-stranded oligonucleotide. In some embodiments, a provided
single-stranded C9orf72 oligonucleotide further comprises one or
more additional strands which are partially or completely
complementary to the single-stranded C9orf72 oligonucleotide.
[0474] In some embodiments, a provided oligonucleotide is a
hybridized oligonucleotide strand. In certain embodiments, a
provided oligonucleotide is a partially hybridized oligonucleotide
strand. In certain embodiments, a provided oligonucleotide is a
completely hybridized oligonucleotide strand. In certain
embodiments, a provided oligonucleotide is a double-stranded
oligonucleotide. In certain embodiments, a provided oligonucleotide
is a triple-stranded oligonucleotide (e.g., a triplex).
[0475] In some embodiments, a provided C9orf72 oligonucleotide is
chimeric. For example, in some embodiments, a provided
oligonucleotide (e.g., a C9orf72 oligonucleotide which has a base
sequence which comprises, consists of, or comprises a portion of a
base sequence of a C9orf72 oligonucleotide disclosed herein) is
DNA-RNA chimera, DNA-LNA chimera, a chimera comprising any two or
more of DNA, RNA, LNA, 2'-modified sugars, etc.
[0476] In some embodiments, a C9orf72 oligonucleotide can comprise
a chemical structure described in WO2012/030683.
[0477] In some embodiments, a provided oligonucleotide is a
therapeutic agent.
[0478] In some embodiments, a provided oligonucleotide comprises a
nucleic acid analog, e.g., GNA, LNA, PNA, TNA, F-HNA (F-THP or
3'-fluoro tetrahydropyran), MNA (mannitol nucleic acid, e.g.,
Leumann 2002 Bioorg. Med. Chem. 10: 841-854), ANA (anitol nucleic
acid), and Morpholino.
[0479] In some embodiments, a provided oligonucleotide is about
2-500 nucleotide units in length. In some embodiments, a provided
oligonucleotide is about 5-500 nucleotide units in length. In some
embodiments, a provided oligonucleotide is about 10-50 nucleotide
units in length. In some embodiments, a provided oligonucleotide is
about 15-50 nucleotide units in length. In some embodiments, each
nucleotide unit independently comprises a heteroaryl nucleobase
unit (e.g., adenine, cytosine, guanosine, thymine, and uracil, each
of which is optionally and independently substituted or protected),
a sugar unit comprising a 5-10 membered heterocyclyl ring, and an
internucleotidic linkage having the structure of formula I.
[0480] In some embodiments, a provided oligonucleotide is from
about 15 to about 30 nucleotide units in length. In some
embodiments, a provided oligonucleotide is from about 10 to about
25 nucleotide units in length. In some embodiments, a provided
oligonucleotide is from about 15 to about 22 nucleotide units in
length. In some embodiments, a provided oligonucleotide is from
about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, or 25 nucleotide units in length.
[0481] In some embodiments, an oligonucleotide is at least 15
nucleotide units in length. In some embodiments, an oligonucleotide
is at least 16 nucleotide units in length. In some embodiments, an
oligonucleotide is at least 17 nucleotide units in length. In some
embodiments, an oligonucleotide is at least 18 nucleotide units in
length. In some embodiments, an oligonucleotide is at least 19
nucleotide units in length. In some embodiments, an oligonucleotide
is at least 20 nucleotide units in length. In some embodiments, an
oligonucleotide is at least 21 nucleotide units in length. In some
embodiments, an oligonucleotide is at least 22 nucleotide units in
length. In some embodiments, an oligonucleotide is at least 23
nucleotide units in length. In some embodiments, an oligonucleotide
is at least 24 nucleotide units in length. In some embodiments, an
oligonucleotide is at least 25 nucleotide units in length. In some
other embodiments, an oligonucleotide is at least 30 nucleotide
units in length. In some other embodiments, an oligonucleotide is a
duplex of complementary strands of at least 18 nucleotide units in
length. In some other embodiments, an oligonucleotide is a duplex
of complementary strands of at least 21 nucleotide units in
length.
[0482] In some embodiments, oligonucleotides of an oligonucleotide
type characterized by 1) a common base sequence and length, 2) a
common pattern of backbone linkages, and 3) a common pattern of
backbone chiral centers, have the same chemical structure. For
example, they have the same base sequence, the same pattern of
nucleoside modifications, the same pattern of backbone linkages
(i.e., pattern of internucleotidic linkage types, for example,
phosphate, phosphorothioate, etc), the same pattern of backbone
chiral centers (i.e. pattern of linkage phosphorus stereochemistry
(Rp/Sp)), and the same pattern of backbone phosphorus modifications
(e.g., pattern of "-XLR.sup.1" groups in Formula I).
[0483] Oligonucleotides
[0484] In some embodiments, provided C9orf72 oligonucleotides can
direct a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product. In some embodiments,
provided C9orf72 oligonucleotides can direct a decrease in the
expression, level and/or activity of a C9orf72 target gene or its
gene product and has a base sequence which consists of, comprises,
or comprises a portion (e.g., a span of 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or more contiguous bases) of the base sequence of any
C9orf72 oligonucleotide disclosed herein, and the oligonucleotide
comprises at least one non-naturally-occurring modification of a
base, sugar and/or internucleotidic linkage.
[0485] In some embodiments, a provided composition comprises an
oligonucleotide. In some embodiments, a provided oligonucleotide
comprises one or more carbohydrate moieties. In some embodiments, a
provided oligonucleotide comprises one or more targeting moieties.
Non-limiting examples of additional chemical moieties which can be
conjugated to an oligonucleotide are shown in Example 1.
[0486] In some embodiments, provided oligonucleotides can direct a
decrease in the expression, level and/or activity of a C9orf72
target gene or its gene product. In some embodiments, provided
oligonucleotides can direct a decrease in the expression, level
and/or activity of a C9orf72 target gene or its gene product via
RNase H-mediated knockdown. In some embodiments, provided
oligonucleotides can direct a decrease in the expression, level
and/or activity of a C9orf72 target gene or its gene product by
sterically blocking translation after binding to a C9orf72 target
gene mRNA, and/or by altering or interfering with mRNA splicing. In
some embodiments, a C9orf72 target gene comprises a hexanucleotide
repeat expansion.
[0487] In some embodiments, C9orf72 oligonucleotides include
nucleic acids (including antisense compounds), including but not
limited to antisense oligonucleotides (ASOs), oligonucleotides,
double- and single-stranded siRNAs; and C9orf72 oligonucleotide can
be co-administered or be used as part of a treatment regiment along
with aptamers, antibodies, peptides, small molecules, and/or other
agents capable of inhibiting the expression of C9orf72 antisense
transcript or gene and/or its expression product or gene product,
or a gene or gene product which increases the expression, activity
and/or level of a C9orf72 transcript comprising a repeat expansion
or its gene product, or a gene or gene product which is associated
with a C9orf72-related disorder.
[0488] In some embodiments, a provided oligonucleotide capable of
directing a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product has a base sequence (or a
portion thereof), pattern of chemical modification (or a portion
thereof), structural element or a portion thereof, or a format or
portion thereof described herein. In some embodiments, a provided
oligonucleotide capable of directing a decrease in the expression,
level and/or activity of a C9orf72 target gene or its gene product
has the base sequence (or a portion thereof), pattern of chemical
modification (or a portion thereof), format of any oligonucleotide
disclosed herein, e.g., in Table 1A or in the Figures, or otherwise
disclosed herein, or a structural element or format or portion
thereof described herein.
[0489] In some embodiments, a C9orf72 oligonucleotide can hybridize
to a C9orf72 nucleic acid derived from either DNA strand. In some
embodiments, a C9orf72 oligonucleotide can hybridize to a C9orf72
antisense or sense transcript. In some embodiments, a C9orf72
oligonucleotide can hybridize to a C9orf72 nucleic acid in any
stage of RNA processing, including but not limited to a pre-mRNA or
a mature mRNA. In some embodiments, a C9orf72 oligonucleotide can
hybridize to any element of a C9orf72 nucleic acid or its
complement, including but not limited to: a promoter region, an
enhancer region, a transcriptional stop region, a translational
start signal, a translation stop signal, a coding region, a
non-coding region, an exon, an intron, the 5' UTR, the 3' UTR, a
repeat region, a hexanucleotide repeat expansion, a splice
junction, intron/exon or exon/intron junction, an exon:exon splice
junction, an exonic splicing silencer (ESS), an exonic splicing
enhancer (ESE), exon 1a, exon 1b, exon 1c, exon 1d, exon 1e, exon
2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10,
exon 11, intron 1, intron 2, intron 3, intron 4, intron 5, intron
6, intron 7, intron 8, intron 9, or intron 10 of a C9orf72 nucleic
acid. The introns and exons alternate; intron 1 is between exon 1
(or 1a or 1b or 1c, etc.) and exon 2; intron 2 is between exon 2
and 3; etc. The positions of exons and introns in variant
transcripts of C9orf72 are diagrammed in the literature, e.g., WO
2014/062691.
[0490] In some embodiments, a C9orf72 sequence is represented
by:
TABLE-US-00001 (SEQ ID NO: 1)
CAAAGAAAAGGGGGAGGTTTTGTTAAAAAAGAGAAATGTTACATAGTGCTCTTTGAGAAAATTCATTGGC
ACTATTAAGGATCTGAGGAGCTGGTGAGTTTCAACTGGTGAGTGATGGTGGTAGATAAAATTAGAGCTGC
AGCAGGTCATTTTAGCAACTATTAGATAAAACTGGTCTCAGGTCACAACGGGCAGTTGCAGCAGCTGGAC
TTGGAGAGAATTACACTGTGGGAGCAGTGTCATTTGTCCTAAGTGCTTTTCTACCCCCTACCCCCACTAT
TTTAGTTGGGTATAAAAAGAATGACCCAATTTGTATGATCAACTTTCACAAAGCATAGAACAGTAGGAAA
AGGGTCTGTTTCTGCAGAAGGTGTAGACGTTGAGAGCCATTTTGTGTATTTATTCCTCCCTTTCTTCCTC
GGTGAATGATTAAAACGTTCTGTGTGATTTTTAGTGATGAAAAAGATTAAATGCTACTCACTGTAGTAAG
TGCCATCTCACACTTGCAGATCAAAAGGCACACAGTTTAAAAAACCTTTGTTTTTTTACACATCTGAGTG
GTGTAAATGCTACTCATCTGTAGTAAGTGGAATCTATACACCTGCAGACCAAAAGACGCAAGGTTTCAAA
AATCTTTGTGTTTTTTACACATCAAACAGAATGGTACGTTTTTCAAAAGTTAAAAAAAAACAACTCATCC
ACATATTGCAACTAGCAAAAATGACATTCCCCAGTGTGAAAATCATGCTTGAGAGAATTCTTACATGTAA
AGGCAAAATTGCGATGACTTTGCAGGGGACCGTGGGATTCCCGCCCGCAGTGCCGGAGCTGTCCCCTACC
AGGGTTTGCAGTGGAGTTTTGAATGCACTTAACAGTGTCTTACGGTAAAAACAAAATTTCATCCACCAAT
TATGTGTTGAGCGCCCACTGCCTACCAAGCACAAACAAAACCATTCAAAACCACGAAATCGTCTTCACTT
TCTCCAGATCCAGCAGCCTCCCCTATTAAGGTTCGCACACGCTATTGCGCCAACGCTCCTCCAGAGCGGG
TCTTAAGATAAAAGAACAGGACAAGTTGCCCCGCCCCATTTCGCTAGCCTCGTGAGAAAACGTCATCGCA
CATAGAAAACAGACAGACGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGACAAGT
TCCGCCCACGTAAAAGATGACGCTTGGTGTGTCAGCCGTCCCTGCTGCCCGGTTGCTTCTCTTTTGGGGG
CGGGGTCTAGCAAGAGCAGGTGTGGGTTTAGGAGGTGTGTGTTTTTGTTTTTCCCACCCTCTCTCCCCAC
TACTTGCTCTCACAGTACTCGCTGAGGGTGAACAAGAAAAGACCTGATAAAGATTAACCAGAAGAAAACA
AGGAGGGAAACAACCGCAGCCTGTAGCAAGCTCTGGAACTCAGGAGTCGCGCGCTAGGGGCCGGGGCCGG
GGCCGGGGCGTGGTCGGGGCGGGCCCGGGGGCGGGCCCGGGGCGGGGCTGCGGTTGCGGTGCCTGCGCCC
GCGGCGGCGGAGGCGCAGGCGGTGGCGAGTGGGTGAGTGAGGAGGCGGCATCCTGGCGGGTGGCTGTTTG
GGGTTCGGCTGCCGGGAAGAGGCGCGGGTAGAAGCGGGGGCTCTCCTCAGAGCTCGACGCATTTTTACTT
TCCCTCTCATTTCTCTGACCGAAGCTGGGTGTCGGGCTTTCGCCTCTAGCGACTGGTGGAATTGCCTGCA
TCCGGGCCCCGGGCTTCCCGGCGGCGGCGGCGGCGGCGGCGGCGCAGGGACAAGGGATGGGGATCTGGCC
TCTTCCTTGCTTTCCCGCCCTCAGTACCCGAGCTGTCTCCTTCCCGGGGACCCGCTGGGAGCGCTGCCGC
TGCGGGCTCGAGAAAAGGGAGCCTCGGGTACTGAGAGGCCTCGCCTGGGGGAAGGCCGGAGGGTGGGCGG
CGCGCGGCTTCTGCGGACCAAGTCGGGGTTCGCTAGGAACCCGAGACGGTCCCTGCCGGCGAGGAGATCA
TGCGGGATGAGATGGGGGTGTGGAGACGCCTGCACAATTTCAGCCCAAGCTTCTAGAGAGTGGTGATGAC
TTGCATATGAGGGCAGCAATGCAAGTCGGTGTGCTCCCCATTCTGTGGGACATGACCTGGTTGCTTCACA
GCTCCGAGATGACACAGACTTGCTTAAAGGAAGTGACTATTGTGACTTGGGCATCACTTGACTGATGGTA
ATCAGTTGTCTAAAGAAGTGCACAGATTACATGTCCGTGTGCTCATTGGGTCTATCTGGCCGCGTTGAAC
ACCACCAGGCTTTGTATTCAGAAACAGGAGGGAGGTCCTGCACTTTCCCAGGAGGGGTGGCCCTTTCAGA
TGCAATCGAGATTGTTAGGCTCTGGGAGAGTAGTTGCCTGGTTGTGGCAGTTGGTAAATTTCTATTCAAA
CAGTTGCCATGCACCAGTTGTTCACAACAAGGGTACGTAATCTGTCTGGCATTACTTCTACTTTTGTACA
AAGGATCAAAAAAAAAAAAGATACTGTTAAGATATGATTTTTCTCAGACTTTGGGAAACTTTTAACATAA
TCTGTGAATATCACAGAAACAAGACTATCATATAGGGGATATTAATAACCTGGAGTCAGAATACTTGAAA
TACGGTGTCATTTGACACGGGCATTGTTGTCACCACCTCTGCCAAGGCCTGCCACTTTAGGAAAACCCTG
AATCAGTTGGAAACTGCTACATGCTGATAGTACATCTGAAACAAGAACGAGAGTAATTACCACATTCCAG
ATTGTTCACTAAGCCAGCATTTACCTGCTCCAGGAAAAAATTACAAGCACCTTATGAAGTTGATAAAATA
TTTTGTTTGGCTATGTTGGCACTCCACAATTTGCTTTCAGAGAAACAAAGTAAACCAAGGAGGACTTCTG
TTTTTCAAGTCTGCCCTCGGGTTCTATTCTACGTTAATTAGATAGTTCCCAGGAGGACTAGGTTAGCCTA
CCTATTGTCTGAGAAACTTGGAACTGTGAGAAATGGCCAGATAGTGATATGAACTTCACCTTCCAGTCTT
CCCTGATGTTGAAGATTGAGAAAGTGTTGTGAACTTTCTGGTACTGTAAACAGTTCACTGTCCTTGAAGT
GGTCCTGGGCAGCTCCTGTTGTGGAAAGTGGACGGTTTAGGATCCTGCTTCTCTTTGGGCTGGGAGAAAA
TAAACAGCATGGTTACAAGTATTGAGAGCCAGGTTGGAGAAGGTGGCTTACACCTGTAATGCCAGAGCTT
TGGGAGGCGGAGGCAAGAGGATCACTTGAAGCCAGGAGTTCAAGCTCAACCTGGGCAACGTAGACCCTGT
CTCTACAAAAAATTAAAAACTTAGCCGGGCGTGGTGATGTGCACCTGTAGTCCTAGCTACTTGGGAGGCT
GAGGCAGGAGGGTCATTTGAGCCCAAGAGTTTGAAGTTACCGAGAGCTATGATCCTGCCAGTGCATTCCA
GCCTGGATGACAAAACGAGACCCTGTCTCTAAAAAACAAGAAGTGAGGGCTTTATGATTGTAGAATTTTC
ACTACAATAGCAGTGGACCAACCACCTTTCTAAATACCAATCAGGGAAGAGATGGTTGATTTTTTAACAG
ACGTTTAAAGAAAAAGCAAAACCTCAAACTTAGCACTCTACTAACAGTTTTAGCAGATGTTAATTAATGT
AATCATGTCTGCATGTATGGGATTATTTCCAGAAAGTGTATTGGGAAACCTCTCATGAACCCTGTGAGCA
AGCCACCGTCTCACTCAATTTGAATCTTGGCTTCCCTCAAAAGACTGGCTAATGTTTGGTAACTCTCTGG
AGTAGACAGCACTACATGTACGTAAGATAGGTACATAAACAACTATTGGTTTTGAGCTGATTTTTTTCAG
CTGCATTTGCATGTATGGATTTTTCTCACCAAAGACGATGACTTCAAGTATTAGTAAAATAATTGTACAG
CTCTCCTGATTATACTTCTCTGTGACATTTCATTTCCCAGGCTATTTCTTTTGGTAGGATTTAAAACTAA
GCAATTCAGTATGATCTTTGTCCTTCATTTTCTTTCTTATTCTTTTTGTTTGTTTGTTTGTTTGTTTTTT
TCTTGAGGCAGAGTCTCTCTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCCATCTCAGCTCATTGCAACCT
CTGCCACCTCCGGGTTCAAGAGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGTGTCCACC
ACCACACCCGGCTAATTTTTTGTATTTTTAGTAGAGGTGGGGTTTCACCATGTTGGCCAGGCTGGTCTTG
AGCTCCTGACCTCAGGTGATCCACCTGCCTCGGCCTACCAAAGAGCTGGGATAACAGGTGTGACCCACCA
TGCCCGGCCCATTTTTTTTTTCTTATTCTGTTAGGAGTGAGAGTGTAACTAGCAGTATAATAGTTCAATT
TTCACAACGTGGTAAAAGTTTCCCTATAATTCAATCAGATTTTGCTCCAGGGTTCAGTTCTGTTTTAGGA
AATACTTTTATTTTCAGTTTAATGATGAAATATTAGAGTTGTAATATTGCCTTTATGATTATCCACCTTT
TTAACCTAAAAGAATGAAAGAAAAATATGTTTGCAATATAATTTTATGGTTGTATGTTAACTTAATTCAT
TATGTTGGCCTCCAGTTTGCTGTTGTTAGTTATGACAGCAGTAGTGTCATTACCATTTCAATTCAGATTA
CATTCCTATATTTGATCATTGTAAACTGACTGCTTACATTGTATTAAAAACAGTGGATATTTTAAAGAAG
CTGTACGGCTTATATCTAGTGCTGTCTCTTAAGACTATTAAATTGATACAACATATTTAAAAGTAAATAT
TACCTAAATGAATTTTTGAAATTACAAATACACGTGTTAAAACTGTCGTTGTGTTCAACCATTTCTGTAC
ATACTTAGAGTTAACTGTTTTGCCAGGCTCTGTATGCCTACTCATAATATGATAAAAGCACTCATCTAAT
GCTCTGTAAATAGAAGTCAGTGCTTTCCATCAGACTGAACTCTCTTGACAAGATGTGGATGAAATTCTTT
AAGTAAAATTGTTTACTTTGTCATACATTTACAGATCAAATGTTAGCTCCCAAAGCAATCATATGGCAAA
GATAGGTATATCATAGTTTGCCTATTAGCTGCTTTGTATTGCTATTATTATAAATAGACTTCACAGTTTT
AGACTTGCTTAGGTGAAATTGCAATTCTTTTTACTTTCAGTCTTAGATAACAAGTCTTCAATTATAGTAC
AATCACACATTGCTTAGGAATGCATCATTAGGCGATTTTGTCATTATGCAAACATCATAGAGTGTACTTA
CACAAACCTAGATAGTATAGCCTTTATGTACCTAGGCCGTATGGTATAGTCTGTTGCTCCTAGGCCACAA
ACCTGTACAACTGTTACTGTACTGAATACTATAGACAGTTGTAACACAGTGGTAAATATTTATCTAAATA
TATGCAAACAGAGAAAAGGTACAGTAAAAGTATGGTATAAAAGATAATGGTATACCTGTGTAGGCCACTT
ACCACGAATGGAGCTTGCAGGACTAGAAGTTGCTCTGGGTGAGTCAGTGAGTGAGTGGTGAATTAATGTG
AAGGCCTAGAACACTGTACACCACTGTAGACTATAAACACAGTACGCTGAAGCTACACCAAATTTATCTT
AACAGTTTTTCTTCAATAAAAAATTATAACTTTTTAACTTTGTAAACTTTTTAATTTTTTAACTTTTAAA
ATACTTAGCTTGAAACACAAATACATTGTATAGCTATACAAAAATATTTTTTCTTTGTATCCTTATTCTA
GAAGCTTTTTTCTATTTTCTATTTTAAATTTTTTTTTTTACTTGTTAGTCGTTTTTGTTAAAAACTAAAA
CACACACACTTTCACCTAGGCATAGACAGGATTAGGATCATCAGTATCACTCCCTTCCACCTCACTGCCT
TCCACCTCCACATCTTGTCCCACTGGAAGGTTTTTAGGGGCAATAACACACATGTAGCTGTCACCTATGA
TAACAGTGCTTTCTGTTGAATACCTCCTGAAGGACTTGCCTGAGGCTGTTTTACATTTAACTTAAAAAAA
AAAAAAGTAGAAGGAGTGCACTCTAAAATAACAATAAAAGGCATAGTATAGTGAATACATAAACCAGCAA
TGTAGTAGTTTATTATCAAGTGTTGTACACTGTAATAATTGTATGTGCTATACTTTAAATAACTTGCAAA
ATAGTACTAAGACCTTATGATGGTTACAGTGTCACTAAGGCAATAGCATATTTTCAGGTCCATTGTAATC
TAATGGGACTACCATCATATATGCAGTCTACCATTGACTGAAACGTTACATGGCACATAACTGTATTTGC
AAGAATGATTTGTTTTACATTAATATCACATAGGATGTACCTTTTTAGAGTGGTATGTTTATGTGGATTA
AGATGTACAAGTTGAGCAAGGGGACCAAGAGCCCTGGGTTCTGTCTTGGATGTGAGCGTTTATGTTCTTC
TCCTCATGTCTGTTTTCTCATTAAATTCAAAGGCTTGAACGGGCCCTATTTAGCCCTTCTGTTTTCTACG
TGTTCTAAATAACTAAAGCTTTTAAATTCTAGCCATTTAGTGTAGAACTCTCTTTGCAGTGATGAAATGC
TGTATTGGTTTCTTGGCTAGCATATTAAATATTTTTATCTTTGTCTTGATACTTCAATGTCGTTTTAAAC
ATCAGGATCGGGCTTCAGTATTCTCATAACCAGAGAGTTCACTGAGGATACAGGACTGTTTGCCCATTTT
TTGTTATGGCTCCAGACTTGTGGTATTTCCATGTCTTTTTTTTTTTTTTTTTTTTTGACCTTTTAGCGGC
TTTAAAGTATTTCTGTTGTTAGGTGTTGTATTACTTTTCTAAGATTACTTAACAAAGCACCACAAACTGA
GTGGCTTTAAACAACAGCAATTTATTCTCTCACAATTCTAGAAGCTAGAAGTCCGAAATCAAAGTGTTGA
CAGGGGCATGATCTTCAAGAGAGAAGACTCTTTCCTTGCCTCTTCCTGGCTTCTGGTGGTTACCAGCAAT
CCTGAGTGTTCCTTTCTTGCCTTGTAGTTTCAACAATCCAGTATCTGCCTTTTGTCTTCACATGGCTGTC
TACCATTTGTCTCTGTGTCTCCAAATCTCTCTCCTTATAAACACAGCAGTTATTGGATTAGGCCCCACTC
TAATCCAGTATGACCCCATTTTAACATGATTACACTTATTTCTAGATAAGGTCACATTCACGTACACCAA
GGGTTAGGAATTGAACATATCTTTTTGGGGGACACAATTCAACCCACAAGTGTCAGTCTCTAGCTGAGCC
TTTCCCTTCCTGTTTTTCTCCTTTTTAGTTGCTATGGGTTAGGGGCCAAATCTCCAGTCATACTAGAATT
GCACATGGACTGGATATTTGGGAATACTGCGGGTCTATTCTATGAGCTTTAGTATGTAACATTTAATATC
AGTGTAAAGAAGCCCTTTTTTAAGTTATTTCTTTGAATTTCTAAATGTATGCCCTGAATATAAGTAACAA
GTTACCATGTCTTGTAAAATGATCATATCAACAAACATTTAATGTGCACCTACTGTGCTAGTTGAATGTC
TTTATCCTGATAGGAGATAACAGGATTCCACATCTTTGACTTAAGAGGACAAACCAAATATGTCTAAATC
ATTTGGGGTTTTGATGGATATCTTTAAATTGCTGAACCTAATCATTGGTTTCATATGTCATTGTTTAGAT
ATCTCCGGAGCATTTGGATAATGTGACAGTTGGAATGCAGTGATGTCGACTCTTTGCCCACCGCCATCTC
CAGCTGTTGCCAAGACAGAGATTGCTTTAAGTGGCAAATCACCTTTATTAGCAGCTACTTTTGCTTACTG
GGACAATATTCTTGGTCCTAGAGTAAGGCACATTTGGGCTCCAAAGACAGAACAGGTACTTCTCAGTGAT
GGAGAAATAACTTTTCTTGCCAACCACACTCTAAATGGAGAAATCCTTCGAAATGCAGAGAGTGGTGCTA
TAGATGTAAAGTTTTTTGTCTTGTCTGAAAAGGGAGTGATTATTGTTTCATTAATCTTTGATGGAAACTG
GAATGGGGATCGCAGCACATATGGACTATCAATTATACTTCCACAGACAGAACTTAGTTTCTACCTCCCA
CTTCATAGAGTGTGTGTTGATAGATTAACACATATAATCCGGAAAGGAAGAATATGGATGCATAAGGTAA
GTGATTTTTCAGCTTATTAATCATGTTAACCTATCTGTTGAAAGCTTATTTTCTGGTACATATAAATCTT
ATTTTTTTAATTATATGCAGTGAACATCAAACAATAAATGTTATTTATTTTGCATTTACCCTATTAGATA
CAAATACATCTGGTCTGATACCTGTCATCTTCATATTAACTGTGGAAGGTACGAAATGGTAGCTCCACAT
TATAGATGAAAAGCTAAAGCTTAGACAAATAAAGAAACTTTTAGACCCTGGATTCTTCTTGGGAGCCTTT
GACTCTAATACCTTTTGTTTCCCTTTCATTGCACAATTCTGTCTTTTGCTTACTACTATGTGTAAGTATA
ACAGTTCAAAGTAATAGTTTCATAAGCTGTTGGTCATGTAGCCTTTGGTCTCTTTAACCTCTTTGCCAAG
TTCCCAGGTTCATAAAATGAGGAGGTTGAATGGAATGGTTCCCAAGAGAATTCCTTTTAATCTTACAGAA
ATTATTGTTTTCCTAAATCCTGTAGTTGAATATATAATGCTATTTACATTTCAGTATAGTTTTGATGTAT
CTAAAGAACACATTGAATTCTCCTTCCTGTGTTCCAGTTTGATACTAACCTGAAAGTCCATTAAGCATTA
CCAGTTTTAAAAGGCTTTTGCCCAATAGTAAGGAAAAATAATATCTTTTAAAAGAATAATTTTTTACTAT
GTTTGCAGGCTTACTTCCTTTTTTCTCACATTATGAAACTCTTAAAATCAGGAGAATCTTTTAAACAACA
TCATAATGTTTAATTTGAAAAGTGCAAGTCATTCTTTTCCTTTTTGAAACTATGCAGATGTTACATTGAC
TGTTTTCTGTGAAGTTATCTTTTTTTCACTGCAGAATAAAGGTTGTTTTGATTTTATTTTGTATTGTTTA
TGAGAACATGCATTTGTTGGGTTAATTTCCTACCCCTGCCCCCATTTTTTCCCTAAAGTAGAAAGTATTT
TTCTTGTGAACTAAATTACTACACAAGAACATGTCTATTGAAAAATAAGCAAGTATCAAAATGTTGTGGG
TTGTTTTTTTAAATAAATTTTCTCTTGCTCAGGAAAGACAAGAAAATGTCCAGAAGATTATCTTAGAAGG
CACAGAGAGAATGGAAGATCAGGTATATGCAAATTGCATACTGTCAAATGTTTTTCTCACAGCATGTATC
TGTATAAGGTTGATGGCTACATTTGTCAAGGCCTTGGAGACATACGAATAAGCCTTTAATGGAGCTTTTA
TGGAGGTGTACAGAATAAACTGGAGGAAGATTTCCATATCTTAAACCCAAAGAGTTAAATCAGTAAACAA
AGGAAAATAGTAATTGCATCTACAAATTAATATTTGCTCCCTTTTTTTTTCTGTTTGCCCAGAATAAATT
TTGGATAACTTGTTCATAGTAAAAATAAAAAAAATTGTCTCTGATATGTTCTTTAAGGTACTACTTCTCG
AACCTTTCCCTAGAAGTAGCTGTAACAGAAGGAGAGCATATGTACCCCTGAGGTATCTGTCTGGGGTGTA
GGCCCAGGTCCACACAATATTTCTTCTAAGTCTTATGTTGTATCGTTAAGACTCATGCAATTTACATTTT
ATTCCATAACTATTTTAGTATTAAAATTTGTCAGTGATATTTCTTACCCTCTCCTCTAGGAAAATGTGCC
ATGTTTATCCCTTGGCTTTGAATGCCCCTCAGGAACAGACACTAAGAGTTTGAGAAGCATGGTTACAAGG
GTGTGGCTTCCCCTGCGGAAACTAAGTACAGACTATTTCACTGTAAAGCAGAGAAGTTCTTTTGAAGGAG
AATCTCCAGTGAAGAAAGAGTTCTTCACTTTTACTTCCATTTCCTCTTGTGGGTGACCCTCAATGCTCCT
TGTAAAACTCCAATATTTTAAACATGGCTGTTTTGCCTTTCTTTGCTTCTTTTTAGCATGAATGAGACAG
ATGATACTTTAAAAAAGTAATTAAAAAAAAAAACTTGTGAAAATACATGGCCATAATACAGAACCCAATA
CAATGATCTCCTTTACCAAATTGTTATGTTTGTACTTTTGTAGATAGCTTTCCAATTCAGAGACAGTTAT
TCTGTGTAAAGGTCTGACTTAACAAGAAAAGATTTCCCTTTACCCAAAGAATCCCAGTCCTTATTTGCTG
GTCAATAAGCAGGGTCCCCAGGAATGGGGTAACTTTCAGCACCCTCTAACCCACTAGTTATTAGTAGACT
AATTAAGTAAACTTATCGCAAGTTGAGGAAACTTAGAACCAACTAAAATTCTGCTTTTACTGGGATTTTG
TTTTTTCAAACCAGAAACCTTTACTTAAGTTGACTACTATTAATGAATTTTGGTCTCTCTTTTAAGTGCT
CTTCTTAAAAATGTTATCTTACTGCTGAGAAGTTCAAGTTTGGGAAGTACAAGGAGGAATAGAAACTTAA
GAGATTTTCTTTTAGAGCCTCTTCTGTATTTAGCCCTGTAGGATTTTTTTTTTTTTTTTTTTTTTTGGTG
TTGTTGAGCTTCAGTGAGGCTATTCATTCACTTATACTGATAATGTCTGAGATACTGTGAATGAAATACT
ATGTATGCTTAAACCTAAGAGGAAATATTTTCCCAAAATTATTCTTCCCGAAAAGGAGGAGTTGCCTTTT
GATTGAGTTCTTGCAAATCTCACAACGACTTTATTTTGAACAATACTGTTTGGGGATGATGCATTAGTTT
GAAACAACTTCAGTTGTAGCTGTCATCTGATAAAATTGCTTCACAGGGAAGGAAATTTAACACGGATCTA
GTCATTATTCTTGTTAGATTGAATGTGTGAATTGTAATTGTAAACAGGCATGATAATTATTACTTTAAAA
ACTAAAAACAGTGAATAGTTAGTTGTGGAGGTTACTAAAGGATGGTTTTTTTTTAAATAAAACTTTCAGC
ATTATGCAAATGGGCATATGGCTTAGGATAAAACTTCCAGAAGTAGCATCACATTTAAATTCTCAAGCAA
CTTAATAATATGGGGCTCTGAAAAACTGGTTAAGGTTACTCCAAAAATGGCCCTGGGTCTGACAAAGATT
CTAACTTAAAGATGCTTATGAAGACTTTGAGTAAAATCATTTCATAAAATAAGTGAGGAAAAACAACTAG
TATTAAATTCATCTTAAATAATGTATGATTTAAAAAATATGTTTAGCTAAAAATGCATAGTCATTTGACA
ATTTCATTTATATCTCAAAAAATTTACTTAACCAAGTTGGTCACAAAACTGATGAGACTGGTGGTGGTAG
TGAATAAATGAGGGACCATCCATATTTGAGACACTTTACATTTGTGATGTGTTATACTGAATTTTCAGTT
TGATTCTATAGACTACAAATTTCAAAATTACAATTTCAAGATGTAATAAGTAGTAATATCTTGAAATAGC
TCTAAAGGGAATTTTTCTGTTTTATTGATTCTTAAAATATATGTGCTGATTTTGATTTGCATTTGGGTAG
ATTATACTTTTATGAGTATGGAGGTTAGGTATTGATTCAAGTTTTCCTTACCTATTTGGTAAGGATTTCA
AAGTCTTTTTGTGCTTGGTTTTCCTCATTTTTAAATATGAAATATATTGATGACCTTTAACAAATTTTTT
TTATCTCAAATTTTAAAGGAGATCTTTTCTAAAAGAGGCATGATGACTTAATCATTGCATGTAACAGTAA
ACGATAAACCAATGATTCCATACTCTCTAAAGAATAAAAGTGAGCTTTAGGGCCGGGCATGGTCAGAAAT
TTGACACCAACCTGGCCAACATGGCGAAACCCCGTCTCTACTAAAAATACAAAAATCAGCCGGGCATGGT
GGCGGCACCTATAGTCCCAGCTACTTGGGAGGATGAGACAGGAGAGTCACTTGAACCTGGGAGGAGAGGT
TGCAGTGAGCTGAGATCACGCCATTGCACTCCAGCCTGAGCAATGAAAGCAAAACTCCATCTCAAAAAAA
AAAAAAGAAAAGAAAGAATAAAAGTGAGCTTTGGATTGCATATAAATCCTTTAGACATGTAGTAGACTTG
TTTGATACTGTGTTTGAACAAATTACGAAGTATTTTCATCAAAGAATGTTATTGTTTGATGTTATTTTTA
TTTTTTATTGCCCAGCTTCTCTCATATTACGTGATTTTCTTCACTTCATGTCACTTTATTGTGCAGGGTC
AGAGTATTATTCCAATGCTTACTGGAGAAGTGATTCCTGTAATGGAACTGCTTTCATCTATGAAATCACA
CAGTGTTCCTGAAGAAATAGATGTAAGTTTAAATGAGAGCAATTATACACTTTATGAGTTTTTTGGGGTT
ATAGTATTATTATGTATATTATTAATATTCTAATTTTAATAGTAAGGACTTTGTCATACATACTATTCAC
ATACAGTATTAGCCACTTTAGCAAATAAGCACACACAAAATCCTGGATTTTATGGCAAAACAGAGGCATT
TTTGATCAGTGATGACAAAATTAAATTCATTTTGTTTATTTCATTACTTTTATAATTCCTAAAAGTGGGA
GGATCCCAGCTCTTATAGGAGCAATTAATATTTAATGTAGTGTCTTTTGAAACAAAACTGTGTGCCAAAG
TAGTAACCATTAATGGAAGTTTACTTGTAGTCACAAATTTAGTTTCCTTAATCATTTGTTGAGGACGTTT
TGAATCACACACTATGAGTGTTAAGAGATACCTTTAGGAAACTATTCTTGTTGTTTTCTGATTTTGTCAT
TTAGGTTAGTCTCCTGATTCTGACAGCTCAGAAGAGGAAGTTGTTCTTGTAAAAATTGTTTAACCTGCTT
GACCAGCTTTCACATTTGTTCTTCTGAAGTTTATGGTAGTGCACAGAGATTGTTTTTTGGGGAGTCTTGA
TTCTCGGAAATGAAGGCAGTGTGTTATATTGAATCCAGACTTCCGAAAACTTGTATATTAAAAGTGTTAT
TTCAACACTATGTTACAGCCAGACTAATTTTTTTATTTTTTGATGCATTTTAGATAGCTGATACAGTACT
CAATGATGATGATATTGGTGACAGCTGTCATGAAGGCTTTCTTCTCAAGTAAGAATTTTTCTTTTCATAA
AAGCTGGATGAAGCAGATACCATCTTATGCTCACCTATGACAAGATTTGGAAGAAAGAAAATAACAGACT
GTCTACTTAGATTGTTCTAGGGACATTACGTATTTGAACTGTTGCTTAAATTTGTGTTATTTTTCACTCA
TTATATTTCTATATATATTTGGTGTTATTCCATTTGCTATTTAAAGAAACCGAGTTTCCATCCCAGACAA
GAAATCATGGCCCCTTGCTTGATTCTGGTTTCTTGTTTTACTTCTCATTAAAGCTAACAGAATCCTTTCA
TATTAAGTTGTACTGTAGATGAACTTAAGTTATTTAGGCGTAGAACAAAATTATTCATATTTATACTGAT
CTTTTTCCATCCAGCAGTGGAGTTTAGTACTTAAGAGTTTGTGCCCTTAAACCAGACTCCCTGGATTAAT
GCTGTGTACCCGTGGGCAAGGTGCCTGAATTCTCTATACACCTATTTCCTCATCTGTAAAATGGCAATAA
TAGTAATAGTACCTAATGTGTAGGGTTGTTATAAGCATTGAGTAAGATAAATAATATAAAGCACTTAGAA
CAGTGCCTGGAACATAAAAACACTTAATAATAGCTCATAGCTAACATTTCCTATTTACATTTCTTCTAGA
AATAGCCAGTATTTGTTGAGTGCCTACATGTTAGTTCCTTTACTAGTTGCTTTACATGTATTATCTTATA
TTCTGTTTTAAAGTTTCTTCACAGTTACAGATTTTCATGAAATTTTACTTTTAATAAAAGAGAAGTAAAA
GTATAAAGTATTCACTTTTATGTTCACAGTCTTTTCCTTTAGGCTCATGATGGAGTATCAGAGGCATGAG
TGTGTTTAACCTAAGAGCCTTAATGGCTTGAATCAGAAGCACTTTAGTCCTGTATCTGTTCAGTGTCAGC
CTTTCATACATCATTTTAAATCCCATTTGACTTTAAGTAAGTCACTTAATCTCTCTACATGTCAATTTCT
TCAGCTATAAAATGATGGTATTTCAATAAATAAATACATTAATTAAATGATATTATACTGACTAATTGGG
CTGTTTTAAGGCTCAATAAGAAAATTTCTGTGAAAGGTCTCTAGAAAATGTAGGTTCCTATACAAATAAA
AGATAACATTGTGCTTATAGCTTCGGTGTTTATCATATAAAGCTATTCTGAGTTATTTGAAGAGCTCACC
TACTTTTTTTTGTTTTTAGTTTGTTAAATTGTTTTATAGGCAATGTTTTTAATCTGTTTTCTTTAACTTA
CAGTGCCATCAGCTCACACTTGCAAACCTGTGGCTGTTCCGTTGTAGTAGGTAGCAGTGCAGAGAAAGTA
AATAAGGTAGTTTATTTTATAATCTAGCAAATGATTTGACTCTTTAAGACTGATGATATATCATGGATTG
TCATTTAAATGGTAGGTTGCAATTAAAATGATCTAGTAGTATAAGGAGGCAATGTAATCTCATCAAATTG
CTAAGACACCTTGTGGCAACAGTGAGTTTGAAATAAACTGAGTAAGAATCATTTATCAGTTTATTTTGAT
AGCTCGGAAATACCAGTGTCAGTAGTGTATAAATGGTTTTGAGAATATATTAAAATCAGATATATAAAAA
AAATTACTCTTCTATTTCCCAATGTTATCTTTAACAAATCTGAAGATAGTCATGTACTTTTGGTAGTAGT
TCCAAAGAAATGTTATTTGTTTATTCATCTTGATTTCATTGTCTTCGCTTTCCTTCTAAATCTGTCCCTT
CTAGGGAGCTATTGGGATTAAGTGGTCATTGATTATTATACTTTATTCAGTAATGTTTCTGACCCTTTCC
TTCAGTGCTACTTGAGTTAATTAAGGATTAATGAACAGTTACATTTCCAAGCATTAGCTAATAAACTAAA
GGATTTTGCACTTTTCTTCACTGACCATTAGTTAGAAAGAGTTCAGAGATAAGTATGTGTATCTTTCAAT
TTCAGCAAACCTAATTTTTTAAAAAAAGTTTTACATAGGAAATATGTTGGAAATGATACTTTACAAAGAT
ATTCATAATTTTTTTTTGTAATCAGCTACTTTGTATATTTACATGAGCCTTAATTTATATTTCTCATATA
ACCATTTATGAGAGCTTAGTATACCTGTGTCATTATATTGCATCTACGAACTAGTGACCTTATTCCTTCT
GTTACCTCAAACAGGTGGCTTTCCATCTGTGATCTCCAAAGCCTTAGGTTGCACAGAGTGACTGCCGAGC
TGCTTTATGAAGGGAGAAAGGCTCCATAGTTGGAGTGTTTTTTTTTTTTTTTTTAAACATTTTTCCCATC
CTCCATCCTCTTGAGGGAGAATAGCTTACCTTTTATCTTGTTTTAATTTGAGAAAGAAGTTGCCACCACT
CTAGGTTGAAAACCACTCCTTTAACATAATAACTGTGGATATGGTTTGAATTTCAAGATAGTTACATGCC
TTTTTATTTTTCCTAATAGAGCTGTAGGTCAAATATTATTAGAATCAGATTTCTAAATCCCACCCAATGA
CCTGCTTATTTTAAATCAAATTCAATAATTAATTCTCTTCTTTTTGGAGGATCTGGACATTCTTTGATAT
TTCTTACAACGAATTTCATGTGTAGACCCACTAAACAGAAGCTATAAAAGTTGCATGGTCAAATAAGTCT
GAGAAAGTCTGCAGATGATATAATTCACCTGAAGAGTCACAGTATGTAGCCAAATGTTAAAGGTTTTGAG
ATGCCATACAGTAAATTTACCAAGCATTTTCTAAATTTATTTGACCACAGAATCCCTATTTTAAGCAACA
ACTGTTACATCCCATGGATTCCAGGTGACTAAAGAATACTTATTTCTTAGGATATGTTTTATTGATAATA
ACAATTAAAATTTCAGATATCTTTCATAAGCAAATCAGTGGTCTTTTTACTTCATGTTTTAATGCTAAAA
TATTTTCTTTTATAGATAGTCAGAACATTATGCCTTTTTCTGACTCCAGCAGAGAGAAAATGCTCCAGGT
TATGTGAAGCAGAATCATCATTTAAATATGAGTCAGGGCTCTTTGTACAAGGCCTGCTAAAGGTATAGTT
TCTAGTTATCACAAGTGAAACCACTTTTCTAAAATCATTTTTGAGACTCTTTATAGACAAATCTTAAATA
TTAGCATTTAATGTATCTCATATTGACATGCCCAGAGACTGACTTCCTTTACACAGTTCTGCACATAGAC
TATATGTCTTATGGATTTATAGTTAGTATCATCAGTGAAACACCATAGAATACCCTTTGTGTTCCAGGTG
GGTCCCTGTTCCTACATGTCTAGCCTCAGGACTTTTTTTTTTTTAACACATGCTTAAATCAGGTTGCACA
TCAAAAATAAGATCATTTCTTTTTAACTAAATAGATTTGAATTTTATTGAAAAAAAATTTTAAACATCTT
TAAGAAGCTTATAGGATTTAAGCAATTCCTATGTATGTGTACTAAAATATATATATTTCTATATATAATA
TATATTAGAAAAAAATTGTATTTTTCTTTTATTTGAGTCTACTGTCAAGGAGCAAAACAGAGAAATGTAA
ATTAGCAATTATTTATAATACTTAAAGGGAAGAAAGTTGTTCACCTTGTTGAATCTATTATTGTTATTTC
AATTATAGTCCCAAGACGTGAAGAAATAGCTTTCCTAATGGTTATGTGATTGTCTCATAGTGACTACTTT
CTTGAGGATGTAGCCACGGCAAAATGAAATAAAAAAATTTAAAAATTGTTGCAAATACAAGTTATATTAG
GCTTTTGTGCATTTTCAATAATGTGCTGCTATGAACTCAGAATGATAGTATTTAAATATAGAAACTAGTT
AAAGGAAACGTAGTTTCTATTTGAGTTATACATATCTGTAAATTAGAACTTCTCCTGTTAAAGGCATAAT
AAAGTGCTTAATACTTTTGTTTCCTCAGCACCCTCTCATTTAATTATATAATTTTAGTTCTGAAAGGGAC
CTATACCAGATGCCTAGAGGAAATTTCAAAACTATGATCTAATGAAAAAATATTTAATAGTTCTCCATGC
AAATACAAATCATATAGTTTTCCAGAAAATACCTTTGACATTATACAAAGATGATTATCACAGCATTATA
ATAGTAAAAAAATGGAAATAGCCTCTTTCTTCTGTTCTGTTCATAGCACAGTGCCTCATACGCAGTAGGT
TATTATTACATGGTAACTGGCTACCCCAACTGATTAGGAAAGAAGTAAATTTGTTTTATAAAAATACATA
CTCATTGAGGTGCATAGAATAATTAAGAAATTAAAAGACACTTGTAATTTTGAATCCAGTGAATACCCAC
TGTTAATATTTGGTATATCTCTTTCTAGTCTTTTTTTCCCTTTTGCATGTATTTTCTTTAAGACTCCCAC
CCCCACTGGATCATCTCTGCATGTTCTAATCTGCTTTTTTCACAGCAGATTCTAAGCCTCTTTGAATATC
AACACAAACTTCAACAACTTCATCTATAGATGCCAAATAATAAATTCATTTTTATTTACTTAACCACTTC
CTTTGGATGCTTAGGTCATTCTGATGTTTTGCTATTGAAACCAATGCTATACTGAACACTTCTGTCACTA
AAACTTTGCACACACTCATGAATAGCTTCTTAGGATAAATTTTTAGAGATGGATTTGCTAAATCAGAGAC
CATTTTTTAAAATTAAAAAACAATTATTCATATCGTTTGGCATGTAAGACAGTAAATTTTCCTTTTATTT
TGACAGGATTCAACTGGAAGCTTTGTGCTGCCTTTCCGGCAAGTCATGTATGCTCCATATCCCACCACAC
ACATAGATGTGGATGTCAATACTGTGAAGCAGATGCCACCCTGTCATGAACATATTTATAATCAGCGTAG
ATACATGAGATCCGAGCTGACAGCCTTCTGGAGAGCCACTTCAGAAGAAGACATGGCTCAGGATACGATC
ATCTACACTGACGAAAGCTTTACTCCTGATTTGTACGTAATGCTCTGCCTGCTGGTACTGTAGTCAAGCA
ATATGAAATTGTGTCTTTTACGAATAAAAACAAAACAGAAGTTGCATTTAAAAAGAAAGAAATATTACCA
GCAGAATTATGCTTGAAGAAACATTTAATCAAGCATTTTTTTCTTAAATGTTCTTCTTTTTCCATACAAT
TGTGTTTACCCTAAAATAGGTAAGATTAACCCTTAAAGTAAATATTTAACTATTTGTTTAATAAATATAT
ATTGAGCTCCTAGGCACTGTTCTAGGTACCGGGCTTAATAGTGGCCAACCAGACAGCCCCAGCCCCAGCC
CCTACATTGTGTATAGTCTATTATGTAACAGTTATTGAATGGACTTATTAACAAAACCAAAGAAGTAATT
CTAAGTCTTTTTTTTCTTGACATATGAATATAAAATACAGCAAAACTGTTAAAATATATTAATGGAACAT
TTTTTTACTTTGCATTTTATATTGTTATTCACTTCTTATTTTTTTTTAAAAAAAAAAGCCTGAACAGTAA
ATTCAAAAGGAAAAGTAATGATAATTAATTGTTGAGCATGGACCCAACTTGAAAAAAAAAATGATGATGA
TAAATCTATAATCCTAAAACCCTAAGTAAACACTTAAAAGATGTTCTGAAATCAGGAAAAGAATTATAGT
ATACTTTTGTGTTTCTCTTTTATCAGTTGAAAAAAGGCACAGTAGCTCATGCCTGTAAGAACAGAGCTTT
GGGAGTGCAAGGCAGGCGGATCACTTGAGGCCAGGAGTTCCAGACCAGCCTGGGCAACATAGTGAAACCC
CATCTCTACAAAAAATAAAAAAGAATTATTGGAATGTGTTTCTGTGTGCCTGTAATCCTAGCTATTCCGA
AAGCTGAGGCAGGAGGATCTTTTGAGCCCAGGAGTTTGAGGTTACAGGGAGTTATGATGTGCCAGTGTAC
TCCAGCCTGGGGAACACCGAGACTCTGTCTTATTTAAAAAAAAAAAAAAAAAAATGCTTGCAATAATGCC
TGGCACATAGAAGGTAACAGTAAGTGTTAACTGTAATAACCCAGGTCTAAGTGTGTAAGGCAATAGAAAA
ATTGGGGCAAATAAGCCTGACCTATGTATCTACAGAATCAGTTTGAGCTTAGGTAACAGACCTGTGGAGC
ACCAGTAATTACACAGTAAGTGTTAACCAAAAGCATAGAATAGGAATATCTTGTTCAAGGGACCCCCAGC
CTTATACATCTCAAGGTGCAGAAAGATGACTTAATATAGGACCCATTTTTTCCTAGTTCTCCAGAGTTTT
TATTGGTTCTTGAGAAAGTAGTAGGGGAATGTTTTAGAAAATGAATTGGTCCAACTGAAATTACATGTCA
GTAAGTTTTTATATATTGGTAAATTTTAGTAGACATGTAGAAGTTTTCTAATTAATCTGTGCCTTGAAAC
ATTTTCTTTTTTCCTAAAGTGCTTAGTATTTTTTCCGTTTTTTGATTGGTTACTTGGGAGCTTTTTTGAG
GAAATTTAGTGAACTGCAGAATGGGTTTGCAACCATTTGGTATTTTTGTTTTGTTTTTTAGAGGATGTAT
GTGTATTTTAACATTTCTTAATCATTTTTAGCCAGCTATGTTTGTTTTGCTGATTTGACAAACTACAGTT
AGACAGCTATTCTCATTTTGCTGATCATGACAAAATAATATCCTGAATTTTTAAATTTTGCATCCAGCTC
TAAATTTTCTAAACATAAAATTGTCCAAAAAATAGTATTTTCAGCCACTAGATTGTGTGTTAAGTCTATT
GTCACAGAGTCATTTTACTTTTAAGTATATGTTTTTACATGTTAATTATGTTTGTTATTTTTAATTTTAA
CTTTTTAAAATAATTCCAGTCACTGCCAATACATGAAAAATTGGTCACTGGAATTTTTTTTTTGACTTTT
ATTTTAGGTTCATGTGTACATGTGCAGGTGTGTTATACAGGTAAATTGCGTGTCATGAGGGTTTGGTGTA
CAGGTGATTTCATTACCCAGGTAATAAGCATAGTACCCAATAGGTAGTTTTTTGATCCTCACCCTTCTCC
CACCCTCAAGTAGGCCCTGGTGTTGCTGTTTCCTTCTTTGTGTCCATGTATACTCAGTGTTTAGCTCCCA
CTTAGAAGTGAGAACATGCGGTAGTTGGTTTTCTGTTCCTGGATTAGTTCACTTAGGATAATGACCTCTA
GCTCCATCTGGTTTTTATGGCTGCATAGTATTCCATGGTGTATATGTATCACATTTTCTTTATCCAGTCT
ACCATTGATAGGCATTTAGGTTGATTCCCTGTCTTTGTTATCATGAATAGTGCTGTGATGAACATACACA
TGCATGTGTCTTTATGGTAGAAAAATTTGTATTCCTTTAGGTACATATAGAATAATGGGGTTGCTAGGGT
GAATGGTAGTTCTATTTTCAGTTATTTGAGAAATCTTCAAACTGCTTTTCATAATAGCTAAACTAATTTA
CAGTCCCGCCAGCAGTGTATAAGTGTTCCCTTTTCTCCACAACCTTGCCAACATCTGTGATTTTTTGACT
TTTTAATAATAGCCATTCCTAGAGAATTGATTTGCAATTCTCTATTAGTGATATTAAGCATTTTTTCATA
TGCTTTTTAGCTGTCTGTATATATTCTTCTGAAAAATTTTCATGTCCTTTGCCCAGTTTGTAGTGGGGTG
GGTTGTTTTTTGCTTGTTAATTAGTTTTAAGTTCCTTCCAGATTCTGCATATCCCTTTGTTGGATACATG
GTTTGCAGATATTTTTCTCCCATTGTGTAGGTTGTCTTTTACTCTGTTGATAGTTTCTTTTGCCATGCAG
GAGCTCGTTAGGTCCCATTTGTGTTTGTTTTTGTTGCAGTTGCTTTTGGCGTCTTCATCATAAAATCTGT
GCCAGGGCCTATGTCCAGAATGGTATTTCCTAGGTTGTCTTCCAGGGTTTTTACAATTTTAGATTTTACG
TTTATGTCTTTAATCCATCTTGAGTTGATTTTTGTATATGGCACAAGGAAGGGGTCCAGTTTCACTCCAA
TTCCTATGGCTAGCAATTATCCCAGCACCATTTATTGAATACGGAGTCCTTTCCCCATTGCTTGTTTTTT
GTCAACTTTGTTGAAGATCAGATGGTTGTAAGTGTGTGGCTTTATTTCTTGGCTCTCTATTCTCCATTGG
TCTATGTGTCTGTTTTTATAACAGTACCCTGCTGTTCAGGTTCCTATAGCCTTTTAGTATAAAATCGGCT
AATGTGATGCCTCCAGCTTTGTTCTTTTTGCTTAGGATTGCTTTGGCTATTTGGGCTCCTTTTTGGGTCC
ATATTAATTTTAAAACAGTTTTTTCTGGTTTTGTGAAGGATATCATTGGTAGTTTATAGGAATAGCATTG
AATCTGTAGATTGCTTTGGGCAGTATGGCCATTTTAACAATATTAATTCTTCCTATCTATGAATATGGAA
TGTTTTTCCATGTGTTTGTGTCATCTCTTTATACCTGATGTATAAAGAAAAGCTGGTATTATTCCTACTC
AATCTGTTCCAAAAAATTGAGGAGGAGGAACTCTTCCCTAATGAGGCCAGCATCATTCTGATACCAAAAC
CTGGCAGAGACACAACAGAAAAAAGAAAACTTCAGGCCAATATCCTTGATGAATATAGATGCAAAAATCC
TCAACAAAATACTAGCAAACCAAATCCAGCAGCACATCAAAAAGCTGATCTACTTTGATCAAGTAGGCTT
TATCCCTGGGATGCAAGGTTGGTTCAACATACACAAATCAATAAGTGTGATTCATCACATAAACAGAGCT
AAAAACAAAAACCACAAGATTATCTCAATAGGTAGAGAAAAGGTTGTCAATAAAATTTAACATCCTCCAT
GTTAAAAACCTTCAGTAGGTCAGGTGTAGTGACTCACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGC
GGGCATATCTCTTAAGCCCAGGAGTTCAAGACGAGCCTAGGCAGCATGGTGAAACCCCATCTCTACAAAA
AAAAAAAAAAAAAAAAATTAGCTTGGTATGGTGACATGCACCTATAGTCCCAGCTATTCAGGAGGTTGAG
GTGGGAGGATTGTTTGAGCCCGGGAGGCAGAGGTTGGCAGCGAGCTGAGATCATGCCACCGCACTCCAGC
CTGGGCAACGGAGTGAGACCCTGTCTCAAAAAAGAAAAATCACAAACAATCCTAAACAAACTAGGCATTG
AAGGAACATGCCTCAAAAAAATAAGAACCATCTATGACAGACCCATAGCCAATATCTTACCAAATGGGCA
AAAGCTGGAAGTATTCTCCTTGAGAACCGTAACAAGACAAGGATGTCCACTCTCACCACTCCTTTTCAGC
ATAGTTCTGGAAGTCCTAGCCAGAGCAATCAGGAAAGAGAAAGAAAGAAAGACATTCAGATAGGAAGAGA
AGAAGTCAAACTATTTCTGTTTGCAGGCAGTATAATTCTGTACCTAGAAAATCTCATAGTCTCTGCCCAG
AAACTCCTAAATCTGTTAAAAATTTCAGCAAAGTTTTGGCATTCTCTATACTCCAACACCTTCCAAAGTG
AGAGCAAAATCAAGAACACAGTCCCATTCACAATAGCCGCAAAACGAATAAAATACCTAGGAATCCAGCT
AACCAGGGAGGTGAAAGATCTCTATGAGAATTACAAAACACTGCTGAAAGAAATCAGAGATGACACAAAC
AAATGGAAATGTTCTTTTTTAACACCTTGCTTTATCTAATTCACTTATGATGAAGATACTCATTCAGTGG
AACAGGTATAATAAGTCCACTCGATTAAATATAAGCCTTATTCTCTTTCCAGAGCCCAAGAAGGGGCACT
ATCAGTGCCCAGTCAATAATGACGAAATGCTAATATTTTTCCCCTTTACGGTTTCTTTCTTCTGTAGTGT
GGTACACTCGTTTCTTAAGATAAGGAAACTTGAACTACCTTCCTGTTTGCTTCTACACATACCCATTCTC
TTTTTTTGCCACTCTGGTCAGGTATAGGATGATCCCTACCACTTTCAGTTAAAAACTCCTCCTCTTACTA
AATGTTCTCTTACCCTCTGGCCTGAGTAGAACCTAGGGAAAATGGAAGAGAAAAAGATGAAAGGGAGGTG
GGGCCTGGGAAGGGAATAAGTAGTCCTGTTTGTTTGTGTGTTTGCTTTAGCACCTGCTATATCCTAGGTG
CTGTGTTAGGCACACATTATTTTAAGTGGCCATTATATTACTACTACTCACTCTGGTCGTTGCCAAGGTA
GGTAGTACTTTCTTGGATAGTTGGTTCATGTTACTTACAGATGGTGGGCTTGTTGAGGCAAACCCAGTGG
ATAATCATCGGAGTGTGTTCTCTAATCTCACTCAAATTTTTCTTCACATTTTTTGGTTTGTTTTGGTTTT
TGATGGTAGTGGCTTATTTTTGTTGCTGGTTTGTTTTTTGTTTTTTTTTGAGATGGCAAGAATTGGTAGT
TTTATTTATTAATTGCCTAAGGGTCTCTACTTTTTTTAAAAGATGAGAGTAGTAAAATAGATTGATAGAT
ACATACATACCCTTACTGGGGACTGCTTATATTCTTTAGAGAAAAAATTACATATTAGCCTGACAAACAC
CAGTAAAATGTAAATATATCCTTGAGTAAATAAATGAATGTATATTTTGTGTCTCCAAATATATATATCT
ATATTCTTACAAATGTGTTTATATGTAATATCAATTTATAAGAACTTAAAATGTTGGCTCAAGTGAGGGA
TTGTGGAAGGTAGCATTATATGGCCATTTCAACATTTGAACTTTTTTCTTTTCTTCATTTTCTTCTTTTC
TTCAGGAATATTTTTCAAGATGTCTTACACAGAGACACTCTAGTGAAAGCCTTCCTGGATCAGGTAAATG
TTGAACTTGAGATTGTCAGAGTGAATGATATGACATGTTTTCTTTTTTAATATATCCTACAATGCCTGTT
CTATATATTTATATTCCCCTGGATCATGCCCCAGAGTTCTGCTCAGCAATTGCAGTTAAGTTAGTTACAC
TACAGTTCTCAGAAGAGTCTGTGAGGGCATGTCAAGTGCATCATTACATTGGTTGCCTCTTGTCCTAGAT
TTATGCTTCGGGAATTCAGACCTTTGTTTACAATATAATAAATATTATTGCTATCTTTTAAAGATATAAT
AATAAGATATAAAGTTGACCACAACTACTGTTTTTTGAAACATAGAATTCCTGGTTTACATGTATCAAAG
TGAAATCTGACTTAGCTTTTACAGATATAATATATACATATATATATCCTGCAATGCTTGTACTATATAT
GTAGTACAAGTATATATATATGTTTGTGTGTGTATATATATATAGTACGAGCATATATACATATTACCAG
CATTGTAGGATATATATATGTTTATATATTAAAAAAAAGTTATAAACTTAAAACCCTATTATGTTATGTA
GAGTATATGTTATATATGATATGTAAAATATATAACATATACTCTATGATAGAGTGTAATATATTTTTTA
TATATATTTTAACATTTATAAAATGATAGAATTAAGAATTGAGTCCTAATCTGTTTTATTAGGTGCTTTT
TGTAGTGTCTGGTCTTTCTAAAGTGTCTAAATGATTTTTCCTTTTGACTTATTAATGGGGAAGAGCCTGT
ATATTAACAATTAAGAGTGCAGCATTCCATACGTCAAACAACAAACATTTTAATTCAAGCATTAACCTAT
AACAAGTAAGTTTTTTTTTTTTTTTTGAGAAAGGGAGGTTGTTTATTTGCCTGAAATGACTCAAAAATAT
TTTTGAAACATAGTGTACTTATTTAAATAACATCTTTATTGTTTCATTCTTTTAAAAAATATCTACTTAA
TTACACAGTTGAAGGAAATCGTAGATTATATGGAACTTATTTCTTAATATATTACAGTTTGTTATAATAA
CATTCTGGGGATCAGGCCAGGAAACTGTGTCATAGATAAAGCTTTGAAATAATGAGATCCTTATGTTTAC
TAGAAATTTTGGATTGAGATCTATGAGGTCTGTGACATATTGCGAAGTTCAAGGAAAATTCGTAGGCCTG
GAATTTCATGCTTCTCAAGCTGACATAAAATCCCTCCCACTCTCCACCTCATCATATGCACACATTCTAC
TCCTACCCACCCACTCCACCCCCTGCAAAAGTACAGGTATATGAATGTCTCAAAACCATAGGCTCATCTT
CTAGGAGCTTCAATGTTATTTGAAGATTTGGGCAGAAAAAATTAAGTAATACGAAATAACTTATGTATGA
GTTTTAAAAGTGAAGTAAACATGGATGTATTCTGAAGTAGAATGCAAAATTTGAATGCATTTTTAAAGAT
AAATTAGAAAACTTCTAAAAACTGTCAGATTGTCTGGGCCTGGTGGCTTATGCCTGTAATCCCAGCACTT
TGGGAGTCCGAGGTGGGTGGATCACAAGGTCAGGAGATCGAGACCATCCTGCCAACATGGTGAAACCCCG
TCTCTACTAAGTATACAAAAATTAGCTGGGCGTGGCAGCGTGTGCCTGTAATCCCAGCTACCTGGGAGGC
TGAGGCAGGAGAATCGCTTGAACCCAGGAGGTGTAGGTTGCAGTGAGTCAAGATCGCGCCACTGCACTTT
AGCCTGGTGACAGAGCTAGACTCCGTCTCAAAAAAAAAAAAAAATATCAGATTGTTCCTACACCTAGTGC
TTCTATACCACACTCCTGTTAGGGGGCATCAGTGGAAATGGTTAAGGAGATGTTTAGTGTGTATTGTCTG
CCAAGCACTGTCAACACTGTCATAGAAACTTCTGTACGAGTAGAATGTGAGCAAATTATGTGTTGAAATG
GTTCCTCTCCCTGCAGGTCTTTCAGCTGAAACCTGGCTTATCTCTCAGAAGTACTTTCCTTGCACAGTTT
CTACTTGTCCTTCACAGAAAAGCCTTGACACTAATAAAATATATAGAAGACGATACGTGAGTAAAACTCC
TACACGGAAGAAAAACCTTTGTACATTGTTTTTTTGTTTTGTTTCCTTTGTACATTTTCTATATCATAAT
TTTTGCGCTTCTTTTTTTTTTTTTTTTTTTTTTTTTTCCATTATTTTTAGGCAGAAGGGAAAAAAGCCCT
TTAAATCTCTTCGGAACCTGAAGATAGACCTTGATTTAACAGCAGAGGGCGATCTTAACATAATAATGGC
TCTGGCTGAGAAAATTAAACCAGGCCTACACTCTTTTATCTTTGGAAGACCTTTCTACACTAGTGTGCAA
GAACGAGATGTTCTAATGACTTTTTAAATGTGTAACTTAATAAGCCTATTCCATCACAATCATGATCGCT
GGTAAAGTAGCTCAGTGGTGTGGGGAAACGTTCCCCTGGATCATACTCCAGAATTCTGCTCTCAGCAATT
GCAGTTAAGTAAGTTACACTACAGTTCTCACAAGAGCCTGTGAGGGGATGTCAGGTGCATCATTACATTG
GGTGTCTCTTTTCCTAGATTTATGCTTTTGGGATACAGACCTATGTTTACAATATAATAAATATTATTGC
TATCTTTTAAAGATATAATAATAGGATGTAAACTTGACCACAACTACTGTTTTTTTGAAATACATGATTC
ATGGTTTACATGTGTCAAGGTGAAATCTGAGTTGGCTTTTACAGATAGTTGACTTTCTATCTTTTGGCAT
TCTTTGGTGTGTAGAATTACTGTAATACTTCTGCAATCAACTGAAAACTAGAGCCTTTAAATGATTTCAA
TTCCACAGAAAGAAAGTGAGCTTGAACATAGGATGAGCTTTAGAAAGAAAATTGATCAAGCAGATGTTTA
ATTGGAATTGATTATTAGATCCTACTTTGTGGATTTAGTCCCTGGGATTCAGTCTGTAGAAATGTCTAAT
AGTTCTCTATAGTCCTTGTTCCTGGTGAACCACAGTTAGGGTGTTTTGTTTATTTTATTGTTCTTGCTAT
TGTTGATATTCTATGTAGTTGAGCTCTGTAAAAGGAAATTGTATTTTATGTTTTAGTAATTGTTGCCAAC
TTTTTAAATTAATTTTCATTATTTTTGAGCCAAATTGAAATGTGCACCTCCTGTGCCTTTTTTCTCCTTA
GAAAATCTAATTACTTGGAACAAGTTCAGATTTCACTGGTCAGTCATTTTCATCTTGTTTTCTTCTTGCT
AAGTCTTACCATGTACCTGCTTTGGCAATCATTGCAACTCTGAGATTATAAAATGCCTTAGAGAATATAC
TAACTAATAAGATCTTTTTTTCAGAAACAGAAAATAGTTCCTTGAGTACTTCCTTCTTGCATTTCTGCCT
ATGTTTTTGAAGTTGTTGCTGTTTGCCTGCAATAGGCTATAAGGAATAGCAGGAGAAATTTTACTGAAGT
GCTGTTTTCCTAGGTGCTACTTTGGCAGAGCTAAGTTATCTTTTGTTTTCTTAATGCGTTTGGACCATTT
TGCTGGCTATAAAATAACTGATTAATATAATTCTAACACAATGTTGACATTGTAGTTACACAAACACAAA
TAAATATTTTATTTAAAATTCTGGAAGTAATATAAAAGGGAAAATATATTTATAAGAAAGGGATAAAGGT
AATAGAGCCCTTCTGCCCCCCACCCACCAAATTTACACAACAAAATGACATGTTCGAATGTGAAAGGTCA
TAATAGCTTTCCCATCATGAATCAGAAAGATGTGGACAGCTTGATGTTTTAGACAACCACTGAACTAGAT
GACTGTTGTACTGTAGCTCAGTCATTTAAAAAATATATAAATACTACCTTGTAGTGTCCCATACTGTGTT
TTTTACATGGTAGATTCTTATTTAAGTGCTAACTGGTTATTTTCTTTGGCTGGTTTATTGTACTGTTATA
CAGAATGTAAGTTGTACAGTGAAATAAGTTATTAAAGCATGTGTAAACATTGTTATATATCTTTTCTCCT
AAATGGAGAATTTTGAATAAAATATATTTGAAATTTTGCCTCTTTCAGTTGTTCATTCAGAAAAAAATAC
TATGATATTTGAAGACTGATCAGCTTCTGTTCAGCTGACAGTCATGCTGGATCTAAACTTTTTTTAAAAT
TAATTTTGTCTTTTCAAAGAAAAAATATTTAAAGAAGCTTTATAATATAATCTTATGTTAAAAAAACTTT
CTGCTTAACTCTCTGGATTTCATTTTGATTTTTCAAATTATATATTAATATTTCAAATGTAAAATACTAT
TTAGATAAATTGTTTTTAAACATTCTTATTATTATAATATTAATATAACCTAAACTGAAGTTATTCATCC
CAGGTATCTAATACATGTATCCAAAGTAAAAATCCAAGGAATCTGAACACTTTCATCTGCAAAGCTAGGA
ATAGGTTTGACATTTTCACTCCAAGAAAAAGTTTTTTTTTGAAAATAGAATAGTTGGGATGAGAGGTTTC
TTTAAAAGAAGACTAACTGATCACATTACTATGATTCTCAAAGAAGAAACCAAAACTTCATATAATACTA
TAAAGTAAATATAAAATAGTTCCTTCTATAGTATATTTCTATAATGCTACAGTTTAAACAGATCACTCTT
ATATAATACTATTTTGATTTTGATGTAGAATTGCACAAATTGATATTTCTCCTATGATCTGCAGGGTATA
GCTTAAAGTAACAAAAACAGTCAACCACCTCCATTTAACACACAGTAACACTATGGGACTAGTTTTATTA
CTTCCATTTTACAAATGAGGAAACTAAAGCTTAAAGATGTGTAATACACCGCCCAAGGTCACACAGCTGG
TAAAGGTGGATTTCATCCCAGACAGTTACAGTCATTGCCATGGGCACAGCTCCTAACTTAGTAACTCCAT
GTAACTGGTACTCAGTGTAGCTGAATTGAAAGGAGAGTAAGGAAGCAGGTTTTACAGGTCTACTTGCACT
ATTCAGAGCCCGAGTGTGAATCCCTGCTGTGCTGCTTGGAGAAGTTACTTAACCTATGCAAGGTTCATTT
TGTAAATATTGGAAATGGAGTGATAATACGTACTTCACCAGAGGATTTAATGAGACCTTATACGATCCTT
AGTTCAGTACCTGACTAGTGCTTCATAAATGCTTTTTCATCCAATCTGACAATCTCCAGCTTGTAATTGG
GGCATTTAGAACATTTAATATGATTATTGGCATGGTAGGTTAAAGCTGTCATCTTGCTGTTTTCTATTTG
TTCTTTTTGTTTTCTCCTTACTTTTGGATTTTTTTATTCTACTATGTCTTTTCTATTGTCTTATTAACTA
TACTCTTTGATTTATTTTAGTGGTTGTTTTAGGGTTATACCTCTTTCTAATTTACCAGTTTATAACCAGT
TTATATACTACTTGACATATAGCTTAAGAAACTTACTGTTGTTGTCTTTTTGCTGTTATGGTCTTAACGT
TTTTATTTCTACAAACATTATAAACTCCACACTTTATTGTTTTTTAATTTTACTTATACAGTCAATTATC
TTTTAAAGATATTTAAATATAAACATTCAAAACACCCCAAT.
[0491] In some embodiments, a sequence of exon 1a is represented by
nt 1137-1216; exon 1b, 1510-1572; exon 1c, 1137-1294; exon 1d,
1241-1279; and exon 1e, 1135-1169 of SEQ ID NO: 1. In some
embodiments, intron 1 is represented by 1217-7838 (if the
transcript includes exon 1a), 1573-7838 (1b), 1295-7838 (1c),
1280-7838 (1d), or 1170-7838 (1e) of SEQ ID NO: 1. In some
embodiments, a sequence of exon 2 is represented by nt 7839-8326;
exon 3, 9413-9472; exon 4, 12527-12622; exon 5, 13354-13418; exon
6, 14704-14776; exon 7, 16396-16512; exon 8, 18207-18442; exon 9,
24296-24353; exon 10, 26337-26446; and exon 11, 26581-28458 of SEQ
ID NO: 1. In some embodiments, introns lie between the exons. The
portion upstream (5') of exon 1a, 1b, 1c, 1d, or 1e includes the
5'-UTR. The portion downstream (3') of exon 11 is the 3'-UTR.
[0492] In some embodiments, a C9orf72 oligonucleotide recognizes a
site within a C9orf72 Intron 1 nearby the repeat expansion and is
selected from: WV-6969, WV-3690, WV-6976, WV-7002, WV-6970,
WV-3689, WV-6960, WV-7001, WV-6974, WV-6978, WV-6952, WV-6989,
WV-3704, WV-7007, WV-7004, WV-6951, WV-6474, WV-3688, WV-7006,
WV-6977, WV-6955, WV-6995, WV-6972, WV-7003, WV-6982, WV-6996,
WV-7005, WV-6986, WV-6979, WV-6971, WV-6985, WV-6488, WV-6489,
WV-6980, WV-6981, or any oligonucleotide having the same base
sequence of any of these oligonucleotides. In some embodiments, a
C9orf72 oligonucleotide recognizes a site within C9orf72 Exon 1a
and is selected from: WV-3677, WV-6940, WV-3683, WV-6931, WV-3679,
WV-6927, WV-6922, WV-6937, WV-6926, WV-3685, WV-6930, WV-6932,
WV-6928, WV-6933, WV-6936, WV-7027, WV-3678, WV-8114, WV-8122,
WV-8311, WV-8315, WV-8312, WV-8313, WV-8314, WV-8316, WV-8317, or
WV-8318, or any oligonucleotide having the same base sequence of
any of these oligonucleotides. In some embodiments, a C9orf72
oligonucleotide recognizes a site within C9orf72 Antisense (AS)
transcript and is selected from: WV-3723, WV-3737, WV-3719,
WV-3730, WV-3722, WV-3743, WV-3745, WV-3739, WV-3724, WV-3732,
WV-3734, WV-3733, WV-3720, WV-3721, WV-3731, or any oligonucleotide
having the same base sequence of any of these oligonucleotides. In
some embodiments, a C9orf72 oligonucleotide recognizes a site
within C9orf72 Exon 2 transcript and is selected from: WV-3662 and
WV-7118, or any oligonucleotide having the same base sequence of
any of these oligonucleotides. In some embodiments, a C9orf72
oligonucleotide can hybridize to a portion of the C9orf72 sequence
represented in GENBANK Accession No. NT_008413.18 or a complement
thereof. In some embodiments, a C9orf72 oligonucleotide can
hybridize to a portion of the C9orf72 pre-mRNA represented by the
region which begins in the region from the start site of exon 1a to
the start site of exon 1b. In some embodiments, a C9orf72
oligonucleotide can hybridize to a portion of the C9orf72 pre-mRNA
represented by the region which begins in the region from the end
site of exon 1a to the start site of exon 1b. In some embodiments,
a c9orf72 oligonucleotide recognizes a site which straddles the
junction between an intron and an exon.
[0493] In some embodiments, a c9orf72 oligonucleotide straddles the
junction between exon 1b and intron 1. In some embodiments, a
c9orf72 oligonucleotide straddles the junction between exon 1b and
intron 1, and has a base sequence which is, comprises or comprises
15 contiguous bases of the sequence CCTCACTCACCCACTCGCCA.
[0494] Without wishing to be bound by any particular scientific
theory, the present disclosure notes that the sequence
CCTCACTCACCCACTCGCCA straddles the junction of exon 1b and intron 1
reported for c9orf72 mRNA Variant 2 or V2 (which lacks the
hexanucleotide repeat), and that the site may be blocked by the
splicing machinery from being bound by an oligonucleotide having a
sequence which is, comprises or comprises 15 contiguous bases of
the sequence CCTCACTCACCCACTCGCCA. This sequence exists in c9orf72
mRNA variants V1, V2 and V3, but an oligonucleotide having a
sequence which is, comprises or comprises 15 contiguous bases of
the sequence CCTCACTCACCCACTCGCCA is particularly efficacious in
degrading disease-associated variants V1 and V3 relative to
non-disease-associated V2. Without wishing to be bound by any
particular theory, the present disclosure suggests that the
sequence CCTCACTCACCCACTCGCCA is in the middle of an intron
reported in V1 and V3; that the sequence CCTCACTCACCCACTCGCCA
straddles the junction of an exon (1b) and an intron (1) reported
for V2, and access to this site may be sterically blocked by the
splicing machinery. In some embodiments, a C9orf72 oligonucleotide
comprises a base sequence complementary to a 5' branching site at
an intron-exon junction. In some embodiments, a C9orf72
oligonucleotide comprises a sequence complementary to a 5'
branching site at the junction of a C9orf72 exon 1 and a C9orf72
intron 1. In some embodiments, a 5' branching site at the junction
of C9orf72 exon 1 and intron 1 comprises the base sequence of
GTGAGT. In some embodiments, a C9orf72 oligonucleotide comprises a
base sequence complementary to GTGAGT. In some embodiments, an
oligonucleotide is capable of preferentially decreasing the
expression, level and/or activity of a disease-associated allele of
a gene or a gene product thereof relative to that of a
corresponding wild-type allele of the gene or the gene product
thereof, wherein the oligonucleotide has a base sequence
complementary to both the disease-associated allele and the
wild-type allele, and wherein the binding site of the
oligonucleotide in a mRNA or DNA of the disease-associated allele
is less accessible to the oligonucleotide than the binding site of
the oligonucleotide in a mRNA or DNA expressing the wild-type
allele. In some embodiments, the accessibility of the
oligonucleotide to a binding site in a mRNA or DNA of the
disease-associated allele is decreased by binding of splicing
machinery and/or other nucleic acids or proteins to the mRNA or DNA
of the disease-associate allele. In some embodiments, the present
disclosure pertains to: an oligonucleotide capable of
preferentially decreasing (knocking down) the expression, level
and/or activity of a mutant or disease-associated allele of a gene
or a gene product thereof relative to that of a corresponding
wild-type or non-disease-associated allele of the gene or the gene
product thereof, wherein the oligonucleotide has a base sequence
complementary to both the mutant or disease-associated allele and
the wild-type or non-disease-associated allele, and wherein the
binding site of (e.g., sequence complementary to) the
oligonucleotide in a nucleic acid (e.g., chromosomal DNA, mRNA,
pre-mRNA, etc.) of the mutant or disease-associated allele is less
accessible to the oligonucleotide (e.g., due to increased binding
of splicing machinery and/or other nucleic acids or proteins) than
the binding site of the oligonucleotide in a nucleic acid of the
wild-type or non-disease-associated allele.
[0495] In some embodiments, a C9orf72 oligonucleotide can hybridize
to a portion of the C9orf72 pre-mRNA represented by GENBANK
Accession No. NT_008413.18, nucleosides 27535000 to 27565000 or a
complement thereof.
[0496] In some embodiments, a C9orf72 oligonucleotide can hybridize
to an intron. In some embodiments, a C9orf72 oligonucleotide can
hybridize to an intron comprising a hexanucleotide repeat.
[0497] In some embodiments, a C9orf72 oligonucleotide hybridizes to
all variants of C9orf72 derived from the sense strand. In some
embodiments, the antisense oligonucleotides described herein
selectively hybridize to a variant of C9orf72 derived from the
sense strand, including but not limited to that comprising a
hexanucleotide repeat expansion. In some embodiments, a
hexanucleotide repeat expansion comprises at least 24 repeats of
any hexanucleotide. In some embodiments, a hexanucleotide repeat
expansion comprises at least 30 repeats of any hexanucleotide. In
some embodiments, a hexanucleotide repeat expansion comprises at
least 50 repeats of any of a hexanucleotide. In some embodiments, a
hexanucleotide repeat expansion comprises at least 100 repeats of
any of a hexanucleotide. In some embodiments, a hexanucleotide
repeat expansion comprises at least 200 repeats of any
hexanucleotide. In some embodiments, a hexanucleotide repeat
expansion comprises at least 500 repeats of any hexanucleotide. In
some embodiments, a hexanucleotide is GGGGCC, GGGGGG, GGGGGC,
GGGGCG, CCCCGG, CCCCCC, GCCCCC, and/or CGCCCC. In some embodiments,
a hexanucleotide GGGGCC is designated GGGGCCexp or (GGGGCC), or is
a repeat of the hexanucleotide GGGGCC.
[0498] In some embodiments, a C9orf72 target of a C9orf72
oligonucleotide is a C9orf72 RNA which is not a mRNA. In some
embodiments, provided oligonucleotides in provided compositions,
e.g., oligonucleotides of a first plurality, comprise base
modifications, sugar modifications, and/or internucleotidic linkage
modifications. In some embodiments, provided oligonucleotides
comprise base modifications and sugar modifications. In some
embodiments, provided oligonucleotides comprise base modifications
and internucleotidic linkage modifications. In some embodiments,
provided oligonucleotides comprise sugar modifications and
internucleotidic modifications. In some embodiments, provided
compositions comprise base modifications, sugar modifications, and
internucleotidic linkage modifications. Example chemical
modifications, such as base modifications, sugar modifications,
internucleotidic linkage modifications, etc. are widely known in
the art including but not limited to those described in this
disclosure. In some embodiments, a modified base is substituted A,
T, C, G or U. In some embodiments, a sugar modification is
2'-modification. In some embodiments, a 2'-modification is 2-F
modification. In some embodiments, a 2'-modification is
2'-OR.sup.1. In some embodiments, a 2'-modification is 2'-OR.sup.1,
wherein R is optionally substituted alkyl. In some embodiments, a
2'-modification is 2'-OMe. In some embodiments, a 2'-modification
is 2'-MOE. In some embodiments, a modified sugar moiety is a
bridged bicyclic or polycyclic ring. In some embodiments, a
modified sugar moiety is a bridged bicyclic or polycyclic ring
having 5-20 ring atoms wherein one or more ring atoms are
optionally and independently heteroatoms. Example ring structures
are widely known in the art, such as those found in BNA, LNA, etc.
In some embodiments, provided oligonucleotides comprise both one or
more modified internucleotidic linkages and one or more natural
phosphate linkages. In some embodiments, oligonucleotides
comprising both modified internucleotidic linkage and natural
phosphate linkage and compositions thereof provide improved
properties, e.g., activities, etc. In some embodiments, a modified
internucleotidic linkage is a chiral internucleotidic linkage. In
some embodiments, a modified internucleotidic linkage is a
phosphorothioate linkage. In some embodiments, a modified
internucleotidic linkage is a substituted phosphorothioate
linkage.
[0499] In some embodiments, the present disclosure provides a
stereorandom oligonucleotide having a base sequence which is,
comprises or comprises a portion of the base sequence of any
oligonucleotide described herein. In some embodiments, a portion of
a base sequence is at least 15 contiguous bases thereof. In some
embodiments, the present disclosure provides an oligonucleotide
having a base sequence which is, comprises or comprises a portion
of the base sequence of any oligonucleotide described herein,
wherein the oligonucleotide comprises one or more stereorandom
internucleotidic linkages. In some embodiments, the present
disclosure provides an oligonucleotide having a base sequence which
is, comprises or comprises a portion of the base sequence of any
oligonucleotide described herein, wherein the oligonucleotide
comprises one or more stereorandom phosphorothioate
internucleotidic linkages.
[0500] In some embodiments, oligonucleotide properties can be
adjusted by optimizing stereochemistry (pattern of backbone chiral
centers) and chemical modifications (modifications of base, sugar,
and/or internucleotidic linkage) or patterns thereof.
[0501] In some embodiments, a pattern of backbone chiral centers in
a C9orf72 oligonucleotide provides increased stability. In some
embodiments, a pattern of backbone chiral centers provides
surprisingly increased activity. In some embodiments, a pattern of
backbone chiral centers provides increased stability and activity.
In some embodiments, a pattern of backbone chiral centers provides
surprisingly increased binding to certain proteins. In some
embodiments, a pattern of backbone chiral centers provides
surprisingly enhanced delivery.
[0502] In some embodiments, the present disclosure pertains to a
c9orf72 oligonucleotide wherein the oligonucleotide comprises a
backbone comprising at least one chiral center. In some
embodiments, the present disclosure pertains to a c9orf72
oligonucleotide wherein the oligonucleotide comprises a backbone
comprising at least one chiral center which is a phosphorothioate
in the Rp or Sp configuration.
[0503] In some embodiments, a C9orf72 oligonucleotide has a pattern
of backbone chiral centers.
[0504] In some embodiments, a pattern of backbone chiral centers of
a provided oligonucleotide or a region thereof (e.g., a core)
comprises or is (Sp)m(Rp)n, (Rp)n(Sp)m, (Np)t[(Op)n(Sp)m]y,
(Sp)t[(Op)n(Sp)m]y, (Np)t[(Rp)n(Sp)m]y, or (Sp)t[(Rp)n(Sp)m]y,
wherein each variable is as described in the present disclosure. In
some embodiments, y is 1. In some embodiments, a pattern of
backbone chiral centers comprises or is (Sp)m(Rp)n, (Rp)n(Sp)m,
(Np)t(Rp)n(Sp)m, (Sp)t(Rp)n(Sp)m, (Np)t[(Rp)n(Sp)m]2,
(Sp)t[(Rp)n(Sp)m]2, (Np)t(Op)n(Sp)m, (Sp)t(Op)n(Sp)m,
(Np)t[(Op)n(Sp)m]2, or (Sp)t[(Op)n(Sp)m]2. In some embodiments, y
is 2. In some embodiments, a pattern is
(Np)t(Op/Rp)n(Sp)m(Op/Rp)n(Sp)m. In some embodiments, a pattern is
(Np)t(Op/Rp)n(Sp)1-5(Op/Rp)n(Sp)m. In some embodiments, a pattern
is (Np)t(Op/Rp)n(Sp)2-5(Op/Rp)n(Sp)m. In some embodiments, a
pattern is (Np)t(Op/Rp)n(Sp)2(Op/Rp)n(Sp)m. In some embodiments, a
pattern is (Np)t(Op/Rp)n(Sp)3(Op/Rp)n(Sp)m. In some embodiments, a
pattern is (Np)t(Op/Rp)n(Sp)4(Op/Rp)n(Sp)m. In some embodiments, a
pattern is (Np)t(Op/Rp)n(Sp)5(Op/Rp)n(Sp)m. In some embodiments, Np
is Sp. In some embodiments, (Op/Rp) is Op. In some embodiments,
(Op/Rp) is Rp. In some embodiments, Np is Sp and (Op/Rp) is Rp. In
some embodiments, Np is Sp and (Op/Rp) is Op. In some embodiments,
Np is Sp and at least one (Op/Rp) is Rp, and at least one (Op/Rp)
is Op. In some embodiments, a pattern of backbone chiral centers
comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m,
wherein m>2. In some embodiments, a pattern of backbone chiral
centers comprises or is (Rp)n(Sp)m, (Np)t(Rp)n(Sp)m, or
(Sp)t(Rp)n(Sp)m, wherein n is 1, at least one t>1, and at least
one m>2. In some embodiments, at one n is 1, at least one t is
no less than 1, and at least one m is no less than 2. In some
embodiments, at one n is 1, at least one t is no less than 2, and
at least one m is no less than 3. In some embodiments, each n is 1.
In some embodiments, at least one t>1. In some embodiments, at
least one t>2. In some embodiments, at least one t>3. In some
embodiments, at least one t>4. In some embodiments, at least one
m>1. In some embodiments, at least one m>2. In some
embodiments, at least one m>3. In some embodiments, at least one
m>4. In some embodiments, a pattern of backbone chiral centers
comprises one or more achiral natural phosphate linkages. In some
embodiments, the sum of m, t, and n (or m and n if no t in a
pattern) is no less than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20. In some embodiments, the sum is 5. In some
embodiments, the sum is 6. In some embodiments, the sum is 7. In
some embodiments, the sum is 8. In some embodiments, the sum is 9
In some embodiments, the sum is 10. In some embodiments, the sum is
11. In some embodiments, the sum is 12. In some embodiments, the
sum is 13. In some embodiments, the sum is 14. In some embodiments,
the sum is 15.
[0505] In some embodiments, a nucleotidic unit comprising Op is
Nu.sup.O as described in the present disclosure. For example, in
some embodiments, Nu.sup.O comprises a 5'-substitution/modification
as described in the present disclosure, e.g., --C(R.sup.5s).sub.2--
as described in the present disclosure. In some embodiments,
--C(R.sup.5s).sub.2-- is 5MRd as described in the present
disclosure. In some embodiments, --C(R.sup.5s).sub.2-- is 5MSd as
described in the present disclosure.
[0506] In some embodiments, a pattern of backbone chiral centers
comprises or is (Rp)n(Sp)m. In some embodiments, a pattern of
backbone chiral centers comprises or is (Sp)t(Rp)n. In some
embodiments, a pattern of backbone chiral centers comprises or is
(Np)t(Rp)n(Sp)m. In some embodiments, a pattern of backbone chiral
centers comprises or is (Sp)t(Sp)m, optionally with n achiral
phosphate diester internucleotidic linkages and/or stereorandom
(non-chirally controlled) chiral internucleotidic linkages between
the section having (Sp)t and the section having (Sp)m. In some
embodiments, there are n achiral phosphate diester internucleotidic
linkages in between. In some embodiments, there are n stereorandom
chiral internucleotidic linkages in between. In some embodiments, a
pattern of backbone chiral centers comprises or is (Sp)t(Rp)n(Sp)m.
In some embodiments, each of t and m is independently equal to or
greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19 or 20.
[0507] In some embodiments, a common pattern of backbone chiral
centers in a provided oligonucleotide comprises a pattern of
i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.o,
i.sup.o-i.sup.s-i.sup.s-i.sup.s-i.sup.o,
i.sup.o-i.sup.s-i.sup.s-i.sup.s-i.sup.o-i.sup.s-,
i.sup.s-i.sup.o-i.sup.s-i.sup.o-, i.sup.s-i.sup.o-i.sup.s-i.sup.o-,
i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-,
i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.o-,
i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.o-,
i.sup.s-i.sup.o-i.sup.s-i.sup.s-i.sup.s-i.sup.o-, i.sup.s-i.sup.s-
i.sup.o-i.sup.s-i.sup.s-i.sup.s-i.sup.o-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.o-i.sup.s-i.sup.o-i.sup.s-i.sup.s-i-
.sup.s-i.sup.s-, i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.s- i.sup.s-i.sup.s-i.sup.s-i.sup.s-,
i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-i.sup.s-,
or i.sup.r-i.sup.r-i.sup.r, wherein i.sup.s represents an
internucleotidic linkage in the Sp configuration; i.sup.o
represents an achiral internucleotidic linkage; and i.sup.r
represents an internucleotidic linkage in the Rp configuration.
[0508] In some embodiments, a common pattern of backbone chiral
centers (e.g., a pattern of backbone chiral centers in a C9orf72
oligonucleotide or in a core or a wing or in two wings thereof)
comprises a pattern of OSOSO, OSSSO, OSSSOS, SOSO, SOSO, SOSOS,
SOSOSO, SOSOSOSO, SOSSSO, SSOSSSOSS, SSSOSOSSS, SSSSOSOSSSS, SSSSS,
SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS, or RRR, wherein S represents
a phosphorothioate of the Sp configuration, O represents a
phosphodiester, and R represents a phosphorothioate of the Rp
configuration.
[0509] In some embodiments, the non-chiral center is a linkage
phosphorus of a phosphodiester linkage. In some embodiments, the
chiral center in a Sp configuration is a linkage phosphorus of a
phosphorothioate linkage. In some embodiments, the chiral center in
a Rp configuration is a linkage phosphorus of a phosphorothioate
linkage.
[0510] In some embodiments, 5% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 10% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 15% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 20% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 25% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 30% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 35% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages. In some embodiments, 40% or more of the internucleotidic
linkages of provided oligonucleotides are modified internucleotidic
linkages.
[0511] In some embodiments, expression or level of a C9orf72 target
gene or its gene product is decreased by at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by
administration of an oligonucleotide. In some embodiments,
expression or level of a C9orf72 target gene or its gene product is
decreased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 60%, 70%, or 80% total by RNase H-mediated knockdown directed
by an oligonucleotide. In some embodiments, expression or level of
a C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by administration of an oligonucleotide in a cell(s) in vitro. In
some embodiments, expression or level of a C9orf72 target gene or
its gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by RNase H-mediated
knockdown directed by an oligonucleotide in a cell(s) in vitro. In
some embodiments, expression or level of a C9orf72 target gene or
its gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by administration of an
oligonucleotide at a concentration of 25 nm or less in a cell(s) in
vitro. In some embodiments, expression or level of a C9orf72 target
gene or its gene product is decreased by at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by
administration of an oligonucleotide at a concentration of 10 nm or
less in a cell(s) in vitro. In some embodiments, expression or
level of a C9orf72 target gene or its gene product is decreased by
at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
70%, or 80% by administration of an oligonucleotide at a
concentration of 5 nm or less in a cell(s) in vitro. In some
embodiments, expression or level of a C9orf72 target gene or its
gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by RNase H-mediated
knockdown directed by an oligonucleotide at a concentration of 5 nm
or less in a cell(s) in vitro. In some embodiments, a cell(s) is a
mammalian cell(s). In some embodiments, a cell(s) is a human
cell(s). In some embodiments, a cell(s) is a hepatic cell(s). In
some embodiments, a cell(s) is a Huh7 or Hep3B cell(s). In some
embodiments, a C9orf72 oligonucleotide is capable of decreasing
expression or level of a C9orf72 target gene or its gene product by
at least about 20% in a cell(s) in vitro at a concentration of 25
nM or less. In some embodiments, a C9orf72 oligonucleotide is
capable of decreasing expression or level of a C9orf72 target gene
or its gene product by at least about 30% in a cell(s) in vitro at
a concentration of 25 nM or less. In some embodiments, a C9orf72
oligonucleotide is capable of decreasing expression or level of a
C9orf72 target gene or its gene product by at least about 40% in a
cell(s) in vitro at a concentration of 25 nM or less. In some
embodiments, a C9orf72 oligonucleotide is capable of decreasing
expression or level of a C9orf72 target gene or its gene product by
at least about 50% in a cell(s) in vitro at a concentration of 25
nM or less. In some embodiments, a C9orf72 oligonucleotide is
capable of decreasing expression or level of a C9orf72 target gene
or its gene product by at least about 60% in a cell(s) in vitro at
a concentration of 25 nM or less. In some embodiments, a C9orf72
oligonucleotide is capable of decreasing expression or level of a
C9orf72 target gene or its gene product by at least about 70% in a
cell(s) in vitro at a concentration of 25 nM or less. In some
embodiments, a C9orf72 oligonucleotide is capable of decreasing
expression or level of a C9orf72 target gene or its gene product by
at least about 80% in a cell(s) in vitro at a concentration of 25
nM or less. In some embodiments, a C9orf72 oligonucleotide is
capable of decreasing expression or level of a C9orf72 target gene
or its gene product by at least about 90% in a cell(s) in vitro at
a concentration of 25 nM or less. In some embodiments, an
oligonucleotide is capable of decreasing expression or level of a
C9orf72 target gene or its gene product by at least about 20% in a
cell(s) in vitro at a concentration of 25 nM or less. In some
embodiments, an oligonucleotide is capable of decreasing expression
or level of a C9orf72 target gene or its gene product by at least
about 30% in a cell(s) in vitro at a concentration of 25 nM or
less. In some embodiments, an oligonucleotide is capable of
decreasing expression or level of a C9orf72 target gene or its gene
product by at least about 40% in a cell(s) in vitro at a
concentration of 25 nM or less. In some embodiments, an
oligonucleotide is capable of decreasing expression or level of a
C9orf72 target gene or its gene product by at least about 50% in a
cell(s) in vitro at a concentration of 25 nM or less. In some
embodiments, an oligonucleotide is capable of decreasing expression
or level of a C9orf72 target gene or its gene product by at least
about 60% in a cell(s) in vitro at a concentration of 25 nM or
less. In some embodiments, an oligonucleotide is capable of
decreasing expression or level of a C9orf72 target gene or its gene
product by at least about 70% in a cell(s) in vitro at a
concentration of 25 nM or less. In some embodiments, an
oligonucleotide is capable of decreasing expression or level of a
C9orf72 target gene or its gene product by at least about 80% in a
cell(s) in vitro at a concentration of 25 nM or less. In some
embodiments, an oligonucleotide is capable of decreasing expression
or level of a C9orf72 target gene or its gene product by at least
about 90% in a cell(s) in vitro at a concentration of 25 nM or
less. In some embodiments, IC50 is inhibitory concentration to
decrease expression or level or a C9orf72 target gene or its gene
product by 50% in a cell(s) in vitro.
[0512] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)mRp or Rp(Sp)m. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises Rp(Sp)m. In some embodiments, the
present disclosure provides a C9orf72 oligonucleotide of an
oligonucleotide type whose pattern of backbone chiral centers
comprises (Sp)mRp. In some embodiments, m is 2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises Rp(Sp).sub.2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp).sub.2Rp(Sp).sub.2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Rp).sub.2Rp(Sp).sub.2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises RpSpRp(Sp).sub.2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises SpRpRp(Sp).sub.2. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp).sub.2Rp.
[0513] As defined herein, m is 1-50. In some embodiments, m is 1.
In some embodiments, m is 2-50. In some embodiments, m is 2, 3, 4,
5, 6, 7 or 8. In some embodiments, m is 3, 4, 5, 6, 7 or 8. In some
embodiments, m is 4, 5, 6, 7 or 8. In some embodiments, m is 5, 6,
7 or 8. In some embodiments, m is 6, 7 or 8. In some embodiments, m
is 7 or 8. In some embodiments, m is 2. In some embodiments, m is
3. In some embodiments, m is 4. In some embodiments, m is 5. In
some embodiments, m is 6. In some embodiments, m is 7. In some
embodiments, m is 8. In some embodiments, m is 9. In some
embodiments, m is 10. In some embodiments, m is 11. In some
embodiments, m is 12. In some embodiments, m is 13. In some
embodiments, m is 14. In some embodiments, m is 15. In some
embodiments, m is 16. In some embodiments, m is 17. In some
embodiments, m is 18. In some embodiments, m is 19. In some
embodiments, m is 20. In some embodiments, m is 21. In some
embodiments, m is 22. In some embodiments, m is 23. In some
embodiments, m is 24. In some embodiments, m is 25. In some
embodiments, m is greater than 25.
[0514] In some embodiments, a repeating pattern is (Sp)m(Rp)n,
wherein n is 1-10, and m is independently described in the present
disclosure. In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)m(Rp)n. In some embodiments,
the present disclosure provides a C9orf72 oligonucleotide of an
oligonucleotide type whose pattern of backbone chiral centers
comprises (Sp)m(Rp)n. In some embodiments, a repeating pattern is
(Rp)n(Sp)m, wherein n is 1-10, and m is independently described in
the present disclosure. In some embodiments, the present disclosure
provides a C9orf72 oligonucleotide of an oligonucleotide type whose
pattern of backbone chiral centers comprises (Rp)n(Sp)m. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Rp)n(Sp)m. In some embodiments,
(Rp)n(Sp)m is (Rp)(Sp).sub.2. In some embodiments, (Sp)n(Rp)m is
(Sp).sub.2(Rp).
[0515] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)m(Rp)n(Sp)t. In some
embodiments, a repeating pattern is (Sp)m(Rp)n(Sp)t, wherein n is
1-10, t is 1-50, and m is as described in the present disclosure.
In some embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)m(Rp)n(Sp)t. In some
embodiments, a repeating pattern is (Sp)t(Rp)n(Sp)m, wherein n is
1-10, t is 1-50, and m is as described in the present disclosure.
In some embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)t(Rp)n(Sp)m. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Sp)t(Rp)n(Sp)m.
[0516] In some embodiments, a repeating pattern is (Np)t(Rp)n(Sp)m,
wherein n is 1-10, t is 1-50, Np is independently Rp or Sp, and m
is as described in the present disclosure. In some embodiments, the
present disclosure provides a C9orf72 oligonucleotide of an
oligonucleotide type whose pattern of backbone chiral centers
comprises (Np)t(Rp)n(Sp)m. In some embodiments, the present
disclosure provides a C9orf72 oligonucleotide of an oligonucleotide
type whose pattern of backbone chiral centers comprises
(Np)t(Rp)n(Sp)m. In some embodiments, a repeating pattern is
(Np)m(Rp)n(Sp)t, wherein n is 1-10, t is 1-50, Np is independently
Rp or Sp, and m is as described in the present disclosure. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Np)m(Rp)n(Sp)t. In some
embodiments, the present disclosure provides a C9orf72
oligonucleotide of an oligonucleotide type whose pattern of
backbone chiral centers comprises (Np)m(Rp)n(Sp)t. In some
embodiments, Np is Rp. In some embodiments, Np is Sp. In some
embodiments, all Np are the same. In some embodiments, all Np are
Sp. In some embodiments, at least one Np is different from the
other Np. In some embodiments, t is 2.
[0517] As defined herein, n is 1-10. In some embodiments, n is 1,
2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is 1. In some
embodiments, n is 2, 3, 4, 5, 6, 7 or 8. In some embodiments, n is
3, 4, 5, 6, 7 or 8. In some embodiments, n is 4, 5, 6, 7 or 8. In
some embodiments, n is 5, 6, 7 or 8. In some embodiments, n is 6, 7
or 8. In some embodiments, n is 7 or 8. In some embodiments, n is
1. In some embodiments, n is 2. In some embodiments, n is 3. In
some embodiments, n is 4. In some embodiments, n is 5. In some
embodiments, n is 6. In some embodiments, n is 7. In some
embodiments, n is 8. In some embodiments, n is 9. In some
embodiments, n is 10.
[0518] As defined herein, t is 1-50. In some embodiments, t is 1.
In some embodiments, t is 2-50. In some embodiments, t is 2, 3, 4,
5, 6, 7 or 8. In some embodiments, t is 3, 4, 5, 6, 7 or 8. In some
embodiments, t is 4, 5, 6, 7 or 8. In some embodiments, t is 5, 6,
7 or 8. In some embodiments, t is 6, 7 or 8. In some embodiments, t
is 7 or 8. In some embodiments, t is 2. In some embodiments, t is
3. In some embodiments, t is 4. In some embodiments, t is 5. In
some embodiments, t is 6. In some embodiments, t is 7. In some
embodiments, t is 8. In some embodiments, t is 9. In some
embodiments, t is 10. In some embodiments, t is 11. In some
embodiments, t is 12. In some embodiments, t is 13. In some
embodiments, t is 14. In some embodiments, t is 15. In some
embodiments, t is 16. In some embodiments, t is 17. In some
embodiments, t is 18. In some embodiments, t is 19. In some
embodiments, t is 20. In some embodiments, t is 21. In some
embodiments, t is 22. In some embodiments, t is 23. In some
embodiments, t is 24. In some embodiments, t is 25. In some
embodiments, t is greater than 25.
[0519] In some embodiments, at least one of m and t is greater than
2. In some embodiments, at least one of m and t is greater than 3.
In some embodiments, at least one of m and t is greater than 4. In
some embodiments, at least one of m and t is greater than 5. In
some embodiments, at least one of m and t is greater than 6. In
some embodiments, at least one of m and t is greater than 7. In
some embodiments, at least one of m and t is greater than 8. In
some embodiments, at least one of m and t is greater than 9. In
some embodiments, at least one of m and t is greater than 10. In
some embodiments, at least one of m and t is greater than 11. In
some embodiments, at least one of m and t is greater than 12. In
some embodiments, at least one of m and t is greater than 13. In
some embodiments, at least one of m and t is greater than 14. In
some embodiments, at least one of m and t is greater than 15. In
some embodiments, at least one of m and t is greater than 16. In
some embodiments, at least one of m and t is greater than 17. In
some embodiments, at least one of m and t is greater than 18. In
some embodiments, at least one of m and t is greater than 19. In
some embodiments, at least one of m and t is greater than 20. In
some embodiments, at least one of m and t is greater than 21. In
some embodiments, at least one of m and t is greater than 22. In
some embodiments, at least one of m and t is greater than 23. In
some embodiments, at least one of m and t is greater than 24. In
some embodiments, at least one of m and t is greater than 25.
[0520] In some embodiments, each one of m and t is greater than 2.
In some embodiments, each one of m and t is greater than 3. In some
embodiments, each one of m and t is greater than 4. In some
embodiments, each one of m and t is greater than 5. In some
embodiments, each one of m and t is greater than 6. In some
embodiments, each one of m and t is greater than 7. In some
embodiments, each one of m and t is greater than 8. In some
embodiments, each one of m and t is greater than 9. In some
embodiments, each one of m and t is greater than 10. In some
embodiments, each one of m and t is greater than 11. In some
embodiments, each one of m and t is greater than 12. In some
embodiments, each one of m and t is greater than 13. In some
embodiments, each one of m and t is greater than 14. In some
embodiments, each one of m and t is greater than 15. In some
embodiments, each one of m and t is greater than 16. In some
embodiments, each one of m and t is greater than 17. In some
embodiments, each one of m and t is greater than 18. In some
embodiments, each one of m and t is greater than 19. In some
embodiments, each one of m and t is greater than 20.
[0521] In some embodiments, the sum of m and t is greater than 3.
In some embodiments, the sum of m and t is greater than 4. In some
embodiments, the sum of m and t is greater than 5. In some
embodiments, the sum of m and t is greater than 6. In some
embodiments, the sum of m and t is greater than 7. In some
embodiments, the sum of m and t is greater than 8. In some
embodiments, the sum of m and t is greater than 9. In some
embodiments, the sum of m and t is greater than 10. In some
embodiments, the sum of m and t is greater than 11. In some
embodiments, the sum of m and t is greater than 12. In some
embodiments, the sum of m and t is greater than 13. In some
embodiments, the sum of m and t is greater than 14. In some
embodiments, the sum of m and t is greater than 15. In some
embodiments, the sum of m and t is greater than 16. In some
embodiments, the sum of m and t is greater than 17. In some
embodiments, the sum of m and t is greater than 18. In some
embodiments, the sum of m and t is greater than 19. In some
embodiments, the sum of m and t is greater than 20. In some
embodiments, the sum of m and t is greater than 21. In some
embodiments, the sum of m and t is greater than 22. In some
embodiments, the sum of m and t is greater than 23. In some
embodiments, the sum of m and t is greater than 24. In some
embodiments, the sum of m and t is greater than 25.
[0522] In some embodiments, n is 1, and at least one of m and t is
greater than 1. In some embodiments, n is 1 and each of m and t is
independently greater than 1. In some embodiments, m>n and
t>n. In some embodiments, (Sp)m(Rp)n(Sp)t is
(Sp).sub.2Rp(Sp).sub.2. In some embodiments, (Sp)t(Rp)n(Sp)m is
(Sp).sub.2Rp(Sp).sub.2. In some embodiments, (Sp)t(Rp)n(Sp)m is
SpRp(Sp).sub.2. In some embodiments, (Np)t(Rp)n(Sp)m is
(Np)tRp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is
(Np).sub.2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is
(Rp).sub.2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is
(Sp).sub.2Rp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is
RpSpRp(Sp)m. In some embodiments, (Np)t(Rp)n(Sp)m is
SpRpRp(Sp)m.
[0523] In some embodiments, (Sp)t(Rp)n(Sp)m is SpRpSpSp. In some
embodiments, (Sp)t(Rp)n(Sp)m is (Sp).sub.2Rp(Sp).sub.2. In some
embodiments, (Sp)t(Rp)n(Sp)m is (Sp).sub.3Rp(Sp).sub.3. In some
embodiments, (Sp)t(Rp)n(Sp)m is (Sp).sub.4Rp(Sp).sub.4. In some
embodiments, (Sp)t(Rp)n(Sp)m is (Sp)tRp(Sp)s. In some embodiments,
(Sp)t(Rp)n(Sp)m is SpRp(Sp)s. In some embodiments, (Sp)t(Rp)n(Sp)m
is (Sp).sub.2Rp(Sp).sub.5. In some embodiments, (Sp)t(Rp)n(Sp)m is
(Sp).sub.3Rp(Sp).sub.5. In some embodiments, (Sp)t(Rp)n(Sp)m is
(Sp).sub.4Rp(Sp). In some embodiments, (Sp)t(Rp)n(Sp)m is
(Sp).sub.5Rp(Sp).sub.5.
[0524] In some embodiments, provided oligonucleotides are
blockmers. In some embodiments, provided oligonucleotide are
altmers. In some embodiments, provided oligonucleotides are altmers
comprising alternating blocks. In some embodiments, a blockmer or
an altmer can be defined by chemical modifications (including
presence or absence), e.g., base modifications, sugar modification,
internucleotidic linkage modifications, stereochemistry, etc., or
patterns thereof. Example chemical modifications, stereochemistry
and patterns thereof for a block and/or an alternating unit include
but are not limited to those described in this disclosure, such as
those described for an oligonucleotide, etc. In some embodiments, a
blockmer comprises a pattern of . . . SS . . . RR . . . SS . . . RR
. . . . In some embodiments, an altmer comprises a pattern of
SRSRSRSR.
[0525] In some embodiments, a provided pattern of backbone chiral
centers comprises repeating (Sp)m(Rp)n, (Rp)n(Sp)m,
(Np)t(Rp)n(Sp)m, or (Sp)t(Rp)n(Sp)m units. In some embodiments, a
repeating unit is (Sp)m(Rp)n. In some embodiments, a repeating unit
is SpRp. In some embodiments, a repeating unit is SpSpRp. In some
embodiments, a repeating unit is SpRpRp. In some embodiments, a
repeating unit is RpRpSp. In some embodiments, a repeating unit is
(Rp)n(Sp)m. In some embodiments, a repeating unit is
(Np)t(Rp)n(Sp)m. In some embodiments, a repeating unit is
(Sp)t(Rp)n(Sp)m.
[0526] In some embodiments, a provided pattern of backbone chiral
centers is or comprises (Rp/Sp)x-(All Rp or All Sp)-(Rp/Sp)y. In
some embodiments, a provided pattern of backbone chiral centers is
or comprises (Rp/Sp)-(All Rp or All Sp)-(Rp/Sp). In some
embodiments, a provided pattern of backbone chiral centers is or
comprises (Rp)x-(All Sp)-(Rp)y. In some embodiments, a provided
pattern of backbone chiral centers is or comprises (Rp)-(All
Sp)-(Rp). In some embodiments, a provided pattern of backbone
chiral centers is or comprises (Sp)x-(All Rp)-(Sp)y. In some
embodiments, a provided pattern of backbone chiral centers is or
comprises (Sp)-(All Rp)-(Sp). In some embodiments, a provided
pattern of backbone chiral centers is or comprises
(Rp/Sp)x-(repeating (Sp)m(Rp)n)-(Rp/Sp)y. In some embodiments, a
provided pattern of backbone chiral centers is or comprises
(Rp/Sp)-(repeating (Sp)m(Rp)n)-(Rp/Sp). In some embodiments, a
provided pattern of backbone chiral centers is or comprises
(Rp/Sp)x-(repeating SpSpRp)-(Rp/Sp)y. In some embodiments, a
provided pattern of backbone chiral centers is or comprises
(Rp/Sp)-(repeating SpSpRp)-(Rp/Sp).
[0527] In some embodiments, a provided oligonucleotide comprises
any pattern of stereochemistry or any sugar modification described
herein.
[0528] In some embodiments, a modified sugar moiety comprises a
2'-modification. In some embodiments, a modified sugar moiety
comprises a 2'-modification. In some embodiments, a 2'-modification
is 2'-OR.sup.1. In some embodiments, a 2'-modification is a 2'-OMe.
In some embodiments, a 2'-modification is a 2'-MOE. In some
embodiments, a 2'-modification is an LNA sugar modification. In
some embodiments, a 2'-modification is 2'-F. In some embodiments,
each sugar modification is independently a 2'-modification. In some
embodiments, each sugar modification is independently 2'-OR.sup.1
or 2'-F. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl. In some embodiments, each sugar
modification is independently 2'-OR.sup.1 or 2'-F, wherein at least
one is 2'-F. In some embodiments, each sugar modification is
independently 2'-OR' or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl, and wherein at least one is
2'-OR.sup.1. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein at least one is 2'-F,
and at least one is 2'-OR.sup.1. In some embodiments, each sugar
modification is independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1
is optionally substituted C.sub.1-6 alkyl, and wherein at least one
is 2'-F, and at least one is 2'-OR.sup.1.
[0529] In some embodiments, 5% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 10% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 15% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 20% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 25% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 30% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 35% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 40% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 45% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 50% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 55% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 60% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 65% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 70% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 75% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 80% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 85% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, 90% or
more of the sugar moieties of provided oligonucleotides are
modified. In some embodiments, 95% or more of the sugar moieties of
provided oligonucleotides are modified. In some embodiments, each
sugar moiety of provided oligonucleotides is modified. In some
embodiments, a modified sugar moiety comprises a 2'-modification.
In some embodiments, a modified sugar moiety comprises a
2'-modification. In some embodiments, a 2'-modification is
2'-OR.sup.1. In some embodiments, a 2'-modification is a 2'-OMe. In
some embodiments, a 2'-modification is a 2'-MOE. In some
embodiments, a 2'-modification is an LNA sugar modification. In
some embodiments, a 2'-modification is 2'-F. In some embodiments,
each sugar modification is independently a 2'-modification. In some
embodiments, each sugar modification is independently 2'-OR.sup.1
or 2'-F. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl. In some embodiments, each sugar
modification is independently 2'-OR.sup.1 or 2'-F, wherein at least
one is 2'-F. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl, and wherein at least one is
2'-OR.sup.1. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein at least one is 2'-F,
and at least one is 2'-OR.sup.1. In some embodiments, each sugar
modification is independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1
is optionally substituted C.sub.1-6 alkyl, and wherein at least one
is 2'-F, and at least one is 2'-OR.sup.1.
[0530] In some embodiments, a nucleoside comprising a
2'-modification is followed by a modified internucleotidic linkage.
In some embodiments, a nucleoside comprising a 2'-modification is
preceded by a modified internucleotidic linkage. In some
embodiments, a modified internucleotidic linkage is a chiral
internucleotidic linkage. In some embodiments, a modified
internucleotidic linkage is a phosphorothioate. In some
embodiments, a chiral internucleotidic linkage is Sp. In some
embodiments, a nucleoside comprising a 2'-modification is followed
by an Sp chiral internucleotidic linkage. In some embodiments, a
nucleoside comprising a 2'-F is followed by an Sp chiral
internucleotidic linkage. In some embodiments, a nucleoside
comprising a 2'-modification is preceded by an Sp chiral
internucleotidic linkage. In some embodiments, a nucleoside
comprising a 2'-F is preceded by an Sp chiral internucleotidic
linkage. In some embodiments, a chiral internucleotidic linkage is
Rp. In some embodiments, a nucleoside comprising a 2'-modification
is followed by an Rp chiral internucleotidic linkage. In some
embodiments, a nucleoside comprising a 2'-F is followed by an Rp
chiral internucleotidic linkage. In some embodiments, a nucleoside
comprising a 2'-modification is preceded by an Rp chiral
internucleotidic linkage. In some embodiments, a nucleoside
comprising a 2'-F is preceded by an Rp chiral internucleotidic
linkage.
[0531] Provided oligonucleotides can comprise various number of
natural phosphate linkages. In some embodiments, provided
oligonucleotides comprise no natural phosphate linkages. In some
embodiments, provided oligonucleotides comprise one natural
phosphate linkage. In some embodiments, provided oligonucleotides
comprise 1 to 30 or more natural phosphate linkages. In some
embodiments, provided oligonucleotides comprise about 25 or more
consecutive modified sugar moieties. In some embodiments, provided
oligonucleotides comprise about 1 to 20 or more consecutive
modified sugar moieties. In some embodiments, provided
oligonucleotides comprise no more than about 5% to 90% unmodified
sugar moieties. In some embodiments, each sugar moiety of the
oligonucleotides of the first plurality is independently modified.
In some embodiments, provided oligonucleotides are capable of
directing a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product.
[0532] In some embodiments, each oligonucleotide of the first
plurality comprises one or more modified sugar moieties and
modified internucleotidic linkages. In some embodiments, each
oligonucleotide of the first plurality comprises two or more
modified sugar moieties. In some embodiments, each oligonucleotide
of the first plurality comprises three or more modified sugar
moieties. In some embodiments, each oligonucleotide of the first
plurality comprises four or more modified sugar moieties. In some
embodiments, each oligonucleotide of the first plurality comprises
five or more modified sugar moieties. In some embodiments, each
oligonucleotide of the first plurality comprises ten or more
modified sugar moieties. In some embodiments, each oligonucleotide
of the first plurality comprises about 15 or more modified sugar
moieties. In some embodiments, each oligonucleotide of the first
plurality comprises about 20 or more modified sugar moieties. In
some embodiments, each oligonucleotide of the first plurality
comprises about 25 or more modified sugar moieties.
[0533] In some embodiments, each oligonucleotide of the first
plurality comprises two or more modified internucleotidic linkages.
In some embodiments, each oligonucleotide of the first plurality
comprises three or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
four or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
five or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
ten or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
about 15 or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
about 20 or more modified internucleotidic linkages. In some
embodiments, each oligonucleotide of the first plurality comprises
about 25 or more modified internucleotidic linkages.
[0534] In some embodiments, about 5% of the internucleotidic
linkages in each oligonucleotide of the first plurality are
modified internucleotidic linkages. In some embodiments, about 10%
of the internucleotidic linkages in each oligonucleotide of the
first plurality are modified internucleotidic linkages. In some
embodiments, about 20% of the internucleotidic linkages in each
oligonucleotide of the first plurality are modified
internucleotidic linkages. In some embodiments, about 30% of the
internucleotidic linkages in each oligonucleotide of the first
plurality are modified internucleotidic linkages. In some
embodiments, about 40% of the internucleotidic linkages in each
oligonucleotide of the first plurality are modified
internucleotidic linkages. In some embodiments, about 50% of the
internucleotidic linkages in each oligonucleotide of the first
plurality are modified internucleotidic linkages. In some
embodiments, about 60% of the internucleotidic linkages in each
oligonucleotide of the first plurality are modified
internucleotidic linkages. In some embodiments, about 70% of the
internucleotidic linkages in each oligonucleotide of the first
plurality are modified internucleotidic linkages. In some
embodiments, about 80% of the internucleotidic linkages in each
oligonucleotide of the first plurality are modified
internucleotidic linkages. In some embodiments, about 85% of the
internucleotidic linkages in each oligonucleotide of the first
plurality are modified internucleotidic linkages. In some
embodiments, about 90% of the internucleotidic linkages in each
oligonucleotide of the first plurality are modified
internucleotidic linkages. In some embodiments, about 95% of the
internucleotidic linkages in each oligonucleotide of the first
plurality are modified internucleotidic linkages.
[0535] In some embodiments, compared to a reference condition,
provided chirally controlled C9orf72 oligonucleotide compositions
are surprisingly effective. In some embodiments, desired biological
effects (e.g., as measured by decreased levels of undesired mRNA,
proteins, etc.) can be enhanced by more than 5, 10, 15, 20, 25, 30,
40, 50, or 100 fold. In some embodiments, a change is measured by
increase of a desired mRNA level compared to a reference condition.
In some embodiments, a change is measured by decrease of an
undesired mRNA level compared to a reference condition. In some
embodiments, a reference condition is absence of oligonucleotide
treatment. In some embodiments, a reference condition is a
stereorandom composition of oligonucleotides having the same base
sequence and chemical modifications.
[0536] In some embodiments, provided oligonucleotides contain
increased levels of one or more isotopes. In some embodiments,
provided oligonucleotides are labeled, e.g., by one or more
isotopes of one or more elements, e.g., hydrogen, carbon, nitrogen,
etc. In some embodiments, provided oligonucleotides in provided
compositions, e.g., oligonucleotides of a first plurality, comprise
base modifications, sugar modifications, and/or internucleotidic
linkage modifications, wherein the oligonucleotides contain an
enriched level of deuterium. In some embodiments, provided
oligonucleotides are labeled with deuterium (replacing --.sup.1H
with --.sup.2H) at one or more positions. In some embodiments, one
or more .sup.1H of an oligonucleotide or any moiety conjugated to
the oligonucleotide (e.g., a targeting moiety, etc.) is substituted
with .sup.2H. Such oligonucleotides can be used in any composition
or method described herein.
[0537] In some embodiments, the present disclosure provides an
oligonucleotide composition comprising a first plurality of
oligonucleotides which: [0538] 1) have a common base sequence
complementary to a C9orf72 target sequence in a transcript; and
[0539] 2) comprise one or more modified sugar moieties and modified
internucleotidic linkages.
[0540] In some embodiments, each of the consecutive nucleoside
units is independently preceded and/or followed by a modified
internucleotidic linkage. In some embodiments, each of the
consecutive nucleoside units is independently preceded and/or
followed by a phosphorothioate linkage. In some embodiments, each
of the consecutive nucleoside units is independently preceded
and/or followed by a chirally controlled modified internucleotidic
linkage. In some embodiments, each of the consecutive nucleoside
units is independently preceded and/or followed by a chirally
controlled phosphorothioate linkage. In some embodiments, a
modified internucleotidic linkage has a structure of Formula I. In
some embodiments, a modified internucleotidic linkage has a
structure of Formula I-a.
[0541] In some embodiments, a modified internucleotidic linkage has
a structure of Formula I. In some embodiments, a modified
internucleotidic linkage has a structure of Formula I-a.
[0542] In some embodiments, a common base sequence and length may
be referred to as a common base sequence. In some embodiments,
oligonucleotides having a common base sequence may have the same
pattern of nucleoside modifications, e.g., sugar modifications,
base modifications, etc. In some embodiments, a pattern of
nucleoside modifications may be represented by a combination of
locations and modifications. In some embodiments, a pattern of
backbone linkages comprises locations and types (e.g., phosphate,
phosphorothioate, substituted phosphorothioate, etc.) of each
internucleotidic linkages. A pattern of backbone chiral centers of
an oligonucleotide can be designated by a combination of linkage
phosphorus stereochemistry (Rp/Sp) from 5' to 3'. As exemplified
above, locations of non-chiral linkages may be obtained, for
example, from pattern of backbone linkages.
[0543] As understood by a person having ordinary skill in the art,
a stereorandom or racemic preparation of oligonucleotides is
prepared by non-stereoselective and/or low-stereoselective coupling
of nucleotide monomers, typically without using any chiral
auxiliaries, chiral modification reagents, and/or chiral catalysts.
In some embodiments, in a substantially racemic (or chirally
uncontrolled) preparation of oligonucleotides, all or most coupling
steps are not chirally controlled in that the coupling steps are
not specifically conducted to provide enhanced stereoselectivity.
An example substantially racemic preparation of oligonucleotides is
the preparation of phosphorothioate oligonucleotides through
sulfurizing phosphite triesters from commonly used phosphoramidite
oligonucleotide synthesis with either tetraethylthiuram disulfide
or (TETD) or 3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD), a
well-known process in the art. In some embodiments, substantially
racemic preparation of oligonucleotides provides substantially
racemic oligonucleotide compositions (or chirally uncontrolled
oligonucleotide compositions). In some embodiments, at least one
coupling of a nucleotide monomer has a diastereoselectivity lower
than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3,
98:2, or 99:1. In some embodiments, at least two couplings of a
nucleotide monomer have a diastereoselectivity lower than about
60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1.
In some embodiments, at least three couplings of a nucleotide
monomer have a diastereoselectivity lower than about 60:40, 70:30,
80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some
embodiments, at least four couplings of a nucleotide monomer have a
diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15,
90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at
least five couplings of a nucleotide monomer have a
diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15,
90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, each
coupling of a nucleotide monomer independently has a
diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15,
90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, in a
stereorandom or racemic preparations, at least one internucleotidic
linkage has a diastereoselectivity lower than about 60:40, 70:30,
80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some
embodiments, at least two internucleotidic linkages have a
diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15,
90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, at
least three internucleotidic linkages have a diastereoselectivity
lower than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8,
97:3, 98:2, or 99:1. In some embodiments, at least four
internucleotidic linkages have a diastereoselectivity lower than
about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or
99:1. In some embodiments, at least five internucleotidic linkages
have a diastereoselectivity lower than about 60:40, 70:30, 80:20,
85:15, 90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments,
each internucleotidic linkage independently has a
diastereoselectivity lower than about 60:40, 70:30, 80:20, 85:15,
90:10, 91:9, 92:8, 97:3, 98:2, or 99:1. In some embodiments, a
diastereoselectivity is lower than about 60:40. In some
embodiments, a diastereoselectivity is lower than about 70:30. In
some embodiments, a diastereoselectivity is lower than about 80:20.
In some embodiments, a diastereoselectivity is lower than about
90:10. In some embodiments, a diastereoselectivity is lower than
about 91:9. In some embodiments, a diastereoselectivity is lower
than about 92:8. In some embodiments, a diastereoselectivity is
lower than about 93:7. In some embodiments, a diastereoselectivity
is lower than about 94:6. In some embodiments, a
diastereoselectivity is lower than about 95:5. In some embodiments,
a diastereoselectivity is lower than about 96:4. In some
embodiments, a diastereoselectivity is lower than about 97:3. In
some embodiments, a diastereoselectivity is lower than about 98:2.
In some embodiments, a diastereoselectivity is lower than about
99:1. In some embodiments, at least one coupling has a
diastereoselectivity lower than about 90:10. In some embodiments,
at least two couplings have a diastereoselectivity lower than about
90:10. In some embodiments, at least three couplings have a
diastereoselectivity lower than about 90:10. In some embodiments,
at least four couplings have a diastereoselectivity lower than
about 90:10. In some embodiments, at least five couplings have a
diastereoselectivity lower than about 90:10. In some embodiments,
each coupling independently has a diastereoselectivity lower than
about 90:10. In some embodiments, at least one internucleotidic
linkage has a diastereoselectivity lower than about 90:10. In some
embodiments, at least two internucleotidic linkages have a
diastereoselectivity lower than about 90:10. In some embodiments,
at least three internucleotidic linkages have a
diastereoselectivity lower than about 90:10. In some embodiments,
at least four internucleotidic linkages have a diastereoselectivity
lower than about 90:10. In some embodiments, at least five
internucleotidic linkages have a diastereoselectivity lower than
about 90:10. In some embodiments, each internucleotidic linkage
independently has a diastereoselectivity lower than about
90:10.
[0544] In some embodiments, a chirally controlled internucleotidic
linkage, such as those of oligonucleotides of chirally controlled
C9orf72 oligonucleotide compositions, has a diastereoselectivity of
90:10 or more. In some embodiments, each chirally controlled
internucleotidic linkage, such as those of oligonucleotides of
chirally controlled C9orf72 oligonucleotide compositions, has a
diastereoselectivity of 90:10 or more. In some embodiments, the
selectivity is 91:9 or more. In some embodiments, the selectivity
is 92:8 or more. In some embodiments, the selectivity is 97:3 or
more. In some embodiments, the selectivity is 94:6 or more. In some
embodiments, the selectivity is 95:5 or more. In some embodiments,
the selectivity is 96:4 or more. In some embodiments, the
selectivity is 97:3 or more. In some embodiments, the selectivity
is 98:2 or more. In some embodiments, the selectivity is 99:1 or
more.
[0545] As understood by a person having ordinary skill in the art,
in some embodiments, diastereoselectivity of a coupling or a
linkage can be assessed through the diastereoselectivity of a dimer
formation under the same or comparable conditions, wherein the
dimer has the same 5'- and 3'-nucleosides and internucleotidic
linkage.
[0546] In some embodiments, the present disclosure provides
chirally controlled (and/or stereochemically pure) oligonucleotide
compositions comprising a first plurality of oligonucleotides
defined by having: [0547] 1) a common base sequence and length;
[0548] 2) a common pattern of backbone linkages; and [0549] 3) a
common pattern of backbone chiral centers, which composition is a
substantially pure preparation of a single oligonucleotide in that
at least about 10% of the oligonucleotides in the composition have
the common base sequence and length, the common pattern of backbone
linkages, and the common pattern of backbone chiral centers.
[0550] In some embodiments, the present disclosure provides
chirally controlled C9orf72 oligonucleotide composition of a first
plurality of oligonucleotides in that the composition is enriched,
relative to a substantially racemic preparation of the same
oligonucleotides, for oligonucleotides of a single oligonucleotide
type. In some embodiments, the present disclosure provides chirally
controlled C9orf72 oligonucleotide composition of a first plurality
of oligonucleotides in that the composition is enriched, relative
to a substantially racemic preparation of the same
oligonucleotides, for oligonucleotides of a single oligonucleotide
type that share: [0551] 1) a common base sequence and length;
[0552] 2) a common pattern of backbone linkages; and [0553] 3) a
common pattern of backbone chiral centers.
[0554] In some embodiments, oligonucleotides having a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers have a common pattern of
backbone phosphorus modifications and a common pattern of base
modifications. In some embodiments, oligonucleotides having a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers have a
common pattern of backbone phosphorus modifications and a common
pattern of nucleoside modifications. In some embodiments,
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers have identical structures.
[0555] In some embodiments, oligonucleotides of an oligonucleotide
type have a common pattern of backbone phosphorus modifications and
a common pattern of sugar modifications. In some embodiments,
oligonucleotides of an oligonucleotide type have a common pattern
of backbone phosphorus modifications and a common pattern of base
modifications. In some embodiments, oligonucleotides of an
oligonucleotide type have a common pattern of backbone phosphorus
modifications and a common pattern of nucleoside modifications. In
some embodiments, oligonucleotides of an oligonucleotide type are
identical.
[0556] In some embodiments, a C9orf72 oligonucleotide is a
substantially pure preparation of an oligonucleotide type in that
oligonucleotides in the composition that are not of the
oligonucleotide type are impurities form the preparation process of
said oligonucleotide type, in some case, after certain purification
procedures.
[0557] In some embodiments, oligonucleotides having a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers have a common pattern of
backbone phosphorus modifications. In some embodiments,
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers have a common pattern of backbone phosphorus
modifications and a common pattern of nucleoside modifications. In
some embodiments, oligonucleotides having a common base sequence
and length, a common pattern of backbone linkages, and a common
pattern of backbone chiral centers have a common pattern of
backbone phosphorus modifications and a common pattern of sugar
modifications. In some embodiments, oligonucleotides having a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers have a
common pattern of backbone phosphorus modifications and a common
pattern of base modifications. In some embodiments,
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers have a common pattern of backbone phosphorus
modifications and a common pattern of nucleoside modifications. In
some embodiments, oligonucleotides having a common base sequence
and length, a common pattern of backbone linkages, and a common
pattern of backbone chiral centers are identical.
[0558] In some embodiments, oligonucleotides in provided
compositions have a common pattern of backbone phosphorus
modifications. In some embodiments, a common base sequence is a
base sequence of an oligonucleotide type. In some embodiments, a
provided composition is an oligonucleotide composition that is
chirally controlled in that the composition contains a non-random
or controlled level of a first plurality of C9orf72
oligonucleotides of an individual oligonucleotide type, wherein an
oligonucleotide type is defined by: [0559] 1) base sequence; [0560]
2) pattern of backbone linkages; [0561] 3) pattern of backbone
chiral centers; and [0562] 4) pattern of backbone phosphorus
modifications.
[0563] As noted above and understood in the art, in some
embodiments, the base sequence of an oligonucleotide may refer to
the identity and/or modification status of nucleoside residues
(e.g., of sugar and/or base components, relative to standard
naturally occurring nucleotides such as adenine, cytosine,
guanosine, thymine, and uracil) in the oligonucleotide and/or to
the hybridization character (i.e., the ability to hybridize with
particular complementary residues) of such residues.
[0564] In some embodiments, a particular oligonucleotide type may
be defined by [0565] 1A) base identity; [0566] 1B) pattern of base
modification; [0567] 1C) pattern of sugar modification; [0568] 2)
pattern of backbone linkages; [0569] 3) pattern of backbone chiral
centers; and [0570] 4) pattern of backbone phosphorus
modifications. Thus, in some embodiments, oligonucleotides of a
particular type may share identical bases but differ in their
pattern of base modifications and/or sugar modifications. In some
embodiments, oligonucleotides of a particular type may share
identical bases and pattern of base modifications (including, e.g.,
absence of base modification), but differ in pattern of sugar
modifications.
[0571] In some embodiments, purity of a C9orf72 oligonucleotide can
be controlled by stereoselectivity of each coupling step in its
preparation process. In some embodiments, a coupling step has a
stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the
new internucleotidic linkage formed from the coupling step has the
intended stereochemistry). After such a coupling step, the new
internucleotidic linkage formed may be referred to have a 60%
purity. In some embodiments, each coupling step has a
stereoselectivity of at least 60%. In some embodiments, each
coupling step has a stereoselectivity of at least 70%. In some
embodiments, each coupling step has a stereoselectivity of at least
80%. In some embodiments, each coupling step has a
stereoselectivity of at least 85%. In some embodiments, each
coupling step has a stereoselectivity of at least 90%. In some
embodiments, each coupling step has a stereoselectivity of at least
91%. In some embodiments, each coupling step has a
stereoselectivity of at least 92%. In some embodiments, each
coupling step has a stereoselectivity of at least 93%. In some
embodiments, each coupling step has a stereoselectivity of at least
94%. In some embodiments, each coupling step has a
stereoselectivity of at least 95%. In some embodiments, each
coupling step has a stereoselectivity of at least 96%. In some
embodiments, each coupling step has a stereoselectivity of at least
97%. In some embodiments, each coupling step has a
stereoselectivity of at least 98%. In some embodiments, each
coupling step has a stereoselectivity of at least 99%. In some
embodiments, each coupling step has a stereoselectivity of at least
99.5%. In some embodiments, each coupling step has a
stereoselectivity of virtually 100%. In some embodiments, a
coupling step has a stereoselectivity of virtually 100% in that all
detectable product from the coupling step by an analytical method
(e.g., NMR, HPLC, etc) has the intended stereoselectivity.
[0572] Among other things, the present disclosure recognizes that
combinations of oligonucleotide structural elements (e.g., patterns
of chemical modifications, backbone linkages, backbone chiral
centers, and/or backbone phosphorus modifications) can provide
surprisingly improved properties such as bioactivities.
[0573] In some embodiments, provided chirally controlled (and/or
stereochemically pure) preparations are of oligonucleotides that
include one or more modified backbone linkages, bases, and/or
sugars.
[0574] In some embodiments, provided chirally controlled (and/or
stereochemically pure) preparations are of oligonucleotides having
a common base sequence of at least 15 bases. In some embodiments,
provided chirally controlled (and/or stereochemically pure)
preparations are of oligonucleotides having a common base sequence
of at least 16 bases. In some embodiments, provided chirally
controlled (and/or stereochemically pure) preparations are of
oligonucleotides having a common base sequence of at least 17
bases. In some embodiments, provided chirally controlled (and/or
stereochemically pure) preparations are of oligonucleotides having
a common base sequence of at least 18 bases. In some embodiments,
provided chirally controlled (and/or stereochemically pure)
preparations are of oligonucleotides having a common base sequence
of at least 19 bases. In some embodiments, provided chirally
controlled (and/or stereochemically pure) preparations are of
oligonucleotides having a common base sequence of at least 20
bases. In some embodiments, provided chirally controlled (and/or
stereochemically pure) preparations are of oligonucleotides having
a common base sequence of at least 21 bases. In some embodiments,
provided chirally controlled (and/or stereochemically pure)
preparations are of oligonucleotides having a common base sequence
of at least 22 bases. In some embodiments, provided chirally
controlled (and/or stereochemically pure) preparations are of
oligonucleotides having a common base sequence of at least 23
bases. In some embodiments, provided chirally controlled (and/or
stereochemically pure) preparations are of oligonucleotides having
a common base sequence of at least 24 bases. In some embodiments,
provided chirally controlled (and/or stereochemically pure)
preparations are of oligonucleotides having a common base sequence
of at least 25 bases. In some embodiments, provided chirally
controlled (and/or stereochemically pure) preparations are of
oligonucleotides having a common base sequence of at least 30, 35,
40, 45, 50, 55, 60, 65, 70, or 75 bases.
[0575] In some embodiments, provided compositions comprise
oligonucleotides containing one or more residues which are modified
at the sugar moiety. In some embodiments, provided compositions
comprise oligonucleotides containing one or more residues which are
modified at the 2' position of the sugar moiety (referred to herein
as a "2'-modification"). Examples of such modifications are
described above and herein and include, but are not limited to,
2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, 5'-vinyl, Morpholino,
S-cEt, etc. In some embodiments, provided compositions comprise
oligonucleotides containing one or more residues which are
2'-modified. For example, in some embodiments, provided
oligonucleotides contain one or more residues which are
2'-O-methoxyethyl (2'-MOE)-modified residues. In some embodiments,
provided compositions comprise oligonucleotides which do not
contain any 2'-modifications. In some embodiments, provided
compositions are oligonucleotides which do not contain any 2'-MOE
residues. That is, in some embodiments, provided oligonucleotides
are not MOE-modified. Additional example sugar modifications are
described in the present disclosure.
[0576] In some embodiments, one or more is one. In some
embodiments, one or more is two. In some embodiments, one or more
is three. In some embodiments, one or more is four. In some
embodiments, one or more is five. In some embodiments, one or more
is six. In some embodiments, one or more is seven. In some
embodiments, one or more is eight. In some embodiments, one or more
is nine. In some embodiments, one or more is ten. In some
embodiments, one or more is at least one. In some embodiments, one
or more is at least two. In some embodiments, one or more is at
least three. In some embodiments, one or more is at least four. In
some embodiments, one or more is at least five. In some
embodiments, one or more is at least six. In some embodiments, one
or more is at least seven. In some embodiments, one or more is at
least eight. In some embodiments, one or more is at least nine. In
some embodiments, one or more is at least ten.
[0577] As a person having ordinary skill in the art understands,
provided oligonucleotide compositions and methods have various uses
as known by a person having ordinary skill in the art. Methods for
assessing provided compositions, and properties and uses thereof,
are also widely known and practiced by a person having ordinary
skill in the art. Example properties, uses, and/or methods include
but are not limited to those described in WO/2014/012081 and
WO/2015/107425.
[0578] In some embodiments, a chiral internucleotidic linkage has
the structure of Formula I. In some embodiments, a chiral
internucleotidic linkage is phosphorothioate. In some embodiments,
each chiral internucleotidic linkage in a single oligonucleotide of
a provided composition independently has the structure of Formula
I. In some embodiments, each chiral internucleotidic linkage in a
single oligonucleotide of a provided composition is a
phosphorothioate.
[0579] In some embodiments, oligonucleotides of the present
disclosure comprise one or more modified sugar moieties. In some
embodiments, C9orf72 oligonucleotides of the present disclosure
comprise one or more modified base moieties. As known by a person
of ordinary skill in the art and described in the disclosure,
various modifications can be introduced to a sugar and/or moiety.
For example, in some embodiments, a modification is a modification
described in U.S. Pat. No. 9,006,198, WO2014/012081 and
WO/2015/107425, the sugar and base modifications of each of which
are incorporated herein by reference.
[0580] In some embodiments, a sugar modification is a
2'-modification. Commonly used 2'-modifications include but are not
limited to 2'-OR.sup.1, wherein R.sup.1 is not hydrogen. In some
embodiments, a modification is 2'-OR, wherein R is optionally
substituted aliphatic. In some embodiments, a modification is
2'-OMe. In some embodiments, a modification is 2'-O-MOE. In some
embodiments, the present disclosure demonstrates that inclusion
and/or location of particular chirally pure internucleotidic
linkages can provide stability improvements comparable to or better
than those achieved through use of modified backbone linkages,
bases, and/or sugars. In some embodiments, a provided single
oligonucleotide of a provided composition has no modifications on
the sugars. In some embodiments, a provided single oligonucleotide
of a provided composition has no modifications on 2'-positions of
the sugars (i.e., the two groups at the 2'-position are either
--H/--H or -H/--OH). In some embodiments, a provided single
oligonucleotide of a provided composition does not have any 2'-MOE
modifications.
[0581] In some embodiments, a 2'-modification is --O-L- or -L-which
connects the 2'-carbon of a sugar moiety to another carbon of a
sugar moiety. In some embodiments, a 2'-modification is --O-L- or
-L-which connects the 2'-carbon of a sugar moiety to the 4'-carbon
of a sugar moiety. In some embodiments, a 2'-modification is S-cEt.
In some embodiments, a modified sugar moiety is an LNA moiety.
[0582] In some embodiments, a 2'-modification is --F. In some
embodiments, a 2'-modification is FANA. In some embodiments, a
2'-modification is FRNA.
[0583] In some embodiments, a sugar modification is a
5'-modification, e.g., R-5'-Me, S-5'-Me, etc.
[0584] In some embodiments, a sugar modification changes the size
of the sugar ring. In some embodiments, a sugar modification is the
sugar moiety in FHNA.
[0585] In some embodiments, a sugar modification replaces a sugar
moiety with another cyclic or acyclic moiety. Examples of such
moieties are widely known in the art, including but not limited to
those used in morpholino (optionally with its phosphorodiamidate
linkage), glycol nucleic acids, etc.
[0586] In some embodiments, a single oligonucleotide in a provided
composition has at least about 25% of its internucleotidic linkages
in Sp configuration. In some embodiments, a single oligonucleotide
in a provided composition has at least about 30% of its
internucleotidic linkages in Sp configuration. In some embodiments,
a single oligonucleotide in a provided composition has at least
about 35% of its internucleotidic linkages in Sp configuration. In
some embodiments, a single oligonucleotide in a provided
composition has at least about 40% of its internucleotidic linkages
in Sp configuration. In some embodiments, a single oligonucleotide
in a provided composition has at least about 45% of its
internucleotidic linkages in Sp configuration. In some embodiments,
a single oligonucleotide in a provided composition has at least
about 50% of its internucleotidic linkages in Sp configuration. In
some embodiments, a single oligonucleotide in a provided
composition has at least about 55% of its internucleotidic linkages
in Sp configuration. In some embodiments, a single oligonucleotide
in a provided composition has at least about 60% of its
internucleotidic linkages in Sp configuration. In some embodiments,
a single oligonucleotide in a provided composition has at least
about 65% of its internucleotidic linkages in Sp configuration. In
some embodiments, a single oligonucleotide in a provided
composition has at least about 70% of its internucleotidic linkages
in Sp configuration. In some embodiments, a single oligonucleotide
in a provided composition has at least about 75% of its
internucleotidic linkages in Sp configuration. In some embodiments,
a single oligonucleotide in a provided composition has at least
about 80% of its internucleotidic linkages in Sp configuration. In
some embodiments, a single oligonucleotide in a provided
composition has at least about 85% of its internucleotidic linkages
in Sp configuration. In some embodiments, a single oligonucleotide
in a provided composition has at least about 90% of its
internucleotidic linkages in Sp configuration.
[0587] In some embodiments, a C9orf72 oligonucleotide is or
comprises a C9orf72 oligonucleotide selected from the group
consisting of WV-3536, WV-3537, WV-3538, WV-3539, WV-3540, WV-3541,
WV-3542, WV-3561, WV-3562, WV-3563, WV-3564, WV-3565, WV-3566,
WV-3567, WV-3568, WV-3569, WV-3570, WV-3571, WV-3572, WV-3573,
WV-3574, WV-3575, WV-3576, WV-3577, WV-3578, WV-3579, WV-3580,
WV-3581, WV-3582, WV-3583, WV-3584, WV-3585, WV-3586, WV-3587,
WV-3588, WV-3589, WV-3590, WV-3591, WV-3592, WV-3593, WV-3594,
WV-3595, WV-3596, WV-3597, WV-3598, WV-3599, WV-3600, WV-3601,
WV-3602, WV-3603, WV-3604, WV-3605, WV-3606, WV-3607, WV-3608,
WV-3609, WV-3610, WV-3611, WV-3612, WV-3613, WV-3614, WV-3615,
WV-3616, WV-3617, WV-3618, WV-3619, WV-3620, WV-3621, WV-3622,
WV-3623, WV-3624, WV-3625, WV-3626, WV-3627, WV-3628, WV-3629,
WV-3630, WV-3631, WV-3632, WV-3633, WV-3634, WV-3635, WV-3636,
WV-3637, WV-3638, WV-3639, WV-3640, WV-3641, WV-3642, WV-3643,
WV-3644, WV-3645, WV-3646, WV-3647, WV-3648, WV-3649, WV-3650,
WV-3651, WV-3652, WV-3653, WV-3654, WV-3655, WV-3656, WV-3657,
WV-3658, WV-3659, WV-3660, WV-3661, WV-3662, WV-3663, WV-3664,
WV-3665, WV-3666, WV-3667, WV-3668, WV-3669, WV-3670, WV-3671,
WV-3672, WV-3673, WV-3674, WV-3675, WV-3676, WV-3677, WV-3678,
WV-3679, WV-3680, WV-3681, WV-3682, WV-3683, WV-3684, WV-3685,
WV-3686, WV-3687, WV-3688, WV-3689, WV-3690, WV-3691, WV-3692,
WV-3693, WV-3694, WV-3695, WV-3696, WV-3697, WV-3698, WV-3699,
WV-3700, WV-3701, WV-3702, WV-3703, WV-3704, WV-3705, WV-3706,
WV-3707, WV-3708, WV-3709, WV-3710, WV-3711, WV-3712, WV-3713,
WV-3714, WV-3715, WV-3716, WV-3717, WV-3718, WV-3719, WV-3720,
WV-3721, WV-3722, WV-3723, WV-3724, WV-3725, WV-3726, WV-3727,
WV-3728, WV-3729, WV-3730, WV-3731, WV-3732, WV-3733, WV-3734,
WV-3735, WV-3736, WV-3737, WV-3738, WV-3739, WV-3740, WV-3741,
WV-3742, WV-3743, WV-3744, WV-3745, WV-3746, WV-3747, WV-3748,
WV-3749, WV-3750, WV-3751, WV-3752, WV-5905, WV-5906, WV-5907,
WV-5908, WV-5909, WV-5910, WV-5911, WV-5912, WV-5913, WV-5914,
WV-5915, WV-5916, WV-5917, WV-5918, WV-5919, WV-5920, WV-5921,
WV-5922, WV-5923, WV-5924, WV-5925, WV-5926, WV-5927, WV-5928,
WV-5929, WV-5930, WV-5931, WV-5932, WV-5933, WV-5934, WV-5935,
WV-5936, WV-5937, WV-5938, WV-5939, WV-5940, WV-5941, WV-5942,
WV-5943, WV-5944, WV-5945, WV-5946, WV-5947, WV-5948, WV-5949,
WV-5950, WV-5951, WV-5952, WV-5953, WV-5954, WV-5955, WV-5956,
WV-5957, WV-5958, WV-5959, WV-5960, WV-5961, WV-5962, WV-5963,
WV-5964, WV-5965, WV-5966, WV-5967, WV-5968, WV-5969, WV-5970,
WV-5971, WV-5972, WV-5973, WV-5974, WV-5975, WV-5976, WV-5977,
WV-5978, WV-5979, WV-5980, WV-5981, WV-5982, WV-5983, WV-5984,
WV-5985, WV-5986, WV-5987, WV-5988, WV-5989, WV-5990, WV-5991,
WV-5992, WV-5993, WV-5994, WV-5995, WV-5996, WV-5997, WV-5998,
WV-5999, WV-6000, WV-6408, WV-6471, WV-6472, WV-6473, WV-6474,
WV-6475, WV-6476, WV-6477, WV-6478, WV-6479, WV-6480, WV-6481,
WV-6482, WV-6483, WV-6484, WV-6485, WV-6486, WV-6487, WV-6488,
WV-6489, WV-6490, WV-6491, WV-6492, WV-6831, WV-6832, WV-6833,
WV-6834, WV-6835, WV-6836, WV-6837, WV-6838, WV-6839, WV-6840,
WV-6841, WV-6842, WV-6843, WV-6844, WV-6845, WV-6846, WV-6847,
WV-6848, WV-6849, WV-6850, WV-6851, WV-6852, WV-6853, WV-6854,
WV-6855, WV-6856, WV-6857, WV-6858, WV-6859, WV-6860, WV-6861,
WV-6862, WV-6863, WV-6864, WV-6865, WV-6866, WV-6867, WV-6868,
WV-6869, WV-6870, WV-6871, WV-6872, WV-6873, WV-6874, WV-6875,
WV-6876, WV-6877, WV-6878, WV-6879, WV-6880, WV-6881, WV-6882,
WV-6883, WV-6884, WV-6885, WV-6886, WV-6887, WV-6888, WV-6889,
WV-6890, WV-6891, WV-6892, WV-6893, WV-6894, WV-6895, WV-6896,
WV-6897, WV-6898, WV-6899, WV-6900, WV-6901, WV-6902, WV-6903,
WV-6904, WV-6905, WV-6906, WV-6907, WV-6908, WV-6909, WV-6910,
WV-6911, WV-6912, WV-6913, WV-6914, WV-6915, WV-6916, WV-6917,
WV-6918, WV-6919, WV-6920, WV-6921, WV-6922, WV-6923, WV-6924,
WV-6925, WV-6926, WV-6927, WV-6928, WV-6929, WV-6930, WV-6931,
WV-6932, WV-6933, WV-6934, WV-6935, WV-6936, WV-6937, WV-6938,
WV-6939, WV-6940, WV-6941, WV-6942, WV-6943, WV-6944, WV-6945,
WV-6946, WV-6947, WV-6948, WV-6949, WV-6950, WV-6951, WV-6952,
WV-6953, WV-6954, WV-6955, WV-6956, WV-6957, WV-6958, WV-6959,
WV-6960, WV-6961, WV-6962, WV-6963, WV-6964, WV-6965, WV-6966,
WV-6967, WV-6968, WV-6969, WV-6970, WV-6971, WV-6972, WV-6973,
WV-6974, WV-6975, WV-6976, WV-6977, WV-6978, WV-6979, WV-6980,
WV-6981, WV-6982, WV-6983, WV-6984, WV-6985, WV-6986, WV-6987,
WV-6988, WV-6989, WV-6990, WV-6991, WV-6992, WV-6993, WV-6994,
WV-6995, WV-6996, WV-6997, WV-6998, WV-6999, WV-7000, WV-7001,
WV-7002, WV-7003, WV-7004, WV-7005, WV-7006, WV-7007, WV-7008,
WV-7009, WV-7010, WV-7011, WV-7012, WV-7013, WV-7014, WV-7015,
WV-7016, WV-7017, WV-7018, WV-7019, WV-7020, WV-7021, WV-7022,
WV-7023, WV-7024, WV-7025, WV-7026, WV-7027, WV-7028, WV-7029,
WV-7030, WV-7031, WV-7032, WV-7033, WV-7034, WV-7035, WV-7036,
WV-7037, WV-7038, WV-7039, WV-7040, WV-7041, WV-7042, WV-7043,
WV-7044, WV-7045, WV-7046, WV-7047, WV-7048, WV-7049, WV-7050,
WV-7051, WV-7052, WV-7053, WV-7054, WV-7055, WV-7056, WV-7057,
WV-7058, WV-7059, WV-7060, WV-7061, WV-7062, WV-7063, WV-7064,
WV-7065, WV-7066, WV-7067, WV-7068, WV-7069, WV-7070, WV-7071,
WV-7072, WV-7073, WV-7074, WV-7075, WV-7076, WV-7077, WV-7078,
WV-7079, WV-7080, WV-7081, WV-7082, WV-7083, WV-7084, WV-7085,
WV-7086, WV-7087, WV-7088, WV-7089, WV-7090, WV-7091, WV-7092,
WV-7093, WV-7094, WV-7095, WV-7096, WV-7097, WV-7098, WV-7099,
WV-7100, WV-7101, WV-7102, WV-7103, WV-7117, WV-7118, WV-7119,
WV-7120, WV-7121, WV-7122, WV-7123, WV-7124, WV-7125, WV-7126,
WV-7127, WV-7128, WV-7129, WV-7130, WV-7131, WV-7132, WV-7405,
WV-7434, WV-7435, WV-7601, WV-7602, WV-7603, WV-7604, WV-7605,
WV-7606, WV-7657, WV-7658, WV-7659, WV-7773, WV-7774, WV-7775,
WV-7866, WV-8005, WV-8006, WV-8007, WV-8008, WV-8009, WV-8010,
WV-8011, WV-8012, WV-8114, WV-8115, WV-8116, WV-8117, WV-8118,
WV-8119, WV-8120, WV-8121, WV-8122, WV-8123, WV-8124, WV-8125,
WV-8126, WV-8127, WV-8128, WV-8129, WV-8311, WV-8312, WV-8313,
WV-8314, WV-8315, WV-8316, WV-8317, WV-8318, WV-8319, WV-8320,
WV-8321, WV-8322, WV-8329, WV-8444, WV-8445, WV-8446, WV-8447,
WV-8452, WV-8453, WV-8454, WV-8455, WV-8456, WV-8457, WV-8458,
WV-8459, WV-8460, WV-8461, WV-8462, WV-8463, WV-8464, WV-8465,
WV-8466, WV-8467, WV-8468, WV-8469, WV-8470, WV-8471, WV-8472,
WV-8473, WV-8474, WV-8475, WV-8476, WV-8477, WV-8547, WV-8548,
WV-8549, WV-8550, WV-8551, WV-8568, WV-8569, WV-8594, WV-8595,
WV-8691, WV-8692, WV-8693, WV-8694, WV-8695, WV-8696, WV-9062,
WV-9063, WV-9228, WV-9285, WV-9286, WV-9380, WV-9381, WV-9394,
WV-9395, WV-9396, WV-9397, WV-9398, WV-9399, and WV-9421, and any
C9orf72 oligonucleotide described herein.
[0588] Those skilled in the art, reading the present specification,
will appreciate that the present disclosure specifically does not
exclude the possibility that any C9orf72 or other oligonucleotide
described herein which is labeled as an antisense oligonucleotide
(ASO) may also or alternatively operate through another mechanism
(e.g., as a ssRNAi utilizing RISC); the disclosure also notes that
various oligonucleotides may operate via different mechanisms
(utilizing RNase H, sterically blocking translation or other
post-transcriptional processes, changing the conformation of a
C9orf72 target nucleic acid, etc.).
Chirally Controlled Oligonucleotides and Chirally Controlled
Oligonucleotide Compositions
[0589] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, provided C9orf72 oligonucleotides are capable of
directing a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product by sterically blocking
translation after annealing to a C9orf72 target gene mRNA, and/or
by altering or interfering with mRNA splicing. In some embodiments,
a C9orf72 target gene comprises a repeat expansion. In some
embodiments, provided C9orf72 oligonucleotides are chirally
controlled.
[0590] The present disclosure provides chirally controlled C9orf72
oligonucleotides, and chirally controlled C9orf72 oligonucleotide
compositions which are of high crude purity and of high
diastereomeric purity. In some embodiments, the present disclosure
provides chirally controlled C9orf72 oligonucleotides, and chirally
controlled C9orf72 oligonucleotide compositions which are of high
crude purity. In some embodiments, the present disclosure provides
chirally controlled C9orf72 oligonucleotides, and chirally
controlled C9orf72 oligonucleotide compositions which are of high
diastereomeric purity.
[0591] In some embodiments, a C9orf72 oligonucleotide is a
substantially pure preparation of a C9orf72 oligonucleotide type in
that oligonucleotides in the composition that are not of the
oligonucleotide type are impurities form the preparation process of
said oligonucleotide type, in some case, after certain purification
procedures.
[0592] In some embodiments, the present disclosure provides C9orf72
oligonucleotides comprising one or more diastereomerically pure
internucleotidic linkages with respect to the chiral linkage
phosphorus. In some embodiments, the present disclosure provides
C9orf72 oligonucleotides comprising one or more diastereomerically
pure internucleotidic linkages having the structure of Formula I.
In some embodiments, the present disclosure provides C9orf72
oligonucleotides comprising one or more diastereomerically pure
internucleotidic linkages with respect to the chiral linkage
phosphorus, and one or more phosphate diester linkages. In some
embodiments, the present disclosure provides C9orf72
oligonucleotides comprising one or more diastereomerically pure
internucleotidic linkages having the structure of Formula I, and
one or more phosphate diester linkages. In some embodiments, the
present disclosure provides C9orf72 oligonucleotides comprising one
or more diastereomerically pure internucleotidic linkages having
the structure of Formula I-c, and one or more phosphate diester
linkages. In some embodiments, such oligonucleotides are prepared
by using stereoselective oligonucleotide synthesis, as described in
this application, to form pre-designed diastereomerically pure
internucleotidic linkages with respect to the chiral linkage
phosphorus. Example internucleotidic linkages, including those
having structures of Formula I, are further described below.
[0593] Internucleotidic Linkages
[0594] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, provided C9orf72 oligonucleotides comprise any
internucleotidic linkage described herein or known in the art.
[0595] A non-limiting example of an internucleotidic linkage or
unmodified internucleotidic linkage is a phosphodiester;
non-limiting examples of modified internucleotidic linkages include
those in which one or more oxygen of a phosphodiester has been
replaced by, as non-limiting examples, sulfur (as in a
phosphorothioate), H, alkyl, or another moiety or element which is
not oxygen. A non-limiting example of an internucleotidic linkage
is a moiety which does not a comprise a phosphorus but serves to
link two sugars. A non-limiting example of an internucleotidic
linkage is a moiety which does not a comprise a phosphorus but
serves to link two sugars in the backbone of a C9orf72
oligonucleotide. Disclosed herein are additional non-limiting
examples of nucleotides, modified nucleotides, nucleotide analogs,
internucleotidic linkages, modified internucleotidic linkages,
bases, modified bases, and base analogs, sugars, modified sugars,
and sugar analogs, and nucleosides, modified nucleosides, and
nucleoside analogs.
[0596] In certain embodiments, a internucleotidic linkage has the
structure of Formula I
##STR00103##
wherein each variable is as defined and described below. In some
embodiments, a linkage of Formula I is chiral. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide comprising one or more modified internucleotidic
linkages of Formula I. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising
one or more modified internucleotidic linkages of Formula I, and
wherein individual internucleotidic linkages of Formula I within
the oligonucleotide have different P-modifications relative to one
another. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising one or more
modified internucleotidic linkages of Formula I, and wherein
individual internucleotidic linkages of Formula I within the
oligonucleotide have different --X-L-R.sup.1 relative to one
another. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising one or more
modified internucleotidic linkages of Formula I, and wherein
individual internucleotidic linkages of Formula I within the
oligonucleotide have different X relative to one another. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising one or more modified
internucleotidic linkages of Formula I, and wherein individual
internucleotidic linkages of Formula I within the oligonucleotide
have different -L-R.sup.1 relative to one another. In some
embodiments, a chirally controlled C9orf72 oligonucleotide is a
C9orf72 oligonucleotide in a provided composition that is of the
particular oligonucleotide type. In some embodiments, a chirally
controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in
a provided composition that has the common base sequence and
length, the common pattern of backbone linkages, and the common
pattern of backbone chiral centers. In some embodiments, a chirally
controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in
a chirally controlled composition that is of the particular
oligonucleotide type, and the chirally controlled C9orf72
oligonucleotide is of the type. In some embodiments, a chirally
controlled C9orf72 oligonucleotide is a C9orf72 oligonucleotide in
a provided composition that comprises a non-random or controlled
level of a plurality of oligonucleotides that share a common base
sequence, a common pattern of backbone linkages, and a common
pattern of backbone chiral centers, and the chirally controlled
C9orf72 oligonucleotide shares the common base sequence, the common
pattern of backbone linkages, and the common pattern of backbone
chiral centers.
[0597] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different stereochemistry and/or different
P-modifications relative to one another. In some embodiments, the
present disclosure provides a chirally controlled C9orf72
oligonucleotide, wherein at least two of the individual
internucleotidic linkages within the oligonucleotide have different
stereochemistry relative to one another, and wherein at least a
portion of the structure of the chirally controlled C9orf72
oligonucleotide is characterized by a repeating pattern of
alternating stereochemistry.
[0598] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different P-modifications relative to one
another, in that they have different X atoms in their --XLR.sup.1
moieties, and/or in that they have different L groups in their
--XLR.sup.1 moieties, and/or that they have different R.sup.1 atoms
in their --XLR.sup.1 moieties, wherein XLR.sup.1 is equivalent to
X-L-R.sup.1 and X, L, and R.sup.1 are as defined in Formula I,
disclosed herein.
[0599] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different stereochemistry and/or different
P-modifications relative to one another and the oligonucleotide has
a structure represented by the following formula:
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . S.sup.BnxR.sup.Bny]
wherein: each R.sup.B independently represents a block of
nucleotide units having the R configuration at the linkage
phosphorus; each S.sup.B independently represents a block of
nucleotide units having the S configuration at the linkage
phosphorus; each of n1-ny is zero or an integer, with the
requirement that at least one odd n and at least one even n must be
non-zero so that the oligonucleotide includes at least two
individual internucleotidic linkages with different stereochemistry
relative to one another; and wherein the sum of n1-ny is between 2
and 200, and in some embodiments is between a lower limit selected
from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more and an
upper limit selected from the group consisting of 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 130, 140, 150, 160, 170, 180, 190, and 200, the upper
limit being larger than the lower limit.
[0600] In some such embodiments, each n has the same value; in some
embodiments, each even n has the same value as each other even n;
in some embodiments, each odd n has the same value each other odd
n; in some embodiments, at least two even ns have different values
from one another; in some embodiments, at least two odd ns have
different values from one another.
[0601] In some embodiments, at least two adjacent ns are equal to
one another, so that a provided C9orf72 oligonucleotide includes
adjacent blocks of S stereochemistry linkages and R stereochemistry
linkages of equal lengths. In some embodiments, provided C9orf72
oligonucleotides include repeating blocks of S and R
stereochemistry linkages of equal lengths. In some embodiments,
provided C9orf72 oligonucleotides include repeating blocks of S and
R stereochemistry linkages, where at least two such blocks are of
different lengths from one another; in some such embodiments each S
stereochemistry block is of the same length, and is of a different
length from each R stereochemistry length, which may optionally be
of the same length as one another.
[0602] In some embodiments, at least two skip-adjacent ns are equal
to one another, so that a provided C9orf72 oligonucleotide includes
at least two blocks of linkages of a first stereochemistry that are
equal in length to one another and are separated by a block of
linkages of the other stereochemistry, which separating block may
be of the same length or a different length from the blocks of
first stereochemistry.
[0603] In some embodiments, ns associated with linkage blocks at
the ends of a provided C9orf72 oligonucleotide are of the same
length. In some embodiments, provided C9orf72 oligonucleotides have
terminal blocks of the same linkage stereochemistry. In some such
embodiments, the terminal blocks are separated from one another by
a middle block of the other linkage stereochemistry.
[0604] In some embodiments, a provided C9orf72 oligonucleotide of
formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . .
.sup.BnxR.sup.Bny] is a stereoblockmer. In some embodiments, a
provided C9orf72 oligonucleotide of formula
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . S.sup.BnxR.sup.Bny] is
a stereoskipmer. In some embodiments, a provided C9orf72
oligonucleotide of formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 .
. . S.sup.BnxR.sup.Bny] is a stereoaltmer. In some embodiments, a
provided C9orf72 oligonucleotide of formula
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4.sup.BnxR.sup.Bny] is a
gapmer.
[0605] In some embodiments, a provided C9orf72 oligonucleotide of
formula [S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . .
S.sup.BnxR.sup.Bny] is of any of the above described patterns and
further comprises patterns of P-modifications. For instance, in
some embodiments, a provided C9orf72 oligonucleotide of formula
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4S.sup.BnxR.sup.Bny] and is a
stereoskipmer and P-modification skipmer. In some embodiments, a
provided C9orf72 oligonucleotide of formula
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . S.sup.BnxR.sup.Bny] and
is a stereoblockmer and P-modification altmer. In some embodiments,
a provided C9orf72 oligonucleotide of formula
[S.sup.Bn1R.sup.Bn2S.sup.Bn3R.sup.Bn4 . . . S.sup.BnxR.sup.Bny] and
is a stereoaltmer and P-modification blockmer.
[0606] In some embodiments, a provided C9orf72 oligonucleotide of
formula [S.sup.Bn1R.sup.Bn2.sup.Bn3R.sup.Bn4 . . .
S.sup.BnxR.sup.Bny] is a chirally controlled C9orf72
oligonucleotide comprising one or more modified internucleotidic
linkages independently having the structure of Formula I:
##STR00104##
wherein: P* is a symmetric phosphorus atom, or asymmetric
phosphorus atom that is either Rp or Sp;
W is O, S or Se;
[0607] each of X, Y and Z is independently --O--, --S--,
--N(-L-R')--, or L; [0608] L is a covalent bond or an optionally
substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein
one or more methylene units of L are optionally and independently
replaced by an optionally substituted group selected from
C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene,
--C.ident.C--, a C.sub.1-C.sub.6 heteroaliphatic moiety,
--C(R').sub.2--, --Cy-, --O--, --S--, --S--S--, --N(R')--,
--C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--,
--N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--, --S(O)--,
--S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--SC(O)--, --C(O)S--, --OC(O)--, and --C(O)O--; [0609] R.sup.1 is
halogen, R, or an optionally substituted C.sub.1-C.sub.50 aliphatic
wherein one or more methylene units are optionally and
independently replaced by an optionally substituted group selected
from C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene, --CC, a
C.sub.1-C.sub.6 heteroaliphatic moiety, --C(R').sub.2--, --Cy-,
--O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R')--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--N(R')S(O).sub.2--, --SC(O)--, --C(O)S--, --OC(O)--, and --C(O)O--
[0610] each R' is independently --R, --C(O)R, --CO.sub.2R, or
--SO.sub.2R, or: [0611] two R' are taken together with their
intervening atoms to form an optionally substituted aryl,
carbocyclic, heterocyclic, or heteroaryl ring; [0612] Cy-is an
optionally substituted bivalent ring selected from phenylene,
carbocyclylene, arylene, heteroarylene, and heterocyclylene; [0613]
each R is independently hydrogen, or an optionally substituted
group selected from C.sub.1-C.sub.6 aliphatic, carbocyclyl, aryl,
heteroaryl, and heterocyclyl; and [0614] each independently
represents a connection to a nucleoside.
[0615] In some embodiments, L is a covalent bond or an optionally
substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein
one or more methylene units of L are optionally and independently
replaced by an optionally substituted C.sub.1-C.sub.6 alkylene,
C.sub.1-C.sub.6 alkenylene, --C.ident.C--, --C(R').sub.2--, --Cy-,
--O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R')--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--N(R')S(O).sub.2--, --SC(O)--, --C(O)S--, --OC(O)--, or --C(O)O--;
[0616] R.sup.1 is halogen, R, or an optionally substituted
C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are
optionally and independently replaced by an optionally substituted
C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene,
--C.ident.C--, --C(R').sub.2--, --Cy-, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--SC(O)--, --C(O)S--, --OC(O)--, or --C(O)O--; [0617] each R' is
independently --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: [0618]
two R' on the same nitrogen are taken together with their
intervening atoms to form an optionally substituted heterocyclic or
heteroaryl ring, or [0619] two R' on the same carbon are taken
together with their intervening atoms to form an optionally
substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring;
[0620] --Cy- is an optionally substituted bivalent ring selected
from phenylene, carbocyclylene, arylene, heteroarylene, and
heterocyclylene; [0621] each R is independently hydrogen, or an
optionally substituted group selected from C.sub.1-C.sub.6
aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, and heterocyclyl;
and
[0622] each
##STR00105##
independently represents a connection to a nucleoside. In some
embodiments, a chirally controlled C9orf72 oligonucleotide
comprises one or more modified internucleotidic phosphorus
linkages. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises, e.g., a phosphorothioate or a
phosphorothioate triester linkage. In some embodiments, a chirally
controlled C9orf72 oligonucleotide comprises a phosphorothioate
triester linkage. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least two phosphorothioate
triester linkages. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least three phosphorothioate
triester linkages. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least four phosphorothioate
triester linkages. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least five phosphorothioate
triester linkages. Examples of such modified internucleotidic
phosphorus linkages are described further herein.
[0623] In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises different internucleotidic phosphorus
linkages. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least one modified internucleotidic
linkage. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least one phosphorothioate triester
linkage. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least two phosphorothioate triester
linkages. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least three phosphorothioate
triester linkages. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least four phosphorothioate
triester linkages. In some embodiments, a chirally controlled
C9orf72 oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least five phosphorothioate
triester linkages. Examples of such modified internucleotidic
phosphorus linkages are described further herein.
[0624] In some embodiments, a phosphorothioate triester linkage
comprises a chiral auxiliary, which, for example, is used to
control the stereoselectivity of a reaction. In some embodiments, a
phosphorothioate triester linkage does not comprise a chiral
auxiliary. In some embodiments, a phosphorothioate triester linkage
is intentionally maintained until and/or during the administration
to a subject.
[0625] In some embodiments, a chirally controlled C9orf72
oligonucleotide is linked to a solid support. In some embodiments,
a chirally controlled C9orf72 oligonucleotide is cleaved from a
solid support.
[0626] In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least two consecutive modified
internucleotidic linkages. In some embodiments, a chirally
controlled C9orf72 oligonucleotide comprises at least one phosphate
diester internucleotidic linkage and at least two consecutive
phosphorothioate triester internucleotidic linkages.
[0627] In some embodiments, a provided C9orf72 oligonucleotide or a
core or a wing or both wings thereof comprises a pattern of
backbone linkages. In some embodiments, a pattern of backbone
linkages is or comprises a sequence of any of: OOO, OOOO, OOOOO,
OOOOOOO, OOOOOOO, OOOOOOOO, OOOOOOOOO, OOOOOOOOOO, OXOX, OXOX,
OXXO, XOOX, XXOOXX, XOXOXOXX, OXOXOXOO, XXX, XXXX, XXXXX, XXXXXX,
XXXXXXX, XXXXXXXX, XXXXXXXXX, XXXXXXXXXX, OOOOOOOOOOOOOOOOO,
OOOOOOOOOOOOOOOOOO, OOOOOOOOOOOOOOOOOOO, OOOOOOOOOOOOOOOOOOOO,
OOOOOOOOOOOOOOOOOOOOO, OOOOOOOOOOOOOOOOOOOOOO, XOXOXOXOOOXOOXXXXXO,
XOXOXOXOXOXOOOOOOOOXX, XOXOXOXOXOXOOOOOOXX, XOXOXOXOXOXOOOOOXXX,
XOXOXOXOXOXOXOOOOOOXX, XOXOXOXOXOXOXOOOOXX, XOXOXOXOXOXOXXXXXX,
XOXOXOXOXOXOXXXXXXO, XOXOXOXOXOXOXXXXXXX, XOXOXOXOXOXOXXXXXXXXXXO,
XOXOXOXOXOXOXXXXXXXXXXX, XXOXOXOXOOOXOOXXXXXO,
XXOXOXOXOXOXOOOOOOOOXX, XXOXOXOXOXOXOOOOOOXX, XXOXOXOXOXOXOOOOOXXX,
XXOXOXOXOXOXOXOOOOOOXX, XXOXOXOXOXOXOXOOOOXX, XXOXOXOXOXOXOXXXXXX,
XXOXOXOXOXOXOXXXXXXO, XXOXOXOXOXOXOXXXXXXX,
XXOXOXOXOXOXOXXXXXXXXXXO, XXOXOXOXOXOXOXXXXXXXXXXX,
XXOXOXXXOOOXOOXXXXXO, XXOXOXXXOXOXOOOOOOOOXX, XXOXOXXXOXOXOOOOOOXX,
XXOXOXXXOXOXOOOOOXXX, XXOXOXXXOXOXOXOOOOOOXX, XXOXOXXXOXOXOXOOOOXX,
XXOXOXXXOXOXOXXXXXX, XXOXOXXXOXOXOXXXXXXO, XXOXOXXXOXOXOXXXXXXX,
XXOXOXXXOXOXOXXXXXXXXXXO, XXOXOXXXOXOXOXXXXXXXXXXX,
XXOXOXXXOXXXOOOOOOOOXX, XXOXOXXXOXXXOOOOOOXX, XXOXOXXXOXXXOOOOOXXX,
XXOXOXXXOXXXOXOOOOOOXX, XXOXOXXXOXXXOXOOOOXX, XXOXOXXXOXXXOXXXXXX,
XXOXOXXXOXXXOXXXXXXO, XXOXOXXXOXXXOXXXXXXX,
XXOXOXXXOXXXOXXXXXXXXXXO, XXOXOXXXOXXXOXXXXXXXXXXX,
XXOXOXXXXOOXOOXXXXXO, XXOXOXXXXXOXOOOOOOOOXX, XXOXOXXXXXOXOOOOOOXX,
XXOXOXXXXXOXOOOOOXXX, XXOXOXXXXXOXOXOOOOOOXX, XXOXOXXXXXOXOXOOOOXX,
XXOXOXXXXXOXOXXXXXX, XXOXOXXXXXOXOXXXXXXO, XXOXOXXXXXOXOXXXXXXX,
XXOXOXXXXXOXOXXXXXXXXXXO, XXOXOXXXXXOXOXXXXXXXXXXX,
XXOXXXOXOOOXOOXXXXXO, XXOXXXOXOXOXOOOOOOOOXX, XXOXXXOXOXOXOOOOOOXX,
XXOXXXOXOXOXOOOOOXXX, XXOXXXOXOXOXOXOOOOOOXX, XXOXXXOXOXOXOXOOOOXX,
XXOXXXOXOXOXOXXXXXX, XXOXXXOXOXOXOXXXXXXO, XXOXXXOXOXOXOXXXXXXX,
XXOXXXOXOXOXOXXXXXXXXXXO, XXOXXXOXOXOXOXXXXXXXXXXX,
XXOXXXOXOXXXOOOOOOOOXX, XXOXXXOXOXXXOOOOOOXX, XXOXXXOXOXXXOOOOOXXX,
XXOXXXOXOXXXOXOOOOOOXX, XXOXXXOXOXXXOXOOOOXX, XXOXXXOXOXXXOXXXXXX,
XXOXXXOXOXXXOXXXXXXO, XXOXXXOXOXXXOXXXXXXX,
XXOXXXOXOXXXOXXXXXXXXXXO, XXOXXXOXOXXXOXXXXXXXXXXX,
XXOXXXOXXOOXOOXXXXXO, XXOXXXOXXXOXOOOOOOOOXX, XXOXXXOXXXOXOOOOOOXX,
XXOXXXOXXXOXOOOOOXXX, XXOXXXOXXXOXOXOOOOOOXX, XXOXXXOXXXOXOXOOOOXX,
XXOXXXOXXXOXOXXXXXX, XXOXXXOXXXOXOXXXXXXO, XXOXXXOXXXOXOXXXXXXX,
XXOXXXOXXXOXOXXXXXXXXXXO, XXOXXXOXXXOXOXXXXXXXXXXX,
XXOXXXXXOOOXOOXXXXXO, XXOXXXXXOXOXOOOOOOOOXX, XXOXXXXXOXOXOOOOOOXX,
XXOXXXXXOXOXOOOOOXXX, XXOXXXXXOXOXOXOOOOOOXX, XXOXXXXXOXOXOXOOOOXX,
XXOXXXXXOXOXOXXXXXX, XXOXXXXXOXOXOXXXXXXO, XXOXXXXXOXOXOXXXXXXX,
XXOXXXXXOXOXOXXXXXXXXXXO, XXOXXXXXOXOXOXXXXXXXXXXX,
XXXOXOXOXOOOXOOXXXXXO, XXXOXOXOXOXOXOOOOOOOOXX,
XXXOXOXOXOXOXOOOOOOXX, XXXOXOXOXOXOXOOOOOXXX,
XXXOXOXOXOXOXOXOOOOOOXX, XXXOXOXOXOXOXOXOOOOXX,
XXXOXOXOXOXOXOXXXXXX, XXXOXOXOXOXOXOXXXXXXO, XXXOXOXOXOXOXOXXXXXXX,
XXXOXOXOXOXOXOXXXXXXXXXXO, XXXOXOXOXOXOXOXXXXXXXXXXX,
XXXXOXOXOOOXOOXXXXXO, XXXXOXOXOXOXOOOOOOOOXX, XXXXOXOXOXOXOOOOOOXX,
XXXXOXOXOXOXOOOOOXXX, XXXXOXOXOXOXOXOOOOOOXX, XXXXOXOXOXOXOXOOOOXX,
XXXXOXOXOXOXOXXXXXX, XXXXOXOXOXOXOXXXXXXO, XXXXOXOXOXOXOXXXXXXX,
XXXXOXOXOXOXOXXXXXXXXXXO, XXXXOXOXOXOXOXXXXXXXXXXX,
XXXXOXOXOXXXOOOOOOOOXX, XXXXOXOXOXXXOOOOOOXX, XXXXOXOXOXXXOOOOOXXX,
XXXXOXOXOXXXOXOOOOOOXX, XXXXOXOXOXXXOXOOOOXX, XXXXOXOXOXXXOXXXXXX,
XXXXOXOXOXXXOXXXXXXO, XXXXOXOXOXXXOXXXXXXX,
XXXXOXOXOXXXOXXXXXXXXXXO, XXXXOXOXOXXXOXXXXXXXXXXX,
XXXXOXOXXOOXOOXXXXXO, XXXXOXOXXOOXOOXXXXXO, XXXXOXOXXXOXOOOOOOOOXX,
XXXXOXOXXXOXOOOOOOXX, XXXXOXOXXXOXOOOOOXXX, XXXXOXOXXXOXOXOOOOOOXX,
XXXXOXOXXXOXOXOOOOXX, XXXXOXOXXXOXOXXXXXX, XXXXOXOXXXOXOXXXXXXO,
XXXXOXOXXXOXOXXXXXXX, XXXXOXOXXXOXOXXXXXXXXXXO,
XXXXOXOXXXOXOXXXXXXXXXXX, XXXXOXOXXXXXOOOOOOOOXX,
XXXXOXOXXXXXOOOOOOXX, XXXXOXOXXXXXOOOOOXXX, XXXXOXOXXXXXOXOOOOOOXX,
XXXXOXOXXXXXOXOOOOXX, XXXXOXOXXXXXOXXXXXX, XXXXOXOXXXXXOXXXXXXO,
XXXXOXOXXXXXOXXXXXXX, XXXXOXOXXXXXOXXXXXXXXXXO,
XXXXOXOXXXXXOXXXXXXXXXXX, XXXXOXXXOOOXOOXXXXXO,
XXXXOXXXOOOXOOXXXXXO, XXXXOXXXOXOXXOOOOOOOXX, XXXXOXXXOXOXXOOOOOXX,
XXXXOXXXOXOXXOOOOXXX, XXXXOXXXOXOXXXOOOOOOXX, XXXXOXXXOXOXXXOOOOXX,
XXXXOXXXOXOXXXXXXXX, XXXXOXXXOXOXXXXXXXXO, XXXXOXXXOXOXXXXXXXXX,
XXXXOXXXOXOXXXXXXXXXXXXO, XXXXOXXXOXOXXXXXXXXXXXXX,
XXXXOXXXOXXXOOOOOOOOXX, XXXXOXXXOXXXOOOOOOXX, XXXXOXXXOXXXOOOOOXXX,
XXXXOXXXOXXXOXOOOOOOXX, XXXXOXXXOXXXOXOOOOXX, XXXXOXXXOXXXOXXXXXX,
XXXXOXXXOXXXOXXXXXXO, XXXXOXXXOXXXOXXXXXXX,
XXXXOXXXOXXXOXXXXXXXXXXO, XXXXOXXXOXXXOXXXXXXXXXXX,
XXXXXXOXOOOXOOXXXXXO, XXXXXXOXOOOXOOXXXXXO, XXXXXXOXOXOXOOOOOOOOXX,
XXXXXXOXOXOXOOOOOOXX, XXXXXXOXOXOXOOOOOXXX, XXXXXXOXOXOXOXOOOOOOXX,
XXXXXXOXOXOXOXOOOOXX, XXXXXXOXOXOXOXXXXXX, XXXXXXOXOXOXOXXXXXXO,
XXXXXXOXOXOXOXXXXXXX, XXXXXXOXOXOXOXXXXXXXXXXO,
XXXXXXOXOXOXOXXXXXXXXXXX, XXXXXXOXOXXXOOOOOOOOXX,
XXXXXXOXOXXXOOOOOOXX, XXXXXXOXOXXXOOOOOXXX, XXXXXXOXOXXXOXOOOOOOXX,
XXXXXXOXOXXXOXOOOOXX, XXXXXXOXOXXXOXXXXXX, XXXXXXOXOXXXOXXXXXXO,
XXXXXXOXOXXXOXXXXXXX, XXXXXXOXOXXXOXXXXXXXXXXO,
XXXXXXOXOXXXOXXXXXXXXXXX, XXXXXXXXXXXXXXXXXXX,
XXXXXXXXXXXXXXXXXXXX, XXXXXXXXXXXXXXXXXXXXX,
XXXXXXXXXXXXXXXXXXXXXX, XXXXXXXXXXXXXXXXXXXXXXX,
XXXXXXXXXXXXXXXXXXXXXXXX, XXXXXXXXXXXXXXXXXXXXXXXXX,
XXXXXXXXXXXXXXXXXXXXXXXXXX, XXXXXXXXXXXXXXXXXXXXXXXXXXX, or
XXXXXXXXXXXXXXXXXXXXXXXXXXXX, or any span of at least 5 consecutive
internucleotidic linkages thereof, wherein O indicates a
phosphodiester, and X indicates an internucleotidic linkage or
modified internucleotidic linkage which is not phosphodiester; in
some embodiments, a modified internucleotidic linkage is a
phosphorothioate; in some embodiments, a modified internucleotidic
linkage is chirally controlled; in some embodiments, a modified
internucleotidic linkage is a chirally controlled
phosphorothioate.
[0628] In some embodiments, a C9orf72 oligonucleotide can comprise
any internucleotidic linkage described herein or known in the
art.
[0629] In some embodiments, the present disclosure provides C9orf72
oligonucleotides comprising one or more modified internucleotidic
linkages independently having the structure of Formula I, disclosed
herein. In some embodiments, a modified internucleotidic linkage is
phosphorothioate. Examples of internucleotidic linkages having the
structure of Formula I are widely known in the art.
[0630] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage has a chiral linkage phosphorus. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein at least one internucleotidic linkage has
the structure of Formula I. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
having the sequence of any oligonucleotide disclosed herein,
wherein each internucleotidic linkage has the structure of Formula
I. In some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide having the sequence of any
oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage has the structure of Formula I-c. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein each internucleotidic linkage has the
structure of Formula I-c. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
having the sequence of any oligonucleotide disclosed herein,
wherein at least one internucleotidic linkage is
##STR00106##
In some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide having the sequence of any
oligonucleotide disclosed herein, wherein each internucleotidic
linkage is
##STR00107##
[0631] In some embodiments, a modified internucleotidic linkage is
a non-negatively charged internucleotidic linkage. In some
embodiments, a modified internucleotidic linkage is a neutral
internucleotidic linkage. In some embodiments, provided
oligonucleotides comprise one or more non-negatively charged
internucleotidic linkages. In some embodiments, a non-negatively
charged internucleotidic linkage is a positively charged
internucleotidic linkage. In some embodiments, a non-negatively
charged internucleotidic linkage is a neutral internucleotidic
linkage. In some embodiments, a modified internucleotidic linkage
(e.g., a non-negatively charged internucleotidic linkage) comprises
optionally substituted triazolyl. In some embodiments, a modified
internucleotidic linkage (e.g., a non-negatively charged
internucleotidic linkage) comprises optionally substituted alkynyl.
In some embodiments, a modified internucleotidic linkage comprises
a triazole or alkyne moiety. In some embodiments, a triazole
moiety, e.g., a triazolyl group, is optionally substituted. In some
embodiments, a triazole moiety, e.g., a triazolyl group) is
substituted. In some embodiments, a triazole moiety is
unsubstituted. In some embodiments, a modified internucleotidic
linkage comprises an optionally substituted cyclic guanidine
moiety. In some embodiments, a modified internucleotidic linkage
comprises an optionally substituted cyclic guanidine moiety and has
the structure of:
##STR00108##
wherein W is O or S. In some embodiments, W is O. In some
embodiments, W is S. In some embodiments, a non-negatively charged
internucleotidic linkage is stereochemically controlled.
[0632] In some embodiments, an internucleotidic linkage comprising
a triazole moiety (e.g., an optionally substituted triazolyl group)
in a provided oligonucleotide, e.g., a C9orf72 oligonucleotide, has
the structure of:
##STR00109##
In some embodiments, an internucleotidic linkage comprising a
triazole moiety has the formula of
##STR00110##
where W is O or S. In some embodiments, an internucleotidic linkage
comprising an alkyne moiety (e.g., an optionally substituted
alkynyl group) has the formula of:
##STR00111##
wherein W is O or S. In some embodiments, an internucleotidic
linkage comprises a cyclic guanidine moiety. In some embodiments,
an internucleotidic linkage comprising a cyclic guanidine moiety
has the structure of:
##STR00112##
In some embodiments, a neutral internucleotidic linkage or
internucleotidic linkage comprising a cyclic guanidine moiety is
stereochemically controlled.
[0633] In some embodiments, a C9orf72 oligonucleotide comprises a
lipid moiety In some embodiments, an internucleotidic linkage
comprises a Tmg group
##STR00113##
In some embodiments, an internucleotidic linkage comprises a Tmg
group and has the structure of
##STR00114##
(the "Tmg internucleotidic linkage"). In some embodiments, neutral
internucleotidic linkages include internucleotidic linkages of PNA
and PMO, and an Tmg internucleotidic linkage.
[0634] In some embodiments, a non-negatively charged
internucleotidic linkage has the structure of formula I, I-a, I-b,
I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2,
II-c-1, II-c-2, II-d-1, II-d-2, etc., or a salt form thereof. In
some embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted 3-20 membered heterocyclyl or
heteroaryl group having 1-10 heteroatoms. In some embodiments, a
non-negatively charged internucleotidic linkage comprises an
optionally substituted 3-20 membered heterocyclyl or heteroaryl
group having 1-10 heteroatoms, wherein at least one heteroatom is
nitrogen. In some embodiments, such a heterocyclyl or heteroaryl
group is of a 5-membered ring. In some embodiments, such a
heterocyclyl or heteroaryl group is of a 6-membered ring.
[0635] In some embodiments, a non-negatively charged
internucleotidic linkage comprises an optionally substituted 5-20
membered heteroaryl group having 1-10 heteroatoms. In some
embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted 5-20 membered heteroaryl group
having 1-10 heteroatoms, wherein at least one heteroatom is
nitrogen. In some embodiments, a non-negatively charged
internucleotidic linkage comprises an optionally substituted 5-6
membered heteroaryl group having 1-4 heteroatoms, wherein at least
one heteroatom is nitrogen. In some embodiments, a non-negatively
charged internucleotidic linkage comprises an optionally
substituted 5-membered heteroaryl group having 1-4 heteroatoms,
wherein at least one heteroatom is nitrogen. In some embodiments, a
heteroaryl group is directly bonded to a linkage phosphorus. In
some embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted triazolyl group. In some
embodiments, a non-negatively charged internucleotidic linkage
comprises an unsubstituted triazolyl group, e.g.,
##STR00115##
In some embodiments, a non-negatively charged internucleotidic
linkage comprises a substituted triazolyl group, e.g.,
##STR00116##
[0636] In some embodiments, a non-negatively charged
internucleotidic linkage comprises an optionally substituted 5-20
membered heterocyclyl group having 1-10 heteroatoms. In some
embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted 5-20 membered heterocyclyl
group having 1-10 heteroatoms, wherein at least one heteroatom is
nitrogen. In some embodiments, a non-negatively charged
internucleotidic linkage comprises an optionally substituted 5-6
membered heterocyclyl group having 1-4 heteroatoms, wherein at
least one heteroatom is nitrogen. In some embodiments, a
non-negatively charged internucleotidic linkage comprises an
optionally substituted 5-membered heterocyclyl group having 1-4
heteroatoms, wherein at least one heteroatom is nitrogen. In some
embodiments, at least two heteroatoms are nitrogen. In some
embodiments, a heterocyclyl group is directly bonded to a linkage
phosphorus. In some embodiments, a heterocyclyl group is bonded to
a linkage phosphorus through a linker, e.g., .dbd.N-- when the
heterocyclyl group is part of a guanidine moiety who directed
bonded to a linkage phosphorus through its=N--. In some
embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted
##STR00117##
group. In some embodiments, a non-negatively charged
internucleotidic linkage comprises an substituted
##STR00118##
group. In some embodiments, a non-negatively charged
internucleotidic linkage comprises a
##STR00119##
group. In some embodiments, each R.sup.1 is independently
optionally substituted C.sub.1-6 alkyl. In some embodiments, each
R.sup.1 is independently methyl.
[0637] In some embodiments, a modified internucleotidic linkage,
e.g., a non-negatively charged internucleotidic linkage, comprises
a triazole or alkyne moiety, each of which is optionally
substituted. In some embodiments, a modified internucleotidic
linkage comprises a triazole moiety. In some embodiments, a
modified internucleotidic linkage comprises a unsubstituted
triazole moiety. In some embodiments, a modified internucleotidic
linkage comprises a substituted triazole moiety. In some
embodiments, a modified internucleotidic linkage comprises an alkyl
moiety. In some embodiments, a modified internucleotidic linkage
comprises an optionally substituted alkynyl group. In some
embodiments, a modified internucleotidic linkage comprises an
unsubstituted alkynyl group. In some embodiments, a modified
internucleotidic linkage comprises a substituted alkynyl group. In
some embodiments, an alkynyl group is directly bonded to a linkage
phosphorus.
[0638] In some embodiments, an oligonucleotide comprises different
types of internucleotidic phosphorus linkages. In some embodiments,
a chirally controlled oligonucleotide comprises at least one
natural phosphate linkage and at least one modified (non-natural)
internucleotidic linkage. In some embodiments, an oligonucleotide
comprises at least one natural phosphate linkage and at least one
phosphorothioate. In some embodiments, an oligonucleotide comprises
at least one non-negatively charged internucleotidic linkage. In
some embodiments, an oligonucleotide comprises at least one natural
phosphate linkage and at least one non-negatively charged
internucleotidic linkage. In some embodiments, an oligonucleotide
comprises at least one phosphorothioate internucleotidic linkage
and at least one non-negatively charged internucleotidic linkage.
In some embodiments, an oligonucleotide comprises at least one
phosphorothioate internucleotidic linkage, at least one natural
phosphate linkage, and at least one non-negatively charged
internucleotidic linkage. In some embodiments, oligonucleotides
comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20 or more non-negatively charged
internucleotidic linkages. In some embodiments, a non-negatively
charged internucleotidic linkage is not negatively charged in that
at a given pH in an aqueous solution less than 50%, 40%, 40%, 30%,
20%, 10%, 5%, or 1% of the internucleotidic linkage exists in a
negatively charged salt form. In some embodiments, a pH is about pH
7.4. In some embodiments, a pH is about 4-9. In some embodiments,
the percentage is less than 10%. In some embodiments, the
percentage is less than 5%. In some embodiments, the percentage is
less than 1%. In some embodiments, an internucleotidic linkage is a
non-negatively charged internucleotidic linkage in that the neutral
form of the internucleotidic linkage has no pKa that is no more
than about 1, 2, 3, 4, 5, 6, or 7 in water. In some embodiments, no
pKa is 7 or less. In some embodiments, no pKa is 6 or less. In some
embodiments, no pKa is 5 or less. In some embodiments, no pKa is 4
or less. In some embodiments, no pKa is 3 or less. In some
embodiments, no pKa is 2 or less. In some embodiments, no pKa is 1
or less. In some embodiments, pKa of the neutral form of an
internucleotidic linkage can be represented by pKa of the neutral
form of a compound having the structure of CH.sub.3-- the
internucleotidic linkage-CH.sub.3. For example, pKa of the neutral
form of an internucleotidic linkage having the structure of formula
I may be represented by the pKa of the neutral form of a compound
having the structure of
##STR00120##
pKa of
##STR00121##
can be represented by pKa
##STR00122##
In some embodiments, a non-negatively charged internucleotidic
linkage is a neutral internucleotidic linkage. In some embodiments,
a non-negatively charged internucleotidic linkage is a
positively-charged internucleotidic linkage. In some embodiments, a
non-negatively charged internucleotidic linkage comprises a
guanidine moiety. In some embodiments, a non-negatively charged
internucleotidic linkage comprises a heteroaryl base moiety. In
some embodiments, a non-negatively charged internucleotidic linkage
comprises a triazole moiety. In some embodiments, a non-negatively
charged internucleotidic linkage comprises an alkynyl moiety.
[0639] In some embodiments, a non-negatively charged
internucleotidic linkage has the structure of formula I, I-a, I-b,
I-c, I-n-1, I-n-2, I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2,
II-c-1, II-c-2, II-d-1, II-d-2, or a salt form thereof (not
negatively charged). In some embodiments, an internucleotidic
linkage, e.g., a non-negatively charged internucleotidic linkage,
has the structure of formula I-n-1 or a salt form thereof.
##STR00123##
[0640] In some embodiments, X is a covalent bond and --X-Cy-R.sup.1
is --Cy-R.sup.1. In some embodiments, --Cy- is an optionally
substituted bivalent group selected from a 5-20 membered heteroaryl
ring having 1-10 heteroatoms, and a 3-20 membered heterocyclyl ring
having 1-10 heteroatoms. In some embodiments, --Cy- is an
optionally substituted bivalent 5-20 membered heteroaryl ring
having 1-10 heteroatoms. In some embodiments, --Cy-R.sup.1 is
optionally substituted 5-20 membered heteroaryl ring having 1-10
heteroatoms, wherein at least one heteroatom is nitrogen. In some
embodiments, --Cy-R.sup.1 is optionally substituted 5-membered
heteroaryl ring having 1-4 heteroatoms, wherein at least one
heteroatom is nitrogen. In some embodiments, --Cy-R.sup.1 is
optionally substituted 6-membered heteroaryl ring having 1-4
heteroatoms, wherein at least one heteroatom is nitrogen. In some
embodiments, --Cy-R.sup.1 is optionally substituted triazolyl.
[0641] In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage, has the structure
of formula I-n-2 or a salt form thereof:
##STR00124##
[0642] In some embodiments, R.sup.1 is R'. In some embodiments, L
is a covalent bond. In some embodiments, an internucleotidic
linkage, e.g., a non-negatively charged internucleotidic linkage,
has the structure of formula I-n-3 or a salt form thereof
##STR00125##
[0643] In some embodiments, two R' on different nitrogen atoms are
taken together to form a ring as described. In some embodiments, a
formed ring is 5-membered. In some embodiments, a formed ring is
6-membered. In some embodiments, a formed ring is substituted. In
some embodiments, the two R' group that are not taken together to
form a ring are each independently R. In some embodiments, the two
R' group that are not taken together to form a ring are each
independently hydrogen or an optionally substituted C.sub.1-6
aliphatic. In some embodiments, the two R' group that are not taken
together to form a ring are each independently hydrogen or an
optionally substituted C.sub.1-6 alkyl. In some embodiments, the
two R' group that are not taken together to form a ring are the
same. In some embodiments, the two R' group that are not taken
together to form a ring are different. In some embodiments, both of
them are --CH.sub.3.
[0644] In some embodiments, a internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage, has the structure
of formula II or a salt form thereof:
##STR00126##
or a salt form thereof, wherein: [0645] P.sup.L is P(.dbd.W), P, or
P.fwdarw.B(R').sub.3; [0646] W is O, N(-L-R.sup.5), S or Se; each
of X, Y and Z is independently --O--, --S--, --N(-L-R.sup.5)--, or
L; [0647] Ring A.sup.L is an optionally substituted 3-20 membered
monocyclic, bicyclic or polycyclic ring having 0-10 heteroatoms;
[0648] each R.sup.s is independently --H, halogen, --CN, --N.sub.3,
--NO, --NO.sub.2, -L-R', -L-Si(R).sub.3, -L-OR', -L-SR',
-L-N(R').sub.2, --O-L-R', --O-L-Si(R).sub.3, --O-L-OR', --O-L-SR',
or --O-L-N(R').sub.2; [0649] g is 0-20; [0650] each L is
independently a covalent bond, or a bivalent, optionally
substituted, linear or branched group selected from a C.sub.1-30
aliphatic group and a C.sub.1-30 heteroaliphatic group having 1-10
heteroatoms, wherein one or more methylene units are optionally and
independently replaced with C.sub.1-6 alkylene, C.sub.1-6
alkenylene, --C.ident.C--, a bivalent C.sub.1-C.sub.6
heteroaliphatic group having 1-5 heteroatoms, --C(R').sub.2--,
--Cy-, --O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--,
--C(NR')--, --C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)O--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--, --C(O)S--,
--C(O)O--, --P(O)(OR')--, --P(O)(SR')--, --P(O)(R')--,
--P(O)(NR')--, --P(S)(OR')--, --P(S)(SR')--, --P(S)(R')--,
--P(S)(NR')--, --P(R')--, --P(OR')--, --P(SR')--, --P(NR')--,
--P(OR')[B(R').sub.3]--, --OP(O)(OR')O--, --OP(O)(SR')O--,
--OP(O)(R')O--, --OP(O)(NR')O--, --OP(OR')O--, --OP(SR')O--,
--OP(NR')O--, --OP(R')O--, or --OP(OR')[B(R').sub.3]O--, and one or
more CH or carbon atoms are optionally and independently replaced
with Cy.sup.L; [0651] each --Cy- is independently an optionally
substituted bivalent group selected from a C.sub.3-20
cycloaliphatic ring, a C.sub.6-20 aryl ring, a 5-20 membered
heteroaryl ring having 1-10 heteroatoms, and a 3-20 membered
heterocyclyl ring having 1-10 heteroatoms; [0652] each Cy.sup.L is
independently an optionally substituted trivalent or tetravalent
group selected from a C.sub.3-20 cycloaliphatic ring, a C.sub.6-20
aryl ring, a 5-20 membered heteroaryl ring having 1-10 heteroatoms,
and a 3-20 membered heterocyclyl ring having 1-10 heteroatoms;
[0653] each R' is independently --R, --C(O)R, --C(O)OR, or
--S(O).sub.2R; [0654] each R is independently --H, or an optionally
substituted group selected from C.sub.1-30 aliphatic, C.sub.1-30
heteroaliphatic having 1-10 heteroatoms, C.sub.6-30 aryl,
C.sub.6-30 arylaliphatic, C.sub.6-30 arylheteroaliphatic having
1-10 heteroatoms, 5-30 membered heteroaryl having 1-10 heteroatoms,
and 3-30 membered heterocyclyl having 1-10 heteroatoms, or [0655]
two R groups are optionally and independently taken together to
form a covalent bond, or, [0656] two or more R groups on the same
atom are optionally and independently taken together with the atom
to form an optionally substituted, 3-30 membered, monocyclic,
bicyclic or polycyclic ring having, in addition to the atom, 0-10
heteroatoms, or [0657] two or more R groups on two or more atoms
are optionally and independently taken together with their
intervening atoms to form an optionally substituted, 3-30 membered,
monocyclic, bicyclic or polycyclic ring having, in addition to the
intervening atoms, 0-10 heteroatoms.
[0658] In some embodiments, a internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II, has
the structure of formula II-a-1 or a salt form thereof:
##STR00127##
or a salt form thereof.
[0659] In some embodiments, a internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II, has
the structure of formula II-a-2 or a salt form thereof:
##STR00128##
or a salt form thereof.
[0660] In some embodiments, A.sup.L is bonded to --N.dbd. or L
through a carbon atom. In some embodiments, an internucleotidic
linkage, e.g., a non-negatively charged internucleotidic linkage of
formula II or II-a-1, II-a-2, has the structure of formula II-b-1
or a salt form thereof.
##STR00129##
[0661] In some embodiments, a structure of formula II-a-1 or II-a-2
may be referred to a structure of formula II-a. In some
embodiments, a structure of formula II-b-1 or II-b-2 may be
referred to a structure of formula II-b. In some embodiments, a
structure of formula II-c-1 or II-c-2 may be referred to a
structure of formula II-c. In some embodiments, a structure of
formula II-d-1 or II-d-2 may be referred to a structure of formula
II-d.
[0662] In some embodiments, A.sup.L is bonded to --N.dbd. or L
through a carbon atom. In some embodiments, an internucleotidic
linkage, e.g., a non-negatively charged internucleotidic linkage of
formula II or II-a-1, II-a-2, has the structure of formula II-b-2
or a salt form thereof.
##STR00130##
[0663] In some embodiments, Ring A.sup.L is an optionally
substituted 3-20 membered monocyclic ring having 0-10 heteroatoms
(in addition to the two nitrogen atoms for formula II-b). In some
embodiments, Ring A.sup.L is an optionally substituted 5-membered
monocyclic saturated ring.
[0664] In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II,
II-a, or II-b, has the structure of formula II-c-1 or a salt form
thereof
##STR00131##
[0665] In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II,
II-a, or II-b, has the structure of formula II-c-2 or a salt form
thereof:
##STR00132##
[0666] In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II,
II-a, II-b, or II-c has the structure of formula II-d-1 or a salt
form thereof:
##STR00133##
[0667] In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage of formula II,
II-a, II-b, or II-c has the structure of formula II-d-2 or a salt
form thereof:
##STR00134##
[0668] In some embodiments, each R' is independently optionally
substituted C.sub.1-6 aliphatic. In some embodiments, each R' is
independently optionally substituted C.sub.1-6 alkyl. In some
embodiments, each R' is independently --CH.sub.3. In some
embodiments, each R is --H.
[0669] In some embodiments, a non-negatively charged
internucleotidic linkage has the structure of
##STR00135##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00136##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00137##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00138##
In some embodiments, anon-negatively charged internucleotidic
linkage has the structure of
##STR00139##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00140##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00141##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00142##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00143##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00144##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00145##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00146##
In some embodiments, W is O. In some embodiments, W is S.
[0670] In some embodiments, each L.sup.P independently has the
structure of formula I, I-a, I-b, I-c, I-n-1, I-n-2, I-n-3, II,
II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1, II-d-2, or
a salt form thereof.
[0671] In some embodiments, the present disclosure provides
oligonucleotides comprising one or more non-negatively charged
internucleotidic linkages. In some embodiments, a non-negatively
charged internucleotidic linkage is a neutral internucleotidic
linkage. In some embodiments, the present disclosure provides
oligonucleotides comprising one or more neutral internucleotidic
linkages. In some embodiments, a non-negatively charged
internucleotidic linkage has the structure of formula I-n-1, I-n-2,
I-n-3, II, II-a-1, II-a-2, II-b-1, II-b-2, II-c-1, II-c-2, II-d-1,
II-d-2, or a salt form thereof.
[0672] In some embodiments, a non-negatively charged
internucleotidic linkage comprises a triazole moiety. In some
embodiments, a non-negatively charged internucleotidic linkage
comprises an optionally substituted triazolyl group. In some
embodiments, a non-negatively charged internucleotidic linkage has
the structure of
##STR00147##
In some embodiments, a non-negatively charged internucleotidic
linkage has the structure of
##STR00148##
In some embodiments, a non-negatively charged internucleotidic
linkage comprises a substituted triazolyl group. In some
embodiments, a non-negatively charged internucleotidic linkage has
the structure of
##STR00149##
wherein W is O or S. In some embodiments, a non-negatively charged
internucleotidic linkage comprises an optionally substituted
alkynyl group. In some embodiments, a non-negatively charged
internucleotidic linkage has the structure of
##STR00150##
wherein W is O or S.
[0673] In some embodiments, the present disclosure provides
oligonucleotides comprising an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage, which comprises a
cyclic guanidine moiety. In some embodiments, an internucleotidic
linkage comprises a cyclic guanidine and has the structure of:
##STR00151##
In some embodiments, an internucleotidic linkage, e.g., a
non-negatively charged internucleotidic linkage, comprising a
cyclic guanidine is stereochemically controlled.
[0674] In some embodiments, a non-negatively charged
internucleotidic linkage, or a neutral internucleotidic linkage, is
or comprising a structure selected from
##STR00152##
wherein W is O or S. In some embodiments, a non-negatively charged
internucleotidic linkage is a chirally controlled internucleotidic
linkage. In some embodiments, a neutral internucleotidic linkage is
a chirally controlled internucleotidic linkage. In some
embodiments, a nucleic acid or an oligonucleotide comprising a
modified internucleotidic linkage comprising a cyclic guanidine
moiety is a siRNA, double-stranded siRNA, single-stranded siRNA,
gapmer, skipmer, blockmer, antisense oligonucleotide, antagomir,
microRNA, pre-microRNs, antimir, supermir, ribozyme, UI adaptor,
RNA activator, RNAi agent, decoy oligonucleotide, triplex forming
oligonucleotide, aptamer or adjuvant.
[0675] In some embodiments, an oligonucleotide comprises a neutral
internucleotidic linkage and a chirally controlled internucleotidic
linkage. In some embodiments, an oligonucleotide comprises a
neutral internucleotidic linkage and a chirally controlled
internucleotidic linkage which is a phosphorothioate in the Rp or
Sp configuration. In some embodiments, the present disclosure
provides an oligonucleotide comprising one or more non-negatively
charged internucleotidic linkages and one or more phosphorothioate
internucleotidic linkage, wherein each phosphorothioate
internucleotidic linkage in the oligonucleotide is independently a
chirally controlled internucleotidic linkage. In some embodiments,
the present disclosure provides an oligonucleotide comprising one
or more neutral internucleotidic linkages and one or more
phosphorothioate internucleotidic linkage, wherein each
phosphorothioate internucleotidic linkage in the oligonucleotide is
independently a chirally controlled internucleotidic linkage. In
some embodiments, a provided oligonucleotide comprises at least 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
chirally controlled phosphorothioate internucleotidic linkages.
[0676] Without wishing to be bound by any particular theory, the
present disclosure notes that a neutral internucleotidic linkage
can be more hydrophobic than a phosphorothioate internucleotidic
linkage (PS), which is more hydrophobic than a phosphodiester
linkage (natural phosphate linkage, PO). Typically, unlike a PS or
PO, a neutral internucleotidic linkage bears less charge. Without
wishing to be bound by any particular theory, the present
disclosure notes that incorporation of one or more neutral
internucleotidic linkages into an oligonucleotide may increase
oligonucleotides' ability to be taken up by a cell and/or to escape
from endosomes. Without wishing to be bound by any particular
theory, the present disclosure notes that incorporation of one or
more neutral internucleotidic linkages can be utilized to modulate
melting temperature between an oligonucleotide and its target
nucleic acid.
[0677] Without wishing to be bound by any particular theory, the
present disclosure notes that incorporation of one or more
non-negatively charged internucleotidic linkages, e.g., neutral
internucleotidic linkages, into an oligonucleotide may be able to
increase the oligonucleotide's ability to mediate a function such
as exon skipping or gene knockdown. In some embodiments, an
oligonucleotide capable of mediating knockdown of level of a
nucleic acid or a product encoded thereby comprises one or more
non-negatively charged internucleotidic linkages. In some
embodiments, an oligonucleotide capable of mediating knockdown of
expression of a target gene comprises one or more non-negatively
charged internucleotidic linkages. In some embodiments, an
oligonucleotide capable of mediating knockdown of expression of a
target gene comprises one or more neutral internucleotidic
linkages.
[0678] In some embodiments, a non-negatively charged
internucleotidic linkage is not chirally controlled. In some
embodiments, a non-negatively charged internucleotidic linkage is
chirally controlled. In some embodiments, a non-negatively charged
internucleotidic linkage is chirally controlled and its linkage
phosphorus is Rp. In some embodiments, a non-negatively charged
internucleotidic linkage is chirally controlled and its linkage
phosphorus is Sp.
[0679] In some embodiments, a provided oligonucleotide comprises 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-negatively charged
internucleotidic linkages. In some embodiments, a provided
oligonucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
neutral internucleotidic linkages. In some embodiments, each of
non-negatively charged internucleotidic linkage and/or neutral
internucleotidic linkages is optionally and independently chirally
controlled. In some embodiments, each non-negatively charged
internucleotidic linkage in an oligonucleotide is independently a
chirally controlled internucleotidic linkage. In some embodiments,
each neutral internucleotidic linkage in an oligonucleotide is
independently a chirally controlled internucleotidic linkage. In
some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00153##
wherein W is O or S. In some embodiments, at least one
non-negatively charged internucleotidic linkage/neutral
internucleotidic linkage has the structure of
##STR00154##
In some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00155##
In some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00156##
wherein W is O or S. In some embodiments, at least one
non-negatively charged internucleotidic linkage/neutral
internucleotidic linkage has the structure of
##STR00157##
In some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00158##
In some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00159##
wherein W is O or S. In some embodiments, at least one
non-negatively charged internucleotidic linkage/neutral
internucleotidic linkage has the structure of
##STR00160##
In some embodiments, at least one non-negatively charged
internucleotidic linkage/neutral internucleotidic linkage has the
structure of
##STR00161##
In some embodiments, a provided oligonucleotide comprises at least
one non-negatively charged internucleotidic linkage wherein its
linkage phosphorus is in Rp configuration, and at least one
non-negatively charged internucleotidic linkage wherein its linkage
phosphorus is in Sp configuration.
[0680] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein at least one linkage
phosphorus is Rp. It is understood by a person of ordinary skill in
the art that in certain embodiments wherein the chirally controlled
C9orf72 oligonucleotide comprises a base sequence, each T is
independently and optionally replaced with U. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein each linkage phosphorus is Rp. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein at least one linkage phosphorus is Sp. In
some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide having the sequence of any
oligonucleotide disclosed herein, wherein each linkage phosphorus
is Sp. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein the oligonucleotide
is a blockmer. In some embodiments, the present disclosure provides
a chirally controlled C9orf72 oligonucleotide having the sequence
of any oligonucleotide disclosed herein, wherein the
oligonucleotide is a stereoblockmer. In some embodiments, the
present disclosure provides a chirally controlled C9orf72
oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein the oligonucleotide is a P-modification
blockmer. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein the oligonucleotide
is a linkage blockmer. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide having the
sequence of any oligonucleotide disclosed herein, wherein the
oligonucleotide is an altmer. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
having the sequence of any oligonucleotide disclosed herein,
wherein the oligonucleotide is a stereoaltmer. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein the oligonucleotide is a P-modification
altmer. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein the oligonucleotide
is a linkage altmer. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide having the
sequence of any oligonucleotide disclosed herein, wherein the
oligonucleotide is a unimer. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
having the sequence of any oligonucleotide disclosed herein,
wherein the oligonucleotide is a stereounimer. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein the oligonucleotide is a P-modification
unimer. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein the oligonucleotide
is a linkage unimer. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide having the
sequence of any oligonucleotide disclosed herein, wherein the
oligonucleotide is a gapmer. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
having the sequence of any oligonucleotide disclosed herein,
wherein the oligonucleotide is a skipmer.
[0681] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide having the sequence of
any oligonucleotide disclosed herein, wherein each cytosine is
optionally and independently replaced by 5-methylcytosine. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein at least one cytosine is optionally and
independently replaced by 5-methylcytosine. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide having the sequence of any oligonucleotide
disclosed herein, wherein each cytosine is optionally and
independently replaced by 5-methylcytosine.
[0682] In some embodiments, a chirally controlled C9orf72
oligonucleotide is designed such that one or more nucleotides
comprise a phosphorus modification prone to "autorelease" under
certain conditions. That is, under certain conditions, a particular
phosphorus modification is designed such that it self-cleaves from
the oligonucleotide to provide, e.g., a phosphate diester such as
those found in naturally occurring DNA and RNA. In some
embodiments, such a phosphorus modification has a structure of
--O-L-R, wherein each of L and R.sup.1 is independently described
in the present disclosure. In some embodiments, an autorelease
group comprises a morpholino group. In some embodiments, an
autorelease group is characterized by the ability to deliver an
agent to the internucleotidic phosphorus linker, which agent
facilitates further modification of the phosphorus atom such as,
e.g., desulfurization. In some embodiments, the agent is water and
the further modification is hydrolysis to form a phosphate diester
as is found in naturally occurring DNA and RNA.
[0683] In some embodiments, a chirally controlled C9orf72
oligonucleotide is designed such that the resulting pharmaceutical
properties are improved through one or more particular
modifications at phosphorus. It is well documented in the art that
certain oligonucleotides are rapidly degraded by nucleases and
exhibit poor cellular uptake through the cytoplasmic cell membrane
(Poijarvi-Virta et al., Curr. Med. Chem. (2006), 13(28); 3441-65;
Wagner et al., Med. Res. Rev. (2000), 20(6):417-51; Peyrottes et
al., Mini Rev. Med. Chem. (2004), 4(4):395-408; Gosselin et al.,
(1996), 43(1):196-208; Bologna et al., (2002), Antisense &
Nucleic Acid Drug Development 12:33-41). For instance, Vives et
al., (Nucleic Acids Research (1999), 27(20):4071-76) found that
tert-butyl SATE pro-oligonucleotides displayed markedly increased
cellular penetration compared to the parent oligonucleotide.
[0684] Base Sequence of an C9orf72 Oligonucleotide
[0685] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 gene or its gene product. In some
embodiments, a C9orf72 target gene comprises a repeat expansion. In
some embodiments, provided C9orf72 oligonucleotides can comprise
any base sequence described herein, or portion thereof, wherein a
portion is a span of at least 15 contiguous bases, or a span of at
least 15 contiguous bases with 1-5 mismatches.
[0686] In some embodiments, the base sequence of a C9orf72
oligonucleotide has a sufficient length and identity to a C9orf72
transcript target to mediate target-specific knockdown. In some
embodiments, the C9orf72 oligonucleotide is complementary to a
portion of a transcript target sequence.
[0687] In some embodiments, the base sequence of a C9orf72
oligonucleotide is complementary to that of a C9orf72 target
transcript. As used herein, "target transcript sequence," "target
sequence", "target gene", and the like, refer to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of a C9orf72 gene, including mRNA that is
a product of RNA processing of a primary transcription product.
[0688] The terms "complementary," "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between a C9orf72 oligonucleotide and a C9orf72
target sequence, as will be understood from the context of their
use. In some embodiments, the base sequence of a C9orf72
oligonucleotide is complementary to that of a C9orf72 target
sequence when each base of the oligonucleotide is capable of
base-pairing with a sequential base on the target strand, when
maximally aligned. As a non-limiting example, if a target sequence
has, for example, a base sequence of 5'-GCAUAGCGAGCGAGGGAAAAC-3',
an oligonucleotide with a base sequence of
5'GUUUUCCCUCGCUCGCUAUGC-3' is complementary or fully complementary
to such a target sequence. It is noted, of course, that
substitution of T for U, or vice versa, does not alter the amount
of complementarity.
[0689] As used herein, a polynucleotide that is "substantially
complementary" to a C9orf72 target sequence is largely or mostly
complementary but not 100% complementary. In some embodiments, a
sequence (e.g., a C9orf72 oligonucleotide) which is substantially
complementary has 1, 2, 3, 4 or 5 mismatches from a sequence which
is 100% complementary to the target sequence.
[0690] The present disclosure presents, in Table 1A and elsewhere,
various oligonucleotides, each of which has a defined base
sequence. In some embodiments, the disclosure encompasses any
oligonucleotide having a base sequence which is, comprises, or
comprises a portion of the base sequence of any of oligonucleotide
disclosed herein. In some embodiments, the disclosure encompasses
any oligonucleotide having a base sequence which is, comprises, or
comprises a portion of the base sequence of any oligonucleotide
disclosed herein, which has any chemical modification,
stereochemistry, format, structural feature (e.g., any structure or
pattern of modification or portion thereof), and/or any other
modification described herein (e.g., conjugation with another
moiety, such as a targeting moiety, carbohydrate moiety, etc.;
and/or multimerization). In some embodiments, a "portion" (e.g., of
a base sequence or a pattern of modifications), is at least 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 long. In
some embodiments, a "portion" of a base sequence is at least 5 nt
long. In some embodiments, a "portion" of a base sequence is at
least 10 nt long. In some embodiments, a "portion" of a base
sequence is at least 15 nt long. In some embodiments, a "portion"
of a base sequence is at least 20 nt long.
[0691] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCACACCTGCTCTTGCTAG.
[0692] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGGGCAGCAGGGACGGCTG.
[0693] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGGTTGCGGTGCCTGCGCC.
[0694] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGGTTGTTTCCCTCCTTGT.
[0695] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTGTTCACCCTCAGCGAGT.
[0696] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCCCATCTCATCCCGCAT.
[0697] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGACCCGCTGGGAGCGCTGC.
[0698] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCCGCCTCCTCACTCACCC.
[0699] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCTCTTTCCTAGCGGGAC.
[0700] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCCCATTCCAGTTTCCATC.
[0701] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCCTCTCAGTACCCGAGGC.
[0702] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGATGCCGCCTCCTCACTCA.
[0703] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCAGCAGGGACGGCTGACA.
[0704] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TACCCGCGCCTCTTCCCGGC.
[0705] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACAGGCTGCGGTTGTTTCCC.
[0706] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCTTCCCGGCAGCCGAACC.
[0707] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGGAGGTCCTGCACTTTCCC.
[0708] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTGGGTGTCGGGCTTTCGC.
[0709] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTCCTTGCTTTCCCGCCCT.
[0710] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCTTCTACCCGCGCCTCTT.
[0711] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTCTACCCGCGCCTCTTCC.
[0712] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCAGGCGGTGGCGAGTGGGT.
[0713] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCGCCTCCTCACTCACCCA.
[0714] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACATCCCCTCACAGGCTCTT.
[0715] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCTCCTTGTTTTCTTCTGG.
[0716] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGGCTCTCCAGAAGGCTGT.
[0717] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGGCTGTCAGCTCGGATCTC.
[0718] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCCTCCTCACTCACCCACT.
[0719] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCTTTCCTAGCGGGACACC.
[0720] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CACCCACTCGCCACCGCCTG.
[0721] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TACAGGCTGCGGTTGTTTCC.
[0722] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCACCTCTCTTTCCTAGCG.
[0723] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGGCGAGTGGGTGAGTGAGGAGGCGGCATC.
[0724] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCACCCGCCAGGATGCCGC.
[0725] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GATGCACCTGACATCCCCTC.
[0726] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTTGCTACAGGCTGCGGTT.
[0727] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCACTCACCCACTCGCCAC.
[0728] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCCTCACTCACCCACTCGC.
[0729] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGGAAGGCCGGAGGGTGGGC.
[0730] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGGCAGCAGGGACGGCTGAC.
[0731] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGCAGGCGGTGGCGAGTGGGTGAGTGAGG.
[0732] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCTGCGGTTGTTTCCCTCC.
[0733] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCTCAGTACCCGAGGCTCCC.
[0734] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTTGGTGTGTCAGCCGTCC.
[0735] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTGTTCTGTCTTTGGAGCCC.
[0736] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCATAGAATCCAGTACCAT.
[0737] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGCGCGCGACTCCTGAGTTC.
[0738] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGGCTGCGGTTGTTTCCCTC.
[0739] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCAGTACCCGAGGCTCCCT.
[0740] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTCCCGGCAGCCGAACCCC.
[0741] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CACCCGCCAGGATGCCGCCT.
[0742] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCTCTCTTTCCTAGCGGGA.
[0743] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTACAGGCTGCGGTTGTTT.
[0744] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGCGACTCCTGAGTTCCAG.
[0745] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCCGGCAGCCGAACCCCAA.
[0746] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCAGATCCCCATCCCTTGT.
[0747] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCACCCACTCGCCACCGCC.
[0748] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGCAACCGGGCAGCAGGGAC.
[0749] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTGCGGTTGTTTCCCTCCT.
[0750] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AACCGGGCAGCAGGGACGGC.
[0751] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGTTGTTTCCCTCCTTGTTT.
[0752] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTGGTGTGTCAGCCGTCCC.
[0753] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTGGAGATGGCGGTGGGCA.
[0754] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCGCGCCTCTTCCCGGCAG.
[0755] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTGCTAGACCCCGCCCCCA.
[0756] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCTCTCCAGAAGGCTGTCA.
[0757] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCTCACTCACCCACTCGCC.
[0758] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CAGGATGCCGCCTCCTCACT.
[0759] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTGGTTGCTTCACAGCTCC.
[0760] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGGGCAGCAGGGACGGCTGA.
[0761] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGCTGCGATCCCCATTCCA.
[0762] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCGCCAGGATGCCGCCTCC.
[0763] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GATCCCCATCCCTTGTCCCT.
[0764] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCTTACTCTAGGACCAAGA.
[0765] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCGGGCAGCAGGGACGGCT.
[0766] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCCAGGCTGGTTATGACTC.
[0767] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCATCCTGGCGGGTGGCTGT.
[0768] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCTTCCCGGCAGCCGAAC.
[0769] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCCAAACAGCCACCCGCCA.
[0770] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTTGCGGTGCCTGCGCCCGC.
[0771] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCAAACAGCCACCCGCCAG.
[0772] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCGCCAGGATGCCGCCTC.
[0773] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCTTCCCGGCAGCCGAACCC.
[0774] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCCGGCCTTCCCCCAGGC.
[0775] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCATCCGGGCCCCGGGCTTC.
[0776] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CACCCCCATCTCATCCCGCA.
[0777] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CAGAGCTTGCTACAGGCTGC.
[0778] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGCTTCTACCCGCGCCTCT.
[0779] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGCAGGCGTCTCCACACCC.
[0780] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACTCACCCACTCGCCACCGC.
[0781] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCAGGCGTCTCCACACCCCC.
[0782] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTGAGTTCCAGAGCTTGCT.
[0783] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GAGAGCCCCCGCTTCTACCC.
[0784] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCTGAGTTCCAGAGCTTGC.
[0785] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGCTTGCTACAGGCTGCGGT.
[0786] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCAAACAGCCACCCGCCAGG.
[0787] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGCAGGCGGTGGCGAGTGGGTGAGTGAGGAGGCGGCATC.
[0788] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTGCGGTTGTTTCCCTCCTT.
[0789] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGCCGTCCCTGCTGCCCGGT.
[0790] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCTCCTCACTCACCCACTC.
[0791] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCTACAGGCTGCGGTTGTT.
[0792] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCAGGGACGGCTGACACACC.
[0793] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGGATGCCGCCTCCTCACTC.
[0794] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGTCTTTTCTTGTTCACCCT.
[0795] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTGCTCTTGCTAGACCCCG.
[0796] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTTCACCCTCAGCGAGTACT.
[0797] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTAGCGGGACACCGTAGGT.
[0798] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCCTCAGTACCCGAGCTGT.
[0799] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTACCCGCGCCTCTTCCCGG.
[0800] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCTTTTCTTGTTCACCCTC.
[0801] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGGTGTCGGGCTTTCGCCTC.
[0802] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGGTTGTTTCCCTCCTTGTT.
[0803] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCACCCTCCGGCCTTCCCCC.
[0804] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCTCTCAGTACCCGAGGCT.
[0805] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCACTCACCCACTCGCCA.
[0806] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GAGCTTGCTACAGGCTGCGG.
[0807] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTGACATCCCCTCACAGGCT.
[0808] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTGTTTGACGCACCTCTCT.
[0809] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGGAATGGGGATCGCAGCAC.
[0810] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGCCTCCTCACTCACCCAC.
[0811] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCTGCCAAGGCCTGCCAC.
[0812] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGACTTGCATTGCTGCCCT.
[0813] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCACCCTCAGCGAGTACTGT.
[0814] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGCGCCTCTTCCCGGCAGC.
[0815] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCCTCTTCCCGGCAGCCGA.
[0816] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTGCTACAGGCTGCGGTTGT.
[0817] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCGGGAAGAGGCGCGGGTAG.
[0818] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCAACCGGGCAGCAGGGACG.
[0819] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCTTGCTAGACCCCGCCCCC.
[0820] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTAGACCCCGCCCCCAAAA.
[0821] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTGCGATCCCCATTCCAGT.
[0822] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCACTCGCCACCGCCTGCG.
[0823] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGGCAGGCCTTGGCAGAGG.
[0824] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACGCACCTCTCTTTCCTAGC.
[0825] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGGGCCACCCCTCCTGGGAA.
[0826] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCAGGATGCCGCCTCCTCAC.
[0827] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCGCGCCTCTTCCCGGCA.
[0828] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCGAGCTGTCTCCTTCCC.
[0829] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCTCTTCCCGGCAGCCGAA.
[0830] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACTCCTGAGTTCCAGAGCTT.
[0831] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ATTGCCTGCATCCGGGCCCC.
[0832] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTCTACCCGCGCCTCTTCCC.
[0833] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTACCCGAGGCTCCCTTTTC.
[0834] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTGCTGCCCGGTTGCTTCT.
[0835] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGCGCGACTCCTGAGTTCC.
[0836] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCGGTGTGCTCCCCATTCT.
[0837] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCACTCGCCACCGCCTGC.
[0838] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GACATCCCCTCACAGGCTCT.
[0839] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GAGCTGCCCAGGACCACTTC.
[0840] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCGGCATCCTGGCGGGTGGC.
[0841] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCCGTGTGCTCATTGGGTC.
[0842] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGGAATGGGGATCGCAGCACA.
[0843] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCGGAGGCGCAGGCGGTGG.
[0844] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCAGGATGCCGCCTCCTCA.
[0845] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCACTCGCCACCGCCTGCGC.
[0846] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCCTGCATCCGGGCCCCGG.
[0847] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTGCTAGACCCCGCCCCCAA.
[0848] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CAGGCTGCGGTTGTTTCCCT.
[0849] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCCGTCCCTGCTGCCCGGTT.
[0850] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTTCCCCACACCACTGAGCT.
[0851] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTCCAGAGCTTGCTACAGGC.
[0852] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCGGCAGCCGAACCCCAAA.
[0853] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGTGCTGCGATCCCCATTCC.
[0854] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GAGGCCAGATCCCCATCCCT.
[0855] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCGCTGTTTGACGCACCTC.
[0856] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTCTTGCTAGACCCCGCCCC.
[0857] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGCCAGGATGCCGCCTCCT.
[0858] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCCTCACTCACCCACTCG.
[0859] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ATCCCCTCACAGGCTCTTGT.
[0860] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTCTTGCTAGACCCCGCCC.
[0861] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGTCCCTGCCGGCGAGGAGA.
[0862] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTTCCCTGAAGGTTCCTCC.
[0863] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCCCTCACAGGCTCTTGTG.
[0864] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCTCTTGCTAGACCCCGCC.
[0865] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TTCCCGGCAGCCGAACCCCA.
[0866] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCCCTGCTGCCCGGTTGCT.
[0867] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTTCTACCCGCGCCTCTTC.
[0868] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTTTCCTAGCGGGACACCGT.
[0869] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGCAGGACCTCCCTCCTGT.
[0870] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTGCTCCCCATTCTGTGGGA.
[0871] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCAGAGCTTGCTACAGGCT.
[0872] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCCCTCACAGGCTCTTGTGA.
[0873] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCTAGACCCCGCCCCCAAA.
[0874] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCACCCACTCGCCACCGCCT.
[0875] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CTGCTCTTGCTAGACCCCGC.
[0876] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TGCGGTTGTTTCCCTCCTTG.
[0877] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTTGTTTCCCTCCTTGTTTT.
[0878] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCGTCTCCACACCCCCATC.
[0879] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGGCTCTCCTCAGAGCTCGA.
[0880] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCTCCGGCCTTCCCCCAG.
[0881] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCGCAGCCTGTAGCAAGCTC.
[0882] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AACCCACACCTGCTCTTGCT.
[0883] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AGTGGTCCTGGGCAGCTCCT.
[0884] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCCTTGCTTTCCCGCCCTCA.
[0885] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GAGCTCTGAGGAGAGCCCCC.
[0886] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCACCTCTCTTTCCTAGCGG.
[0887] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCCAGGATGCCGCCTCCTC.
[0888] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
AACAGCCACCCGCCAGGATG.
[0889] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CAGGGTGGCATCTGCTTCAC.
[0890] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CACTCACCCACTCGCCACCG.
[0891] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCACCCGCCAGGATGCCGCC.
[0892] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCACACCTGCTCTTGCTA.
[0893] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ATGCCGCCTCCTCACTCACC.
[0894] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CACCTCTCTTTCCTAGCGGG.
[0895] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GATCCCCATTCCAGTTTCCA.
[0896] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GTCAGCCGTCCCTGCTGCCC.
[0897] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
TCACTCACCCACTCGCCACC.
[0898] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCTCCCTTTTCTCGAGCCC.
[0899] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCAGGACCTCCCTCCTGTTT.
[0900] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTTTCCCGCCCTCAGTACC.
[0901] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GATGCCGCCTCCTCACTCAC.
[0902] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
ACCCCAAACAGCCACCCGCC.
[0903] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCTCCTTGTTTTCTTCTGGT.
[0904] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GGCCTTGGCAGAGGTGGTGA.
[0905] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GCTCTGAGGAGAGCCCCCGC.
[0906] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
GACAGGGTGGCATCTGCTTC.
[0907] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CGCGCGACTCCTGAGTTCCA.
[0908] In some embodiments, an oligonucleotide targets C9orf72 and
has a base sequence which is, comprises or comprises a portion of:
CCACACCTGCTCTTGCTAGA.
[0909] In some embodiments, a portion of a base sequence is a span
of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more contiguous
(consecutive) bases.
[0910] In some embodiments, the present disclosure discloses a
C9orf72 oligonucleotide of a sequence recited herein. In some
embodiments, the present disclosure discloses a C9orf72
oligonucleotide of a sequence recited herein, wherein the
oligonucleotide is capable of directing a decrease in the
expression, level and/or activity of a C9orf72 gene or its gene
product. In some embodiments, a C9orf72 oligonucleotide of a
recited sequence comprises any structure described herein. In
various sequences, U can be replaced by T or vice versa, or a
sequence can comprise a mixture of U and T. In some embodiments, a
C9orf72 oligonucleotide has a length of no more than about 49, 45,
40, 30, 35, 25, 23 total nucleotides. In some embodiments, a
portion is a span of at least 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, or 25 total nucleotides with 0-3 mismatches. In some
embodiments, a portion is a span of at least 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, or 25 total nucleotides with 0-3 mismatches,
wherein a span with 0 mismatches is complementary and a span with 1
or more mismatches is a non-limiting example of substantial
complementarity. In some embodiments, wherein the sequence recited
above starts with a U at the 5'-end, the U can be deleted and/or
replaced by another base. In some embodiments, the disclosure
encompasses any oligonucleotide having a base sequence which is or
comprises or comprises a portion of the base sequence of any
oligonucleotide disclosed herein, which has a format or a portion
of a format disclosed herein.
[0911] In some embodiments, a C9orf72 oligonucleotide can comprise
any base sequence described herein. In some embodiments, a C9orf72
oligonucleotide can comprise any base sequence or portion thereof,
described herein. In some embodiments, a C9orf72 oligonucleotide
can comprise any base sequence or portion thereof, described
herein, wherein a portion is a span of 15 contiguous bases, or a
span of 15 contiguous bases with 1-5 mismatches. In some
embodiments, a C9orf72 oligonucleotide can comprise any base
sequence or portion thereof described herein in combination with
any other structural element or modification described herein.
[0912] Non-limiting examples of C9orf72 oligonucleotides having
various base sequences and modifications are disclosed in Table 1A,
below.
TABLE-US-00002 TABLE 1A C9orf72 oligonucleotides. Stereochemistry/
WAVE Internucleotidic ID Modified Sequence Base Sequence Linkages
WV- Teo * Aeo * m5Ceo * Aeo * Geo * G * m5C * T *
TACAGGCTGCGGTTGTTTCC XXXXXXXXXXXXXXXXXXX 3536 G * m5C * G * G * T *
T * G * Teo * Teo * Teo * m5Ceo * m5Ceo WV- Geo * m5Ceo * m5Ceo *
Teo * Teo * A * m5C * GCCTTACTCTAGGACCAAGA XXXXXXXXXXXXXXXXXXX 3537
T * m5C * T * A * G * G * A * m5C * m5Ceo * Aeo * Aeo * Geo * Aeo
WV- m5Ceo * Geo * m5Ceo * Aeo * Teo * A * G * A *
CGCATAGAATCCAGTACCAT XXXXXXXXXXXXXXXXXXX 3538 A * T * m5C * m5C * A
* G * T * Aeo * m5Ceo * m5Ceo * Aeo * Teo WV- mU * mA * mC * mA *
mG * G * C * T * G * C * UACAGGCTGCGGTTGUUUCC XXXXXXXXXXXXXXXXXXX
3539 G * G * T * T * G * mU * mU * mU * mC * mC WV- mG * mC * mC *
mU * mU * A * C * T * C * T * GCCUUACTCTAGGACCAAGA
XXXXXXXXXXXXXXXXXXX 3540 A * G * G * A * C * mC * mA * mA * mG * mA
WV- mC * mG * mC * mA * mU * A * G * A * A * CGCAUAGAATCCAGTACCAU
XXXXXXXXXXXXXXXXXXX 3541 T * C * C * A * G * T * mA * mC * mC * mA
* mU WV- m5Ceo * m5Ceo * Teo * Teo * m5Ceo * m5C *
CCTTCCCTGAAGGTTCCTCC XXXXXXXXXXXXXXXXXXX 3542 m5C * T * G * A * A *
G * G * T * T * m5Ceo * m5Ceo * Teo * m5Ceo * m5Ceo WV- mG * mC *
mU * mG * mG * A * G * A * T * GCUGGAGATGGCGGTGGGCA
XXXXXXXXXXXXXXXXXXX 3561 G * G * C * G * G * T * mG * mG * mG * mC
* mA WV- mC * mU * mG * mU * mU * C * T * G * T * C *
CUGUUCTGTCTTTGGAGCCC XXXXXXXXXXXXXXXXXXX 3562 T * T * T * G * G *
mA * mG * mC * mC * mC WV- mU * mC * mC * mC * mC * A * T * T * C *
C * UCCCCATTCCAGTTTCCAUC XXXXXXXXXXXXXXXXXXX 3563 A * G * T * T * T
* mC * mC * mA * mU * mC WV- mG * mA * mU * mC * mC * C * C * A * T
* T * GAUCCCCATTCCAGTUUCCA XXXXXXXXXXXXXXXXXXX 3564 C * C * A * G *
T * mU * mU * mC * mC * mA WV- mG * mC * mU * mG * mC * G * A * T *
C * C * GCUGCGATCCCCATTCCAGU XXXXXXXXXXXXXXXXXXX 3565 C * C * A * T
* T * mC * mC * mA * mG * mU WV- mG * mU * mG * mC * mU * G * C * G
* A * GUGCUGCGATCCCCAUUCCA XXXXXXXXXXXXXXXXXXX 3566 T * C * C * C *
C * A * mU * mU * mC * mC * mA WV- mU * mG * mU * mG * mC * T * G *
C * G * UGUGCTGCGATCCCCAUUCC XXXXXXXXXXXXXXXXXXX 3567 A * T * C * C
* C * C * mA * mU * mU * mC * mC WV- mC * mA * mG * mG * mG * T * G
* G * C * CAGGGTGGCATCTGCUUCAC XXXXXXXXXXXXXXXXXXX 3568 A * T * C *
T * G * C * mU * mU * mC * mA * mC WV- mG * mA * mC * mA * mG * G *
G * T * G * GACAGGGTGGCATCTGCUUC XXXXXXXXXXXXXXXXXXX 3569 G * C * A
* T * C * T * mG * mC * mU * mU * mC WV- mA * mG * mG * mC * mU * G
* T * C * A * AGGCUGTCAGCTCGGAUCUC XXXXXXXXXXXXXXXXXXX 3570 G * C *
T * C * G * G * mA * mU * mC * mU * mC WV- mG * mG * mC * mU * mC *
T * C * C * A * G * GGCUCTCCAGAAGGCUGUCA XXXXXXXXXXXXXXXXXXX 3571 A
* A * G * G * C * mU * mG * mU * mC * mA WV- mG * mU * mG * mG * mC
* T * C * T * C * C * GUGGCTCTCCAGAAGGCUGU XXXXXXXXXXXXXXXXXXX 3572
A * G * A * A * G * mG * mC * mU * mG * mU WV- mU * mU * mU * mC *
mC * C * C * A * C * A * UUUCCCCACACCACTGAGCU XXXXXXXXXXXXXXXXXXX
3573 C * C * A * C * T * mG * mA * mG * mC * mU WV- mC * mC * mC *
mC * mU * C * A * C * A * CCCCUCACAGGCTCTUGUGA XXXXXXXXXXXXXXXXXXX
3574 G * G * C * T * C * T * mU * mG * mU * mG * mA WV- mU * mC *
mC * mC * mC * T * C * A * C * UCCCCTCACAGGCTCUUGUG
XXXXXXXXXXXXXXXXXXX 3575 A * G * G * C * T * C * mU * mU * mG * mU
* mG WV- mA * mU * mC * mC * mC * C * T * C * A *
AUCCCCTCACAGGCTCUUGU XXXXXXXXXXXXXXXXXXX 3576 C * A * G * G * C * T
* mC * mU * mU * mG * mU WV- mA * mC * mA * mU * mC * C * C * C * T
* ACAUCCCCTCACAGGCUCUU XXXXXXXXXXXXXXXXXXX 3577 C * A * C * A * G *
G * mC * mU * mC * mU * mU WV- mG * mA * mC * mA * mU * C * C * C *
C * GACAUCCCCTCACAGGCUCU XXXXXXXXXXXXXXXXXXX 3578 T * C * A * C * A
* G * mG * mC * mU * mC * mU WV- mC * mU * mG * mA * mC * A * T * C
* C * CUGACATCCCCTCACAGGCU XXXXXXXXXXXXXXXXXXX 3579 C * C * T * C *
A * C * mA * mG * mG * mC * mU WV- mG * mA * mU * mG * mC * A * C *
C * T * GAUGCACCTGACATCCCCUC XXXXXXXXXXXXXXXXXXX 3580 G * A * C * A
* T * C * mC * mC * mC * mU * mC WV- mG * mC * mA * mC * mC * T * C
* T * C * GCACCTCTCTTTCCTAGCGG XXXXXXXXXXXXXXXXXXX 3581 T * T * T *
C * C * T * mA * mG * mC * mG * mG WV- mG * mG * mG * mC * mA * G *
C * A * G * GGGCAGCAGGGACGGCUGAC XXXXXXXXXXXXXXXXXXX 3582 G * G * A
* C * G * G * mC * mU * mG * mA * mC WV- mU * mG * mC * mU * mA * G
* A * C * C * UGCUAGACCCCGCCCCCAAA XXXXXXXXXXXXXXXXXXX 3583 C * C *
G * C * C * C * mC * mC * mA * mA * mA WV- mU * mC * mU * mU * mG *
C * T * A * G * UCUUGCTAGACCCCGCCCCC XXXXXXXXXXXXXXXXXXX 3584 A * C
* C * C * C * G * mC * mC * mC * mC * mC WV- mG * mC * mU * mC * mU
* T * G * C * T * GCUCUTGCTAGACCCCGCCC XXXXXXXXXXXXXXXXXXX 3585 A *
G * A * C * C * C * mC * mG * mC * mC * mC WV- mC * mU * mG * mC *
mU * C * T * T * G * CUGCUCTTGCTAGACCCCGC XXXXXXXXXXXXXXXXXXX 3586
C * T * A * G * A * C * mC * mC * mC * mG * mC WV- mG * mC * mG *
mG * mU * T * G * T * T * GCGGUTGTTTCCCTCCUUGU XXXXXXXXXXXXXXXXXXX
3587 T * C * C * C * T * C * mC * mU * mU * mG * mU WV- mG * mC *
mU * mG * mC * G * G * T * T * GCUGCGGTTGTTTCCCUCCU
XXXXXXXXXXXXXXXXXXX 3588 G * T * T * T * C * C * mC * mU * mC * mC
* mU WV- mG * mG * mC * mU * mG * C * G * G * T *
GGCUGCGGTTGTTTCCCUCC XXXXXXXXXXXXXXXXXXX 3589 T * G * T * T * T * C
* mC * mC * mU * mC * mC WV- mC * mA * mG * mG * mC * T * G * C * G
* CAGGCTGCGGTTGTTUCCCU XXXXXXXXXXXXXXXXXXX 3590 G * T * T * G * T *
T * mU * mC * mC * mC * mU WV- mU * mC * mA * mC * mU * C * A * C *
C * UCACUCACCCACTCGCCACC XXXXXXXXXXXXXXXXXXX 3591 C * A * C * T * C
* G * mC * mC * mA * mC * mC WV- mC * mC * mU * mC * mA * C * T * C
* A * CCUCACTCACCCACTCGCCA XXXXXXXXXXXXXXXXXXX 3592 C * C * C * A *
C * T * mC * mG * mC * mC * mA WV- mG * mG * mA * mU * mG * C * C *
G * C * GGAUGCCGCCTCCTCACUCA XXXXXXXXXXXXXXXXXXX 3593 C * T * C * C
* T * C * mA * mC * mU * mC * mA WV- mG * mC * mC * mA * mG * G * A
* T * G * GCCAGGATGCCGCCTCCUCA XXXXXXXXXXXXXXXXXXX 3594 C * C * G *
C * C * T * mC * mC * mU * mC * mA WV- mC * mC * mC * mC * mA * A *
A * C * A * CCCCAAACAGCCACCCGCCA XXXXXXXXXXXXXXXXXXX 3595 G * C * C
* A * C * C * mC * mG * mC * mC * mA WV- mG * mA * mG * mA * mG * C
* C * C * C * GAGAGCCCCCGCTTCUACCC XXXXXXXXXXXXXXXXXXX 3596 C * G *
C * T * T * C * mU * mA * mC * mC * mC WV- mG * mC * mU * mC * mU *
G * A * G * G * GCUCUGAGGAGAGCCCCCGC XXXXXXXXXXXXXXXXXXX 3597 A * G
* A * G * C * C * mC * mC * mC * mG * mC WV- mG * mA * mG * mC * mU
* C * T * G * A * GAGCUCTGAGGAGAGCCCCC XXXXXXXXXXXXXXXXXXX 3598 G *
G * A * G * A * G * mC * mC * mC * mC * mC WV- mG * mA * mU * mC *
mC * C * C * A * T * GAUCCCCATCCCTTGUCCCU XXXXXXXXXXXXXXXXXXX 3599
C * C * C * T * T * G * mU * mC * mC * mC * mU WV- mG * mC * mC *
mA * mG * A * T * C * C * GCCAGATCCCCATCCCUUGU XXXXXXXXXXXXXXXXXXX
3600 C * C * A * T * C * C * mC * mU * mU * mG * mU WV- mG * mA *
mG * mG * mC * C * A * G * A * GAGGCCAGATCCCCAUCCCU
XXXXXXXXXXXXXXXXXXX 3601 T * C * C * C * C * A * mU * mC * mC * mC
* mU WV- mG * mG * mC * mU * mC * C * C * T * T *
GGCUCCCTTTTCTCGAGCCC XXXXXXXXXXXXXXXXXXX 3602 T * T * C * T * C * G
* mA * mG * mC * mC * mC WV- mG * mU * mA * mC * mC * C * G * A * G
* GUACCCGAGGCTCCCUUUUC XXXXXXXXXXXXXXXXXXX 3603 G * C * T * C * C *
C * mU * mU * mU * mU * mC WV- mC * mU * mC * mA * mG * T * A * C *
C * CUCAGTACCCGAGGCUCCCU XXXXXXXXXXXXXXXXXXX
3604 C * G * A * G * G * C * mU * mC * mC * mC * mU WV- mU * mC *
mU * mC * mA * G * T * A * C * UCUCAGTACCCGAGGCUCCC
XXXXXXXXXXXXXXXXXXX 3605 C * C * G * A * G * G * mC * mU * mC * mC
* mC WV- mG * mC * mC * mU * mC * T * C * A * G *
GCCUCTCAGTACCCGAGGCU XXXXXXXXXXXXXXXXXXX 3606 T * A * C * C * C * G
* mA * mG * mG * mC * mU WV- mG * mG * mC * mC * mU * C * T * C * A
* GGCCUCTCAGTACCCGAGGC XXXXXXXXXXXXXXXXXXX 3607 G * T * A * C * C *
C * mG * mA * mG * mG * mC WV- mC * mC * mU * mC * mC * G * G * C *
C * CCUCCGGCCTTCCCCCAGGC XXXXXXXXXXXXXXXXXXX 3608 T * T * C * C * C
* C * mC * mA * mG * mG * mC WV- mA * mC * mC * mC * mU * C * C * G
* G * ACCCUCCGGCCTTCCCCCAG XXXXXXXXXXXXXXXXXXX 3609 C * C * T * T *
C * C * mC * mC * mC * mA * mG WV- mC * mC * mA * mC * mC * C * T *
C * C * CCACCCTCCGGCCTTCCCCC XXXXXXXXXXXXXXXXXXX 3610 G * G * C * C
* T * T * mC * mC * mC * mC * mC WV- mA * mC * mC * mC * mC * C * A
* T * C * ACCCCCATCTCATCCCGCAU XXXXXXXXXXXXXXXXXXX 3611 T * C * A *
T * C * C * mC * mG * mC * mA * mU WV- mC * mA * mC * mC * mC * C *
C * A * T * CACCCCCATCTCATCCCGCA XXXXXXXXXXXXXXXXXXX 3612 C * T * C
* A * T * C * mC * mC * mG * mC * mA WV- mG * mG * mC * mG * mU * C
* T * C * C * GGCGUCTCCACACCCCCAUC XXXXXXXXXXXXXXXXXXX 3613 A * C *
A * C * C * C * mC * mC * mA * mU * mC WV- mG * mC * mA * mG * mG *
C * G * T * C * GCAGGCGTCTCCACACCCCC XXXXXXXXXXXXXXXXXXX 3614 T * C
* C * A * C * A * mC * mC * mC * mC * mC WV- mG * mU * mG * mC * mA
* G * G * C * G * GUGCAGGCGTCTCCACACCC XXXXXXXXXXXXXXXXXXX 3615 T *
C * T * C * C * A * mC * mA * mC * mC * mC WV- mC * mC * mG * mA *
mC * T * T * G * C * CCGACTTGCATTGCTGCCCU XXXXXXXXXXXXXXXXXXX 3616
A * T * T * G * C * T * mG * mC * mC * mC * mU WV- mG * mC * mA *
mG * mG * A * C * C * T * GCAGGACCTCCCTCCUGUUU XXXXXXXXXXXXXXXXXXX
3617 C * C * C * T * C * C * mU * mG * mU * mU * mU WV- mG * mU *
mG * mC * mA * G * G * A * C * GUGCAGGACCTCCCTCCUGU
XXXXXXXXXXXXXXXXXXX 3618 C * T * C * C * C * T * mC * mC * mU * mG
* mU WV- mA * mG * mG * mG * mC * C * A * C * C *
AGGGCCACCCCTCCTGGGAA XXXXXXXXXXXXXXXXXXX 3619 C * C * T * C * C * T
* mG * mG * mG * mA * mA WV- mG * mG * mC * mC * mU * T * G * G * C
* GGCCUTGGCAGAGGTGGUGA XXXXXXXXXXXXXXXXXXX 3620 A * G * A * G * G *
T * mG * mG * mU * mG * mA WV- mG * mU * mG * mG * mC * A * G * G *
C * GUGGCAGGCCTTGGCAGAGG XXXXXXXXXXXXXXXXXXX 3621 C * T * T * G * G
* C * mA * mG * mA * mG * mG WV- mG * mA * mG * mC * mU * G * C * C
* C * GAGCUGCCCAGGACCACUUC XXXXXXXXXXXXXXXXXXX 3622 A * G * G * A *
C * C * mA * mC * mU * mU * mC WV- mG * mC * mU * mU * mG * G * T *
G * T * GCUUGGTGTGTCAGCCGUCC XXXXXXXXXXXXXXXXXXX 3623 G * T * C * A
* G * C * mC * mG * mU * mC * mC WV- mC * mU * mU * mG * mG * T * G
* T * G * CUUGGTGTGTCAGCCGUCCC XXXXXXXXXXXXXXXXXXX 3624 T * C * A *
G * C * C * mG * mU * mC * mC * mC WV- mG * mU * mC * mA * mG * C *
C * G * T * GUCAGCCGTCCCTGCUGCCC XXXXXXXXXXXXXXXXXXX 3625 C * C * C
* T * G * C * mU * mG * mC * mC * mC WV- mG * mC * mC * mG * mU * C
* C * C * T * GCCGUCCCTGCTGCCCGGUU XXXXXXXXXXXXXXXXXXX 3626 G * C *
T * G * C * C * mC * mG * mG * mU * mU WV- mG * mU * mC * mC * mC *
T * G * C * T * GUCCCTGCTGCCCGGUUGCU XXXXXXXXXXXXXXXXXXX 3627 G * C
* C * C * G * G * mU * mU * mG * mC * mU WV- mC * mC * mU * mG * mC
* T * G * C * C * CCUGCTGCCCGGTTGCUUCU XXXXXXXXXXXXXXXXXXX 3628 C *
G * G * T * T * G * mC * mU * mU * mC * mU WV- mC * mC * mG * mC *
mA * G * C * C * T * CCGCAGCCTGTAGCAAGCUC XXXXXXXXXXXXXXXXXXX 3629
G * T * A * G * C * A * mA * mG * mC * mU * mC WV- mG * mC * mG *
mG * mU * T * G * C * G * GCGGUTGCGGTGCCTGCGCC XXXXXXXXXXXXXXXXXXX
3630 G * T * G * C * C * T * mG * mC * mG * mC * mC WV- mG * mU *
mU * mG * mC * G * G * T * G * GUUGCGGTGCCTGCGCCCGC
XXXXXXXXXXXXXXXXXXX 3631 C * C * T * G * C * G * mC * mC * mC * mG
* mC WV- mG * mG * mC * mG * mG * A * G * G * C *
GGCGGAGGCGCAGGCGGUGG XXXXXXXXXXXXXXXXXXX 3632 G * C * A * G * G * C
* mG * mG * mU * mG * mG WV- mG * mC * mA * mG * mG * C * G * G * T
* GCAGGCGGTGGCGAGUGGGU XXXXXXXXXXXXXXXXXXX 3633 G * G * C * G * A *
G * mU * mG * mG * mG * mU WV- mG * mC * mG * mG * mC * A * T * C *
C * GCGGCATCCTGGCGGGUGGC XXXXXXXXXXXXXXXXXXX 3634 T * G * G * C * G
* G * mG * mU * mG * mG * mC WV- mG * mC * mA * mU * mC * C * T * G
* G * GCAUCCTGGCGGGTGGCUGU XXXXXXXXXXXXXXXXXXX 3635 C * G * G * G *
T * G * mG * mC * mU * mG * mU WV- mG * mG * mG * mC * mU * C * T *
C * C * GGGCUCTCCTCAGAGCUCGA XXXXXXXXXXXXXXXXXXX 3636 T * C * A * G
* A * G * mC * mU * mC * mG * mA WV- mG * mC * mU * mG * mG * G * T
* G * T * GCUGGGTGTCGGGCTUUCGC XXXXXXXXXXXXXXXXXXX 3637 C * G * G *
G * C * T * mU * mU * mC * mG * mC WV- mG * mG * mG * mU * mG * T *
C * G * G * GGGUGTCGGGCTTTCGCCUC XXXXXXXXXXXXXXXXXXX 3638 G * C * T
* T * T * C * mG * mC * mC * mU * mC WV- mA * mU * mU * mG * mC * C
* T * G * C * AUUGCCTGCATCCGGGCCCC XXXXXXXXXXXXXXXXXXX 3639 A * T *
C * C * G * G * mG * mC * mC * mC * mC WV- mU * mG * mC * mC * mU *
G * C * A * T * UGCCUGCATCCGGGCCCCGG XXXXXXXXXXXXXXXXXXX 3640 C * C
* G * G * G * C * mC * mC * mC * mG * mG WV- mG * mC * mA * mU * mC
* C * G * G * G * GCAUCCGGGCCCCGGGCUUC XXXXXXXXXXXXXXXXXXX 3641 C *
C * C * C * G * G * mG * mC * mU * mU * mC WV- mC * mU * mU * mC *
mC * T * T * G * C * CUUCCTTGCTTTCCCGCCCU XXXXXXXXXXXXXXXXXXX 3642
T * T * T * C * C * C * mG * mC * mC * mC * mU WV- mU * mC * mC *
mU * mU * G * C * T * T * UCCUUGCTTTCCCGCCCUCA XXXXXXXXXXXXXXXXXXX
3643 T * C * C * C * G * C * mC * mC * mU * mC * mA WV- mG * mC *
mU * mU * mU * C * C * C * G * GCUUUCCCGCCCTCAGUACC
XXXXXXXXXXXXXXXXXXX 3644 C * C * C * T * C * A * mG * mU * mA * mC
* mC WV- mG * mC * mC * mC * mU * C * A * G * T *
GCCCUCAGTACCCGAGCUGU XXXXXXXXXXXXXXXXXXX 3645 A * C * C * C * G * A
* mG * mC * mU * mG * mU WV- mA * mC * mC * mC * mG * A * G * C * T
* ACCCGAGCTGTCTCCUUCCC XXXXXXXXXXXXXXXXXXX 3646 G * T * C * T * C *
C * mU * mU * mC * mC * mC WV- mG * mG * mA * mC * mC * C * G * C *
T * GGACCCGCTGGGAGCGCUGC XXXXXXXXXXXXXXXXXXX 3647 G * G * G * A * G
* C * mG * mC * mU * mG * mC WV- mG * mG * mG * mA * mA * G * G * C
* C * GGGAAGGCCGGAGGGUGGGC XXXXXXXXXXXXXXXXXXX 3648 G * G * A * G *
G * G * mU * mG * mG * mG * mC WV- mG * mG * mU * mC * mC * C * T *
G * C * GGUCCCTGCCGGCGAGGAGA XXXXXXXXXXXXXXXXXXX 3649 C * G * G * C
* G * A * mG * mG * mA * mG * mA WV- mG * mU * mC * mG * mG * T * G
* T * G * GUCGGTGTGCTCCCCAUUCU XXXXXXXXXXXXXXXXXXX 3650 C * T * C *
C * C * C * mA * mU * mU * mC * mU WV- mG * mU * mG * mC * mU * C *
C * C * C * GUGCUCCCCATTCTGUGGGA XXXXXXXXXXXXXXXXXXX 3651 A * T * T
* C * T * G * mU * mG * mG * mG * mA WV- mC * mC * mU * mG * mG * T
* T * G * C * CCUGGTTGCTTCACAGCUCC XXXXXXXXXXXXXXXXXXX 3652 T * T *
C * A * C * A * mG * mC * mU * mC * mC WV- mG * mU * mC * mC * mG *
T * G * T * G * GUCCGTGTGCTCATTGGGUC XXXXXXXXXXXXXXXXXXX 3653 C * T
* C * A * T * T * mG * mG * mG * mU * mC WV- mG * mG * mG * mA * mG
* G * T * C * C * GGGAGGTCCTGCACTUUCCC XXXXXXXXXXXXXXXXXXX 3654 T *
G * C * A * C * T * mU * mU * mC * mC *
mC WV- mC * mC * mU * mC * mU * G * C * C * A *
CCUCUGCCAAGGCCTGCCAC XXXXXXXXXXXXXXXXXXX 3655 A * G * G * C * C * T
* mG * mC * mC * mA * mC WV- mA * mG * mU * mG * mG * T * C * C * T
* AGUGGTCCTGGGCAGCUCCU XXXXXXXXXXXXXXXXXXX 3656 G * G * G * C * A *
G * mC * mU * mC * mC * mU WV- mG * mCmUmGmG * A * G * A * T * G *
G * GCUGGAGATGGCGGTGGGCA XOOOXXXXXXXXXXXOOOX 3657 C * G * G * T *
mGmGmGmC * mA WV- mC * mUmGmUmU * C * T * G * T * C * T *
CUGUUCTGTCTTTGGAGCCC XOOOXXXXXXXXXXXOOOX 3658 T * T * G * G *
mAmGmCmC * mC WV- mU * mCmCmCmC * A * T * T * C * C * A *
UCCCCATTCCAGTTTCCAUC XOOOXXXXXXXXXXXOOOX 3659 G * T * T * T *
mCmCmAmU * mC WV- mG * mAmUmCmC * C * C * A * T * T * C *
GAUCCCCATTCCAGTUUCCA XOOOXXXXXXXXXXXOOOX 3660 C * A * G * T *
mUmUmCmC * mA WV- mG * mCmUmGmC * G * A * T * C * C * C *
GCUGCGATCCCCATTCCAGU XOOOXXXXXXXXXXXOOOX 3661 C * A * T * T *
mCmCmAmG * mU WV- mG * mUmGmCmU * G * C * G * A * T * C *
GUGCUGCGATCCCCAUUCCA XOOOXXXXXXXXXXXOOOX 3662 C * C * C * A *
mUmUmCmC * mA WV- mU * mGmUmGmC * T * G * C * G * A * T *
UGUGCTGCGATCCCCAUUCC XOOOXXXXXXXXXXXOOOX 3663 C * C * C * C *
mAmUmUmC * mC WV- mC * mAmGmGmG * T * G * G * C * A * T *
CAGGGTGGCATCTGCUUCAC XOOOXXXXXXXXXXXOOOX 3664 C * T * G * C *
mUmUmCmA * mC WV- mG * mAmCmAmG * G * G * T * G * G * C *
GACAGGGTGGCATCTGCUUC XOOOXXXXXXXXXXXOOOX 3665 A * T * C * T *
mGmCmUmU * mC WV- mA * mGmGmCmU * G * T * C * A * G * C *
AGGCUGTCAGCTCGGAUCUC XOOOXXXXXXXXXXXOOOX 3666 T * C * G * G *
mAmUmCmU * mC WV- mG * mGmCmUmC * T * C * C * A * G * A *
GGCUCTCCAGAAGGCUGUCA XOOOXXXXXXXXXXXOOOX 3667 A * G * G * C *
mUmGmUmC * mA WV- mG * mUmGmGmC * T * C * T * C * C * A *
GUGGCTCTCCAGAAGGCUGU XOOOXXXXXXXXXXXOOOX 3668 G * A * A * G *
mGmCmUmG * mU WV- mU * mUmUmCmC * C * C * A * C * A * C *
UUUCCCCACACCACTGAGCU XOOOXXXXXXXXXXXOOOX 3669 C * A * C * T *
mGmAmGmC * mU WV- mC * mCmCmCmU * C * A * C * A * G * G *
CCCCUCACAGGCTCTUGUGA XOOOXXXXXXXXXXXOOOX 3670 C * T * C * T *
mUmGmUmG * mA WV- mU * mCmCmCmC * T * C * A * C * A * G *
UCCCCTCACAGGCTCUUGUG XOOOXXXXXXXXXXXOOOX 3671 G * C * T * C *
mUmUmGmU * mG WV- mA * mUmCmCmC * C * T * C * A * C * A *
AUCCCCTCACAGGCTCUUGU XOOOXXXXXXXXXXXOOOX 3672 G * G * C * T *
mCmUmUmG * mU WV- mA * mCmAmUmC * C * C * C * T * C * A *
ACAUCCCCTCACAGGCUCUU XOOOXXXXXXXXXXXOOOX 3673 C * A * G * G *
mCmUmCmU * mU WV- mG * mAmCmAmU * C * C * C * C * T * C *
GACAUCCCCTCACAGGCUCU XOOOXXXXXXXXXXXOOOX 3674 A * C * A * G *
mGmCmUmC * mU WV- mC * mUmGmAmC * A * T * C * C * C * C *
CUGACATCCCCTCACAGGCU XOOOXXXXXXXXXXXOOOX 3675 T * C * A * C *
mAmGmGmC * mU WV- mG * mAmUmGmC * A * C * C * T * G * A *
GAUGCACCTGACATCCCCUC XOOOXXXXXXXXXXXOOOX 3676 C * A * T * C *
mCmCmCmU * mC WV- mG * mCmAmCmC * T * C * T * C * T * T *
GCACCTCTCTTTCCTAGCGG XOOOXXXXXXXXXXXOOOX 3677 T * C * C * T *
mAmGmCmG * mG WV- mG * mGmGmCmA * G * C * A * G * G * G *
GGGCAGCAGGGACGGCUGAC XOOOXXXXXXXXXXXOOOX 3678 A * C * G * G *
mCmUmGmA * mC WV- mU * mGmCmUmA * G * A * C * C * C * C *
UGCUAGACCCCGCCCCCAAA XOOOXXXXXXXXXXXOOOX 3679 G * C * C * C *
mCmCmAmA * mA WV- mU * mCmUmUmG * C * T * A * G * A * C *
UCUUGCTAGACCCCGCCCCC XOOOXXXXXXXXXXXOOOX 3680 C * C * C * G *
mCmCmCmC * mC WV- mG * mCmUmCmU * T * G * C * T * A * G *
GCUCUTGCTAGACCCCGCCC XOOOXXXXXXXXXXXOOOX 3681 A * C * C * C *
mCmGmCmC * mC WV- mC * mUmGmCmU * C * T * T * G * C * T *
CUGCUCTTGCTAGACCCCGC XOOOXXXXXXXXXXXOOOX 3682 A * G * A * C *
mCmCmCmG * mC WV- mG * mCmGmGmU * T * G * T * T * T * C *
GCGGUTGTTTCCCTCCUUGU XOOOXXXXXXXXXXXOOOX 3683 C * C * T * C *
mCmUmUmG * mU WV- mG * mCmUmGmC * G * G * T * T * G * T *
GCUGCGGTTGTTTCCCUCCU XOOOXXXXXXXXXXXOOOX 3684 T * T * C * C *
mCmUmCmC * mU WV- mG * mGmCmUmG * C * G * G * T * T * G *
GGCUGCGGTTGTTTCCCUCC XOOOXXXXXXXXXXXOOOX 3685 T * T * T * C *
mCmCmUmC * mC WV- mC * mAmGmGmC * T * G * C * G * G * T *
CAGGCTGCGGTTGTTUCCCU XOOOXXXXXXXXXXXOOOX 3686 T * G * T * T *
mUmCmCmC * mU WV- mU * mCmAmCmU * C * A * C * C * C * A *
UCACUCACCCACTCGCCACC XOOOXXXXXXXXXXXOOOX 3687 C * T * C * G *
mCmCmAmC * mC WV- mC * mCmUmCmA * C * T * C * A * C * C *
CCUCACTCACCCACTCGCCA XOOOXXXXXXXXXXXOOOX 3688 C * A * C * T *
mCmGmCmC * mA WV- mG * mGmAmUmG * C * C * G * C * C * T *
GGAUGCCGCCTCCTCACUCA XOOOXXXXXXXXXXXOOOX 3689 C * C * T * C *
mAmCmUmC * mA WV- mG * mCmCmAmG * G * A * T * G * C * C *
GCCAGGATGCCGCCTCCUCA XOOOXXXXXXXXXXXOOOX 3690 G * C * C * T *
mCmCmUmC * mA WV- mC * mCmCmCmA * A * A * C * A * G * C *
CCCCAAACAGCCACCCGCCA XOOOXXXXXXXXXXXOOOX 3691 C * A * C * C *
mCmGmCmC * mA WV- mG * mAmGmAmG * C * C * C * C * C * G *
GAGAGCCCCCGCTTCUACCC XOOOXXXXXXXXXXXOOOX 3692 C * T * T * C *
mUmAmCmC * mC WV- mG * mCmUmCmU * G * A * G * G * A * G *
GCUCUGAGGAGAGCCCCCGC XOOOXXXXXXXXXXXOOOX 3693 A * G * C * C *
mCmCmCmG * mC WV- mG * mAmGmCmU * C * T * G * A * G * G *
GAGCUCTGAGGAGAGCCCCC XOOOXXXXXXXXXXXOOOX 3694 A * G * A * G *
mCmCmCmC * mC WV- mG * mAmUmCmC * C * C * A * T * C * C *
GAUCCCCATCCCTTGUCCCU XOOOXXXXXXXXXXXOOOX 3695 C * T * T * G *
mUmCmCmC * mU WV- mG * mCmCmAmG * A * T * C * C * C * C *
GCCAGATCCCCATCCCUUGU XOOOXXXXXXXXXXXOOOX 3696 A * T * C * C *
mCmUmUmG * mU WV- mG * mAmGmGmC * C * A * G * A * T * C *
GAGGCCAGATCCCCAUCCCU XOOOXXXXXXXXXXXOOOX 3697 C * C * C * A *
mUmCmCmC * mU WV- mG * mGmCmUmC * C * C * T * T * T * T *
GGCUCCCTTTTCTCGAGCCC XOOOXXXXXXXXXXXOOOX 3698 C * T * C * G *
mAmGmCmC * mC WV- mG * mUmAmCmC * C * G * A * G * G * C *
GUACCCGAGGCTCCCUUUUC XOOOXXXXXXXXXXXOOOX 3699 T * C * C * C *
mUmUmUmU * mC WV- mC * mUmCmAmG * T * A * C * C * C * G *
CUCAGTACCCGAGGCUCCCU XOOOXXXXXXXXXXXOOOX 3700 A * G * G * C *
mUmCmCmC * mU WV- mU * mCmUmCmA * G * T * A * C * C * C *
UCUCAGTACCCGAGGCUCCC XOOOXXXXXXXXXXXOOOX 3701 G * A * G * G *
mCmUmCmC * mC WV- mG * mCmCmUmC * T * C * A * G * T * A *
GCCUCTCAGTACCCGAGGCU XOOOXXXXXXXXXXXOOOX 3702 C * C * C * G *
mAmGmGmC * mU WV- mG * mGmCmCmU * C * T * C * A * G * T *
GGCCUCTCAGTACCCGAGGC XOOOXXXXXXXXXXXOOOX 3703 A * C * C * C *
mGmAmGmG * mC WV- mC * mCmUmCmC * G * G * C * C * T * T *
CCUCCGGCCTTCCCCCAGGC XOOOXXXXXXXXXXXOOOX 3704 C * C * C * C *
mCmAmGmG * mC WV- mA * mCmCmCmU * C * C * G * G * C * C *
ACCCUCCGGCCTTCCCCCAG XOOOXXXXXXXXXXXOOOX 3705 T * T * C * C *
mCmCmCmA * mG WV- mC * mCmAmCmC * C * T * C * C * G * G *
CCACCCTCCGGCCTTCCCCC XOOOXXXXXXXXXXXOOOX 3706 C * C * T * T *
mCmCmCmC * mC WV- mA * mCmCmCmC * C * A * T * C * T * C *
ACCCCCATCTCATCCCGCAU XOOOXXXXXXXXXXXOOOX 3707 A * T * C * C *
mCmGmCmA * mU WV- mC * mAmCmCmC * C * C * A * T * C * T *
CACCCCCATCTCATCCCGCA XOOOXXXXXXXXXXXOOOX 3708 C * A * T * C *
mCmCmGmC * mA WV- mG * mGmCmGmU * C * T * C * C * A * C *
GGCGUCTCCACACCCCCAUC XOOOXXXXXXXXXXXOOOX 3709 A * C * C * C *
mCmCmAmU * mC WV- mG * mCmAmGmG * C * G * T * C * T * C *
GCAGGCGTCTCCACACCCCC XOOOXXXXXXXXXXXOOOX 3710 C * A * C * A *
mCmCmCmC * mC WV- mG * mUmGmCmA * G * G * C * G * T * C *
GUGCAGGCGTCTCCACACCC XOOOXXXXXXXXXXXOOOX 3711 T * C * C * A *
mCmAmCmC * mC WV- mC * mCmGmAmC * T * T * G * C * A * T *
CCGACTTGCATTGCTGCCCU XOOOXXXXXXXXXXXOOOX 3712 T * G * C * T *
mGmCmCmC * mU WV- mG * mCmAmGmG * A * C * C * T * C * C *
GCAGGACCTCCCTCCUGUUU XOOOXXXXXXXXXXXOOOX 3713 C * T * C * C *
mUmGmUmU * mU WV- mG * mUmGmCmA * G * G * A * C * C * T *
GUGCAGGACCTCCCTCCUGU XOOOXXXXXXXXXXXOOOX 3714 C * C * C * T *
mCmCmUmG * mU WV- mA * mGmGmGmC * C * A * C * C * C * C *
AGGGCCACCCCTCCTGGGAA XOOOXXXXXXXXXXXOOOX 3715 T * C * C * T *
mGmGmGmA * mA WV- mG * mGmCmCmU * T * G * G * C * A * G *
GGCCUTGGCAGAGGTGGUGA XOOOXXXXXXXXXXXOOOX 3716 A * G * G * T *
mGmGmUmG * mA
WV- mG * mUmGmGmC * A * G * G * C * C * T * GUGGCAGGCCTTGGCAGAGG
XOOOXXXXXXXXXXXOOOX 3717 T * G * G * C * mAmGmAmG * mG WV- mG *
mAmGmCmU * G * C * C * C * A * G * GAGCUGCCCAGGACCACUUC
XOOOXXXXXXXXXXXOOOX 3718 G * A * C * C * mAmCmUmU * mC WV- mG *
mCmUmUmG * G * T * G * T * G * T * GCUUGGTGTGTCAGCCGUCC
XOOOXXXXXXXXXXXOOOX 3719 C * A * G * C * mCmGmUmC * mC WV- mC *
mUmUmGmG * T * G * T * G * T * C * CUUGGTGTGTCAGCCGUCCC
XOOOXXXXXXXXXXXOOOX 3720 A * G * C * C * mGmUmCmC * mC WV- mG *
mUmCmAmG * C * C * G * T * C * C * GUCAGCCGTCCCTGCUGCCC
XOOOXXXXXXXXXXXOOOX 3721 C * T * G * C * mUmGmCmC * mC WV- mG *
mCmCmGmU * C * C * C * T * G * C * GCCGUCCCTGCTGCCCGGUU
XOOOXXXXXXXXXXXOOOX 3722 T * G * C * C * mCmGmGmU * mU WV- mG *
mUmCmCmC * T * G * C * T * G * C * GUCCCTGCTGCCCGGUUGCU
XOOOXXXXXXXXXXXOOOX 3723 C * C * G * G * mUmUmGmC * mU WV- mC *
mCmUmGmC * T * G * C * C * C * G * CCUGCTGCCCGGTTGCUUCU
XOOOXXXXXXXXXXXOOOX 3724 G * T * T * G * mCmUmUmC * mU WV- mC *
mCmGmCmA * G * C * C * T * G * T * CCGCAGCCTGTAGCAAGCUC
XOOOXXXXXXXXXXXOOOX 3725 A * G * C * A * mAmGmCmU * mC WV- mG *
mCmGmGmU * T * G * C * G * G * T * GCGGUTGCGGTGCCTGCGCC
XOOOXXXXXXXXXXXOOOX 3726 G * C * C * T * mGmCmGmC * mC WV- mG *
mUmUmGmC * G * G * T * G * C * C * GUUGCGGTGCCTGCGCCCGC
XOOOXXXXXXXXXXXOOOX 3727 T * G * C * G * mCmCmCmG * mC WV- mG *
mGmCmGmG * A * G * G * C * G * C * GGCGGAGGCGCAGGCGGUGG
XOOOXXXXXXXXXXXOOOX 3728 A * G * G * C * mGmGmUmG * mG WV- mG *
mCmAmGmG * C * G * G * T * G * G * GCAGGCGGTGGCGAGUGGGU
XOOOXXXXXXXXXXXOOOX 3729 C * G * A * G * mUmGmGmG * mU WV- mG *
mCmGmGmC * A * T * C * C * T * G * GCGGCATCCTGGCGGGUGGC
XOOOXXXXXXXXXXXOOOX 3730 G * C * G * G * mGmUmGmG * mC WV- mG *
mCmAmUmC * C * T * G * G * C * G * GCAUCCTGGCGGGTGGCUGU
XOOOXXXXXXXXXXXOOOX 3731 G * G * T * G * mGmCmUmG * mU WV- mG *
mGmGmCmU * C * T * C * C * T * C * GGGCUCTCCTCAGAGCUCGA
XOOOXXXXXXXXXXXOOOX 3732 A * G * A * G * mCmUmCmG * mA WV- mG *
mCmUmGmG * G * T * G * T * C * G * GCUGGGTGTCGGGCTUUCGC
XOOOXXXXXXXXXXXOOOX 3733 G * G * C * T * mUmUmCmG * mC WV- mG *
mGmGmUmG * T * C * G * G * G * C * GGGUGTCGGGCTTTCGCCUC
XOOOXXXXXXXXXXXOOOX 3734 T * T * T * C * mGmCmCmU * mC WV- mA *
mUmUmGmC * C * T * G * C * A * T * AUUGCCTGCATCCGGGCCCC
XOOOXXXXXXXXXXXOOOX 3735 C * C * G * G * mGmCmCmC * mC WV- mU *
mGmCmCmU * G * C * A * T * C * C * UGCCUGCATCCGGGCCCCGG
XOOOXXXXXXXXXXXOOOX 3736 G * G * G * C * mCmCmCmG * mG WV- mG *
mCmAmUmC * C * G * G * G * C * C * GCAUCCGGGCCCCGGGCUUC
XOOOXXXXXXXXXXXOOOX 3737 C * C * G * G * mGmCmUmU * mC WV- mC *
mUmUmCmC * T * T * G * C * T * T * CUUCCTTGCTTTCCCGCCCU
XOOOXXXXXXXXXXXOOOX 3738 T * C * C * C * mGmCmCmC * mU WV- mU *
mCmCmUmU * G * C * T * T * T * C * UCCUUGCTTTCCCGCCCUCA
XOOOXXXXXXXXXXXOOOX 3739 C * C * G * C * mCmCmUmC * mA WV- mG *
mCmUmUmU * C * C * C * G * C * C * GCUUUCCCGCCCTCAGUACC
XOOOXXXXXXXXXXXOOOX 3740 C * T * C * A * mGmUmAmC * mC WV- mG *
mCmCmCmU * C * A * G * T * A * C * GCCCUCAGTACCCGAGCUGU
XOOOXXXXXXXXXXXOOOX 3741 C * C * G * A * mGmCmUmG * mU WV- mA *
mCmCmCmG * A * G * C * T * G * T * ACCCGAGCTGTCTCCUUCCC
XOOOXXXXXXXXXXXOOOX 3742 C * T * C * C * mUmUmCmC * mC WV- mG *
mGmAmCmC * C * G * C * T * G * G * GGACCCGCTGGGAGCGCUGC
XOOOXXXXXXXXXXXOOOX 3743 G * A * G * C * mGmCmUmG * mC WV- mG *
mGmGmAmA * G * G * C * C * G * G * GGGAAGGCCGGAGGGUGGGC
XOOOXXXXXXXXXXXOOOX 3744 A * G * G * G * mUmGmGmG * mC WV- mG *
mGmUmCmC * C * T * G * C * C * G * GGUCCCTGCCGGCGAGGAGA
XOOOXXXXXXXXXXXOOOX 3745 G * C * G * A * mGmGmAmG * mA WV- mG *
mUmCmGmG * T * G * T * G * C * T * GUCGGTGTGCTCCCCAUUCU
XOOOXXXXXXXXXXXOOOX 3746 C * C * C * C * mAmUmUmC * mU WV- mG *
mUmGmCmU * C * C * C * C * A * T * GUGCUCCCCATTCTGUGGGA
XOOOXXXXXXXXXXXOOOX 3747 T * C * T * G * mUmGmGmG * mA WV- mC *
mCmUmGmG * T * T * G * C * T * T * CCUGGTTGCTTCACAGCUCC
XOOOXXXXXXXXXXXOOOX 3748 C * A * C * A * mGmCmUmC * mC WV- mG *
mUmCmCmG * T * G * T * G * C * T * GUCCGTGTGCTCATTGGGUC
XOOOXXXXXXXXXXXOOOX 3749 C * A * T * T * mGmGmGmU * mC WV- mG *
mGmGmAmG * G * T * C * C * T * G * GGGAGGTCCTGCACTUUCCC
XOOOXXXXXXXXXXXOOOX 3750 C * A * C * T * mUmUmCmC * mC WV- mC *
mCmUmCmU * G * C * C * A * A * G * CCUCUGCCAAGGCCTGCCAC
XOOOXXXXXXXXXXXOOOX 3751 G * C * C * T * mGmCmCmA * mC WV- mA *
mGmUmGmG * T * C * C * T * G * G * AGUGGTCCTGGGCAGCUCCU
XOOOXXXXXXXXXXXOOOX 3752 G * C * A * G * mCmUmCmC * mU WV- Geo *
m5Ceo * Teo * Geo * Geo * A * G * A * GCTGGAGATGGCGGTGGGCA
XXXXXXXXXXXXXXXXXXX 5905 T * G * G * C * G * G * T * Geo * Geo *
Geo * m5Ceo * Aeo WV- m5Ceo * Teo * Geo * Teo * Teo * C * T * G *
CTGTTCTGTCTTTGGAGCCC XXXXXXXXXXXXXXXXXXX 5906 T * C * T * T * T * G
* G * Aeo * Geo * m5Ceo * m5Ceo * m5Ceo WV- Teo * m5Ceo * m5Ceo *
m5Ceo * m5Ceo * A * TCCCCATTCCAGTTTCCATC XXXXXXXXXXXXXXXXXXX 5907 T
* T * C * C * A * G * T * T * T * m5Ceo * m5Ceo * Aeo * Teo * m5Ceo
WV- Geo * Aeo * Teo * m5Ceo * m5Ceo * C * C * GATCCCCATTCCAGTTTCCA
XXXXXXXXXXXXXXXXXXX 5908 A * T * T * C * C * A * G * T * Teo * Teo
* m5Ceo * m5Ceo * Aeo WV- Geo * m5Ceo * Teo * Geo * m5Ceo * G * A *
GCTGCGATCCCCATTCCAGT XXXXXXXXXXXXXXXXXXX 5909 T * C * C * C * C * A
* T * T * m5Ceo * m5Ceo * Aeo * Geo * Teo WV- Geo * Teo * Geo *
m5Ceo * Teo * G * C * G * GTGCTGCGATCCCCATTCCA XXXXXXXXXXXXXXXXXXX
5910 A * T * C * C * C * C * A * Teo * Teo * m5Ceo * m5Ceo * Aeo
WV- Teo * Geo * Teo * Geo * m5Ceo * T * G * C *
TGTGCTGCGATCCCCATTCC XXXXXXXXXXXXXXXXXXX 5911 G * A * T * C * C * C
* C * Aeo * Teo * Teo * m5Ceo * m5Ceo WV- m5Ceo * Aeo * Geo * Geo *
Geo * T * G * G * CAGGGTGGCATCTGCTTCAC XXXXXXXXXXXXXXXXXXX 5912 C *
A * T * C * T * G * C * Teo * Teo * m5Ceo * Aeo * m5Ceo WV- Geo *
Aeo * m5Ceo * Aeo * Geo * G * G * T * GACAGGGTGGCATCTGCTTC
XXXXXXXXXXXXXXXXXXX 5913 G * G * C * A * T * C * T * Geo * m5Ceo *
Teo * Teo * m5Ceo WV- Aeo * Geo * Geo * m5Ceo * Teo * G * T * C *
AGGCTGTCAGCTCGGATCTC XXXXXXXXXXXXXXXXXXX 5914 A * G * C * T * C * G
* G * Aeo * Teo * m5Ceo * Teo * m5Ceo WV- Geo * Geo * m5Ceo * Teo *
m5Ceo * T * C * GGCTCTCCAGAAGGCTGTCA XXXXXXXXXXXXXXXXXXX 5915 C * A
* G * A * A * G * G * C * Teo * Geo * Teo * m5Ceo * Aeo WV- Geo *
Teo * Geo * Geo * m5Ceo * T * C * T * GTGGCTCTCCAGAAGGCTGT
XXXXXXXXXXXXXXXXXXX 5916 C * C * A * G * A * A * G * Geo * m5Ceo *
Teo * Geo * Teo WV- Teo * Teo * Teo * m5Ceo * m5Ceo * C * C *
TTTCCCCACACCACTGAGCT XXXXXXXXXXXXXXXXXXX 5917 A * C * A * C * C * A
* C * T * Geo * Aeo * Geo * m5Ceo * Teo WV- m5Ceo * m5Ceo * m5Ceo *
m5Ceo * Teo * C * CCCCTCACAGGCTCTTGTGA XXXXXXXXXXXXXXXXXXX 5918 A *
C * A * G * G * C * T * C * T * Teo * Geo * Teo * Geo * Aeo WV- Teo
* m5Ceo * m5Ceo * m5Ceo * m5Ceo * T * TCCCCTCACAGGCTCTTGTG
XXXXXXXXXXXXXXXXXXX 5919 C * A * C * A * G * G * C * T * C * Teo *
Teo * Geo * Teo * Geo WV- Aeo * Teo * m5Ceo * m5Ceo * m5Ceo * C *
ATCCCCTCACAGGCTCTTGT XXXXXXXXXXXXXXXXXXX 5920 T * C * A * C * A * G
* G * C * T * m5Ceo * Teo * Teo * Geo * Teo WV- Aeo * m5Ceo * Aeo *
Teo * m5Ceo * C * C * ACATCCCCTCACAGGCTCTT XXXXXXXXXXXXXXXXXXX 5921
C * T * C * A * C * A * G * G * m5Ceo * Teo * m5Ceo * Teo * Teo WV-
Geo * Aeo * m5Ceo * Aeo * Teo * C * C * C * GACATCCCCTCACAGGCTCT
XXXXXXXXXXXXXXXXXXX 5922 C * T * C * A * C * A * G * Geo * m5Ceo *
Teo * m5Ceo * Teo WV- m5Ceo * Teo * Geo * Aeo * m5Ceo * A * T *
CTGACATCCCCTCACAGGCT XXXXXXXXXXXXXXXXXXX 5923 C * C * C * C * T * C
* A * C * Aeo * Geo * Geo * m5Ceo * Teo WV- Geo * Aeo * Teo * Geo *
m5Ceo * A * C * C * GATGCACCTGACATCCCCTC XXXXXXXXXXXXXXXXXXX 5924 T
* G * A * C * A * T * C * m5Ceo * m5Ceo * m5Ceo * Teo * m5Ceo WV-
Geo * m5Ceo * Aeo * m5Ceo * m5Ceo * T * C * GCACCTCTCTTTCCTAGCGG
XXXXXXXXXXXXXXXXXXX 5925 T * C * T * T * T * C * C * T * Aeo * Geo
* m5Ceo * Geo * Geo WV- Geo * Geo * Geo * m5Ceo * Aeo * G * C * A *
GGGCAGCAGGGACGGCTGAC
XXXXXXXXXXXXXXXXXXX 5926 G * G * G * A * C * G * G * m5Ceo * Teo *
Geo * Aeo * m5Ceo WV- Teo * Geo * m5Ceo * Teo * Aeo * G * A * C *
TGCTAGACCCCGCCCCCAAA XXXXXXXXXXXXXXXXXXX 5927 C * C * C * G * C * C
* C * m5Ceo * m5Ceo * Aeo * Aeo * Aeo WV- Teo * m5Ceo * Teo * Teo *
Geo * C * T * A * TCTTGCTAGACCCCGCCCCC XXXXXXXXXXXXXXXXXXX 5928 G *
A * C * C * C * C * G * m5Ceo * m5Ceo * m5Ceo * m5Ceo * m5Ceo WV-
Geo * m5Ceo * Teo * m5Ceo * Teo * T * G * GCTCTTGCTAGACCCCGCCC
XXXXXXXXXXXXXXXXXXX 5929 C * T * A * G * A * C * C * C * m5Ceo *
Geo * m5Ceo * m5Ceo * m5Ceo WV- m5Ceo * Teo * Geo * m5Ceo * Teo * C
* T * CTGCTCTTGCTAGACCCCGC XXXXXXXXXXXXXXXXXXX 5930 T * G * C * T *
A * G * A * C * m5Ceo * m5Ceo * m5Ceo * Geo * m5Ceo WV- Geo * m5Ceo
* Geo * Geo * Teo * T * G * T * GCGGTTGTTTCCCTCCTTGT
XXXXXXXXXXXXXXXXXXX 5931 T * T * C * C * C * T * C * m5Ceo * Teo *
Teo * Geo * Teo WV- Geo * m5Ceo * Teo * Geo * m5Ceo * G * G *
GCTGCGGTTGTTTCCCTCCT XXXXXXXXXXXXXXXXXXX 5932 T * T * G * T * T * T
* C * C * m5Ceo * Teo * m5Ceo * m5Ceo * Teo WV- Geo * Geo * m5Ceo *
Teo * Geo * C * G * G * GGCTGCGGTTGTTTCCCTCC XXXXXXXXXXXXXXXXXXX
5933 T * T * G * T * T * T * C * m5Ceo * m5Ceo * Teo * m5Ceo *
m5Ceo WV- m5Ceo * Aeo * Geo * Geo * m5Ceo * T * G *
CAGGCTGCGGTTGTTTCCCT XXXXXXXXXXXXXXXXXXX 5934 C * G * G * T * T * G
* T * T * Teo * m5Ceo * m5Ceo * m5Ceo * Teo WV- Teo * m5Ceo * Aeo *
m5Ceo * Teo * C * A * TCACTCACCCACTCGCCACC XXXXXXXXXXXXXXXXXXX 5935
C * C * C * A * C * T * C * G * m5Ceo * m5Ceo * Aeo * m5Ceo * m5Ceo
WV- m5Ceo * m5Ceo * Teo * m5Ceo * Aeo * C * T *
CCTCACTCACCCACTCGCCA XXXXXXXXXXXXXXXXXXX 5936 C * A * C * C * C * A
* C * T * m5Ceo * Geo * m5Ceo * m5Ceo * Aeo WV- Geo * Geo * Aeo *
Teo * Geo * C * C * G * C * GGATGCCGCCTCCTCACTCA
XXXXXXXXXXXXXXXXXXX 5937 C * T * C * C * T * C * Aeo * m5Ceo * Teo
* m5Ceo * Aeo WV- Geo * m5Ceo * m5Ceo * Aeo * Geo * G * A *
GCCAGGATGCCGCCTCCTCA XXXXXXXXXXXXXXXXXXX 5938 T * G * C * C * G * C
* C * T * m5Ceo * m5Ceo * Teo * m5Ceo * Aeo WV- m5Ceo * m5Ceo *
m5Ceo * m5Ceo * Aeo * A * CCCCAAACAGCCACCCGCCA XXXXXXXXXXXXXXXXXXX
5939 A * C * A * G * C * C * A * C * C * m5Ceo * Geo * m5Ceo *
m5Ceo * Aeo WV- Geo * Aeo * Geo * Aeo * Geo * C * C * C * C *
GAGAGCCCCCGCTTCTACCC XXXXXXXXXXXXXXXXXXX 5940 C * G * C * T * T * C
* Teo * Aeo * m5Ceo * m5Ceo * m5Ceo WV- Geo * m5Ceo * Teo * m5Ceo *
Teo * G * A * GCTCTGAGGAGAGCCCCCGC XXXXXXXXXXXXXXXXXXX 5941 G * G *
A * G * A * G * C * C * m5Ceo * m5Ceo * m5Ceo * Geo * m5Ceo WV- Geo
* Aeo * Geo * m5Ceo * Teo * C * T * G * GAGCTCTGAGGAGAGCCCCC
XXXXXXXXXXXXXXXXXXX 5942 A * G * G * A * G * A * G * m5Ceo * m5Ceo
* m5Ceo * m5Ceo * m5Ceo WV- Geo * Aeo * Teo * m5Ceo * m5Ceo * C * C
* GATCCCCATCCCTTGTCCCT XXXXXXXXXXXXXXXXXXX 5943 A * T * C * C * C *
T * T * G * Teo * m5Ceo * m5Ceo * m5Ceo * Teo WV- Geo * m5Ceo *
m5Ceo * Aeo * Geo * A * T * GCCAGATCCCCATCCCTTGT
XXXXXXXXXXXXXXXXXXX 5944 C * C * C * C * A * T * C * C * m5Ceo *
Teo * Teo * Geo * Teo WV- Geo * Aeo * Geo * Geo * m5Ceo * C * A * G
* GAGGCCAGATCCCCATCCCT XXXXXXXXXXXXXXXXXXX 5945 A * T * C * C * C *
C * A * Teo * m5Ceo * m5Ceo * m5Ceo * Teo WV- Geo * Geo * m5Ceo *
Teo * m5Ceo * C * C * GGCTCCCTTTTCTCGAGCCC XXXXXXXXXXXXXXXXXXX 5946
T * T * T * T * C * T * C * G * Aeo * Geo * m5Ceo * m5Ceo * m5Ceo
WV- Geo * Teo * Aeo * m5Ceo * m5Ceo * C * G * GTACCCGAGGCTCCCTTTTC
XXXXXXXXXXXXXXXXXXX 5947 A * G * G * C * T * C * C * C * Teo * Teo
* Teo * Teo * m5Ceo WV- m5Ceo * Teo * m5Ceo * Aeo * Geo * T * A *
CTCAGTACCCGAGGCTCCCT XXXXXXXXXXXXXXXXXXX 5948 C * C * C * G * A * G
* G * C * Teo * m5Ceo * m5Ceo * m5Ceo * Teo WV- Teo * m5Ceo * Teo *
m5Ceo * Aeo * G * T * TCTCAGTACCCGAGGCTCCC XXXXXXXXXXXXXXXXXXX 5949
A * C * C * C * G * A * G * G * m5Ceo * Teo * m5Ceo * m5Ceo * m5Ceo
WV- Geo * m5Ceo * m5Ceo * Teo * m5Ceo * T * C *
GCCTCTCAGTACCCGAGGCT XXXXXXXXXXXXXXXXXXX 5950 A * G * T * A * C * C
* C * G * Aeo * Geo * Geo * m5Ceo * Teo WV- Geo * Geo * m5Ceo *
m5Ceo * Teo * C * T * GGCCTCTCAGTACCCGAGGC XXXXXXXXXXXXXXXXXXX 5951
C * A * G * T * A * C * C * C * Geo * Aeo * Geo * Geo * m5Ceo WV-
m5Ceo * m5Ceo * Teo * m5Ceo * m5Ceo * G * CCTCCGGCCTTCCCCCAGGC
XXXXXXXXXXXXXXXXXXX 5952 G * C * C * T * T * C * C * C * C * m5Ceo
* Aeo * Geo * Geo * m5Ceo WV- Aeo * m5Ceo * m5Ceo * m5Ceo * Teo * C
* ACCCTCCGGCCTTCCCCCAG XXXXXXXXXXXXXXXXXXX 5953 C * G * G * C * C *
T * T * C * C * m5Ceo * m5Ceo * m5Ceo * Aeo * Geo WV- m5Ceo * m5Ceo
* Aeo * m5Ceo * m5Ceo * C * CCACCCTCCGGCCTTCCCCC
XXXXXXXXXXXXXXXXXXX 5954 T * C * C * G * G * C * C * T * T * m5Ceo
* m5Ceo * m5Ceo * m5Ceo * m5Ceo WV- Aeo * m5Ceo * m5Ceo * m5Ceo *
m5Ceo * C * ACCCCCATCTCATCCCGCAT XXXXXXXXXXXXXXXXXXX 5955 A * T * C
* T * C * A * T * C * C * m5Ceo * Geo * m5Ceo * Aeo * Teo WV- m5Ceo
* Aeo * m5Ceo * m5Ceo * m5Ceo * C * CACCCCCATCTCATCCCGCA
XXXXXXXXXXXXXXXXXXX 5956 C * A * T * C * T * C * A * T * C * m5Ceo
* m5Ceo * Geo * m5Ceo * Aeo WV- Geo * Geo * m5Ceo * Geo * Teo * C *
T * C * GGCGTCTCCACACCCCCATC XXXXXXXXXXXXXXXXXXX 5957 C * A * C * A
* C * C * C * m5Ceo * m5Ceo * Aeo * Teo * m5Ceo WV- Geo * m5Ceo *
Aeo * Geo * Geo * C * G * T * GCAGGCGTCTCCACACCCCC
XXXXXXXXXXXXXXXXXXX 5958 C * T * C * C * A * C * A * m5Ceo * m5Ceo
* m5Ceo * m5Ceo * m5Ceo WV- Geo * Teo * Geo * m5Ceo * Aeo * G * G *
C * GTGCAGGCGTCTCCACACCC XXXXXXXXXXXXXXXXXXX 5959 G * T * C * T * C
* C * A * m5Ceo * Aeo * m5Ceo * m5Ceo * m5Ceo WV- m5Ceo * m5Ceo *
Geo * Aeo * m5Ceo * T * T * CCGACTTGCATTGCTGCCCT
XXXXXXXXXXXXXXXXXXX 5960 G * C * A * T * T * G * C * T * Geo *
m5Ceo * m5Ceo * m5Ceo * Teo WV- Geo * m5Ceo * Aeo * Geo * Geo * A *
C * C * GCAGGACCTCCCTCCTGTTT XXXXXXXXXXXXXXXXXXX 5961 T * C * C * C
* T * C * C * Teo * Geo * Teo * Teo * Teo WV- Geo * Teo * Geo *
m5Ceo * Aeo * G * G * A * GTGCAGGACCTCCCTCCTGT XXXXXXXXXXXXXXXXXXX
5962 C * C * T * C * C * C * T * m5Ceo * m5Ceo * Teo * Geo * Teo
WV- Aeo * Geo * Geo * Geo * m5Ceo * C * A * C *
AGGGCCACCCCTCCTGGGAA XXXXXXXXXXXXXXXXXXX 5963 C * C * C * T * C * C
* T * Geo * Geo * Geo * Aeo * Aeo WV- Geo * Geo * m5Ceo * m5Ceo *
Teo * T * G * GGCCTTGGCAGAGGTGGTGA XXXXXXXXXXXXXXXXXXX 5964 G * C *
A * G * A * G * G * T * Geo * Geo * Teo * Geo * Aeo WV- Geo * Teo *
Geo * Geo * m5Ceo * A * G * G * GTGGCAGGCCTTGGCAGAGG
XXXXXXXXXXXXXXXXXXX 5965 C * C * T * T * G * G * C * Aeo * Geo *
Aeo * Geo * Geo WV- Geo * Aeo * Geo * m5Ceo * Teo * G * C * C *
GAGCTGCCCAGGACCACTTC XXXXXXXXXXXXXXXXXXX 5966 C * A * G * G * A * C
* C * Aeo * m5Ceo * Teo * Teo * m5Ceo WV- Geo * m5Ceo * Teo * Teo *
Geo * G * T * G * GCTTGGTGTGTCAGCCGTCC XXXXXXXXXXXXXXXXXXX 5967 T *
G * T * C * A * G * C * m5Ceo * Geo * Teo * m5Ceo * m5Ceo WV- m5Ceo
* Teo * Teo * Geo * Geo * T * G * T * CTTGGTGTGTCAGCCGTCCC
XXXXXXXXXXXXXXXXXXX 5968 G * T * C * A * G * C * C * Geo * Teo *
m5Ceo * m5Ceo * m5Ceo WV- Geo * Teo * m5Ceo * Aeo * Geo * C * C * G
* GTCAGCCGTCCCTGCTGCCC XXXXXXXXXXXXXXXXXXX 5969 T * C * C * C * T *
G * C * Teo * Geo * m5Ceo * m5Ceo * m5Ceo WV- Geo * m5Ceo * m5Ceo *
Geo * Teo * C * C * GCCGTCCCTGCTGCCCGGTT XXXXXXXXXXXXXXXXXXX 5970 C
* T * G * C * T * G * C * C * m5Ceo * Geo * Geo * Teo * Teo WV- Geo
* Teo * m5Ceo * m5Ceo * m5Ceo * T * G * GTCCCTGCTGCCCGGTTGCT
XXXXXXXXXXXXXXXXXXX 5971 C * T * G * C * C * C * G * G * Teo * Teo
* Geo * m5Ceo * Teo WV- m5Ceo * m5Ceo * Teo * Geo * m5Ceo * T * G *
CCTGCTGCCCGGTTGCTTCT XXXXXXXXXXXXXXXXXXX 5972 C * C * C * G * G * T
* T * G * m5Ceo * Teo * Teo * m5Ceo * Teo WV- m5Ceo * m5Ceo * Geo *
m5Ceo * Aeo * G * C * CCGCAGCCTGTAGCAAGCTC XXXXXXXXXXXXXXXXXXX 5973
C * T * G * T * A * G * C * A * Aeo * Geo * m5Ceo * Teo * m5Ceo WV-
Geo * m5Ceo * Geo * Geo * Teo * T * G * C * GCGGTTGCGGTGCCTGCGCC
XXXXXXXXXXXXXXXXXXX 5974 G * G * T * G * C * C * T * Geo * m5Ceo *
Geo * m5Ceo * m5Ceo WV- Geo * Teo * Teo * Geo * m5Ceo * G * G * T *
GTTGCGGTGCCTGCGCCCGC XXXXXXXXXXXXXXXXXXX 5975 G * C * C * T * G * C
* G * m5Ceo * m5Ceo * m5Ceo * Geo * m5Ceo WV- Geo * Geo * m5Ceo *
Geo * Geo * A * G * G * GGCGGAGGCGCAGGCGGTGG
XXXXXXXXXXXXXXXXXXX
5976 C * G * C * A * G * G * C * Geo * Geo * Teo * Geo * Geo WV-
Geo * m5Ceo * Aeo * Geo * Geo * C * G * G * GCAGGCGGTGGCGAGTGGGT
XXXXXXXXXXXXXXXXXXX 5977 T * G * G * C * G * A * G * Teo * Geo *
Geo * Geo * Teo WV- Geo * m5Ceo * Geo * Geo * m5Ceo * A * T *
GCGGCATCCTGGCGGGTGGC XXXXXXXXXXXXXXXXXXX 5978 C * C * T * G * G * C
* G * G * Geo * Teo * Geo * Geo * m5Ceo WV- Geo * m5Ceo * Aeo * Teo
* m5Ceo * C * T * GCATCCTGGCGGGTGGCTGT XXXXXXXXXXXXXXXXXXX 5979 G *
G * C * G * G * G * T * G * Geo * m5Ceo * Teo * Geo * Teo WV- Geo *
Geo * Geo * m5Ceo * Teo * C * T * C * GGGCTCTCCTCAGAGCTCGA
XXXXXXXXXXXXXXXXXXX 5980 C * T * C * A * G * A * G * m5Ceo * Teo *
m5Ceo * Geo * Aeo WV- Geo * m5Ceo * Teo * Geo * Geo * G * T * G *
GCTGGGTGTCGGGCTTTCGC XXXXXXXXXXXXXXXXXXX 5981 T * C * G * G * G * C
* T * Teo * Teo * m5Ceo * Geo * m5Ceo WV- Geo * Geo * Geo * Teo *
Geo * T * C * G * G * GGGTGTCGGGCTTTCGCCTC XXXXXXXXXXXXXXXXXXX 5982
G * C * T * T * T * C * Geo * m5Ceo * m5Ceo * Teo * m5Ceo WV- Aeo *
Teo * Teo * Geo * m5Ceo * C * T * G * ATTGCCTGCATCCGGGCCCC
XXXXXXXXXXXXXXXXXXX 5983 C * A * T * C * C * G * G * Geo * m5Ceo *
m5Ceo * m5Ceo * m5Ceo WV- Teo * Geo * m5Ceo * m5Ceo * Teo * G * C *
TGCCTGCATCCGGGCCCCGG XXXXXXXXXXXXXXXXXXX 5984 A * T * C * C * G * G
* G * C * m5Ceo * m5Ceo * m5Ceo * Geo * Geo WV- Geo * m5Ceo * Aeo *
Teo * m5Ceo * C * G * GCATCCGGGCCCCGGGCTTC XXXXXXXXXXXXXXXXXXX 5985
G * G * C * C * C * C * G * G * Geo * m5Ceo * Teo * Teo * m5Ceo WV-
m5Ceo * Teo * Teo * m5Ceo * m5Ceo * T * T * CTTCCTTGCTTTCCCGCCCT
XXXXXXXXXXXXXXXXXXX 5986 G * C * T * T * T * C * C * C * Geo *
m5Ceo * m5Ceo * m5Ceo * Teo WV- Teo * m5Ceo * m5Ceo * Teo * Teo * G
* C * TCCTTGCTTTCCCGCCCTCA XXXXXXXXXXXXXXXXXXX 5987 T * T * T * C *
C * C * G * C * m5Ceo * m5Ceo * Teo * m5Ceo * Aeo WV- Geo * m5Ceo *
Teo * Teo * Teo * C * C * C * GCTTTCCCGCCCTCAGTACC
XXXXXXXXXXXXXXXXXXX 5988 G * C * C * C * T * C * A * Geo * Teo *
Aeo * m5Ceo * m5Ceo WV- Geo * m5Ceo * m5Ceo * m5Ceo * Teo * C * A *
GCCCTCAGTACCCGAGCTGT XXXXXXXXXXXXXXXXXXX 5989 G * T * A * C * C * C
* G * A * Geo * m5Ceo * Teo * Geo * Teo WV- Aeo * m5Ceo * m5Ceo *
m5Ceo * Geo * A * G * ACCCGAGCTGTCTCCTTCCC XXXXXXXXXXXXXXXXXXX 5990
C * T * G * T * C * T * C * C * Teo * Teo * m5Ceo * m5Ceo * m5Ceo
WV- Geo * Geo * Aeo * m5Ceo * m5Ceo * C * G * GGACCCGCTGGGAGCGCTGC
XXXXXXXXXXXXXXXXXXX 5991 C * T * G * G * G * A * G * C * Geo *
m5Ceo * Teo * Geo * m5Ceo WV- Geo * Geo * Geo * Aeo * Aeo * G * G *
C * C * GGGAAGGCCGGAGGGTGGGC XXXXXXXXXXXXXXXXXXX 5992 G * G * A * G
* G * G * Teo * Geo * Geo * Geo * m5Ceo WV- Geo * Geo * Teo * m5Ceo
* m5Ceo * C * T * G * GGTCCCTGCCGGCGAGGAGA XXXXXXXXXXXXXXXXXXX 5993
C * C * G * G * C * G * A * Geo * Geo * Aeo * Geo * Aeo WV- Geo *
Teo * m5Ceo * Geo * Geo * T * G * T * GTCGGTGTGCTCCCCATTCT
XXXXXXXXXXXXXXXXXXX 5994 G * C * T * C * C * C * C * Aeo * Teo *
Teo * m5Ceo * Teo WV- Geo * Teo * Geo * m5Ceo * Teo * C * C * C *
GTGCTCCCCATTCTGTGGGA XXXXXXXXXXXXXXXXXXX 5995 C * A * T * T * C * T
* G * Teo * Geo * Geo * Geo * Aeo WV- m5Ceo * m5Ceo * Teo * Geo *
Geo * T * T * CCTGGTTGCTTCACAGCTCC XXXXXXXXXXXXXXXXXXX 5996 G * C *
T * T * C * A * C * A * Geo * m5Ceo * Teo * m5Ceo * m5Ceo WV- Geo *
Teo * m5Ceo * m5Ceo * Geo * T * G * GTCCGTGTGCTCATTGGGTC
XXXXXXXXXXXXXXXXXXX 5997 T * G * C * T * C * A * T * T * Geo * Geo
* Geo * Teo * m5Ceo WV- Geo * Geo * Geo * Aeo * Geo * G * T * C * C
* GGGAGGTCCTGCACTTTCCC XXXXXXXXXXXXXXXXXXX 5998 T * G * C * A * C *
T * Teo * Teo * m5Ceo * m5Ceo * m5Ceo WV- m5Ceo * m5Ceo * Teo *
m5Ceo * Teo * G * C * CCTCTGCCAAGGCCTGCCAC XXXXXXXXXXXXXXXXXXX 5999
C * A * A * G * G * C * C * T * Geo * m5Ceo * m5Ceo * Aeo * m5Ceo
WV- Aeo * Geo * Teo * Geo * Geo * T * C * C * T *
AGTGGTCCTGGGCAGCTCCT XXXXXXXXXXXXXXXXXXX 6000 G * G * G * C * A * G
* m5Ceo * Teo * m5Ceo * m5Ceo * Teo WV- m5Ceo * m5CeoTeom5CeoAeo *
C * T * C * A * CCTCACTCACCCACTCGCCA XOOOXXXXXXXXXXXOOOX 6408 C * C
* C * A * C * T * m5CeoGeom5Ceom5Ceo * Aeo WV- mG * mAmUmGmC * C *
G * C * C * T * C * GAUGCCGCCTCCTCACUCAC XOOOXXXXXXXXXXXOOOX 6471 C
* T * C * A * mCmUmCmA * mC WV- mA * mUmGmCmC * G * C * C * T * C *
C * AUGCCGCCTCCTCACUCACC XOOOXXXXXXXXXXXOOOX 6472 T * C * A * C *
mUmCmAmC * mC WV- mU * mGmCmCmG * C * C * T * C * C * T *
UGCCGCCTCCTCACTCACCC XOOOXXXXXXXXXXXOOOX 6473 C * A * C * T *
mCmAmCmC * mC WV- mG * mCmCmGmC * C * T * C * C * T * C *
GCCGCCTCCTCACTCACCCA XOOOXXXXXXXXXXXOOOX 6474 A * C * T * C *
mAmCmCmC * mA WV- mC * mCmGmCmC * T * C * C * T * C * A *
CCGCCTCCTCACTCACCCAC XOOOXXXXXXXXXXXOOOX 6475 C * T * C * A *
mCmCmCmA * mC WV- mC * mGmCmCmU * C * C * T * C * A * C *
CGCCUCCTCACTCACCCACU XOOOXXXXXXXXXXXOOOX 6476 T * C * A * C *
mCmCmAmC * mU WV- mG * mCmCmUmC * C * T * C * A * C * T *
GCCUCCTCACTCACCCACUC XOOOXXXXXXXXXXXOOOX 6477 C * A * C * C *
mCmAmCmU * mC WV- mC * mCmUmCmC * T * C * A * C * T * C *
CCUCCTCACTCACCCACUCG XOOOXXXXXXXXXXXOOOX 6478 A * C * C * C *
mAmCmUmC * mG WV- mC * mUmCmCmU * C * A * C * T * C * A *
CUCCUCACTCACCCACUCGC XOOOXXXXXXXXXXXOOOX 6479 C * C * C * A *
mCmUmCmG * mC WV- mU * mCmCmUmC * A * C * T * C * A * C *
UCCUCACTCACCCACUCGCC XOOOXXXXXXXXXXXOOOX 6480 C * C * A * C *
mUmCmGmC * mC WV- mC * mUmCmAmC * T * C * A * C * C * C *
CUCACTCACCCACTCGCCAC XOOOXXXXXXXXXXXOOOX 6481 A * C * T * C *
mGmCmCmA * mC WV- mC * mAmCmUmC * A * C * C * C * A * C *
CACUCACCCACTCGCCACCG XOOOXXXXXXXXXXXOOOX 6482 T * C * G * C *
mCmAmCmC * mG WV- mA * mCmUmCmA * C * C * C * A * C * T *
ACUCACCCACTCGCCACCGC XOOOXXXXXXXXXXXOOOX 6483 C * G * C * C *
mAmCmCmG * mC WV- mC * mUmCmAmC * C * C * A * C * T * C *
CUCACCCACTCGCCACCGCC XOOOXXXXXXXXXXXOOOX 6484 G * C * C * A *
mCmCmGmC * mC WV- mU * mCmAmCmC * C * A * C * T * C * G *
UCACCCACTCGCCACCGCCU XOOOXXXXXXXXXXXOOOX 6485 C * C * A * C *
mCmGmCmC * mU WV- mC * mAmCmCmC * A * C * T * C * G * C *
CACCCACTCGCCACCGCCUG XOOOXXXXXXXXXXXOOOX 6486 C * A * C * C *
mGmCmCmU * mG WV- mA * mCmCmCmA * C * T * C * G * C * C *
ACCCACTCGCCACCGCCUGC XOOOXXXXXXXXXXXOOOX 6487 A * C * C * G *
mCmCmUmG * mC WV- mC * mCmCmAmC * T * C * G * C * C * A *
CCCACTCGCCACCGCCUGCG XOOOXXXXXXXXXXXOOOX 6488 C * C * G * C *
mCmUmGmC * mG WV- mC * mCmAmCmU * C * G * C * C * A * C *
CCACUCGCCACCGCCUGCGC XOOOXXXXXXXXXXXOOOX 6489 C * G * C * C *
mUmGmCmG * mC WV- R GR CR GR CR AR GR GR CR GR GR UR
GCGCAGGCGGUGGCGAGUGG OOOOOOOOOOOOOOOOOOO 6490 GR GR CR GR AR GR UR
GR GR GR UR GR GUGAGUGAGGAGGCGGCAUC OOOOOOOOOOO OOOOOOO AR GR UR GR
AR GR GR AR GR GR CR GR OO GR CR AR UR C WV- R GR CR GR CR AR GR GR
CR GR GR UR GCGCAGGCGGUGGCGAGUGG OOOOOOOOOOOOOOOOOOO 6491 GR GR CR
GR AR GR UR GR GR GR UR GR GUGAGUGAGG OOOOOOOOOO AR GR UR GR AR GR
G WV- R UR GR GR CR GR AR GR UR GR GR GR UGGCGAGUGGGUGAGUGAGG
OOOOOOOOOOOOOOOOOOO 6492 UR GR AR GR UR GR AR GR GR AR GR GR
AGGCGGCAUC OOOOOOOOOO CR GR GR CR AR UR C WV- mC * mC * mU * mA *
mG * C * G * G * G * CCUAGCGGGACACCGUAGGU XXXXXXXXXXXXXXXXXXX 6831
A * C * A * C * C * G * mU * mA * mG * mG * mU WV- mC * mU * mU *
mU * mC * C * T * A * G * CUUUCCTAGCGGGACACCGU XXXXXXXXXXXXXXXXXXX
6832 C * G * G * G * A * C * mA * mC * mC * mG * mU WV- mC * mU *
mC * mU * mU * T * C * C * T * CUCUUTCCTAGCGGGACACC
XXXXXXXXXXXXXXXXXXX 6833 A * G * C * G * G * G * mA * mC * mA * mC
* mC WV- mC * mC * mU * mC * mU * C * T * T * T *
CCUCUCTTTCCTAGCGGGAC XXXXXXXXXXXXXXXXXXX 6834 C * C * T * A * G * C
* mG * mG * mG * mA * mC WV- mA * mC * mC * mU * mC * T * C * T * T
* ACCUCTCTTTCCTAGCGGGA XXXXXXXXXXXXXXXXXXX 6835 T * C * C * T * A *
G * mC * mG * mG * mG * mA WV- mC * mA * mC * mC * mU * C * T * C *
T * CACCUCTCTTTCCTAGCGGG XXXXXXXXXXXXXXXXXXX 6836 T * T * C * C * T
* A * mG * mC * mG * mG * mG
WV- mC * mG * mC * mA * mC * C * T * C * T * CGCACCTCTCTTTCCUAGCG
XXXXXXXXXXXXXXXXXXX 6837 C * T * T * T * C * C * mU * mA * mG * mC
* mG WV- mA * mC * mG * mC * mA * C * C * T * C *
ACGCACCTCTCTTTCCUAGC XXXXXXXXXXXXXXXXXXX 6838 T * C * T * T * T * C
* mC * mU * mA * mG * mC WV- mG * mC * mU * mG * mU * T * T * G * A
* GCUGUTTGACGCACCUCUCU XXXXXXXXXXXXXXXXXXX 6839 C * G * C * A * C *
C * mU * mC * mU * mC * mU WV- mG * mU * mC * mG * mC * T * G * T *
T * GUCGCTGTTTGACGCACCUC XXXXXXXXXXXXXXXXXXX 6840 T * G * A * C * G
* C * mA * mC * mC * mU * mC WV- mG * mC * mA * mG * mG * G * A * C
* G * GCAGGGACGGCTGACACACC XXXXXXXXXXXXXXXXXXX 6841 G * C * T * G *
A * C * mA * mC * mA * mC * mC WV- mG * mG * mC * mA * mG * C * A *
G * G * GGCAGCAGGGACGGCUGACA XXXXXXXXXXXXXXXXXXX 6842 G * A * C * G
* G * C * mU * mG * mA * mC * mA WV- mC * mG * mG * mG * mC * A * G
* C * A * CGGGCAGCAGGGACGGCUGA XXXXXXXXXXXXXXXXXXX 6843 G * G * G *
A * C * G * mG * mC * mU * mG * mA WV- mC * mC * mG * mG * mG * C *
A * G * C * CCGGGCAGCAGGGACGGCUG XXXXXXXXXXXXXXXXXXX 6844 A * G * G
* G * A * C * mG * mG * mC * mU * mG WV- mA * mC * mC * mG * mG * G
* C * A * G * ACCGGGCAGCAGGGACGGCU XXXXXXXXXXXXXXXXXXX 6845 C * A *
G * G * G * A * mC * mG * mG * mC * mU WV- mA * mA * mC * mC * mG *
G * G * C * A * AACCGGGCAGCAGGGACGGC XXXXXXXXXXXXXXXXXXX 6846 G * C
* A * G * G * G * mA * mC * mG * mG * mC WV- mG * mC * mA * mA * mC
* C * G * G * G * GCAACCGGGCAGCAGGGACG XXXXXXXXXXXXXXXXXXX 6847 C *
A * G * C * A * G * mG * mG * mA * mC * mG WV- mA * mG * mC * mA *
mA * C * C * G * G * AGCAACCGGGCAGCAGGGAC XXXXXXXXXXXXXXXXXXX 6848
G * C * A * G * C * A * mG * mG * mG * mA * mC WV- mG * mC * mU *
mA * mG * A * C * C * C * GCUAGACCCCGCCCCCAAAA XXXXXXXXXXXXXXXXXXX
6849 C * G * C * C * C * C * mC * mA * mA * mA * mA WV- mU * mU *
mG * mC * mU * A * G * A * C * UUGCUAGACCCCGCCCCCAA
XXXXXXXXXXXXXXXXXXX 6850 C * C * C * G * C * C * mC * mC * mC * mA
* mA WV- mC * mU * mU * mG * mC * T * A * G * A *
CUUGCTAGACCCCGCCCCCA XXXXXXXXXXXXXXXXXXX 6851 C * C * C * C * G * C
* mC * mC * mC * mC * mA WV- mC * mU * mC * mU * mU * G * C * T * A
* CUCUUGCTAGACCCCGCCCC XXXXXXXXXXXXXXXXXXX 6852 G * A * C * C * C *
C * mG * mC * mC * mC * mC WV- mU * mG * mC * mU * mC * T * T * G *
C * UGCUCTTGCTAGACCCCGCC XXXXXXXXXXXXXXXXXXX 6853 T * A * G * A * C
* C * mC * mC * mG * mC * mC WV- mC * mC * mU * mG * mC * T * C * T
* T * CCUGCTCTTGCTAGACCCCG XXXXXXXXXXXXXXXXXXX 6854 G * C * T * A *
G * A * mC * mC * mC * mC * mG WV- mC * mC * mA * mC * mA * C * C *
T * G * CCACACCTGCTCTTGCUAGA XXXXXXXXXXXXXXXXXXX 6855 C * T * C * T
* T * G * mC * mU * mA * mG * mA WV- mC * mC * mC * mA * mC * A * C
* C * T * CCCACACCTGCTCTTGCUAG XXXXXXXXXXXXXXXXXXX 6856 G * C * T *
C * T * T * mG * mC * mU * mA * mG WV- mA * mC * mC * mC * mA * C *
A * C * C * ACCCACACCTGCTCTUGCUA XXXXXXXXXXXXXXXXXXX 6857 T * G * C
* T * C * T * mU * mG * mC * mU * mA WV- mA * mA * mC * mC * mC * A
* C * A * C * AACCCACACCTGCTCUUGCU XXXXXXXXXXXXXXXXXXX 6858 C * T *
G * C * T * C * mU * mU * mG * mC * mU WV- mU * mC * mA * mC * mC *
C * T * C * A * UCACCCTCAGCGAGTACUGU XXXXXXXXXXXXXXXXXXX 6859 G * C
* G * A * G * T * mA * mC * mU * mG * mU WV- mG * mU * mU * mC * mA
* C * C * C * T * GUUCACCCTCAGCGAGUACU XXXXXXXXXXXXXXXXXXX 6860 C *
A * G * C * G * A * mG * mU * mA * mC * mU WV- mC * mU * mU * mG *
mU * T * C * A * C * CUUGUTCACCCTCAGCGAGU XXXXXXXXXXXXXXXXXXX 6861
C * C * T * C * A * G * mC * mG * mA * mG * mU WV- mG * mU * mC *
mU * mU * T * T * C * T * GUCUUTTCTTGTTCACCCUC XXXXXXXXXXXXXXXXXXX
6862 T * G * T * T * C * A * mC * mC * mC * mU * mC WV- mG * mG *
mU * mC * mU * T * T * T * C * GGUCUTTTCTTGTTCACCCU
XXXXXXXXXXXXXXXXXXX 6863 T * T * G * T * T * C * mA * mC * mC * mC
* mU WV- mC * mC * mU * mC * mC * T * T * G * T *
CCUCCTTGTTTTCTTCUGGU XXXXXXXXXXXXXXXXXXX 6864 T * T * T * C * T * T
* mC * mU * mG * mG * mU WV- mC * mC * mC * mU * mC * C * T * T * G
* CCCUCCTTGTTTTCTUCUGG XXXXXXXXXXXXXXXXXXX 6865 T * T * T * T * C *
T * mU * mC * mU * mG * mG WV- mG * mU * mU * mG * mU * T * T * C *
C * GUUGUTTCCCTCCTTGUUUU XXXXXXXXXXXXXXXXXXX 6866 C * T * C * C * T
* T * mG * mU * mU * mU * mU WV- mG * mG * mU * mU * mG * T * T * T
* C * GGUUGTTTCCCTCCTUGUUU XXXXXXXXXXXXXXXXXXX 6867 C * C * T * C *
C * T * mU * mG * mU * mU * mU WV- mC * mG * mG * mU * mU * G * T *
T * T * CGGUUGTTTCCCTCCUUGUU XXXXXXXXXXXXXXXXXXX 6868 C * C * C * T
* C * C * mU * mU * mG * mU * mU WV- mU * mG * mC * mG * mG * T * T
* G * T * UGCGGTTGTTTCCCTCCUUG XXXXXXXXXXXXXXXXXXX 6869 T * T * C *
C * C * T * mC * mC * mU * mU * mG WV- mC * mU * mG * mC * mG * G *
T * T * G * CUGCGGTTGTTTCCCUCCUU XXXXXXXXXXXXXXXXXXX 6870 T * T * T
* C * C * C * mU * mC * mC * mU * mU WV- mA * mG * mG * mC * mU * G
* C * G * G * AGGCUGCGGTTGTTTCCCUC XXXXXXXXXXXXXXXXXXX 6871 T * T *
G * T * T * T * mC * mC * mC * mU * mC WV- mA * mC * mA * mG * mG *
C * T * G * C * ACAGGCTGCGGTTGTUUCCC XXXXXXXXXXXXXXXXXXX 6872 G * G
* T * T * G * T * mU * mU * mC * mC * mC WV- mG * mC * mU * mA * mC
* A * G * G * C * GCUACAGGCTGCGGTUGUUU XXXXXXXXXXXXXXXXXXX 6873 T *
G * C * G * G * T * mU * mG * mU * mU * mU WV- mU * mG * mC * mU *
mA * C * A * G * G * UGCUACAGGCTGCGGUUGUU XXXXXXXXXXXXXXXXXXX 6874
C * T * G * C * G * G * mU * mU * mG * mU * mU WV- mU * mU * mG *
mC * mU * A * C * A * G * UUGCUACAGGCTGCGGUUGU XXXXXXXXXXXXXXXXXXX
6875 G * C * T * G * C * G * mG * mU * mU * mG * mU WV- mG * mC *
mU * mU * mG * C * T * A * C * GCUUGCTACAGGCTGCGGUU
XXXXXXXXXXXXXXXXXXX 6876 A * G * G * C * T * G * mC * mG * mG * mU
* mU WV- mA * mG * mC * mU * mU * G * C * T * A *
AGCUUGCTACAGGCTGCGGU XXXXXXXXXXXXXXXXXXX 6877 C * A * G * G * C * T
* mG * mC * mG * mG * mU WV- mG * mA * mG * mC * mU * T * G * C * T
* GAGCUTGCTACAGGCUGCGG XXXXXXXXXXXXXXXXXXX 6878 A * C * A * G * G *
C * mU * mG * mC * mG * mG WV- mC * mA * mG * mA * mG * C * T * T *
G * CAGAGCTTGCTACAGGCUGC XXXXXXXXXXXXXXXXXXX 6879 C * T * A * C * A
* G * mG * mC * mU * mG * mC WV- mU * mC * mC * mA * mG * A * G * C
* T * UCCAGAGCTTGCTACAGGCU XXXXXXXXXXXXXXXXXXX 6880 T * G * C * T *
A * C * mA * mG * mG * mC * mU WV- mU * mU * mC * mC * mA * G * A *
G * C * UUCCAGAGCTTGCTACAGGC XXXXXXXXXXXXXXXXXXX 6881 T * T * G * C
* T * A * mC * mA * mG * mG * mC WV- mC * mC * mU * mG * mA * G * T
* T * C * CCUGAGTTCCAGAGCUUGCU XXXXXXXXXXXXXXXXXXX 6882 C * A * G *
A * G * C * mU * mU * mG * mC * mU WV- mU * mC * mC * mU * mG * A *
G * T * T * UCCUGAGTTCCAGAGCUUGC XXXXXXXXXXXXXXXXXXX 6883 C * C * A
* G * A * G * mC * mU * mU * mG * mC WV- mA * mC * mU * mC * mC * T
* G * A * G * ACUCCTGAGTTCCAGAGCUU XXXXXXXXXXXXXXXXXXX 6884 T * T *
C * C * A * G * mA * mG * mC * mU * mU WV- mG * mC * mG * mC * mG *
A * C * T * C * GCGCGACTCCTGAGTUCCAG XXXXXXXXXXXXXXXXXXX 6885 C * T
* G * A * G * T * mU * mC * mC * mA * mG WV- mC * mG * mC * mG * mC
* G * A * C * T * CGCGCGACTCCTGAGUUCCA XXXXXXXXXXXXXXXXXXX 6886 C *
C * T * G * A * G * mU * mU * mC * mC * mA WV- mG * mC * mG * mC *
mG * C * G * A * C * GCGCGCGACTCCTGAGUUCC
XXXXXXXXXXXXXXXXXXX 6887 T * C * C * T * G * A * mG * mU * mU * mC
* mC WV- mA * mG * mC * mG * mC * G * C * G * A *
AGCGCGCGACTCCTGAGUUC XXXXXXXXXXXXXXXXXXX 6888 C * T * C * C * T * G
* mA * mG * mU * mU * mC WV- mA * mG * mG * mA * mU * G * C * C * G
* AGGAUGCCGCCTCCTCACUC XXXXXXXXXXXXXXXXXXX 6889 C * C * T * C * C *
T * mC * mA * mC * mU * mC WV- mC * mA * mG * mG * mA * T * G * C *
C * CAGGATGCCGCCTCCUCACU XXXXXXXXXXXXXXXXXXX 6890 G * C * C * T * C
* C * mU * mC * mA * mC * mU WV- mC * mC * mA * mG * mG * A * T * G
* C * CCAGGATGCCGCCTCCUCAC XXXXXXXXXXXXXXXXXXX 6891 C * G * C * C *
T * C * mC * mU * mC * mA * mC WV- mC * mG * mC * mC * mA * G * G *
A * T * CGCCAGGATGCCGCCUCCUC XXXXXXXXXXXXXXXXXXX 6892 G * C * C * G
* C * C * mU * mC * mC * mU * mC WV- mC * mC * mG * mC * mC * A * G
* G * A * CCGCCAGGATGCCGCCUCCU XXXXXXXXXXXXXXXXXXX 6893 T * G * C *
C * G * C * mC * mU * mC * mC * mU WV- mC * mC * mC * mG * mC * C *
A * G * G * CCCGCCAGGATGCCGCCUCC XXXXXXXXXXXXXXXXXXX 6894 A * T * G
* C * C * G * mC * mC * mU * mC * mC WV- mA * mC * mC * mC * mG * C
* C * A * G * ACCCGCCAGGATGCCGCCUC XXXXXXXXXXXXXXXXXXX 6895 G * A *
T * G * C * C * mG * mC * mC * mU * mC WV- mC * mA * mC * mC * mC *
G * C * C * A * CACCCGCCAGGATGCCGCCU XXXXXXXXXXXXXXXXXXX 6896 G * G
* A * T * G * C * mC * mG * mC * mC * mU WV- mC * mC * mA * mC * mC
* C * G * C * C * CCACCCGCCAGGATGCCGCC XXXXXXXXXXXXXXXXXXX 6897 A *
G * G * A * T * G * mC * mC * mG * mC * mC WV- mG * mC * mC * mA *
mC * C * C * G * C * GCCACCCGCCAGGATGCCGC XXXXXXXXXXXXXXXXXXX 6898
C * A * G * G * A * T * mG * mC * mC * mG * mC WV- mA * mA * mC *
mA * mG * C * C * A * C * AACAGCCACCCGCCAGGAUG XXXXXXXXXXXXXXXXXXX
6899 C * C * G * C * C * A * mG * mG * mA * mU * mG WV- mC * mC *
mA * mA * mA * C * A * G * C * CCAAACAGCCACCCGCCAGG
XXXXXXXXXXXXXXXXXXX 6900 C * A * C * C * C * G * mC * mC * mA * mG
* mG WV- mC * mC * mC * mA * mA * A * C * A * G *
CCCAAACAGCCACCCGCCAG XXXXXXXXXXXXXXXXXXX 6901 C * C * A * C * C * C
* mG * mC * mC * mA * mG WV- mA * mC * mC * mC * mC * A * A * A * C
* ACCCCAAACAGCCACCCGCC XXXXXXXXXXXXXXXXXXX 6902 A * G * C * C * A *
C * mC * mC * mG * mC * mC WV- mC * mC * mC * mG * mG * C * A * G *
C * CCCGGCAGCCGAACCCCAAA XXXXXXXXXXXXXXXXXXX 6903 C * G * A * A * C
* C * mC * mC * mA * mA * mA WV- mU * mC * mC * mC * mG * G * C * A
* G * UCCCGGCAGCCGAACCCCAA XXXXXXXXXXXXXXXXXXX 6904 C * C * G * A *
A * C * mC * mC * mC * mA * mA WV- mU * mU * mC * mC * mC * G * G *
C * A * UUCCCGGCAGCCGAACCCCA XXXXXXXXXXXXXXXXXXX 6905 G * C * C * G
* A * A * mC * mC * mC * mC * mA WV- mC * mU * mU * mC * mC * C * G
* G * C * CUUCCCGGCAGCCGAACCCC XXXXXXXXXXXXXXXXXXX 6906 A * G * C *
C * G * A * mA * mC * mC * mC * mC WV- mU * mC * mU * mU * mC * C *
C * G * G * UCUUCCCGGCAGCCGAACCC XXXXXXXXXXXXXXXXXXX 6907 C * A * G
* C * C * G * mA * mA * mC * mC * mC WV- mC * mU * mC * mU * mU * C
* C * C * G * CUCUUCCCGGCAGCCGAACC XXXXXXXXXXXXXXXXXXX 6908 G * C *
A * G * C * C * mG * mA * mA * mC * mC WV- mC * mC * mU * mC * mU *
T * C * C * C * CCUCUTCCCGGCAGCCGAAC XXXXXXXXXXXXXXXXXXX 6909 G * G
* C * A * G * C * mC * mG * mA * mA * mC WV- mG * mC * mC * mU * mC
* T * T * C * C * GCCUCTTCCCGGCAGCCGAA XXXXXXXXXXXXXXXXXXX 6910 C *
G * G * C * A * G * mC * mC * mG * mA * mA WV- mC * mG * mC * mC *
mU * C * T * T * C * CGCCUCTTCCCGGCAGCCGA XXXXXXXXXXXXXXXXXXX 6911
C * C * G * G * C * A * mG * mC * mC * mG * mA WV- mC * mC * mG *
mC * mG * C * C * T * C * CCGCGCCTCTTCCCGGCAGC XXXXXXXXXXXXXXXXXXX
6912 T * T * C * C * C * G * mG * mC * mA * mG * mC WV- mC * mC *
mC * mG * mC * G * C * C * T * CCCGCGCCTCTTCCCGGCAG
XXXXXXXXXXXXXXXXXXX 6913 C * T * T * C * C * C * mG * mG * mC * mA
* mG WV- mA * mC * mC * mC * mG * C * G * C * C *
ACCCGCGCCTCTTCCCGGCA XXXXXXXXXXXXXXXXXXX 6914 T * C * T * T * C * C
* mC * mG * mG * mC * mA WV- mU * mA * mC * mC * mC * G * C * G * C
* UACCCGCGCCTCTTCCCGGC XXXXXXXXXXXXXXXXXXX 6915 C * T * C * T * T *
C * mC * mC * mG * mG * mC WV- mC * mU * mA * mC * mC * C * G * C *
G * CUACCCGCGCCTCTTCCCGG XXXXXXXXXXXXXXXXXXX 6916 C * C * T * C * T
* T * mC * mC * mC * mG * mG WV- mU * mU * mC * mU * mA * C * C * C
* G * UUCUACCCGCGCCTCUUCCC XXXXXXXXXXXXXXXXXXX 6917 C * G * C * C *
T * C * mU * mU * mC * mC * mC WV- mC * mU * mU * mC * mU * A * C *
C * C * CUUCUACCCGCGCCTCUUCC XXXXXXXXXXXXXXXXXXX 6918 G * C * G * C
* C * T * mC * mU * mU * mC * mC WV- mG * mC * mU * mU * mC * T * A
* C * C * GCUUCTACCCGCGCCUCUUC XXXXXXXXXXXXXXXXXXX 6919 C * G * C *
G * C * C * mU * mC * mU * mU * mC WV- mC * mG * mC * mU * mU * C *
T * A * C * CGCUUCTACCCGCGCCUCUU XXXXXXXXXXXXXXXXXXX 6920 C * C * G
* C * G * C * mC * mU * mC * mU * mU WV- mC * mC * mG * mC * mU * T
* C * T * A * CCGCUTCTACCCGCGCCUCU XXXXXXXXXXXXXXXXXXX 6921 C * C *
C * G * C * G * mC * mC * mU * mC * mU WV- mC * mCmUmAmG * C * G *
G * G * A * C * CCUAGCGGGACACCGUAGGU XOOOXXXXXXXXXXXOOOX 6922 A * C
* C * G * mUmAmGmG * mU WV- mC * mUmUmUmC * C * T * A * G * C * G *
CUUUCCTAGCGGGACACCGU XOOOXXXXXXXXXXXOOOX 6923 G * G * A * C *
mAmCmCmG * mU WV- mC * mUmCmUmU * T * C * C * T * A * G *
CUCUUTCCTAGCGGGACACC XOOOXXXXXXXXXXXOOOX 6924 C * G * G * G *
mAmCmAmC * mC WV- mC * mCmUmCmU * C * T * T * T * C * C *
CCUCUCTTTCCTAGCGGGAC XOOOXXXXXXXXXXXOOOX 6925 T * A * G * C *
mGmGmGmA * mC WV- mA * mCmCmUmC * T * C * T * T * T * C *
ACCUCTCTTTCCTAGCGGGA XOOOXXXXXXXXXXXOOOX 6926 C * T * A * G *
mCmGmGmG * mA WV- mC * mAmCmCmU * C * T * C * T * T * T *
CACCUCTCTTTCCTAGCGGG XOOOXXXXXXXXXXXOOOX 6927 C * C * T * A *
mGmCmGmG * mG WV- mC * mGmCmAmC * C * T * C * T * C * T *
CGCACCTCTCTTTCCUAGCG XOOOXXXXXXXXXXXOOOX 6928 T * T * C * C *
mUmAmGmC * mG WV- mA * mCmGmCmA * C * C * T * C * T * C *
ACGCACCTCTCTTTCCUAGC XOOOXXXXXXXXXXXOOOX 6929 T * T * T * C *
mCmUmAmG * mC WV- mG * mCmUmGmU * T * T * G * A * C * G *
GCUGUTTGACGCACCUCUCU XOOOXXXXXXXXXXXOOOX 6930 C * A * C * C *
mUmCmUmC * mU WV- mG * mUmCmGmC * T * G * T * T * T * G *
GUCGCTGTTTGACGCACCUC XOOOXXXXXXXXXXXOOOX 6931 A * C * G * C *
mAmCmCmU * mC WV- mG * mCmAmGmG * G * A * C * G * G * C *
GCAGGGACGGCTGACACACC XOOOXXXXXXXXXXXOOOX 6932 T * G * A * C *
mAmCmAmC * mC WV- mG * mGmCmAmG * C * A * G * G * G * A *
GGCAGCAGGGACGGCUGACA XOOOXXXXXXXXXXXOOOX 6933 C * G * G * C *
mUmGmAmC * mA WV- mC * mGmGmGmC * A * G * C * A * G * G *
CGGGCAGCAGGGACGGCUGA XOOOXXXXXXXXXXXOOOX 6934 G * A * C * G *
mGmCmUmG * mA WV- mC * mCmGmGmG * C * A * G * C * A * G *
CCGGGCAGCAGGGACGGCUG XOOOXXXXXXXXXXXOOOX 6935 G * G * A * C *
mGmGmCmU * mG WV- mA * mCmCmGmG * G * C * A * G * C * A *
ACCGGGCAGCAGGGACGGCU XOOOXXXXXXXXXXXOOOX 6936 G * G * G * A *
mCmGmGmC * mU WV- mA * mAmCmCmG * G * G * C * A * G * C *
AACCGGGCAGCAGGGACGGC XOOOXXXXXXXXXXXOOOX 6937 A * G * G * G *
mAmCmGmG * mC WV- mG * mCmAmAmC * C * G * G * G * C * A *
GCAACCGGGCAGCAGGGACG XOOOXXXXXXXXXXXOOOX 6938 G * C * A * G *
mGmGmAmC * mG WV- mA * mGmCmAmA * C * C * G * G * G * C *
AGCAACCGGGCAGCAGGGAC XOOOXXXXXXXXXXXOOOX 6939 A * G * C * A *
mGmGmGmA * mC WV- mG * mCmUmAmG * A * C * C * C * C * G *
GCUAGACCCCGCCCCCAAAA XOOOXXXXXXXXXXXOOOX 6940 C * C * C * C *
mCmAmAmA * mA WV- mU * mUmGmCmU * A * G * A * C * C * C *
UUGCUAGACCCCGCCCCCAA
XOOOXXXXXXXXXXXOOOX 6941 C * G * C * C * mCmCmCmA * mA WV- mC *
mUmUmGmC * T * A * G * A * C * C * CUUGCTAGACCCCGCCCCCA
XOOOXXXXXXXXXXXOOOX 6942 C * C * G * C * mCmCmCmC * mA WV- mC *
mUmCmUmU * G * C * T * A * G * A * CUCUUGCTAGACCCCGCCCC
XOOOXXXXXXXXXXXOOOX 6943 C * C * C * C * mGmCmCmC * mC WV- mU *
mGmCmUmC * T * T * G * C * T * A * UGCUCTTGCTAGACCCCGCC
XOOOXXXXXXXXXXXOOOX 6944 G * A * C * C * mCmCmGmC * mC WV- mC *
mCmUmGmC * T * C * T * T * G * C * CCUGCTCTTGCTAGACCCCG
XOOOXXXXXXXXXXXOOOX 6945 T * A * G * A * mCmCmCmC * mG WV- mC *
mCmAmCmA * C * C * T * G * C * T * CCACACCTGCTCTTGCUAGA
XOOOXXXXXXXXXXXOOOX 6946 C * T * T * G * mCmUmAmG * mA WV- mC *
mCmCmAmC * A * C * C * T * G * C * CCCACACCTGCTCTTGCUAG
XOOOXXXXXXXXXXXOOOX 6947 T * C * T * T * mGmCmUmA * mG WV- mA *
mCmCmCmA * C * A * C * C * T * G * ACCCACACCTGCTCTUGCUA
XOOOXXXXXXXXXXXOOOX 6948 C * T * C * T * mUmGmCmU * mA WV- mA *
mAmCmCmC * A * C * A * C * C * T * AACCCACACCTGCTCUUGCU
XOOOXXXXXXXXXXXOOOX 6949 G * C * T * C * mUmUmGmC * mU WV- mU *
mCmAmCmC * C * T * C * A * G * C * UCACCCTCAGCGAGTACUGU
XOOOXXXXXXXXXXXOOOX 6950 G * A * G * T * mAmCmUmG * mU WV- mG *
mUmUmCmA * C * C * C * T * C * A * GUUCACCCTCAGCGAGUACU
XOOOXXXXXXXXXXXOOOX 6951 G * C * G * A * mGmUmAmC * mU WV- mC *
mUmUmGmU * T * C * A * C * C * C * CUUGUTCACCCTCAGCGAGU
XOOOXXXXXXXXXXXOOOX 6952 T * C * A * G * mCmGmAmG * mU WV- mG *
mUmCmUmU * T * T * C * T * T * G * GUCUUTTCTTGTTCACCCUC
XOOOXXXXXXXXXXXOOOX 6953 T * T * C * A * mCmCmCmU * mC WV- mG *
mGmUmCmU * T * T * T * C * T * T * GGUCUTTTCTTGTTCACCCU
XOOOXXXXXXXXXXXOOOX 6954 G * T * T * C * mAmCmCmC * mU WV- mC *
mCmUmCmC * T * T * G * T * T * T * CCUCCTTGTTTTCTTCUGGU
XOOOXXXXXXXXXXXOOOX 6955 T * C * T * T * mCmUmGmG * mU WV- mC *
mCmCmUmC * C * T * T * G * T * T * CCCUCCTTGTTTTCTUCUGG
XOOOXXXXXXXXXXXOOOX 6956 T * T * C * T * mUmCmUmG * mG WV- mG *
mUmUmGmU * T * T * C * C * C * T * GUUGUTTCCCTCCTTGUUUU
XOOOXXXXXXXXXXXOOOX 6957 C * C * T * T * mGmUmUmU * mU WV- mG *
mGmUmUmG * T * T * T * C * C * C * GGUUGTTTCCCTCCTUGUUU
XOOOXXXXXXXXXXXOOOX 6958 T * C * C * T * mUmGmUmU * mU WV- mC *
mGmGmUmU * G * T * T * T * C * C * CGGUUGTTTCCCTCCUUGUU
XOOOXXXXXXXXXXXOOOX 6959 C * T * C * C * mUmUmGmU * mU WV- mU *
mGmCmGmG * T * T * G * T * T * T * UGCGGTTGTTTCCCTCCUUG
XOOOXXXXXXXXXXXOOOX 6960 C * C * C * T * mCmCmUmU * mG WV- mC *
mUmGmCmG * G * T * T * G * T * T * CUGCGGTTGTTTCCCUCCUU
XOOOXXXXXXXXXXXOOOX 6961 T * C * C * C * mUmCmCmU * mU WV- mA *
mGmGmCmU * G * C * G * G * T * T * AGGCUGCGGTTGTTTCCCUC
XOOOXXXXXXXXXXXOOOX 6962 G * T * T * T * mCmCmCmU * mC WV- mA *
mCmAmGmG * C * T * G * C * G * G * ACAGGCTGCGGTTGTUUCCC
XOOOXXXXXXXXXXXOOOX 6963 T * T * G * T * mUmUmCmC * mC WV- mG *
mCmUmAmC * A * G * G * C * T * G * GCUACAGGCTGCGGTUGUUU
XOOOXXXXXXXXXXXOOOX 6964 C * G * G * T * mUmGmUmU * mU WV- mU *
mGmCmUmA * C * A * G * G * C * T * UGCUACAGGCTGCGGUUGUU
XOOOXXXXXXXXXXXOOOX 6965 G * C * G * G * mUmUmGmU * mU WV- mU *
mUmGmCmU * A * C * A * G * G * C * UUGCUACAGGCTGCGGUUGU
XOOOXXXXXXXXXXXOOOX 6966 T * G * C * G * mGmUmUmG * mU WV- mG *
mCmUmUmG * C * T * A * C * A * G * GCUUGCTACAGGCTGCGGUU
XOOOXXXXXXXXXXXOOOX 6967 G * C * T * G * mCmGmGmU * mU WV- mA *
mGmCmUmU * G * C * T * A * C * A * AGCUUGCTACAGGCTGCGGU
XOOOXXXXXXXXXXXOOOX 6968 G * G * C * T * mGmCmGmG * mU WV- mG *
mAmGmCmU * T * G * C * T * A * C * GAGCUTGCTACAGGCUGCGG
XOOOXXXXXXXXXXXOOOX 6969 A * G * G * C * mUmGmCmG * mG WV- mC *
mAmGmAmG * C * T * T * G * C * T * CAGAGCTTGCTACAGGCUGC
XOOOXXXXXXXXXXXOOOX 6970 A * C * A * G * mGmCmUmG * mC WV- mU *
mCmCmAmG * A * G * C * T * T * G * UCCAGAGCTTGCTACAGGCU
XOOOXXXXXXXXXXXOOOX 6971 C * T * A * C * mAmGmGmC * mU WV- mU *
mUmCmCmA * G * A * G * C * T * T * UUCCAGAGCTTGCTACAGGC
XOOOXXXXXXXXXXXOOOX 6972 G * C * T * A * mCmAmGmG * mC WV- mC *
mCmUmGmA * G * T * T * C * C * A * CCUGAGTTCCAGAGCUUGCU
XOOOXXXXXXXXXXXOOOX 6973 G * A * G * C * mUmUmGmC * mU WV- mU *
mCmCmUmG * A * G * T * T * C * C * UCCUGAGTTCCAGAGCUUGC
XOOOXXXXXXXXXXXOOOX 6974 A * G * A * G * mCmUmUmG * mC WV- mA *
mCmUmCmC * T * G * A * G * T * T * ACUCCTGAGTTCCAGAGCUU
XOOOXXXXXXXXXXXOOOX 6975 C * C * A * G * mAmGmCmU * mU WV- mG *
mCmGmCmG * A * C * T * C * C * T * GCGCGACTCCTGAGTUCCAG
XOOOXXXXXXXXXXXOOOX 6976 G * A * G * T * mUmCmCmA * mG WV- mC *
mGmCmGmC * G * A * C * T * C * C * CGCGCGACTCCTGAGUUCCA
XOOOXXXXXXXXXXXOOOX 6977 T * G * A * G * mUmUmCmC * mA WV- mG *
mCmGmCmG * C * G * A * C * T * C * GCGCGCGACTCCTGAGUUCC
XOOOXXXXXXXXXXXOOOX 6978 C * T * G * A * mGmUmUmC * mC WV- mA *
mGmCmGmC * G * C * G * A * C * T * AGCGCGCGACTCCTGAGUUC
XOOOXXXXXXXXXXXOOOX 6979 C * C * T * G * mAmGmUmU * mC WV- mA *
mGmGmAmU * G * C * C * G * C * C * AGGAUGCCGCCTCCTCACUC
XOOOXXXXXXXXXXXOOOX 6980 T * C * C * T * mCmAmCmU * mC WV- mC *
mAmGmGmA * T * G * C * C * G * C * CAGGATGCCGCCTCCUCACU
XOOOXXXXXXXXXXXOOOX 6981 C * T * C * C * mUmCmAmC * mU WV- mC *
mCmAmGmG * A * T * G * C * C * G * CCAGGATGCCGCCTCCUCAC
XOOOXXXXXXXXXXXOOOX 6982 C * C * T * C * mCmUmCmA * mC WV- mC *
mGmCmCmA * G * G * A * T * G * C * CGCCAGGATGCCGCCUCCUC
XOOOXXXXXXXXXXXOOOX 6983 C * G * C * C * mUmCmCmU * mC WV- mC *
mCmGmCmC * A * G * G * A * T * G * CCGCCAGGATGCCGCCUCCU
XOOOXXXXXXXXXXXOOOX 6984 C * C * G * C * mCmUmCmC * mU WV- mC *
mCmCmGmC * C * A * G * G * A * T * CCCGCCAGGATGCCGCCUCC
XOOOXXXXXXXXXXXOOOX 6985 G * C * C * G * mCmCmUmC * mC WV- mA *
mCmCmCmG * C * C * A * G * G * A * ACCCGCCAGGATGCCGCCUC
XOOOXXXXXXXXXXXOOOX 6986 T * G * C * C * mGmCmCmU * mC WV- mC *
mAmCmCmC * G * C * C * A * G * G * CACCCGCCAGGATGCCGCCU
XOOOXXXXXXXXXXXOOOX 6987 A * T * G * C * mCmGmCmC * mU WV- mC *
mCmAmCmC * C * G * C * C * A * G * CCACCCGCCAGGATGCCGCC
XOOOXXXXXXXXXXXOOOX 6988 G * A * T * G * mCmCmGmC * mC WV- mG *
mCmCmAmC * C * C * G * C * C * A * GCCACCCGCCAGGATGCCGC
XOOOXXXXXXXXXXXOOOX 6989 G * G * A * T * mGmCmCmG * mC WV- mA *
mAmCmAmG * C * C * A * C * C * C * AACAGCCACCCGCCAGGAUG
XOOOXXXXXXXXXXXOOOX 6990 G * C * C * A * mGmGmAmU * mG WV- mC *
mCmAmAmA * C * A * G * C * C * A * CCAAACAGCCACCCGCCAGG
XOOOXXXXXXXXXXXOOOX 6991 C * C * C * G * mCmCmAmG * mG WV- mC *
mCmCmAmA * A * C * A * G * C * C * CCCAAACAGCCACCCGCCAG
XOOOXXXXXXXXXXXOOOX 6992 A * C * C * C * mGmCmCmA * mG WV- mA *
mCmCmCmC * A * A * A * C * A * G * ACCCCAAACAGCCACCCGCC
XOOOXXXXXXXXXXXOOOX 6993 C * C * A * C * mCmCmGmC * mC WV- mC *
mCmCmGmG * C * A * G * C * C * G * CCCGGCAGCCGAACCCCAAA
XOOOXXXXXXXXXXXOOOX 6994 A * A * C * C * mCmCmAmA * mA WV- mU *
mCmCmCmG * G * C * A * G * C * C * UCCCGGCAGCCGAACCCCAA
XOOOXXXXXXXXXXXOOOX 6995 G * A * A * C * mCmCmCmA * mA WV- mU *
mUmCmCmC * G * G * C * A * G * C * UUCCCGGCAGCCGAACCCCA
XOOOXXXXXXXXXXXOOOX 6996 C * G * A * A * mCmCmCmC * mA WV- mC *
mUmUmCmC * C * G * G * C * A * G * CUUCCCGGCAGCCGAACCCC
XOOOXXXXXXXXXXXOOOX 6997 C * C * G * A * mAmCmCmC * mC WV- mU *
mCmUmUmC * C * C * G * G * C * A * UCUUCCCGGCAGCCGAACCC
XOOOXXXXXXXXXXXOOOX 6998 G * C * C * G * mAmAmCmC * mC WV- mC *
mUmCmUmU * C * C * C * G * G * C * CUCUUCCCGGCAGCCGAACC
XOOOXXXXXXXXXXXOOOX 6999 A * G * C * C * mGmAmAmC * mC WV- mC *
mCmUmCmU * T * C * C * C * G * G * CCUCUTCCCGGCAGCCGAAC
XOOOXXXXXXXXXXXOOOX 7000 C * A * G * C * mCmGmAmA * mC WV- mG *
mCmCmUmC * T * T * C * C * C * G * GCCUCTTCCCGGCAGCCGAA
XOOOXXXXXXXXXXXOOOX 7001 G * C * A * G * mCmCmGmA * mA WV- mC *
mGmCmCmU * C * T * T * C * C * C * CGCCUCTTCCCGGCAGCCGA
XOOOXXXXXXXXXXXOOOX 7002 G * G * C * A * mGmCmCmG * mA WV- mC *
mCmGmCmG * C * C * T * C * T * T * CCGCGCCTCTTCCCGGCAGC
XOOOXXXXXXXXXXXOOOX 7003 C * C * C * G * mGmCmAmG * mC
WV- mC * mCmCmGmC * G * C * C * T * C * T * CCCGCGCCTCTTCCCGGCAG
XOOOXXXXXXXXXXXOOOX 7004 T * C * C * C * mGmGmCmA * mG WV- mA *
mCmCmCmG * C * G * C * C * T * C * ACCCGCGCCTCTTCCCGGCA
XOOOXXXXXXXXXXXOOOX 7005 T * T * C * C * mCmGmGmC * mA WV- mU *
mAmCmCmC * G * C * G * C * C * T * UACCCGCGCCTCTTCCCGGC
XOOOXXXXXXXXXXXOOOX 7006 C * T * T * C * mCmCmGmG * mC WV- mC *
mUmAmCmC * C * G * C * G * C * C * CUACCCGCGCCTCTTCCCGG
XOOOXXXXXXXXXXXOOOX 7007 T * C * T * T * mCmCmCmG * mG WV- mU *
mUmCmUmA * C * C * C * G * C * G * UUCUACCCGCGCCTCUUCCC
XOOOXXXXXXXXXXXOOOX 7008 C * C * T * C * mUmUmCmC * mC WV- mC *
mUmUmCmU * A * C * C * C * G * C * CUUCUACCCGCGCCTCUUCC
XOOOXXXXXXXXXXXOOOX 7009 G * C * C * T * mCmUmUmC * mC WV- mG *
mCmUmUmC * T * A * C * C * C * G * GCUUCTACCCGCGCCUCUUC
XOOOXXXXXXXXXXXOOOX 7010 C * G * C * C * mUmCmUmU * mC WV- mC *
mGmCmUmU * C * T * A * C * C * C * CGCUUCTACCCGCGCCUCUU
XOOOXXXXXXXXXXXOOOX 7011 G * C * G * C * mCmUmCmU * mU WV- mC *
mCmGmCmU * T * C * T * A * C * C * CCGCUTCTACCCGCGCCUCU
XOOOXXXXXXXXXXXOOOX 7012 C * G * C * G * mCmCmUmC * mU WV- m5Ceo *
m5CeoTeoAeoGeo * m5C * G * G * CCTAGCGGGACACCGTAGGT
XOOOXXXXXXXXXXXOOOX 7013 G * A * m5C * A * m5C * m5C * G *
TeoAeoGeoGeo * Teo WV- m5Ceo * TeoTeoTeom5Ceo * m5C * T * A *
CTTTCCTAGCGGGACACCGT XOOOXXXXXXXXXXXOOOX 7014 G * m5C * G * G * G *
A * m5C * Aeom5Ceom5CeoGeo * Teo WV- m5Ceo * Teom5CeoTeoTeo * T *
m5C * m5C * CTCTTTCCTAGCGGGACACC XOOOXXXXXXXXXXXOOOX 7015 T * A * G
* m5C * G * G * G * Aeom5CeoAeom5Ceo * m5Ceo WV- m5Ceo *
m5CeoTeom5CeoTeo * m5C * T * T * CCTCTCTTTCCTAGCGGGAC
XOOOXXXXXXXXXXXOOOX 7016 T * m5C * m5C * T * A * G * m5C *
GeoGeoGeoAeo * m5Ceo WV- Aeo * m5Ceom5CeoTeom5Ceo * T * m5C * T *
ACCTCTCTTTCCTAGCGGGA XOOOXXXXXXXXXXXOOOX 7017 T * T * m5C * m5C * T
* A * G * m5CeoGeoGeoGeo * Aeo WV- m5Ceo * Aeom5Ceom5CeoTeo * m5C *
T * CACCTCTCTTTCCTAGCGGG XOOOXXXXXXXXXXXOOOX 7018 m5C * T * T * T *
m5C * m5C * T * A * Geom5CeoGeoGeo * Geo WV- m5Ceo *
Geom5CeoAeom5Ceo * m5C * T * CGCACCTCTCTTTCCTAGCG
XOOOXXXXXXXXXXXOOOX 7019 m5C * T * m5C * T * T * T * m5C * m5C *
TeoAeoGeom5Ceo * Geo WV- Aeo * m5CeoGeom5CeoAeo * m5C * m5C *
ACGCACCTCTCTTTCCTAGC XOOOXXXXXXXXXXXOOOX 7020 T * m5C * T * m5C * T
* T * T * m5C * m5CeoTeoAeoGeo * m5Ceo WV- Geo * m5CeoTeoGeoTeo * T
* T * G * A * GCTGTTTGACGCACCTCTCT XOOOXXXXXXXXXXXOOOX 7021 m5C * G
* m5C * A * m5C * m5C * Teom5CeoTeom5Ceo * Teo WV- Geo *
Teom5CeoGeom5Ceo * T * G * T * T * GTCGCTGTTTGACGCACCTC
XOOOXXXXXXXXXXXOOOX 7022 T * G * A * m5C * G * m5C *
Aeom5Ceom5CeoTeo * m5Ceo WV- Geo * m5CeoAeoGeoGeo * G * A * m5C * G
* GCAGGGACGGCTGACACACC XOOOXXXXXXXXXXXOOOX 7023 G * m5C * T * G * A
* m5C * Aeom5CeoAeom5Ceo * m5Ceo WV- Geo * Geom5CeoAeoGeo * m5C * A
* G * G * GGCAGCAGGGACGGCTGACA XOOOXXXXXXXXXXXOOOX 7024 G * A * m5C
* G * G * m5C * TeoGeoAeom5Ceo * Aeo WV- m5Ceo * GeoGeoGeom5Ceo * A
* G * m5C * CGGGCAGCAGGGACGGCTGA XOOOXXXXXXXXXXXOOOX 7025 A * G * G
* G * A * m5C * G * Geom5CeoTeoGeo * Aeo WV- m5Ceo * m5CeoGeoGeoGeo
* m5C * A * G * CCGGGCAGCAGGGACGGCTG XOOOXXXXXXXXXXXOOOX 7026 m5C *
A * G * G * G * A * m5C * GeoGeom5CeoTeo * Geo WV- Aeo *
m5Ceom5CeoGeoGeo * G * m5C * A * ACCGGGCAGCAGGGACGGCT
XOOOXXXXXXXXXXXOOOX 7027 G * m5C * A * G * G * G * A *
m5CeoGeoGeom5Ceo * Teo WV- Aeo * Aeom5Ceom5CeoGeo * G * G * m5C *
AACCGGGCAGCAGGGACGGC XOOOXXXXXXXXXXXOOOX 7028 A * G * m5C * A * G *
G * G * Aeom5CeoGeoGeo * m5Ceo WV- Geo * m5CeoAeoAeom5Ceo * m5C * G
* G * GCAACCGGGCAGCAGGGACG XOOOXXXXXXXXXXXOOOX 7029 G * m5C * A * G
* m5C * A * G * GeoGeoAeom5Ceo * Geo WV- Aeo * Geom5CeoAeoAeo * m5C
* m5C * G * AGCAACCGGGCAGCAGGGAC XOOOXXXXXXXXXXXOOOX 7030 G * G *
m5C * A * G * m5C * A * GeoGeoGeoAeo * m5Ceo WV- Geo *
m5CeoTeoAeoGeo * A * m5C * m5C * GCTAGACCCCGCCCCCAAAA
XOOOXXXXXXXXXXXOOOX 7031 m5C * m5C * G * m5C * m5C * m5C * m5C *
m5CeoAeoAeoAeo * Aeo WV- Teo * TeoGeom5CeoTeo * A * G * A * m5C *
TTGCTAGACCCCGCCCCCAA XOOOXXXXXXXXXXXOOOX 7032 m5C * m5C * m5C * G *
m5C * m5C * m5Ceom5Ceom5CeoAeo * Aeo WV- m5Ceo * TeoTeoGeom5Ceo * T
* A * G * A * CTTGCTAGACCCCGCCCCCA XOOOXXXXXXXXXXXOOOX 7033 m5C *
m5C * m5C * m5C * G * m5C * m5Ceom5Ceom5Ceom5Ceo * Aeo WV- m5Ceo *
Teom5CeoTeoTeo * G * m5C * T * CTCTTGCTAGACCCCGCCCC
XOOOXXXXXXXXXXXOOOX 7034 A * G * A * m5C * m5C * m5C * m5C *
Geom5Ceom5Ceom5Ceo * m5Ceo WV- Teo * Geom5CeoTeom5Ceo * T * T * G *
TGCTCTTGCTAGACCCCGCC XOOOXXXXXXXXXXXOOOX 7035 m5C * T * A * G * A *
m5C * m5C * m5Ceom5CeoGeom5Ceo * m5Ceo WV- m5Ceo * m5CeoTeoGeom5Ceo
* T * m5C * T * CCTGCTCTTGCTAGACCCCG XOOOXXXXXXXXXXXOOOX 7036 T * G
* m5C * T * A * G * A * m5Ceom5Ceom5Ceom5Ceo * Geo WV- m5Ceo *
m5CeoAeom5CeoAeo * m5C * m5C * CCACACCTGCTCTTGCTAGA
XOOOXXXXXXXXXXXOOOX 7037 T * G * m5C * T * m5C * T * T * G *
m5CeoTeoAeoGeo * Aeo WV- m5Ceo * m5Ceom5CeoAeom5Ceo * A * m5C *
CCCACACCTGCTCTTGCTAG XOOOXXXXXXXXXXXOOOX 7038 m5C * T * G * m5C * T
* m5C * T * T * Geom5CeoTeoAeo * Geo WV- Aeo * m5Ceom5Ceom5CeoAeo *
m5C * A * ACCCACACCTGCTCTTGCTA XOOOXXXXXXXXXXXOOOX 7039 m5C * m5C *
T * G * m5C * T * m5C * T * TeoGeom5CeoTeo * Aeo WV- Aeo *
Aeom5Ceom5Ceom5Ceo * A * m5C * AACCCACACCTGCTCTTGCT
XOOOXXXXXXXXXXXOOOX 7040 A * m5C * m5C * T * G * m5C * T * m5C *
TeoTeoGeom5Ceo * Teo WV- Teo * m5CeoAeom5Ceom5Ceo * m5C * T *
TCACCCTCAGCGAGTACTGT XOOOXXXXXXXXXXXOOOX 7041 m5C * A * G * m5C * G
* A * G * T * Aeom5CeoTeoGeo * Teo WV- Geo * TeoTeom5CeoAeo * m5C *
m5C * GTTCACCCTCAGCGAGTACT XOOOXXXXXXXXXXXOOOX 7042 m5C * T * m5C *
A * G * m5C * G * A * GeoTeoAeom5Ceo * Teo WV- m5Ceo * TeoTeoGeoTeo
* T * m5C * A * CTTGTTCACCCTCAGCGAGT XOOOXXXXXXXXXXXOOOX 7043 m5C *
m5C * m5C * T * m5C * A * G * m5CeoGeoAeoGeo * Teo WV- Geo *
Teom5CeoTeoTeo * T * T * m5C * GTCTTTTCTTGTTCACCCTC
XOOOXXXXXXXXXXXOOOX 7044 T * T * G * T * T * m5C * A *
m5Ceom5Ceom5CeoTeo * m5Ceo WV- Geo * GeoTeom5CeoTeo * T * T * T *
GGTCTTTTCTTGTTCACCCT XOOOXXXXXXXXXXXOOOX 7045 m5C * T * T * G * T *
T * m5C * Aeom5Ceom5Ceom5Ceo * Teo WV- m5Ceo * m5CeoTeom5Ceom5Ceo *
T * T * G * CCTCCTTGTTTTCTTCTGGT XOOOXXXXXXXXXXXOOOX 7046 T * T * T
* T * m5C * T * T * m5CeoTeoGeoGeo * Teo WV- m5Ceo *
m5Ceom5CeoTeom5Ceo * m5C * T * CCCTCCTTGTTTTCTTCTGG
XOOOXXXXXXXXXXXOOOX 7047 T * G * T * T * T * T * m5C * T *
Teom5CeoTeoGeo * Geo WV- Geo * TeoTeoGeoTeo * T * T * m5C * m5C *
GTTGTTTCCCTCCTTGTTTT XOOOXXXXXXXXXXXOOOX 7048 m5C * T * m5C * m5C *
T * T * GeoTeoTeoTeo * Teo WV- Geo * GeoTeoTeoGeo * T * T * T * m5C
* GGTTGTTTCCCTCCTTGTTT XOOOXXXXXXXXXXXOOOX 7049 m5C * m5C * T * m5C
* m5C * T * TeoGeoTeoTeo * Teo WV- m5Ceo * GeoGeoTeoTeo * G * T * T
* T * CGGTTGTTTCCCTCCTTGTT XOOOXXXXXXXXXXXOOOX 7050 m5C * m5C * m5C
* T * m5C * m5C * TeoTeoGeoTeo * Teo WV- Teo * Geom5CeoGeoGeo * T *
T * G * T * T * TGCGGTTGTTTCCCTCCTTG XOOOXXXXXXXXXXXOOOX 7051 T *
m5C * m5C * m5C * T * m5Ceom5CeoTeoTeo * Geo WV- m5Ceo *
TeoGeom5CeoGeo * G * T * T * G * CTGCGGTTGTTTCCCTCCTT
XOOOXXXXXXXXXXXOOOX 7052 T * T * T * m5C * m5C * m5C *
Teom5Ceom5CeoTeo * Teo WV- Aeo * GeoGeom5CeoTeo * G * m5C * G * G *
AGGCTGCGGTTGTTTCCCTC XOOOXXXXXXXXXXXOOOX 7053 T * T * G * T * T * T
* m5Ceom5Ceom5CeoTeo * m5Ceo WV- Aeo * m5CeoAeoGeoGeo * m5C * T * G
* ACAGGCTGCGGTTGTTTCCC XOOOXXXXXXXXXXXOOOX 7054 m5C * G * G * T * T
* G * T * TeoTeom5Ceom5Ceo * m5Ceo WV- Geo * m5CeoTeoAeom5Ceo * A *
G * G * GCTACAGGCTGCGGTTGTTT XOOOXXXXXXXXXXXOOOX 7055 m5C * T * G *
m5C * G * G * T * TeoGeoTeoTeo * Teo
WV- Teo * Geom5CeoTeoAeo * m5C * A * G * G * TGCTACAGGCTGCGGTTGTT
XOOOXXXXXXXXXXXOOOX 7056 m5C * T * G * m5C * G * G * TeoTeoGeoTeo *
Teo WV- Teo * TeoGeom5CeoTeo * A * m5C * A * G *
TTGCTACAGGCTGCGGTTGT XOOOXXXXXXXXXXXOOOX 7057 G * m5C * T * G * m5C
* G * GeoTeoTeoGeo * Teo WV- Geo * m5CeoTeoTeoGeo * m5C * T * A *
GCTTGCTACAGGCTGCGGTT XOOOXXXXXXXXXXXOOOX 7058 m5C * A * G * G * m5C
* T * G * m5CeoGeoGeoTeo * Teo WV- Aeo * Geom5CeoTeoTeo * G * m5C *
T * A * AGCTTGCTACAGGCTGCGGT XOOOXXXXXXXXXXXOOOX 7059 m5C * A * G *
G * m5C * T * Geom5CeoGeoGeo * Teo WV- Geo * AeoGeom5CeoTeo * T * G
* m5C * T * GAGCTTGCTACAGGCTGCGG XOOOXXXXXXXXXXXOOOX 7060 A * m5C *
A * G * G * m5C * TeoGeom5CeoGeo * Geo WV- m5Ceo * AeoGeoAeoGeo *
m5C * T * T * G * CAGAGCTTGCTACAGGCTGC XOOOXXXXXXXXXXXOOOX 7061 m5C
* T * A * m5C * A * G * Geom5CeoTeoGeo * m5Ceo WV- Teo *
m5Ceom5CeoAeoGeo * A * G * m5C * TCCAGAGCTTGCTACAGGCT
XOOOXXXXXXXXXXXOOOX 7062 T * T * G * m5C * T * A * m5C *
AeoGeoGeom5Ceo * Teo WV- Teo * Teom5Ceom5CeoAeo * G * A * G *
TTCCAGAGCTTGCTACAGGC XOOOXXXXXXXXXXXOOOX 7063 m5C * T * T * G * m5C
* T * A * m5CeoAeoGeoGeo * m5Ceo WV- m5Ceo * m5CeoTeoGeoAeo * G * T
* T * CCTGAGTTCCAGAGCTTGCT XOOOXXXXXXXXXXXOOOX 7064 m5C * m5C * A *
G * A * G * m5C * TeoTeoGeom5Ceo * Teo WV- Teo * m5Ceom5CeoTeoGeo *
A * G * T * T * TCCTGAGTTCCAGAGCTTGC XOOOXXXXXXXXXXXOOOX 7065 m5C *
m5C * A * G * A * G * m5CeoTeoTeoGeo * m5Ceo WV- Aeo *
m5CeoTeom5Ceom5Ceo * T * G * A * ACTCCTGAGTTCCAGAGCTT
XOOOXXXXXXXXXXXOOOX 7066 G * T * T * m5C * m5C * A * G *
AeoGeom5CeoTeo * Teo WV- Geo * m5CeoGeom5CeoGeo * A * m5C * T *
GCGCGACTCCTGAGTTCCAG XOOOXXXXXXXXXXXOOOX 7067 m5C * m5C * T * G * A
* G * T * Teom5Ceom5CeoAeo * Geo WV- m5Ceo * Geom5CeoGeom5Ceo * G *
A * CGCGCGACTCCTGAGTTCCA XOOOXXXXXXXXXXXOOOX 7068 m5C * T * m5C *
m5C * T * G * A * G * TeoTeom5Ceom5Ceo * Aeo WV- Geo *
m5CeoGeom5CeoGeo * m5C * G * A * GCGCGCGACTCCTGAGTTCC
XOOOXXXXXXXXXXXOOOX 7069 m5C * T * m5C * m5C * T * G * A *
GeoTeoTeom5Ceo * m5Ceo WV- Aeo * Geom5CeoGeom5Ceo * G * m5C * G *
AGCGCGCGACTCCTGAGTTC XOOOXXXXXXXXXXXOOOX 7070 A * m5C * T * m5C *
m5C * T * G * AeoGeoTeoTeo * m5Ceo WV- Aeo * GeoGeoAeoTeo * G * m5C
* m5C * G * AGGATGCCGCCTCCTCACTC XOOOXXXXXXXXXXXOOOX 7071 m5C * m5C
* T * m5C * m5C * T * m5CeoAeom5CeoTeo * m5Ceo WV- m5Ceo *
AeoGeoGeoAeo * T * G * m5C * CAGGATGCCGCCTCCTCACT
XOOOXXXXXXXXXXXOOOX 7072 m5C * G * m5C * m5C * T * m5C * m5C *
Teom5CeoAeom5Ceo * Teo WV- m5Ceo * m5CeoAeoGeoGeo * A * T * G *
CCAGGATGCCGCCTCCTCAC XOOOXXXXXXXXXXXOOOX 7073 m5C * m5C * G * m5C *
m5C * T * m5C * m5CeoTeom5CeoAeo * m5Ceo WV- m5Ceo *
Geom5Ceom5CeoAeo * G * G * A * CGCCAGGATGCCGCCTCCTC
XOOOXXXXXXXXXXXOOOX 7074 T * G * m5C * m5C * G * m5C * m5C *
Teom5Ceom5CeoTeo * m5Ceo WV- m5Ceo * m5CeoGeom5Ceom5Ceo * A * G *
CCGCCAGGATGCCGCCTCCT XOOOXXXXXXXXXXXOOOX 7075 G * A * T * G * m5C *
m5C * G * m5C * m5CeoTeom5Ceom5Ceo * Teo WV- m5Ceo *
m5Ceom5CeoGeom5Ceo * m5C * A * CCCGCCAGGATGCCGCCTCC
XOOOXXXXXXXXXXXOOOX 7076 G * G * A * T * G * m5C * m5C * G *
m5Ceom5CeoTeom5Ceo * m5Ceo WV- Aeo * m5Ceom5Ceom5CeoGeo * m5C * m5C
* ACCCGCCAGGATGCCGCCTC XOOOXXXXXXXXXXXOOOX 7077 A * G * G * A * T *
G * m5C * m5C * Geom5Ceom5CeoTeo * m5Ceo WV- m5Ceo *
Aeom5Ceom5Ceom5Ceo * G * m5C * CACCCGCCAGGATGCCGCCT
XOOOXXXXXXXXXXXOOOX 7078 m5C * A * G * G * A * T * G * m5C *
m5CeoGeom5Ceom5Ceo * Teo WV- m5Ceo * m5CeoAeom5Ceom5Ceo * m5C * G *
CCACCCGCCAGGATGCCGCC XOOOXXXXXXXXXXXOOOX 7079 m5C * m5C * A * G * G
* A * T * G * m5Ceom5CeoGeom5Ceo * m5Ceo WV- Geo *
m5Ceom5CeoAeom5Ceo * m5C * m5C * GCCACCCGCCAGGATGCCGC
XOOOXXXXXXXXXXXOOOX 7080 G * m5C * m5C * A * G * G * A * T *
Geom5Ceom5CeoGeo * m5Ceo WV- Aeo * Aeom5CeoAeoGeo * m5C * m5C * A *
AACAGCCACCCGCCAGGATG XOOOXXXXXXXXXXXOOOX 7081 m5C * m5C * m5C * G *
m5C * m5C * A * GeoGeoAeoTeo * Geo WV- m5Ceo * m5CeoAeoAeoAeo * m5C
* A * G * CCAAACAGCCACCCGCCAGG XOOOXXXXXXXXXXXOOOX 7082 m5C * m5C *
A * m5C * m5C * m5C * G * m5Ceom5CeoAeoGeo * Geo WV- m5Ceo *
m5Ceom5CeoAeoAeo * A * m5C * CCCAAACAGCCACCCGCCAG
XOOOXXXXXXXXXXXOOOX 7083 A * G * m5C * m5C * A * m5C * m5C * m5C *
Geom5Ceom5CeoAeo * Geo WV- Aeo * m5Ceom5Ceom5Ceom5Ceo * A * A *
ACCCCAAACAGCCACCCGCC XOOOXXXXXXXXXXXOOOX 7084 A * m5C * A * G * m5C
* m5C * A * m5C * m5Ceom5CeoGeom5Ceo * m5Ceo WV- m5Ceo *
m5Ceom5CeoGeoGeo * m5C * A * CCCGGCAGCCGAACCCCAAA
XOOOXXXXXXXXXXXOOOX 7085 G * m5C * m5C * G * A * A * m5C * m5C *
m5Ceom5CeoAeoAeo * Aeo WV- Teo * m5Ceom5Ceom5CeoGeo * G * m5C *
TCCCGGCAGCCGAACCCCAA XOOOXXXXXXXXXXXOOOX 7086 A * G * m5C * m5C * G
* A * A * m5C * m5Ceom5Ceom5CeoAeo * Aeo WV- Teo *
Teom5Ceom5Ceom5Ceo * G * G * TTCCCGGCAGCCGAACCCCA
XOOOXXXXXXXXXXXOOOX 7087 m5C * A * G * m5C * m5C * G * A * A *
m5Ceom5Ceom5Ceom5Ceo * Aeo WV- m5Ceo * TeoTeom5Ceom5Ceo * m5C * G *
CTTCCCGGCAGCCGAACCCC XOOOXXXXXXXXXXXOOOX 7088 G * m5C * A * G * m5C
* m5C * G * A * Aeom5Ceom5Ceom5Ceo * m5Ceo WV- Teo *
m5CeoTeoTeom5Ceo * m5C * m5C * G * TCTTCCCGGCAGCCGAACCC
XOOOXXXXXXXXXXXOOOX 7089 G * m5C * A * G * m5C * m5C * G *
AeoAeom5Ceom5Ceo * m5Ceo WV- m5Ceo * Teom5CeoTeoTeo * m5C * m5C *
CTCTTCCCGGCAGCCGAACC XOOOXXXXXXXXXXXOOOX 7090 m5C * G * G * m5C * A
* G * m5C * m5C * GeoAeoAeom5Ceo * m5Ceo WV- m5Ceo *
m5CeoTeom5CeoTeo * T * m5C * CCTCTTCCCGGCAGCCGAAC
XOOOXXXXXXXXXXXOOOX 7091 m5C * m5C * G * G * m5C * A * G * m5C *
m5CeoGeoAeoAeo * m5Ceo WV- Geo * m5Ceom5CeoTeom5Ceo * T * T * m5C *
GCCTCTTCCCGGCAGCCGAA XOOOXXXXXXXXXXXOOOX 7092 m5C * m5C * G * G *
m5C * A * G * m5Ceom5CeoGeoAeo * Aeo WV- m5Ceo * Geom5Ceom5CeoTeo *
m5C * T * T * CGCCTCTTCCCGGCAGCCGA XOOOXXXXXXXXXXXOOOX 7093 m5C *
m5C * m5C * G * G * m5C * A * Geom5Ceom5CeoGeo * Aeo WV- m5Ceo *
m5CeoGeom5CeoGeo * m5C * m5C * CCGCGCCTCTTCCCGGCAGC
XOOOXXXXXXXXXXXOOOX 7094 T * m5C * T * T * m5C * m5C * m5C * G *
Geom5CeoAeoGeo * m5Ceo WV- m5Ceo * m5Ceom5CeoGeom5Ceo * G * m5C *
CCCGCGCCTCTTCCCGGCAG XOOOXXXXXXXXXXXOOOX 7095 m5C * T * m5C * T * T
* m5C * m5C * m5C * GeoGeom5CeoAeo * Geo WV- Aeo *
m5Ceom5Ceom5CeoGeo * m5C * G * ACCCGCGCCTCTTCCCGGCA
XOOOXXXXXXXXXXXOOOX 7096 m5C * m5C * T * m5C * T * T * m5C * m5C *
m5CeoGeoGeom5Ceo * Aeo WV- Teo * Aeom5Ceom5Ceom5Ceo * G * m5C *
TACCCGCGCCTCTTCCCGGC XOOOXXXXXXXXXXXOOOX 7097 G * m5C * m5C * T *
m5C * T * T * m5C * m5Ceom5CeoGeoGeo * m5Ceo WV- m5Ceo *
TeoAeom5Ceom5Ceo * m5C * G * CTACCCGCGCCTCTTCCCGG
XOOOXXXXXXXXXXXOOOX 7098 m5C * G * m5C * m5C * T * m5C * T * T *
m5Ceom5Ceom5CeoGeo * Geo WV- Teo * Teom5CeoTeoAeo * m5C * m5C * m5C
* TTCTACCCGCGCCTCTTCCC XOOOXXXXXXXXXXXOOOX 7099 G * m5C * G * m5C *
m5C * T * m5C * TeoTeom5Ceom5Ceo * m5Ceo WV- m5Ceo * TeoTeom5CeoTeo
* A * m5C * m5C * CTTCTACCCGCGCCTCTTCC XOOOXXXXXXXXXXXOOOX 7100 m5C
* G * m5C * G * m5C * m5C * T * m5CeoTeoTeom5Ceo * m5Ceo WV- Geo *
m5CeoTeoTeom5Ceo * T * A * m5C * GCTTCTACCCGCGCCTCTTC
XOOOXXXXXXXXXXXOOOX 7101 m5C * m5C * G * m5C * G * m5C * m5C *
Teom5CeoTeoTeo * m5Ceo WV- m5Ceo * Geom5CeoTeoTeo * m5C * T * A *
CGCTTCTACCCGCGCCTCTT XOOOXXXXXXXXXXXOOOX 7102 m5C * m5C * m5C * G *
m5C * G * m5C * m5CeoTeom5CeoTeo * Teo WV- m5Ceo * m5CeoGeom5CeoTeo
* T * m5C * T * CCGCTTCTACCCGCGCCTCT XOOOXXXXXXXXXXXOOOX 7103 A *
m5C * m5C * m5C * G * m5C * G * m5Ceom5CeoTeom5Ceo * Teo WV- Geo *
Teo * Geo * m5Ceo * Teo * G * m5C * GTGCTGCGATCCCCATTCCA
XXXXXXXXXXXXXXXXXXX 7117 G * A * T * m5C * m5C * m5C * m5C * A *
Teo * Teo * m5Ceo * m5Ceo * Aeo WV- Geo * TeoGeom5CeoTeo * G * m5C
* G * A * GTGCTGCGATCCCCATTCCA XOOOXXXXXXXXXXXOOOX 7118 T * m5C *
m5C * m5C * m5C * A * TeoTeom5Ceom5Ceo * Aeo WV- Teo * Geo * Teo *
Geo * m5Ceo * T * G * TGTGCTGCGATCCCCATTCC
XXXXXXXXXXXXXXXXXXX 7119 m5C * G * A * T * m5C * m5C * m5C * m5C *
Aeo * Teo * Teo * m5Ceo * m5Ceo WV- Teo * GeoTeoGeom5Ceo * T * G *
m5C * G * TGTGCTGCGATCCCCATTCC XOOOXXXXXXXXXXXOOOX 7120 A * T * m5C
* m5C * m5C * m5C * AeoTeoTeom5Ceo * m5Ceo WV- mC * S mCmUmCmA * S
C * S T * S C * S A * S CCUCACTCACCCACTCGCCA SOOOSSSSSSSRSSSOOOS
7121 C * S C * S C * R A * S C * S T * S mCmGmCmC * S mA WV- mC * S
mCmUmCmA * S C * S T * S C * R A * S CCUCACTCACCCACTCGCCA
SOOOSSSRSSSSSSSOOOS 7122 C * S C * S C * S A * S C * S T * S
mCmGmCmC * S mA WV- mC * S mCmUmCmA * S C * S T * S C * R A * S
CCUCACTCACCCACTCGCCA SOOOSSSRSSSRSSSOOOS 7123 C * S C * S C * R A *
S C * S T * S mCmGmCmC * S mA WV- mC * S mCmUmCmA * S C * S T * S C
* R A * S CCUCACTCACCCACTCGCCA SOOOSSSRSSRSSSSOOOS 7124 C * S C * R
C * S A * S C * S T * S mCmGmCmC * S mA WV- mC * S mC * S mU * S mC
* S mA * S C * S T * CCUCACTCACCCACTCGCCA SSSSSSSRSSSRSSSSSSS 7125
S C * R A * S C * S C * S C * R A * S C * S T * S mC * S mG * S mC
* S mC * S mA WV- mC * S mC * S mU * S mC * S mA * S C * S T *
CCUCACTCACCCACTCGCCA SSSSSSSRSSRSSSSSSSS 7126 S C * R A * S C * S C
* R C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA WV-
m5Ceo * S m5CeoTeom5CeoAeo * S C * S T * CCTCACTCACCCACTCGCCA
SOOOSSSSSSSRSSSOOOS 7127 S C * S A * S C * S C * S C * R A * S C *
S T * S m5CeoGeom5Ceom5Ceo * S Aeo WV- m5Ceo * S m5CeoTeom5CeoAeo *
S C * S T * CCTCACTCACCCACTCGCCA SOOOSSSRSSSSSSSOOOS 7128 S C * R A
* S C * S C * S C * S A * S C * S T * S m5CeoGeom5Ceom5Ceo * S Aeo
WV- m5Ceo * S m5CeoTeom5CeoAeo * S C * S T * CCTCACTCACCCACTCGCCA
SOOOSSSRSSSRSSSOOOS 7129 S C * R A * S C * S C * S C * R A * S C *
S T * S m5CeoGeom5Ceom5Ceo * S Aeo WV- m5Ceo * S m5CeoTeom5CeoAeo *
S C * S T * CCTCACTCACCCACTCGCCA SOOOSSSRSSRSSSSOOOS 7130 S C * R A
* S C * S C * R C * S A * S C * S T * S m5CeoGeom5Ceom5Ceo * S Aeo
WV- m5Ceo * S m5Ceo * S Teo * S m5Ceo * S Aeo *
CCTCACTCACCCACTCGCCA SSSSSSSRSSSRSSSSSSS 7131 S C * S T * S C * R A
* S C * S C * S C * R A * S C * S T * S m5Ceo * S Geo * S m5Ceo * S
m5Ceo * S Aeo WV- m5Ceo * S m5Ceo * S Teo * S m5Ceo * S Aeo *
CCTCACTCACCCACTCGCCA SSSSSSSRSSRSSSSSSSS 7132 S C * S T * S C * R A
* S C * S C * R C * S A * S C * S T * S m5Ceo * S Geo * S m5Ceo * S
m5Ceo * S Aeo WV- R UR GR GR AR AR UR GR GR GR GR AR
UGGAAUGGGGAUCGCAGCAC OOOOOOOOOOOOO OOOOO 7405 UR CR GR CR AR GR CR
AR C O WV- R GR CR CR GR GR GR AR AR GR AR GR GCCGGGAAGAGGCGCGGGUA
OOOOOOOOOOOOO OOOOO 7434 GR CR GR CR GR GR GR UR AR G G OO WV- R AR
GR CR CR GR UR CR CR CR UR GR AGCCGUCCCUGCUGCCCGGU OOOOOOOOOOOOO
OOOOO 7435 CR UR GR CR CR CR GR GR U O WV- m5Ceo * R m5Ceo * R Teo
* R m5Ceo * R Aeo * CCTCACTCACCCACTCGCCA RRRRRSSRSSRSSSRRRRR 7601 R
C * S T * S C * R A * S C * S C * R C * S A * S C * S T * R m5Ceo *
R Geo * R m5Ceo * R m5Ceo * R Aeo WV- m5Ceo * S m5CeoTeom5CeoAeo *
R C * S T * CCTCACTCACCCACTCGCCA SOOORSSRSSRSSSROOOS 7602 S C * R A
* S C * S C * R C * S A * S C * S T * R m5CeoGeom5Ceom5Ceo * S Aeo
WV- mC * S mCmUmCmA * S C * S T * S C * R A * CCUCACTCACCCACTCGCCA
SOOOSSSRSSSSSSSOOSS 7603 S C * S C * S C * S A * S C * S T * S
mCmGmC * S mC * S mA WV- mC * S mCmUmCmA * S C * S T * S C * R A *
CCUCACTCACCCACTCGCCA SOOOSSSRSSRSSSSOOSS 7604 S C * S C * R C * S A
* S C * S T * S mCmGmC * S mC * S mA WV- mC * S mCmUmCmA * S C * S
T * S C * R A * CCUCACTCACCCACTCGCCA SOOOSSSRSSSSSSSSSSS 7605 S C *
S C * S C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA WV-
mC * S mCmUmCmA * S C * S T * S C * R A * CCUCACTCACCCACTCGCCA
SOOOSSSRSSRSSSSSSSS 7606 S C * S C * R C * S A * S C * S T * S mC *
S mG * S mC * S mC * S mA WV- m5Ceo * R m5Ceo * R Teo * R m5Ceo * R
Aeo * CCTCACTCACCCACTCGCCA RRRRRSSSSSRSSSRRRRR 7657 R C * S T * S C
* S A * S C * S C * R C * S A * S C * S T * R m5Ceo * R Geo * R
m5Ceo * R m5Ceo * R Aeo WV- m5Ceo * R m5CeoTeom5CeoAeo * R C * S T
* CCTCACTCACCCACTCGCCA ROOORSSRSSRSSSROOOR 7658 S C * R A * S C * S
C * R C * S A * S C * S T * R m5CeoGeom5Ceom5Ceo * R Aeo WV- m5Ceo
* R m5CeoTeom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
ROOORSSRSSSSSSROOOR 7659 S C * R A * S C * S C * S C * S A * S C *
S T * R m5CeoGeom5Ceom5Ceo * R Aeo WV- R UR GR GR AR AR UR GR GR GR
GR AR UGGAAUGGGGAUCGCAGCAC OOOOOOOOOOOOO OOOOO 7773 UR CR GR CR AR
GR CR AR CR A A OO WV- mC * S mC * S mU * S mC * S mA * S C * S T *
CCUCACTCACCCACTCGCCA SSSSSSSRSSSSSSSOOOS 7774 S C * R A * S C * S C
* S C * S A * S C * S T * S mCmGmCmC * S mA WV- mC * S mC * S mU *
S mC * S mA * S C * S T * CCUCACTCACCCACTCGCCA SSSSSSSRSSRSSSSOOOS
7775 S C * R A * S C * S C * R C * S A * S C * S T * S mCmGmCmC * S
mA WV- Aeo * m5Ceo * m5Ceo * Geo * Geo * G * ACCGGGCAGCAGGGACGGCT
XXXXXXXXXXXXXXXXXXX 7866 m5C * A * G * m5C * A * G * G * G * A *
m5Ceo * Geo * Geo * m5Ceo * Teo WV- m5Ceo * R m5CeoTeom5CeoAeo * R
C * S T * CCTCACTCACCCACTCGCCA ROOORSSRSSSSSSSOOSS 8005 S C * R A *
S C * S C * S C * S A * S C * S T * S mCmGmC * S mC * S mA WV-
m5Ceo * R m5CeoTeom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
ROOORSSRSSRSSSSOOSS 8006 S C * R A * S C * S C * R C * S A * S C *
S T * S mCmGmC * S mC * S mA WV- m5Ceo * R m5CeoTeom5CeoAeo * R C *
S T * CCTCACTCACCCACTCGCCA ROOORSSRSSSSSSSSSSS 8007 S C * R A * S C
* S C * S C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA
WV- m5Ceo * R m5CeoTeom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
ROOORSSRSSRSSSSSSSS 8008 S C * R A * S C * S C * R C * S A * S C *
S T * S mC * S mG * S mC * S mC * S mA WV- mC * S m5CeoTeom5CeomA *
S C * S T * S C * CCTCACTCACCCACTCGCCA SOOOSSSRSSSSSSSOOSS 8009 R A
* S C * S C * S C * S A * S C * S T * S mCmGmC * S mC * S mA WV- mC
* S m5CeoTeom5CeomA * S C * S T * S C * CCTCACTCACCCACTCGCCA
SOOOSSSRSSRSSSSOOSS 8010 R A * S C * S C * R C * S A * S C * S T *
S mCmGmC * S mC * S mA WV- mC * S m5CeoTeom5CeomA * S C * S T * S C
* CCTCACTCACCCACTCGCCA SOOOSSSRSSSSSSSSSSS 8011 R A * S C * S C * S
C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA WV- mC * S
m5CeoTeom5CeomA * S C * S T * S C * CCTCACTCACCCACTCGCCA
SOOOSSSRSSRSSSSSSSS 8012 R A * S C * S C * R C * S A * S C * S T *
S mC * S mG * S mC * S mC * S mA WV- mA * S mC * S mC * S mGmG * S
G * S C * ACCGGGCAGCAGGGACGGCU SSSOSSSSSRSSRSSSOOS 8114 S A * S G *
S C * R A * S G * S G * R G * S A * S mC * S mGmGmC * S mU WV- mA *
S m5CeomC * S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU
SOSOSSSSSRSSRSSSOOS 8115 S A * S G * S C * R A * S G * S G * R G *
S A * S mC * S mGmGmC * S mU WV- mA * S mC * S m5CeomGmG * S G * S
C * ACCGGGCAGCAGGGACGGCU SSOOSSSSSRSSRSSSOOS 8116 S A * S G * S C *
R A * S G * S G * R G * S A * S mC * S mGmGmC * S mU WV- mA * S mC
* S mC * S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU
SSSOSSSSSRSSRSSOOOS 8117 S A * S G * S C * R A * S G * S G * R G *
S A * S m5CeomGmGmC * S mU WV- mA * S m5Ceom5CeomGmG * S G * S C *
ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSSOOS 8118 S A * S G * S C * R A
* S G * S G * R G * S A * S mC * S mGmGmC * S mU WV- mA * S m5CeomC
* S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU SOSOSSSSSRSSRSSOOOS
8119 S A * S G * S C * R A * S G * S G * R G * S A * S m5CeomGmGmC
* S mU WV- mA * S mC * S m5CeomGmG * S G * S C *
ACCGGGCAGCAGGGACGGCU SSOOSSSSSRSSRSSOOOS 8120 S A * S G * S C * R A
* S G * S G * R G * S A * S m5CeomGmGmC * S mU WV- mA * S m5Ceo * S
m5CeomGmG * S G * S C * ACCGGGCAGCAGGGACGGCU SSOOSSSSSRSSRSSOOOS
8121 S A * S G * S C * R A * S G * S G * R G * S A * S m5CeomGmGmC
* S mU WV- mA * S mC * S mC * S mGmG * S G * S C *
ACCGGGCAGCAGGGACGGCU SSSOSSSSRSSRSSSSOOS 8122 S A * S G * R C * S A
* S G * R G * S G * S A * S mC * S mGmGmC * S mU WV- mA * S m5CeomC
* S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU SOSOSSSSRSSRSSSSOOS
8123 S A * S G * R C * S A * S G * R G * S G * S A * S mC * S
mGmGmC * S mU WV- mA * S mC * S m5CeomGmG * S G * S C *
ACCGGGCAGCAGGGACGGCU SSOOSSSSRSSRSSSSOOS 8124 S A * S G * R C * S A
* S G * R G * S G * S A * S mC * S mGmGmC * S mU WV- mA * S mC * S
mC * S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU SSSOSSSSRSSRSSSOOOS
8125 S A * S G * R C * S A * S G * R G * S G * S A * S m5CeomGmGmC
* S mU WV- mA * S m5Ceom5CeomGmG * S G * S C * S A *
ACCGGGCAGCAGGGACGGCU SOOOSSSSRSSRSSSSOOS
8126 S G * R C * S A * S G * R G * S G * S A * S mC * S mGmGmC * S
mU WV- mA * S m5CeomC * S mGmG * S G * S C * ACCGGGCAGCAGGGACGGCU
SOSOSSSSRSSRSSSOOOS 8127 S A * S G * R C * S A * S G * R G * S G *
S A * S m5CeomGmGmC * S mU WV- mA * S mC * S m5CeomGmG * S G * S C
* ACCGGGCAGCAGGGACGGCU SSOOSSSSRSSRSSSOOOS 8128 S A * S G * R C * S
A * S G * R G * S G * S A * S m5CeomGmGmC * S mU WV- mA * S m5Ceo *
S m5CeomGmG * S G * S C * ACCGGGCAGCAGGGACGGCU SSOOSSSSRSSRSSSOOOS
8129 S A * S G * R C * S A * S G * R G * S G * S A * S m5CeomGmGmC
* S mU WV- mA * S mC * S mC * S mG * S mG * S G * S C *
ACCGGGCAGCAGGGACGGCU SSSSSSSSSRSSRSSSSSS 8311 S A * S G * S C * R A
* S G * S G * R G * S A * S mC * S mG * S mG * S mC * S mU WV- Aeo
* R m5Ceom5CeoGeoGeo * R G * S C * ACCGGGCAGCAGGGACGGCT
ROOORSSSSRSSRSSOOOR 8312 S A * S G * S C * R A * S G * S G * R G *
S A * S m5CeoGeoGeom5Ceo * R Teo WV- Aeo * R m5Ceo * R m5Ceo * R
Geo * R Geo * ACCGGGCAGCAGGGACGGCT RRRRRSSSSRSSRSSRRRR 8313 R G * S
C * S A * S G * S C * R A * S G * S G * R G * S A * S m5Ceo * R Geo
* R Geo * R m5Ceo * R Teo WV- mA * S mC * S mC * S mGmG * S G * S C
* ACCGGGCAGCAGGGACGGCT SSSOSSSSSRSSRSSOOOR 8314 S A * S G * S C * R
A * S G * S G * R G * S A * S m5CeoGeoGeom5Ceo * R Teo WV- mA * S
mC * S mC * S mG * S mG * S G * S C * ACCGGGCAGCAGGGACGGCU
SSSSSSSSRSSRSSSSSSS 8315 S A * S G * R C * S A * S G * R G * S G *
S A * S mC * S mG * S mG * S mC * S mU WV- Aeo * R m5Ceom5CeoGeoGeo
* R G * S C * ACCGGGCAGCAGGGACGGCT ROOORSSSRSSRSSROOOR 8316 S A * S
G * R C * S A * S G * R G * S G * S A * R m5CeoGeoGeom5Ceo * R Teo
WV- Aeo * R m5Ceo * R m5Ceo * R Geo * R Geo * R G *
ACCGGGCAGCAGGGACGGCT RRRRRSSSRSSRSSRRRRR 8317 S C * S A * S G * R C
* S A * S G * R G * S G * S A * R m5Ceo * R Geo * R Geo * R m5Ceo *
R Teo WV- mA * S mC * S mC * S mG * S mG * S G * S C *
ACCGGGCAGCAGGGACGGCT SSSSSSSSRSSRSSROOOR 8318 S A * S G * R C * S A
* S G * R G * S G * S A * R m5CeoGeoGeom5Ceo * R Teo WV- m5Ceo * R
m5CeoTeo * R m5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
RORORSSRSSRSSSROROR 8319 S C * R A * S C * S C * R C * S A * S C *
S T * R m5CeoGeo * R m5Ceom5Ceo * R Aeo WV- m5Ceo * R m5CeoTeo * R
m5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA RORORSSSSSRSSSROROR
8320 S C * S A * S C * S C * R C * S A * S C * S T * R m5CeoGeo * R
m5Ceom5Ceo * R Aeo WV- m5Ceo * R m5Ceo * R Teom5CeoAeo * R C * S T
* CCTCACTCACCCACTCGCCA RROORSSRSSRSSSROORR 8321 S C * R A * S C * S
C * R C * S A * S C * S T * R m5CeoGeom5Ceo * R m5Ceo * R Aeo WV-
m5Ceo * R m5Ceo * R Teo * R m5CeoAeo * R C * CCTCACTCACCCACTCGCCA
RRRORSSSSSRSSSROORR 8322 S T * S C * S A * S C * S C * R C * S A *
S C * S T * R m5CeoGeom5Ceo * R m5Ceo * R Aeo WV- m5Ceo * R m5Ceo *
R Teom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
RROORSSSSSRSSSROORR 8329 S C * S A * S C * S C * R C * S A * S C *
S T * R m5CeoGeom5Ceo * R m5Ceo * R Aeo WV- L001mA * mCmCmGmG * G *
C * A * G * C * ACCGGGCAGCAGGGACGGCU OXOOOXXXXXXXXXXXOOOX 8444 A *
G * G * G * A * mCmGmGmC * mU WV- Mod024L001mA * mCmCmGmG * G * C *
A * ACCGGGCAGCAGGGACGGCU OXOOOXXXXXXXXXXXOOOX 8445 G * C * A * G *
G * G * A * mCmGmGmC * mU WV- Mod059L001mA * mCmCmGmG * G * C * A *
ACCGGGCAGCAGGGACGGCU OXOOOXXXXXXXXXXXOOOX 8446 G * C * A * G * G *
G * A * mCmGmGmC * mU WV- Mod007L001mA * mCmCmGmG * G * C * A *
ACCGGGCAGCAGGGACGGCU OXOOOXXXXXXXXXXXOOOX 8447 G * C * A * G * G *
G * A * mCmGmGmC * mU WV- mC * S m5CeoTeom5CeomA * S C * S T * S C
* CCTCACTCACCCACTCGCCA SOOOSSSRSSSSSSSOOOS 8452 R A * S C * S C * S
C * S A * S C * S T * S m5CeomGm5CeomC * S mA WV- mC * S
m5CeoTeom5CeomA * S C * S T * S C * CCTCACTCACCCACTCGCCA
SOOOSSSRSSRSSSSOOOS 8453 R A * S C * S C * R C * S A * S C * S T *
S m5CeomGm5CeomC * S mA WV- mC * S m5CeoTeom5CeomA * S C * S T * S
C * CCTCACTCACCCACTCGCCA SOOOSSSRSSSSSSSSOSS 8454 R A * S C * S C *
S C * S A * S C * S T * S mC * S mGmC * S mC * S mA WV- mC * S
m5CeoTeom5CeomA * S C * S T * S C * CCTCACTCACCCACTCGCCA
SOOOSSSRSSRSSSSSOSS 8455 R A * S C * S C * R C * S A * S C * S T *
S mC * S mGmC * S mC * S mA WV- m5Ceo * R m5CeoTeom5CeoAeo * R C *
S T * CCTCACTCACCCACTCGCCA ROOORSSRSSSSSSSOOOS 8456 S C * R A * S C
* S C * S C * S A * S C * S T * S m5CeomGm5CeomC * S mA WV- m5Ceo *
R m5CeoTeom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
ROOORSSRSSRSSSSOOOS 8457 S C * R A * S C * S C * R C * S A * S C *
S T * S m5CeomGm5CeomC * S mA WV- m5Ceo * R m5CeoTeom5CeoAeo * R C
* S T * CCTCACTCACCCACTCGCCA ROOORSSRSSSSSSSSOSS 8458 S C * R A * S
C * S C * S C * S A * S C * S T * S mC * S mGmC * S mC * S mA WV-
m5Ceo * R m5CeoTeom5CeoAeo * R C * S T * CCTCACTCACCCACTCGCCA
ROOORSSRSSRSSSSSOSS 8459 S C * R A * S C * S C * R C * S A * S C *
S T * S mC * S mGmC * S mC * S mA WV- m5Ceo * R m5Ceo * R Teo * R
m5Ceo * R Aeo * CCTCACTCACCCACTCGCCA RRRRRSSRSSSSSSRRRRR 8460 R C *
S T * S C * R A * S C * S C * S C * S A * S C * S T * R m5Ceo * R
Geo * R m5Ceo * R m5Ceo * R Aeo WV- m5Ceo * R m5Ceo * R Teom5CeoAeo
* R C * CCTCACTCACCCACTCGCCA RROORSSRSSSSSSROORR 8461 S T * S C * R
A * S C * S C * S C * S A * S C * S T * R m5CeoGeom5Ceo * R m5Ceo *
R Aeo WV- mA * S mCmCmGmG * S G * S C * S A * S G *
ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSOOOS 8462 S C * R A * S G * S G
* R G * S A * S mCmGmGmC * S mU WV- mA * S mCmCmGmG * S G * S C * S
A * S G * ACCGGGCAGCAGGGACGGCU SOOOSSSSRSSRSSSOOOS 8463 R C * S A *
S G * R G * S G * S A * S mCmGmGmC * S mU WV- mA * S mCmCmGmG * S G
* S C * S A * S G * ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSSSSS 8464 S
C * R A * S G * S G * R G * S A * S mC * S mG * S mG * S mC * S mU
WV- mA * S mCmCmGmG * S G * S C * S A * S G * ACCGGGCAGCAGGGACGGCU
SOOOSSSSRSSRSSSSSSS 8465 R C * S A * S G * R G * S G * S A * S mC *
S mG * S mG * S mC * S mU WV- Aeo * R m5Ceom5CeoGeoGeo * R G * S C
* ACCGGGCAGCAGGGACGGCU ROOORSSSSRSSRSSOOOS 8466 S A * S G * S C * R
A * S G * S G * R G * S A * S mCmGmGmC * S mU WV- Aeo * R
m5Ceom5CeoGeoGeo * R G * S C * ACCGGGCAGCAGGGACGGCU
ROOORSSSRSSRSSSOOOS 8467 S A * S G * R C * S A * S G * R G * S G *
S A * S mCmGmGmC * S mU WV- Aeo * R m5Ceom5CeoGeoGeo * R G * S C *
ACCGGGCAGCAGGGACGGCU ROOORSSSSRSSRSSSOOS 8468 S A * S G * S C * R A
* S G * S G * R G * S A * S mC * S mGmGmC * S mU WV- Aeo * R
m5Ceom5CeoGeoGeo * R G * S C * ACCGGGCAGCAGGGACGGCU
ROOORSSSRSSRSSSSOOS 8469 S A * S G * R C * S A * S G * R G * S G *
S A * S mC * S mGmGmC * S mU WV- Aeo * R m5Ceom5CeoGeoGeo * R G * S
C * ACCGGGCAGCAGGGACGGCU ROOORSSSSRSSRSSSSSS 8470 S A * S G * S C *
R A * S G * S G * R G * S A * S mC * S mG * S mG * S mC * S mU WV-
Aeo * R m5Ceom5CeoGeoGeo * R G * S C * ACCGGGCAGCAGGGACGGCU
ROOORSSSRSSRSSSSSSS 8471 S A * S G * R C * S A * S G * R G * S G *
S A * S mC * S mG * S mG * S mC * S mU WV- mA * S m5Ceom5CeomG * S
mG * S G * S C * ACCGGGCAGCAGGGACGGCU SOOSSSSSSRSSRSSOOOS 8472 S A
* S G * S C * R A * S G * S G * R G * S A * S mCmGmGmC * S mU WV-
mA * S m5Ceom5CeomG * S mG * S G * S C * ACCGGGCAGCAGGGACGGCU
SOOSSSSSRSSRSSSOOOS 8473 S A * S G * R C * S A * S G * R G * S G *
S A * S mCmGmGmC * S mU WV- mA * S m5Ceom5CeomG * S mG * S G * S C
* ACCGGGCAGCAGGGACGGCU SOOSSSSSSRSSRSSSOOS 8474 S A * S G * S C * R
A * S G * S G * R G * S A * S mC * S mGmGmC * S mU WV- mA * S
m5Ceom5CeomG * S mG * S G * S C * ACCGGGCAGCAGGGACGGCU
SOOSSSSSRSSRSSSSOOS 8475 S A * S G * R C * S A * S G * R G * S G *
S A * S mC * S mGmGmC * S mU WV- mA * S m5Ceom5CeomG * S mG * S G *
S C * ACCGGGCAGCAGGGACGGCU SOOSSSSSSRSSRSSSSSS 8476 S A * S G * S C
* R A * S G * S G * R G * S A * S mC * S mG * S mG * S mC * S mU
WV- mA * S m5Ceom5CeomG * S mG * S G * S C * ACCGGGCAGCAGGGACGGCU
SOOSSSSSRSSRSSSSSSS 8477 S A * S G * R C * S A * S G * R G * S G *
S A * S mC * S mG * S mG * S mC * S mU WV- m5Ceo * m5CeoTeom5CeoAeo
* C * T * C * CCTCACTCACCCACTCGCCA XOOOXXXXXXXXXXXOOXX 8547 A * C *
C * C * A * C * T * mCmGmC * mC * mA WV- m5Ceo * m5CeoTeom5CeoAeo *
C * T * C * CCTCACTCACCCACTCGCCA XOOOXXXXXXXXXXXXXXX 8548 A * C * C
* C * A * C * T * mC * mG * mC * mC * mA WV- mC * m5CeoTeom5CeomA *
C * T * C * A * CCTCACTCACCCACTCGCCA XOOOXXXXXXXXXXXOOXX 8549 C * C
* C * A * C * T * mCmGmC * mC * mA WV- mC * m5CeoTeom5CeomA * C * T
* C * A * CCTCACTCACCCACTCGCCA XOOOXXXXXXXXXXXXXXX 8550 C * C * C *
A * C * T * mC * mG * mC * mC *
mA WV- mC * m5CeoTeom5CeomA * C * T * C * A * CCTCACTCACCCACTCGCCA
XOOOXXXXXXXXXXXXOXX 8551 C * C * C * A * C * T * mC * mGmC * mC *
mA WV- mA * S m5Ceom5CeoGeomG * S G * S C * S A *
ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSSSSS 8568 S G * S C * R A * S G
* S G * R G * S A * S mC * S mG * S mG * S mC * S mU WV- mA * S
m5Ceom5CeoGeomG * S G * S C * S A * ACCGGGCAGCAGGGACGGCU
SOOOSSSSRSSRSSSSSSS 8569 S G * R C * S A * S G * R G * S G * S A *
S mC * S mG * S mG * S mC * S mU WV- Aeo * m5Ceom5CeoGeoGeo * G * C
* A * G * ACCGGGCAGCAGGGACGGCU XOOOXXXXXXXXXXXXXXX 8594 C * A * G *
G * G * A * mC * mG * mG * mC * mU WV- mA * m5Ceom5CeoGeomG * G * C
* A * G * ACCGGGCAGCAGGGACGGCU XOOOXXXXXXXXXXXXXXX 8595 C * A * G *
G * G * A * mC * mG * mG * mC * mU WV- mA * S m5Ceom5CeoGeomG * S G
* S C * S A * ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSSOSS 8691 S G * S
C * R A * S G * S G * R G * S A * S mC * S mGmG * S mC * S mU WV-
mA * S m5Ceom5CeoGeomG * S G * S C * S A * ACCGGGCAGCAGGGACGGCU
SOOOSSSSRSSRSSSSOSS 8692 S G * R C * S A * S G * R G * S G * S A *
S mC * S mGmG * S mC * S mU WV- mA * m5Ceom5CeoGeomG * G * C * A *
G * ACCGGGCAGCAGGGACGGCU XOOOXXXXXXXXXXXXOXX 8693 C * A * G * G * G
* A * mC * mGmG * mC * mU WV- mA * S m5Ceom5CeoGeomG * S G * S C *
S A * ACCGGGCAGCAGGGACGGCU SOOOSSSSSRSSRSSOOSS 8694 S G * S C * R A
* S G * S G * R G * S A * S mCmGmG * S mC * S mU WV- mA * S
m5Ceom5CeoGeomG * S G * S C * S A * ACCGGGCAGCAGGGACGGCU
SOOOSSSSRSSRSSSOOSS 8695 S G * R C * S A * S G * R G * S G * S A *
S mCmGmG * S mC * S mU WV- mA * m5Ceom5CeoGeomG * G * C * A * G *
ACCGGGCAGCAGGGACGGCU XOOOXXXXXXXXXXXOOXX 8696 C * A * G * G * G * A
* mCmGmG * mC * mU WV- L001mC * S m5CeoTeom5CeomA * S C * S T *
CCTCACTCACCCACTCGCCA OSOOOSSSRSSSSSSSSSSS 9062 S C * R A * S C * S
C * S C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA WV-
Mod007L001mC * S m5CeoTeom5CeomA * S C * CCTCACTCACCCACTCGCCA
OSOOOSSSRSSSSSSSSSSS 9063 S T * S C * R A * S C * S C * S C * S A *
S C * S T * S mC * S mG * S mC * S mC * S mA WV- R GR GR UR GR GR
CR GR AR GR UR GR GGUGGCGAGUGGGUGAGUG OOOOOOOOOOOOOOOOO 9228 GR GR
UR GR AR GR UR GR AR GR GR AR AGGAG OOOOOO G WV- L001mC * S
m5CeoTeom5CeomA * S C * S T * CCTCACTCACCCACTCGCCA
OSOOOSSSRSSRSSSSSOSS 9285 S C * R A * S C * S C * R C * S A * S C *
S T * S mC * S mGmC * S mC * S mA WV- Mod007L001mC * S
m5CeoTeom5CeomA * S C * CCTCACTCACCCACTCGCCA OSOOOSSSRSSRSSSSSOSS
9286 S T * S C * R A * S C * S C * R C * S A * S C * S T * S mC * S
mGmC * S mC * S mA WV- L001mC * S m5CeoTeom5CeomA * S C * S T *
CCTCACTCACCCACTCGCCA OSOOOSSSRSSRSSSSSSSS 9380 S C * R A * S C * S
C * R C * S A * S C * S T * S mC * S mG * S mC * S mC * S mA WV-
Mod007L001mC * S m5CeoTeom5CeomA * S C * CCTCACTCACCCACTCGCCA
OSOOOSSSRSSRSSSSSSSS 9381 S T * S C * R A * S C * S C * R C * S A *
S C * S T * S mC * S mG * S mC * S mC * S mA WV- mC * S
m5CeoTeom5CeomA * S C * S T * S CA * CCTCACTCACCCACTCGCCA
SOOOSSSOSSSSSSSSSSS 9394 S C * S C * S C * S A * S C * S T * S mC *
S mG * S mC * S mC * S mA WV- mC * S m5CeoTeom5CeomA * S C * S T *
S CA * CCTCACTCACCCACTCGCCA SOOOSSSOSSOSSSSSSSS 9395 S C * S CC * S
A * S C * S T * S mC * S mG * S mC * S mC * S mA WV- mC * S
m5CeoTeom5CeomA * S C * S T * CCTCACTCACCCACTCGCCA
SOOOSSSOSSSSSSSSSSS 9396 S C5MSdA * S C * S C * S C * S A * S C * S
T * S mC * S mG * S mC * S mC * S mA WV- mC * S m5CeoTeom5CeomA * S
C * S T * CCTCACTCACCCACTCGCCA SOOOSSSOSSOSSSSSSSS 9397 S C5MSdA *
S C * S C5MSdC * S A * S C * S T * S mC * S mG * S mC * S mC * S mA
WV- mC * S m5CeoTeom5CeomA * S C * S T * CCTCACTCACCCACTCGCCA
SOOOSSSOSSSSSSSSSSS 9398 S C5MRdA * S C * S C * S C * S A * S C * S
T * S mC * S mG * S mC * S mC * S mA WV- mC * S m5CeoTeom5CeomA * S
C * S T * CCTCACTCACCCACTCGCCA SOOOSSSOSSOSSSSSSSS 9399 S C5MRdA *
S C * S C5MRdC * S A * S C * S T * S mC * S mG * S mC * S mC * S mA
WV- Mod059L001mC * S m5CeoTeom5CeomA * S C * CCTCACTCACCCACTCGCCA
OSOOOSSSRSSRSSSSSSSS 9421 S T * S C * R A * S C * S C * R C * S A *
S C * S T * S mC * S mG * S mC * S mC * S mA WV- mU *
Aeom5Ceom5CeomC * G * C * G * C * C * UACCCGCGCCTCTTCCCGGC
XOOOXXXXXXX 9486 T * C * T * T * C * mC * mC * mG * mG * mC
XXXXXXXX WV- mC * TeoAeom5CeomC * C * G * C * G * C * C *
CTACCCGCGCCTCTTCCCGG XOOOXXXXXXX 9487 T * C * T * T * mC * mC * mC
* mG * mG XXXXXXXX WV- mG * GeoGeom5CeomU * C * T * C * C * T * C *
GGGCUCTCCTCAGAGCUCGA XOOOXXXXXXX 9488 A * G * A * G * mC * mU * mC
* mG * mA XXXXXXXX WV- mG * GeoGeoTeomG * T * C * G * G * G * C *
GGGTGTCGGGCTTTCGCCUC XOOOXXXXXXX 9489 T * T * T * C * mG * mC * mC
* mU * mC XXXXXXXX WV- mG * m5CeoAeoTeomC * C * G * G * G * C *
GCATCCGGGCCCCGGGCUUC XOOOXXXXXXX 9490 C * C * C * G * G * mG * mC *
mU * mU * mC XXXXXXXX WV- mC * m5CeoTeoTeomC * C * C * T * G * A *
CCTTCCCTGAAGGTTCCUCC XOOOXXXXXXX 9491 A * G * G * T * T * mC * mC *
mU * mC * mC XXXXXXXX WV- mC * m5Ceom5CeoGeomG * C * C * C * C * T
* CCCGGCCCCTAGCGCGCGAC XOOOXXXXXXX 9492 A * G * C * G * C * mG * mC
* mG * mA * mC XXXXXXXX WV- m5Ceo * m5Ceom5CeoGeoGeo * C * C * C *
C * CCCGGCCCCTAGCGCGCGAC XOOOXXXXXXX 9493 T * A * G * C * G * C *
Geom5CeoGeoAeo * XXXXOOOX m5Ceo WV- mG * TeoGeom5CeomU * G * C * G
* A * T * GTGCUGCGATCCCCAUUCCA XOOOXXXXXXX 9494 C * C * C * C * A *
mU * mU * mC * mC * mA XXXXXXXX WV- mC * Sm5CeoTeom5CeomA * SC * ST
* SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS 9505 SC * SC * SC * RA *
SC * ST * SmC * SmG * RSSSRSSSSSSS SmC * SmC * SmA WV- mC *
Sm5CeoTeom5CeomA * SC * ST * SC * SA * CCTCACTCACCCACTCGCCA SOOOSSS
9506 SC * SC * SC * RA * SC * ST * SmC * SmG * SSSSRSSSSSSS SmC *
SmC * SmA WV- mC * Sm5CeoTeom5CeomA * SC * ST * SC * SA *
CCTCACTCACCCACTCGCCA SOOOSSS 9507 SC * SC * RC * SA * SC * ST * SmC
* SmG * SSSRSSSSSSSS SmC * SmC * SmA WV- mC * Sm5CeoTeom5CeomA * SC
* ST * SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS 9508 SC * SC * RC *
SA * Sc * ST * SfC * SfG * RSSRSSSSSSSS SfC * SfC * SfA WV- mC *
Sm5CeoTeom5CeomA * SC * ST * SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS
9509 Sc * Sc * SC * RA * Sc * ST * SfC * SfG * RSSSRSSSSSSS SfC *
SfC * SfA WV- mC * m5CeoTeom5CeomA * C * T * C * A * C *
CCTCACTCACCCACTCGCCA XOOOXXXXXXX 9510 C * C * A * C * T * fC * fG *
fC * fC * fA XXXXXXXX WV- mC * mCmCmGmG * C * C * C * C * T * A * G
* CCCGGCCCCTAGCGCGCGAC XOOOXXXXXXX 9694 C * G * C * mGmCmGmA * mC
XXXXOOOX WV- mU * mAmCmAmG * G * C * T * G * C * G * G *
UACAGGCTGCGGTTGUUUCC XOOOXXXXXXX 9695 T * T * G * mUmUmUmC * mC
XXXXOOOX WV- mG * AeoTeoGeomC * C * G * C * C * T * C *
GATGCCGCCTCCTCACUCAC XOOOXXXXXXX 10406 C * T * C * A * mC * mU * mC
* mA * mC XXXXXXXX WV- mA * TeoGeom5CeomC * G * C * C * T * C *
ATGCCGCCTCCTCACUCACC XOOOXXXXXXX 10407 C * T * C * A * C * mU * mC
* mA * mC * mC XXXXXXXX WV- mU * Geom5Ceom5CeomG * C * C * T * C *
C * UGCCGCCTCCTCACTCACCC XOOOXXXXXXX 10408 T * C * A * C * T * mC *
mA * mC * mC * mC XXXXXXXX WV- mG * m5Ceom5CeoGeomC * C * T * C * C
* T * GCCGCCTCCTCACTCACCCA XOOOXXXXXXX 10409 C * A * C * T * C * mA
* mC * mC * mC * mA XXXXXXXX WV- mC * m5CeoGeom5CeomC * T * C * C *
T * C * CCGCCTCCTCACTCACCCAC XOOOXXXXXXX 10410 A * C * T * C * A *
mC * mC * mC * mA * mC XXXXXXXX WV- mC * Geom5Ceom5CeomU * C * C *
T * C * A * CGCCUCCTCACTCACCCACU XOOOXXXXXXX 10411 C * T * C * A *
C * mC * mC * mA * mC * mU XXXXXXXX WV- mG * m5Ceom5CeoTeomC * C *
T * C * A * C * GCCTCCTCACTCACCCACUC XOOOXXXXXXX 10412 T * C * A *
C * C * mC * mA * mC * mU * mC XXXXXXXX WV- mC * m5CeoTeom5CeomC *
T * C * A * C * T * CCTCCTCACTCACCCACUCG XOOOXXXXXXX 10413 C * A *
C * C * C * mA * mC * mU * mC * mG XXXXXXXX WV- mC *
Teom5Ceom5CeomU * C * A * C * T * C * CTCCUCACTCACCCACUCGC
XOOOXXXXXXX 10414 A * C * C * C * A * mC * mU * mC * mG * mC
XXXXXXXX WV- mU * m5Ceom5CeoTeomC * A * C * T * C * A *
UCCTCACTCACCCACUCGCC XOOOXXXXXXX 10415 C * C * C * A * C * mU * mC
* mG * mC * mC XXXXXXXX WV- mC * Teom5CeoAeomC * T * C * A * C * C
* CTCACTCACCCACTCGCCAC XOOOXXXXXXX 10416 C * A * C * T * C * mG *
mC * mC * mA * mC XXXXXXXX WV- mC * Aeom5CeoTeomC * A * C * C * C *
A * CACTCACCCACTCGCCACCG XOOOXXXXXXX 10417 C * T * C * G * C * mC *
mA * mC * mC * mG XXXXXXXX WV- mA * m5CeoTeom5CeomA * C * C * C * A
* C * ACTCACCCACTCGCCACCGC XOOOXXXXXXX 10418 T * C * G * C * C * mA
* mC * mC * mG * mC XXXXXXXX
WV- mC * Teom5CeoAeomC * C * C * A * C * T * CTCACCCACTCGCCACCGCC
XOOOXXXXXXX 10419 C * G * C * C * A * mC * mC * mG * mC * mC
XXXXXXXX WV- mU * m5CeoAeom5CeomC * C * A * C * T * C *
UCACCCACTCGCCACCGCCU XOOOXXXXXXX 10420 G * C * C * A * C * mC * mG
* mC * mC * mU XXXXXXXX WV- mC * Aeom5Ceom5CeomC * A * C * T * C *
G * CACCCACTCGCCACCGCCUG XOOOXXXXXXX 10421 C * C * A * C * C * mG *
mC * mC * mU * mG XXXXXXXX WV- mA * m5Ceom5Ceom5CeomA * C * T * C *
G * ACCCACTCGCCACCGCCUGC XOOOXXXXXXX 10422 C * C * A * C * C * G *
mC * mC * mU * mG * XXXXXXXX mC WV- mC * m5Ceom5CeoAeomC * T * C *
G * C * C * CCCACTCGCCACCGCCUGCG XOOOXXXXXXX 10423 A * C * C * G *
C * mC * mU * mG * mC * mG XXXXXXXX WV- mC * m5CeoAeom5CeomU * C *
G * C * C * A * CCACUCGCCACCGCCUGCGC XOOOXXXXXXX 10424 C * C * G *
C * C * mU * mG * mC * mG * mC XXXXXXXX WV- mU * m5CeoAeom5CeomU *
C * A * C * C * C * UCACUCACCCACTCGCCACC XOOOXXXXXXX 10425 A * C *
T * C * G * mC * mC * mA * mC * mC XXXXXXXX WV- fC * fC * fU * fC *
fA * fC * mU * mC * CCUCACUCACCCACUCGCCA XXXXXXXXXXX 10426 mA * mC
* mC * mC * mA * mC * fU * fC * XXXXXXXX fG * fC * fC * fA WV-
m5Ceo * m5Ceo * m5Ceo * Geo * Geo * C * C * CCCGGCCCCTAGCGCGCGAC
XXXXXXXXXXX 10427 C * C * T * A * G * C * G * C * Geo * m5Ceo *
XXXXXXXX Geo * Aeo * m5Ceo WV- Geo * m5Ceo * m5Ceo * m5Ceo * C * T
* A * GCCCCTAGCGCGCGACTC XXXXXXXXXXX 10428 G * C * G * C * G * C *
G * Aeo * m5Ceo * XXXXXX Teo * m5Ceo WV- Geo * m5Ceo * m5Ceo * C *
C * T * A * G * GCCCCTAGCGCGCGACTC XXXXXXXXXXX 10429 C * G * C * G
* C * G * A * m5Ceo * Teo * XXXXXX m5Ceo WV- Geo * m5Ceo * m5Ceo *
m5Ceo * m5Ceo * T * GCCCCTAGCGCGCGACTC XXXXXXXXXXX 10430 A * G * C
* G * C * G * C * Geo * Aeo * XXXXXX m5Ceo * Teo * m5Ceo WV- Geo *
m5Ceom5Ceom5Ceom5Ceo * T * A * G * GCCCCTAGCGCGCGACTC XOOOXXXXXXX
10431 C * G * C * G * C * GeoAeom5Ceo * Teo * XXOOXX m5Ceo WV- mG *
m5CeoTeoTeomG * G * T * G * T * G * GCTTGGTGTGTCAGCCGUCC
XOOOXXXXXXX 10844 T * C * A * G * C * mC * mG * mU * mC * mC
XXXXXXXX WV- mC * TeoTeoGeomG * T * G * T * G * T * C *
CTTGGTGTGTCAGCCGUCCC XOOOXXXXXXX 10845 A * G * C * C * mG * mU * mC
* mC * mC XXXXXXXX WV- mG * Teom5CeoAeomG * C * C * G * T * C *
GTCAGCCGTCCCTGCUGCCC XOOOXXXXXXX 10846 C * C * T * G * C * mU * mG
* mC * mC * mC XXXXXXXX WV- mG * m5Ceom5CeoGeomU * C * C * C * T *
G * GCCGUCCCTGCTGCCCGGUU XOOOXXXXXXX 10847 C * T * G * C * C * mC *
mG * mG * mU * mU XXXXXXXX WV- mG * Teom5Ceom5CeomC * T * G * C * T
* G * GTCCCTGCTGCCCGGUUGCU XOOOXXXXXXX 10848 C * C * C * G * G * mU
* mU * mG * mC * mU XXXXXXXX WV- mC * m5CeoTeoGeomC * T * G * C * C
* C * CCTGCTGCCCGGTTGCUUCU XOOOXXXXXXX 10849 G * G * T * T * G * mC
* mU * mU * mC * mU XXXXXXXX WV- mC * m5CeoGeom5CeomA * G * C * C *
T * G * CCGCAGCCTGTAGCAAGCUC XOOOXXXXXXX 10850 T * A * G * C * A *
mA * mG * mC * mU * mC XXXXXXXX WV- mG * m5CeoGeoGeomU * T * G * C
* G * G * GCGGUTGCGGTGCCTGCGCC XOOOXXXXXXX 10851 T * G * C * C * T
* mG * mC * mG * mC * mC XXXXXXXX WV- mG * TeoTeoGeomC * G * G * T
* G * C * GTTGCGGTGCCTGCGCCCGC XOOOXXXXXXX 10852 C * T * G * C * G
* mC * mC * mC * mG * mC XXXXXXXX WV- mG * Geom5CeoGeomG * A * G *
G * C * G * GGCGGAGGCGCAGGCGGUGG XOOOXXXXXXX 10853 C * A * G * G *
C * mG * mG * mU * mG * mG XXXXXXXX WV- mG * m5CeoAeoGeomG * C * G
* G * T * G * GCAGGCGGTGGCGAGUGGGU XOOOXXXXXXX 10854 G * C * G * A
* G * mU * mG * mG * mG * mU XXXXXXXX WV- mG * m5CeoGeoGeomC * A *
T * C * C * T * GCGGCATCCTGGCGGGUGGC XOOOXXXXXXX 10855 G * G * C *
G * G * mG * mU * mG * mG * mC XXXXXXXX WV- mG * m5CeoAeoTeomC * C
* T * G * G * C * GCATCCTGGCGGGTGGCUGU XOOOXXXXXXX 10856 G * G * G
* T * G * mG * mC * mU * mG * mU XXXXXXXX WV- mG * m5CeoTeoGeomG *
G * T * G * T * C * GCTGGGTGTCGGGCTUUCGC XOOOXXXXXXX 10857 G * G *
G * C * T * mU * mU * mC * mG * mC XXXXXXXX WV- mA * TeoTeoGeomC *
C * T * G * C * A * ATTGCCTGCATCCGGGCCCC XOOOXXXXXXX 10858 T * C *
C * G * G * mG * mC * mC * mC * mC XXXXXXXX WV- mU *
Geom5Ceom5CeomU * G * C * A * T * C * UGCCUGCATCCGGGCCCCGG
XOOOXXXXXXX 10859 C * G * G * G * C * mC * mC * mC * mG * mG
XXXXXXXX WV- mC * TeoTeom5CeomC * T * T * G * C * T *
CTTCCTTGCTTTCCCGCCCU XOOOXXXXXXX 10860 T * T * C * C * C * mG * mC
* mC * mC * mU XXXXXXXX WV- mU * m5Ceom5CeoTeomU * G * C * T * T *
T * UCCTUGCTTTCCCGCCCUCA XOOOXXXXXXX 10861 C * C * C * G * C * mC *
mC * mU * mC * mA XXXXXXXX WV- m5Ceo * m5Ceo * m5Ceo * Geo * Geo *
m5C * CCCGGCCCCTAGCGCGCGAC XXXXXXXXXXX 11039 m5C * m5C * m5C * T *
A * G * m5C * G * m5C * XXXXXXXX Geo * m5Ceo * Geo * Aeo * m5Ceo
WV- Geo * m5Ceo * m5Ceo * m5Ceo * m5Ceo * T * GCCCCTAGCGCGCGACTC
XXXXXXXXXXX 11040 A * G * m5C * G * m5C * G * m5C * Geo * Aeo *
XXXXXX m5Ceo * Teo * m5Ceo WV- Geo * m5Ceom5Ceom5Ceom5Ceo * T * A *
G * GCCCCTAGCGCGCGACTC XOOOXXXXXXX 11041 m5C * G * m5C * G * m5C *
GeoAeom5Ceo * XXOOXX Teo * m5Ceo WV- m5Ceo * Rm5Ceo * Rm5Ceo * RGeo
* RGeo * CCCGGCCCCTAGCGCGCGAC RRRRRSSSSRS 11042 Rm5C * Sm5C * Sm5C
* Sm5C * ST * RA * SG * SSSSRRRR Sm5C * SG * Sm5C * SGeo * Rm5Ceo *
RGeo * RAeo * Rm5Ceo WV- m5Ceo * Rm5Ceo * Rm5Ceo * SGeo * RGeo *
CCCGGCCCCTAGCGCGCGAC RRSRRSSSSRS 11043 Rm5C * Sm5C * Sm5C * Sm5C *
ST * RA * SG * SSSSRRRR Sm5C * SG * Sm5C * SGeo * Rm5Ceo * RGeo *
RAeo * Rm5Ceo WV- m5Ceo * Rm5Ceo * Rm5Ceo * RGeo * RGeo *
CCCGGCCCCTAGCGCGCGAC RRRRRSSSSR 11044 Rm5C * Sm5C * Sm5C * Sm5C *
ST * RA * SG * SSSSSRSRR Sm5C * SG * Sm5C * SGeo * Rm5Ceo * SGeo *
RAeo * Rm5Ceo WV- m5Ceo * Sm5Ceo * Sm5Ceo * RGeo * SGeo *
CCCGGCCCCTAGCGCGCGAC SSRSSSSSSRS 11045 Sm5C * Sm5C * Sm5C * Sm5C *
ST * RA * SG * SSSSSRSS Sm5C * SG * Sm5C * SGeo * Sm5Ceo * RGeo *
SAeo * Sm5Ceo WV- m5Ceo * Sm5Ceo * Sm5Ceo * SGeo * SGeo *
CCCGGCCCCTAGCGCGCGAC SSSSSSSSS 11046 Sm5C * Sm5C * Sm5C * Sm5C * ST
* RA * SG * RSSSSSSSSS Sm5C * SG * Sm5C * SGeo * Sm5Ceo * SGeo *
SAeo * Sm5Ceo WV- mC * Sm5Ceon001Teon001m5Ceon001mA * SC *
CCTCACTCACCCACTCGCCA SnXnXnXSSSR 11532 ST * SC * RA * SC * SC * RC
* SA * SC * ST * SSRSSSSSSSS SmC * SmG * SmC * SmC * SmA WV- mC *
mCmUmCmA * C * T * C * A * C * C * C * CCUCACTCACCCACTCGCCA
XOOOXXXXXXX 11963 A * C * BrdU * mCmGmCmC * mA XXXXOOOX WV- m5Ceo *
m5CeoTeom5CeoAeo * C * T * C * A * CCTCACTCACCCACTCGCCA XOOOXXXXXXX
11964 C * C * C * A * C * BrdU * XXXXOOOX m5CeoGeom5Ceom5Ceo * Aeo
WV- m5Ceo * Sm5CeoTeom5CeoAeo * SC * ST * SC * CCTCACTCACCCACTCGCCA
SOOOSSS 11965 RA * SC * SC * RC * SA * SC * SBrdU * RSSRSSSSOOOS
Sm5CeoGeom5Ceom5Ceo * SAeo WV- mC * m5CeoTeom5CeomA * C * T * C * A
* C * CCTCACTCACCCACTCGCCA XOOOXXXXXXX 11966 C * C * A * C * BrdU *
mC * mG * mC * mC * mA XXXXXXXX WV- mC * Sm5CeoTeom5CeomA * SC * ST
* SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS 11967 SC * SC * RC * SA *
SC * SBrdU * SmC * SmG * RSSRSSSSSSSS SmC * SmC * SmA WV-
rUrGrGrCrGrArGrUrGrGrGrUrGrArGrUrGrArGrG UGGCGAGUGGGUGAGUGAGG
OOOOOOOOO 12048 OOOOOOOOOO WV- m5Ceo * Teom5CeoAeom5Ceo * T * C * A
* C * CTCACTCACCCACTCGCCAC XOOOXXXXXXX 12439 C * C * A * C * T * C
* mG * mC * mC * mA * mC XXXXXXXX WV- Teo * m5Ceom5CeoTeom5Ceo * A
* C * T * C * TCCTCACTCACCCACUCGCC XOOOXXXXXXX 12440 A * C * C * C
* A * C * mU * mC * mG * mC * mC XXXXXXXX WV- Teo *
Geom5Ceom5CeoGeo * C * C * T * C * C * TGCCGCCTCCTCACTCACCC
XOOOXXXXXXX 12441 T * C * A * C * T * mC * mA * mC * mC * mC
XXXXXXXX WV- Geo * m5CeoGeom5CeoGeo * A * C * T * C * C *
GCGCGACTCCTGAGTUCCAG XOOOXXXXXXX 12442 T * G * A * G * T * mU * mC
* mC * mA * mG XXXXXXXX WV- Geo * AeoGeom5CeoTeo * T * G * C * T *
A * GAGCTTGCTACAGGCUGCGG XOOOXXXXXXX 12443 C * A * G * G * C * mU *
mG * mC * mG * mG XXXXXXXX WV- m5Ceo * AeoGeoGeoAeo * T * G * C * C
* G * CAGGATGCCGCCTCCUCACU XOOOXXXXXXX 12444 C * C * T * C * C * mU
* mC * mA * mC * mU XXXXXXXX WV- mC * mU * mC * mA * mC * T * C * A
* C * C * CUCACTCACCCACTCGCCAC XXXXXXXXXXX 12445 C * A * C * T * C
* Geom5Ceom5CeoAeo * m5Ceo XXXXOOOX WV- mC * mC * mU * mC * mA * C
* T * C * A * C * CCUCACTCACCCACTCGCCA XXXXXXXXXXX 12446 C * C * A
* C * T * m5CeoGeom5Ceom5Ceo * Aeo XXXXOOOX WV- mU * mC * mC * mU *
mC * A * C * T * C * A * UCCUCACTCACCCACTCGCC XXXXXXXXXXX 12447 C *
C * C * A * C * Teom5CeoGeom5Ceo * m5Ceo XXXXOOOX WV- mU * mG * mC
* mC * mG * C * C * T * C * C * UGCCGCCTCCTCACTCACCC XXXXXXXXXXX
12448 T * C * A * C * T * m5CeoAeom5Ceom5Ceo * m5Ceo XXXXOOOX WV-
mG * mC * mG * mC * mG * A * C * T * C * C * GCGCGACTCCTGAGTTCCAG
XXXXXXXXXXX 12449 T * G * A * G * T * Teom5Ceom5CeoAeo * Geo
XXXXOOOX
WV- mG * mA * mG * mC * mU * T * G * C * T * A *
GAGCUTGCTACAGGCTGCGG XXXXXXXXXXX 12450 C * A * G * G * C *
TeoGeom5CeoGeo * Geo XXXXOOOX WV- mC * mA * mG * mG * mA * T * G *
C * C * G * CAGGATGCCGCCTCCTCACT XXXXXXXXXXX 12451 C * C * T * C *
C * Teom5CeoAeom5Ceo * Teo XXXXOOOX WV- m5Ceo * Teom5CeoAeom5Ceo *
T * C * A * C * CTCACTCACCCACTCGCCAC XOOOXXXXXXX 12480 C * C * A *
C * T * C * Geom5Ceom5CeoAeo * XXXXOOOX m5Ceo WV- Teo *
m5Ceom5CeoTeom5Ceo * A * C * T * C * TCCTCACTCACCCACTCGCC
XOOOXXXXXXX 12481 A * C * C * C * A * C * Teom5CeoGeom5Ceo *
XXXXOOOX m5Ceo WV- Teo * Geom5Ceom5CeoGeo * C * C * T * C * C *
TGCCGCCTCCTCACTCACCC XOOOXXXXXXX 12482 T * C * A * C * T *
m5CeoAeom5Ceom5Ceo * m5Ceo XXXXOOOX WV- Geo * m5CeoGeom5CeoGeo * A
* C * T * C * C * GCGCGACTCCTGAGTTCCAG XOOOXXXXXXX 12483 T * G * A
* G * T * Teom5Ceom5CeoAeo * Geo XXXXOOOX WV- Geo * AeoGeom5CeoTeo
* T * G * C * T * A * GAGCTTGCTACAGGCTGCGG XOOOXXXXXXX 12484 C * A
* G * G * C * TeoGeom5CeoGeo * Geo XXXXOOOX WV- m5Ceo *
AeoGeoGeoAeo * mA * T * G * C * C * CAGGAATGCCGCCTCCTCACT
XOOOXXXXXXX 12485 G * C * C * T * C * C * Teom5CeoAeom5Ceo * Teo
XXXXXOOOX WV- m5Ceo * AeoGeoGeoAeo * T * G * C * C * G *
CAGGATGCCGCCTCCTCACT XOOOXXXXXXX 12486 C * C * T * C * C *
Teom5CeoAeom5Ceo * Teo XXXXOOOX WV- Teo * m5Ceom5CeoTeoTeo * G * C
* T * T * T * TCCTTGCTTTCCCGCCCTCA XOOOXXXXXXX 12487 C * C * C * G
* C * m5Ceom5CeoTeom5Ceo * Aeo XXXXOOOX WV- Geo * m5CeoAeoTeom5Ceo
* C * G * G * G * C * GCATCCGGGCCCCGGGCTTC XOOOXXXXXXX 12488 C * C
* C * G * G * Geom5CeoTeoTeo * m5Ceo XXXXOOOX WV- Teo *
m5Ceom5CeoTeoTeo * G * C * T * T * T * TCCTTGCTTTCCCGCCCUCA
XOOOXXXXXXX 12489 C * C * C * G * C * mC * mC * mU * mC * mA
XXXXXXXX WV- Geo * m5CeoAeoTeom5Ceo * C * G * G * G * C *
GCATCCGGGCCCCGGGCUUC XOOOXXXXXXX 12490 C * C * C * G * G * mG * mC
* mU * mU * mC XXXXXXXX WV- mU * mC * mC * mU * mU * G * C * T * T
* T * UCCUUGCTTTCCCGCCCTCA XXXXXXXXXXX 12491 C * C * C * G * C *
m5Ceom5CeoTeom5Ceo * Aeo XXXXOOOX WV- mG * mC * mA * mU * mC * C *
G * G * G * C * GCAUCCGGGCCCCGGGCTTC XXXXXXXXXXX 12492 C * C * C *
G * G * Geom5CeoTeoTeo * m5Ceo XXXXOOOX WV- m5CeoTeom5CeomA * SC *
ST * SC * RA * SC * CTCACTCACCCACTCGCCA OOOSSSRSSRSS 12497 SC * RC
* SA * SC * ST * SmC * SmG * SmC * SSSSSS SmC * SmA WV- Teom5CeomA
* SC * ST * SC * RA * SC * SC * TCACTCACCCACTCGCCA OOSSSRSSRSS
12498 RC * SA * SC * ST * SmC * SmG * SmC * SmC * SSSSSS SmA WV-
L001mC * Sm5CeoTeom5CeomA * SC * ST * SC * CCTCACTCACCCACTCGCCA
OSOOOSSS 12545 RA * SC * SC * RC * SA * SC * ST * SmC * SmG *
RSSRSSSSSSSS SmC * SmC * SmA WV- Mod085L001mC * Sm5CeoTeom5CeomA *
SC * ST * CCTCACTCACCCACTCGCCA OSOOOSSS 12546 SC * RA * SC * SC *
RC * SA * SC * ST * SmC * RSSRSSSSSSSS SmG * SmC * SmC * SmA WV-
Mod086L001mC * Sm5CeoTeom5CeomA * SC * ST * CCTCACTCACCCACTCGCCA
OSOOOSSS 12547 SC * RA * SC * SC * RC * SA * SC * ST * SmC *
RSSRSSSSSSSS SmG * SmC * SmC * SmA WV- Mod012L001mC *
Sm5CeoTeom5CeomA * SC * ST * CCTCACTCACCCACTCGCCA OSOOOSSS 12548 SC
* RA * SC * SC * RC * SA * SC * ST * SmC * RSSRSSSSSSSS SmG * SmC *
SmC * SmA WV- mC * Sm5CeoTeom5CeomA * SC * ST * SC * RA *
CCTCACTCACCCACTCGCCA SOOOSSS 12549 SC * SC * RC * SA * SC * ST *
SmC * SmG * RSSRSSSSSSSS SmC * SmC * SmAL004 WV- mC *
Sm5CeoTeom5CeomA * SC * ST * SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS
12550 SC * SC * RC * SA * SC * ST * SmC * SmG * RSSRSSSSSSSS SmC *
SmC * SmAL004Mod085 WV- mC * Sm5CeoTeom5CeomA * SC * ST * SC * RA *
CCTCACTCACCCACTCGCCA SOOOSSS 12551 SC * SC * RC * SA * SC * ST *
SmC * SmG * RSSRSSSSSSSS SmC * SmC * SmAL004Mod086 WV- mC *
Sm5CeoTeom5CeomA * SC * ST * SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS
12552 SC * SC * RC * SA * SC * ST * SmC * SmG * RSSRSSSSSSSS SmC *
SmC * SmAL004Mod012 WV- m51C * m5CeoTeom5CeoAeo * C * T * C * A *
CCTCACTCACCCACTCGCCA XOOOXXXXXXX 12575 C * C * C * A * C * T *
m5CeoGeom5Ceom5Ceo * XXXXOOOX 1A WV- m51C * m5CeoTeom5CeoAeo * mC *
mU * mC * mA * CCTCACUCACCCACUCGCCA XOOOXXXXXXX 12576 mC * mC * mC
* mA * mC * mU * XXXXOOOX m5CeoGeom5Ceom5Ceo * 1A WV- fC * fCfUfCfA
* C * T * C * A * C * C * C * CCUCACTCACCCACTCGCCA XOOOXXXXXXX
12577 A * C * T * fCfGfCfC * fA XXXXOOOX WV- fC * fC * fU * fC * fA
* C * T * C * A * C * CCUCACTCACCCACTCGCCA XXXXXXXXXXX 12578 C * C
* A * C * T * fC * fG * fC * fC * fA XXXXXXXX WV- m51C *
m5CeoTeom5CeomA * C * T * C * A * C * CCTCACTCACCCACTCGCCA
XOOOXXXXXXX 12579 C * C * A * C * T * mC * mG * mC * mC * 1A
XXXXXXXX WV- fC * m5CeoTeom5CeofA * C * T * C * A * C *
CCTCACTCACCCACTCGCCA XOOOXXXXXXX 12580 C * C * A * C * T * fC * fG
* fC * fC * fA XXXXXXXX WV- mC * m5CeoTeom5CeomA * mC * mU * mC *
mA * CCTCACUCACCCACUCGCCA XOOOXXXXXXX 12581 mC * mC * mC * mA * mC
* mU * mC * mG * mC * XXXXXXXX mC * mA WV- m51C * m5CeoTeom5CeomA *
mC * mU * mC * mA * CCTCACUCACCCACUCGCCA XOOOXXXXXXX 12582 mC * mC
* mC * mA * mC * mU * mC * mG * mC * XXXXXXXX mC * 1A WV- fC *
m5CeoTeom5CeofA * mC * mU * mC * mA * CCTCACUCACCCACUCGCCA
XOOOXXXXXXX 12583 mC * mC * mC * mA * mC * mU * fC * fG * fC *
XXXXXXXX fC * fA WV- mC * Sm5CeoTeom5CeomA * SC * ST * SC * RA *
CCTCACTCACCCACTCGCCA SOOOSSS 12893 SC * SC * SC * SA * SC * SBrdU *
SmC * SmG * RSSSSSSSSS SS SmC * SmC * SmA WV- m5Ceo *
Rm5Ceon001Teon001m5Ceon001Aeo * RC * CCTCACTCACCCACTCGCCA
RnXnXnXRSSRS 13305 ST * SC * RA * SC * SC * RC * SA * SC * ST *
SRSSSSSSSS SmC * SmG * SmC * SmC * SmA WV- m5Ceo *
Sm5CeoTeom5CeoAeo * RC * ST * SC * CCTCACTCACCCACTCGCCA SOOORSSRSS
13306 RA * SC * SC * RC * SA * SC * ST * SmC * RSSSSSSSS SmG * SmC
* SmC * SmA WV- m5Ceo * Sm5Ceon001Teon001m5Ceon001Aeo * RC *
CCTCACTCACCCACTCGCCA SnXnXnXRSSR 13307 ST * SC * RA * SC * SC * RC
* SA * SC * ST * SSRSSSSSSSS SmC * SmG * SmC * SmC * SmA WV- m5Ceo
* Rm5CeoTeom5CeoAeo * RC * ST * SC * CCTCACTCACCCACTCGCCA
ROOORSSRSSS 13308 RA * SC * SC * SC * RA * SC * ST * SmC * RSSSSSSS
SmG * SmC * SmC * SmA WV- m5Ceo * Rm5Ceon001Teon001m5Ceon001Aeo *
RC * CCTCACTCACCCACTCGCCA RnXnXnXRSSRSSS 13309 ST * SC * RA * SC *
SC * SC * RA * SC * ST * RSSSSSSS SmC * SmG * SmC * SmC * SmA WV-
m5Ceo * Sm5CeoTeom5CeoAeo * RC * ST * SC * CCTCACTCACCCACTCGCCA
SOOORSSRSSSRS 13310 RA * SC * SC * SC * RA * SC * ST * SmC * SSSSSS
SmG * SmC * SmC * SmA WV- m5Ceo * Sm5Ceon001Teon001m5Ceon001Aeo *
RC * CCTCACTCACCCACTCGCCA SnXnXnXRSSRSS 13311 ST * SC * RA * SC *
SC * SC * RA * SC * ST * SRSSSSSSS SmC * SmG * SmC * SmC * SmA WV-
mC * Sm5Ceon001Teon001m5Ceon001mA * SC * ST * CCTCACTCACCCACTCGCCA
SnXnXnXSSSRSS 13312 SC * RA * SC * SC * SC * SA * SC * ST * SmC *
SSSSSSS SS SmG * SmC * SmC * SmA WV- m5Ceo *
Rm5Ceon001Teon001m5Ceon001Aeo * RC * CCTCACTCACCCACTCGCCA
RnXnXnXRSSRS 13313 ST * SC * RA * SC * SC * SC * SA * SC * ST *
SSSSSSSS SS SmC * SmG * SmC * SmC * SmA WV- Teo *
Geon001m5Ceon001m5Ceon001Geo * C * C * TGCCGCCTCCTCACTCACCC
XnXnXnXXXXX XXXXXX 13803 T * C * C * T * C * A * C * T * mC * mA *
XXXXX mC * mC * mC WV- Teo * Geom5Ceom5CeoGeo * C * C * T * C * C *
TGCCGCCTCCTCACTCACCC XOOOXXXXXXX 13804 T * C * A * C * T *
mCn001mAn001mCn001mC * mC XXXXnXnXnXX WV- Teo *
Geon001m5Ceon001m5Ceon001Geo * C * C * TGCCGCCTCCTCACTCACCC
XnXnXnXXXXXX XXXXX 13805 T * C * C * T * C * A * C * T * XnXnXnXX
mCn001mAn001mCn001mC * mC WV- Geo * m5Ceon001Geon001m5Ceon001Geo *
A * C * GCGCGACTCCTGAGTTCCAG XnXnXnXXXXX XXXXXX 13806 T * C * C * T
* G * A * G * T * XOOOX Teom5Ceom5CeoAeo * Geo WV- Geo *
m5CeoGeom5CeoGeo * A * C * T * C * C * GCGCGACTCCTGAGTTCCAG
XOOOXXXXXXX 13807 T * G * A * G * T * XXXXnXnXnXX
Teon001m5Ceon001m5Ceon001Aeo * Geo WV- Geo *
m5Ceon001Geon001m5Ceon001Geo * A * C * GCGCGACTCCTGAGTTCCAG
XnXnXnXXXX XXXXXXX 13808 T * C * C * T * G * A * G * T * XnXnXnXX
Teon001m5Ceon001m5Ceon001Aeo * Geo WV- m5Ceo * Rm5CeoTeom5CeoAeo *
RC * ST * SC * CCTCACTCACCCACTCGCCA ROOORSSRSSR 14552 RA * SC * SC
* RC * SA * SC * ST * Rm5Ceo * SSSRSSSS SmG * SmC * SmC * SmA WV-
m5Ceo * Rm5Ceon001Teon001m5Ceon001Aeo * RC * CCTCACTCACCCACTCGCCA
RnXnXnXRSSRSS 14553 ST * SC * RA * SC * SC * RC * SA * SC * ST *
RSSSRSSSS Rm5Ceo * SmG * SmC * SmC * SmA WV- m5Ceo *
Rm5CeoTeom5CeoAeo * RC * ST * SC * CCTCACTCACCCACTCGCCA ROOORSSRSS
14554 RA * SC * SC * SC * RA * SC * ST * Rm5Ceo * SRSSRSSSS SmG *
SmC * SmC * SmA WV- m5Ceo * Rm5Ceon001Teon001m5Ceon001Aeo * RC *
CCTCACTCACCCACTCGCCA RnXnXnXRSSRS 14555 ST * SC * RA * SC * SC * SC
* RA * SC * ST * SSRSSRSSSS
Rm5Ceo * SmG * SmC * SmC * SmA WV- mC * Sm5CeoTeom5CeomA * SC * ST
* SC * RA * CCTCACTCACCCACTCGCCA SOOOSSS 14758 SC * SC * RC * SA *
SC * ST * SmCmGmCmCmA RSSRSSSSOOOO WV- mC * Sm5CeoTeom5CeomA * SC *
ST * SC * SA * CCTCACTCACCCACTCGCCA SOOOSSS 14772 SC * SC * SC * SA
* SC * ST * SmC * SmG * SSSSSSSSS SSS SmC * SmC * SmA WV- mU *
Sm5CeoTeom5CeomA * SC * ST * SC * RA * UCTCACTCACCCACTUGUUA SOOOSSS
15049 SC * SC * RC * SA * SC * ST * SmU * SmG * RSSRSSSSSSSS SmU *
SmU * SmA WV- mU * Sm5CeoTeom5CeomA * SC * ST * SC * RA *
UCTCACTCACCCACTGACUC SOOOSSS 15050 SC * SC * RC * SA * SC * ST *
SmG * SmA * RSSRSSSSSSSS SmC * SmU * SmC WV- mC * Sm5CeoTeom5CeomA
* SG * SG * RC * ST * CCTCAGGCTGGTTATCGCCA SOOOSSRS 15051 SG * RG *
ST * ST * RA * ST * SmC * SmG * SRSSRSSSSSS SmC * SmC * SmA WV- Aeo
* m5CeoTeom5CeoAeo * C * C * C * A * C * ACTCACCCACTCGCCACCGC
XOOOXXXXXX 15870 T * C * G * C * C * mA * mC * mC * mG * mC
XXXXXXXXX WV- 1A * m5CeoTeom5Ceo1A * C * C * C * A * C *
ACTCACCCACTCGCCACCGC XOOOXXXXXX 15871 T * C * G * C * C * mA * mC *
mC * mG * mC XXXXXXXXX WV- Aeo * m5CeoTeom5CeoAeom5Ceo * C * C * A
* C * ACTCACCCACTCGCCACCGC XOOOOXXXXX 15872 T * C * G * C * C * mA
* mC * mC * mG * mC XXXXXXXXX WV- m5Ceo * Teom5CeoAeom5Ceo * C * C
* A * C * CTCACCCACTCGCCACCGCC XOOOXXXXXX 15873 T * C * G * C * C *
A * mC * mC * mG * mC * mC XXXXXXXXX WV- m51C * Teom5CeoAeom51C * C
* C * A * C * T * CTCACCCACTCGCCACCGCC XOOOXXXXXX 15874 C * G * C *
C * A * mC * mC * mG * mC * mC XXXXXXXXX WV- Aeo *
m5CeoTeom5CeoAeom5Ceo * C * C * A * C * ACTCACCCACTCGCCACCGC
XOOOOXXXXX 15875 T * C * G * C * C * A * mC * mC * mG * mC
XXXXXXXXX WV- Aeo * m5CeoTeom5CeoAeom5Ceo * C * C * A * C *
ACTCACCCACTCGCCACCGCC XOOOOXXXXX 15876 T * C * G * C * C * A * mC *
mC * mG * mC * mC XXXXXXXXXX WV- mG * AeoTeoGeomC * C * G * C * C *
T * C * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15877 T * C * A * m5Ceo
* Teo * m5Ceo * Aeo * m5Ceo XXXXXXXXX WV- mG * AeoTeoGeomC * C * G
* C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15878 C * T * C *
A * m51C * Teo * m5Ceo * Aeo * XXXXXXXXX m51C WV- mG * AeoTeoGeomC
* C * G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15879 C *
T * C * Aeo * m5Ceo * Teo * m5Ceo * Aeo * XXXXXXXXX m5Ceo WV- fA *
fC * fU * fC * fA * fC * mC * mC * mA * ACUCACCCACUCGCCACCGC
XXXXXXXXXX 15880 mC * mU * mC * mG * mC * fC * fA * fC * fC *
XXXXXXXXX fG * fC WV- fG * fA * fU * fG * fC * fC * mG * mC * mC *
GAUGCCGCCUCCUCACUCAC XXXXXXXXXX 15881 mU * mC * mC * mU * mC * fA *
fC * fU * fC * XXXXXXXXX fA * fC WV- 1A * m5CeoTeom5CeoAeom51C * C
* C * A * C * ACTCACCCACTCGCCACCGCC XOOOOXXXXX 15906 T * C * G * C
* C * A * mC * mC * mG * mC * mC XXXXXXXXXX WV- mG * AeoTeoGeomC *
C * G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15907 C * T
* C * A * m5CeoTeom5CeoAeo * m5Ceo XXXXXOOOX WV- mG * AeoTeoGeomC *
C * G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15908 C * T
* C * Aeom5CeoTeom5CeoAeo * m5Ceo XXXXOOOOX WV- mG * AeoTeoGeomC *
C * G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15909 C * T
* C * A * m51CTeom5CeoAeo * m51C XXXXXOOOX WV- mG * AeoTeoGeomC * C
* G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15910 C * T *
C * 1Am5CeoTeom5CeoAeo * m51C XXXXOOOOX WV- mG * AeoTeoGeomC * C *
G * C * C * T * C * GATGCCGCCTCCTCACTCAC XOOOXXXXXX 15911 C * T * C
* 1A * m5Ceo * Teo * m5Ceo * Aeo * XXXXXXXXX m51C Key to Table 1A:
The present disclosure notes that some sequences, due to their
length, are divided into multiple lines in Table lA (e.g., WV-9421,
WV-9399, WV-9398, WV-9397, WV-9396, etc.); however, these
sequences, as are all oligonucleotides in Table 1A, are
single-stranded (unless otherwise noted). Moieties and
modifications listed in the Tables (or compounds used to construct
oligonucleotides comprising these moieties or modifications:
1: LNA sugar moieties (2'-O--CH.sub.2-4'), e.g., 1A
##STR00162##
if between 5'-end group(s) and internucleotidic linkage, or between
internucleotidic linkages;
##STR00163##
if at 5'-end and without 5'-end groups; or
##STR00164##
if at 3'-end (e.g., in WV-12575)]; and m51C[
##STR00165##
if between 5'-end group(s) and internucleotidic linkages, or
between internucleotidic linkages;
##STR00166##
if at 5'-end and without 5'-end groups (e.g., in WV-12575); or
##STR00167##
if at 3'-end]
m: 2'-OMe
[0913] m5: methyl at 5-position of C (nucleobase is
5-methylcytosine) m5Ceo: 5-methyl 2'-O-methoxyethyl C 5MRd:
5'-methyl group wherein the 5'-C is in the Rp configuration,
2'-deoxy 5MSd: 5'-methyl group wherein the 5'-C is in the Sp
configuration, 2'-deoxy. C
OMe: 2'-OMe
[0914] eo: 2'-MOE (2'-OCH.sub.2CH.sub.2OCH.sub.3);
F, f: 2'-F;
r: 2'-OH;
[0915] O, PO: phoshodiester (phosphate); can be an end group, or a
linkage, e.g., a linkage between linker and oligonucleotide chain,
an internucleotidic linkage, etc. Phosphodiesters indicated in the
Stereochemistry/Internucleotidic Linkages column are not reproduced
in the Modified Sequence column; if no internucleotidic linkage is
indicated in the Modified Sequence column, it is a phosphodiester.
*, PS: Phosphorothioate; this can be an end group, or a linkage,
e.g., a linkage between linker and oligonucleotide chain, an
internucleotidic linkage, etc. R, Rp: Phosphorothioate in Rp
conformation; note that *R indicates a single phosphorothioate in
the Rp conformation S, Sp: Phosphorothioate in Sp conformation;
note that *S indicates a single phosphorothioate in the Sp
conformation n001:
##STR00168##
nX: stereorandom n001 X: Stereorandom phosphorothioate L001:
--NH--(CH.sub.2).sub.6-- linker (also known as a C6 linker, C6
amine linker or C6 amino linker), connected to Mod, if any, through
--NH--, and the 5'-end of the oligonucleotide chain through either
a phosphate linkage (O or PO) or phosphorothioate linkage (* if the
phosphorothioate not chirally controlled; can also be Sp if
chirally controlled and has an Sp configuration, and Rp if chirally
controlled and has an Rp configuration) as illustrated. If no Mod
is present, L001 is connected to --H, e.g., in WV-9380 or WV-9285.
For example, in WV-9381, L001 is connected to Mod007 through --NH--
(forming an amide group --C(O)--NH--), and is connected to the
oligonucleotide chain through a phosphate linkage (indicated by
bold underlined in OSOOOSSSRSSRSSSSSSSS); in WV-9062, L001 is not
connected to any Mod, but to --H, through --NH--, and is connected
to the oligonucleotide chain through a phosphate linkage (indicated
by bold underlined in OSOOOSSSRSSSSSSSSSSS).
Mod007: b
[0916] BrdU: a nucleoside unit wherein the nucleobase is BrU (
##STR00169##
and wherein the sugar is 2-deoxyribose (as widely found in natural
DNA; 2'-deoxy (d)); L004: linker having the structure of
--NH(CH.sub.2).sub.4CH(CH.sub.2OH)CH.sub.2--, wherein --NH-- is
connected to Mod (through --C(O)--) or --H, and the
--CH.sub.2-connecting site is connected to a linkage, e.g.,
phosphodiester (--O--P(O)(OH)--O--. May exist as a salt form. May
be illustrated in the Table as O or PO), phosphorothioate
(--O--P(O)(SH)--O--. May exist as a salt form. May be illustrated
in the Table as * if the phosphorothioate not chirally controlled;
*S, S, or Sp, if chirally controlled and has an Sp configuration,
and *R, R, or Rp, if chirally controlled and has an Rp
configuration), or phosphorodithioate (--P(S)(SH)--O--. May exist
as a salt form. May be illustrated in the Table as PS2 or: or D)
linkage, at the 3'-end of an oligonucleotide chain. For example, an
asterisk immediately preceding a L004 (e.g., *L004) indicates that
the linkage is a phosphorothioate linkage, and the absence of an
asterisk immediately preceding L004 indicates that the linkage is a
phosphodiester linkage. For example, in WV-12549, which terminates
in mAL004, the linker L004 is connected (via the --CH.sub.2-- site)
to the phosphodiester linkage at the 3' position at the 3'-terminal
sugar (which is 2'-OMe and connected to the nucleobase A), and the
L004 linker is connected via --NH-- to --H; similarly, in WV-12552,
WV-12550, and WV-12551, the L004 linker is connected (via the
--CH.sub.2-- site) to the phosphodiester linkage at the 3' position
of the 3'-terminal sugar, and the L004 is connected via --NH-- to
Mod012 (WV-12552), Mod085 (WV-12550) or Mod086 (WV-12551);
##STR00170##
In Mod007, n=8. Mod012 (with --C(O)-- connecting to --NH-- of a
linker such as L001):
##STR00171##
Mod024:
##STR00172##
[0917] Mod027:
##STR00173##
[0918] Mod028:
##STR00174##
[0919] Mod059:
##STR00175##
[0920] Mod085 (with --C(O)-- connecting to --NH-- of a linker such
as L001 or L004):
##STR00176##
Mod086 (with --C(O)-- connecting to --NH-- of L001 or L004):
##STR00177##
[0921] Oligonucleotides
[0922] In some embodiments, provided C9orf72 oligonucleotides can
direct a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product. In some embodiments, a
C9orf72 target gene comprises a hexanucleotide repeat
expansion.
[0923] In some embodiments, a provided C9orf72 oligonucleotide has
a structural element or format or portion thereof described
herein.
[0924] In some embodiments, a provided C9orf72 oligonucleotide
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product has a
structural element or format or portion thereof described
herein.
[0925] In some embodiments, a provided C9orf72 oligonucleotide
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product has the
format of any oligonucleotide disclosed herein, e.g., in Table 1A
or in the Figures, or otherwise disclosed herein, or a structural
element or format or portion thereof.
[0926] In some embodiments, a common pattern of backbone chiral
centers (e.g., a pattern of backbone chiral centers in a C9orf72
oligonucleotide) comprises a pattern of OSOSO, OSSSO, OSSSOS, SOSO,
SOSO, SOSOS, SOSOSO, SOSOSOSO, SOSSSO, SSOSSSOSS, SSSOSOSSS,
SSSSOSOSSSS, SSSSS, SSSSSS, SSSSSSS, SSSSSSSS, SSSSSSSSS, or RRR,
wherein S represents a phosphorothioate in the Sp configuration,
and O represents a phosphodiester. wherein R represents a
phosphorothioate in the Rp configuration.
[0927] In some embodiments, provided C9orf72 oligonucleotides
comprise at least two pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages. In some embodiments,
provided C9orf72 oligonucleotides comprise at least 3 pairs of
alternating phosphodiester and phosphorothioate internucleotidic
linkages. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 4 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages. In some embodiments,
provided C9orf72 oligonucleotides comprise at least 5 pairs of
alternating phosphodiester and phosphorothioate internucleotidic
linkages. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 6 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages. In some embodiments,
provided C9orf72 oligonucleotides comprise at least 7 pairs of
alternating phosphodiester and phosphorothioate internucleotidic
linkages. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 8 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages. In some embodiments,
provided C9orf72 oligonucleotides comprise at least 9 pairs of
alternating phosphodiester and phosphorothioate internucleotidic
linkages. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 10 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages. In some embodiments,
provided C9orf72 oligonucleotides comprise at least two pairs of
alternating phosphodiester and phosphorothioate internucleotidic
linkages; and further comprise a block comprising 5 or more
consecutive phosphorothioate internucleotidic linkages, wherein at
least one phosphorothioate linkage is chirally controlled. In some
embodiments, provided C9orf72 oligonucleotides comprise at least 3
pairs of alternating phosphodiester and phosphorothioate
internucleotidic linkages; and further comprise a block comprising
5 or more consecutive phosphorothioate internucleotidic linkages,
wherein at least one phosphorothioate linkage is chirally
controlled. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 4 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages; and further comprise a
block comprising 5 or more consecutive phosphorothioate
internucleotidic linkages, wherein at least one phosphorothioate
linkage is chirally controlled. In some embodiments, provided
C9orf72 oligonucleotides comprise at least 5 pairs of alternating
phosphodiester and phosphorothioate internucleotidic linkages; and
further comprise a block comprising 5 or more consecutive
phosphorothioate internucleotidic linkages, wherein at least one
phosphorothioate linkage is chirally controlled. In some
embodiments, provided C9orf72 oligonucleotides comprise at least 6
pairs of alternating phosphodiester and phosphorothioate
internucleotidic linkages; and further comprise a block comprising
5 or more consecutive phosphorothioate internucleotidic linkages,
wherein at least one phosphorothioate linkage is chirally
controlled. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 7 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages; and further comprise a
block comprising 5 or more consecutive phosphorothioate
internucleotidic linkages, wherein at least one phosphorothioate
linkage is chirally controlled. In some embodiments, provided
C9orf72 oligonucleotides comprise at least 8 pairs of alternating
phosphodiester and phosphorothioate internucleotidic linkages; and
further comprise a block comprising 5 or more consecutive
phosphorothioate internucleotidic linkages, wherein at least one
phosphorothioate linkage is chirally controlled. In some
embodiments, provided C9orf72 oligonucleotides comprise at least 9
pairs of alternating phosphodiester and phosphorothioate
internucleotidic linkages; and further comprise a block comprising
5 or more consecutive phosphorothioate internucleotidic linkages,
wherein at least one phosphorothioate linkage is chirally
controlled. In some embodiments, provided C9orf72 oligonucleotides
comprise at least 10 pairs of alternating phosphodiester and
phosphorothioate internucleotidic linkages; and further comprise a
block comprising 5 or more consecutive phosphorothioate
internucleotidic linkages, wherein at least one phosphorothioate
linkage is chirally controlled. In some embodiments, provided
C9orf72 oligonucleotides comprise at least two pairs of alternating
phosphodiester and phosphorothioate internucleotidic linkages; and
further comprise a block comprising 3, 4, 5, 6, 7 or more
consecutive phosphorothioate internucleotidic linkages, wherein at
least one phosphorothioate linkage is chirally controlled. In some
embodiments, provided C9orf72 oligonucleotides comprise at least
two pairs of alternating phosphodiester and phosphorothioate
internucleotidic linkages; and further comprise a block comprising
5 or more consecutive phosphodiester internucleotidic linkages,
wherein at least one phosphorothioate linkage is chirally
controlled. In some embodiments, provided C9orf72 oligonucleotides
comprise one or more natural phosphate linkages and one or more
modified internucleotidic linkages. Provided oligonucleotides can
comprise various number of natural phosphate linkages. In some
embodiments, provided C9orf72 oligonucleotides are capable of
directing a decrease in the expression, level and/or activity of a
C9orf72 target gene or its gene product. In some embodiments, a
C9orf72 target gene comprises a repeat expansion. In some
embodiments, 5% or more of the internucleotidic linkages of
provided C9orf72 oligonucleotides are natural phosphate linkages.
In some embodiments, 10% or more of the internucleotidic linkages
of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 15% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 20% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 25% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 30% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 35% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages. In some embodiments, 40% or more of the internucleotidic
linkages of provided C9orf72 oligonucleotides are natural phosphate
linkages
[0928] In some embodiments, provided C9orf72 oligonucleotides can
bind to a transcript, and improve C9orf72 knockdown of the
transcript. In some embodiments, provided C9orf72 oligonucleotides
improve C9orf72 knockdown, with efficiency greater than a
comparable oligonucleotide under one or more suitable
conditions.
[0929] In some embodiments, a provided improved C9orf72 knockdown
is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% more than, or
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
30, 40, 50 or more fold of, that of a comparable oligonucleotide
under one or more suitable conditions.
[0930] In some embodiments, expression or level of a C9orf72 target
gene or its gene product is decreased by at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by
administration of a C9orf72 oligonucleotide. In some embodiments,
expression or level of a C9orf72 target gene or its gene product is
decreased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 60%, 70%, or 80% by knockdown directed by a C9orf72
oligonucleotide. In some embodiments, expression or level of a
C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by RNase H-mediated C9orf72 knockdown directed by a C9orf72
oligonucleotide. In some embodiments, expression or level of a
C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by administration of a C9orf72 oligonucleotide in vitro. In some
embodiments, expression or level of a C9orf72 target gene or its
gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by knockdown directed by
a C9orf72 oligonucleotide in vitro. In some embodiments, expression
or level of a C9orf72 target gene or its gene product is decreased
by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
70%, or 80% by RNase H-mediated C9orf72 knockdown directed by a
C9orf72 oligonucleotide in vitro. In some embodiments, expression
or level of a C9orf72 target gene or its gene product is decreased
by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,
70%, or 80% by administration of a C9orf72 oligonucleotide in a
cell(s) in vitro. In some embodiments, expression or level of a
C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by knockdown directed by a C9orf72 oligonucleotide in a cell(s) in
vitro. In some embodiments, expression or level of a C9orf72 target
gene or its gene product is decreased by at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by RNase
H-mediated C9orf72 knockdown directed by a C9orf72 oligonucleotide
in a cell(s) in vitro. In some embodiments, expression or level of
a C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by administration of a C9orf72 oligonucleotide at a concentration
of 1 uM or less in a cell(s) in vitro. In some embodiments,
expression or level of a C9orf72 target gene or its gene product is
decreased by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 60%, 70%, or 80% by knockdown directed by a C9orf72
oligonucleotide at a concentration of 1 uM or less in a cell(s) in
vitro. In some embodiments, expression or level of a C9orf72 target
gene or its gene product is decreased by at least about 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by RNase
H-mediated C9orf72 knockdown directed by a C9orf72 oligonucleotide
at a concentration of 1 uM or less in a cell(s) in vitro. In some
embodiments, expression or level of a C9orf72 target gene or its
gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by administration of a
C9orf72 oligonucleotide at a concentration of 10 uM or less in a
cell(s) in vitro. In some embodiments, expression or level of a
C9orf72 target gene or its gene product is decreased by at least
about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80%
by knockdown directed by a C9orf72 oligonucleotide at a
concentration of 10 uM or less in a cell(s) in vitro. In some
embodiments, expression or level of a C9orf72 target gene or its
gene product is decreased by at least about 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 60%, 70%, or 80% by RNase H-mediated
C9orf72 knockdown directed by a C9orf72 oligonucleotide at a
concentration of 10 uM or less in a cell(s) in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 10%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 10%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 10%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 20%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 20%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 20%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 30%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 30%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 30%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 40%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 40%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 40%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 50%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 50%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 50%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 75%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 75%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 75%
at a concentration of 10 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 90%
at a concentration of 1 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 90%
at a concentration of 5 nm or less in a cell in vitro. In some
embodiments, a C9orf72 oligonucleotide is capable of mediating a
decrease in the expression, level and/or activity of a repeat
expansion-containing C9orf72 transcript relative to that of a
non-repeat expansion-containing C9orf72 transcript by at least 90%
at a concentration of 10 nm or less in a cell in vitro.
[0931] In some embodiments, IC50 is inhibitory concentration to
decrease expression or level or a C9orf72 target gene or its gene
product by 50% in a cell(s) in vitro. In some embodiments, a
C9orf72 oligonucleotide has an IC50 of no more than about 10 nM in
a cell(s) in vitro. In some embodiments, a C9orf72 oligonucleotide
has an IC50 of no more than about 5 nM in a cell(s) in vitro. In
some embodiments, a C9orf72 oligonucleotide has an IC50 of no more
than about 2 nM in a cell(s) in vitro. In some embodiments, a
C9orf72 oligonucleotide has an IC50 of no more than about 1 nM in a
cell(s) in vitro. In some embodiments, a C9orf72 oligonucleotide
has an IC50 of no more than about 0.5 nM in a cell(s) in vitro. In
some embodiments, a C9orf72 oligonucleotide has an IC50 of no more
than about 0.1 nM in a cell(s) in vitro. In some embodiments, a
C9orf72 oligonucleotide has an IC50 of no more than about 0.01 nM
in a cell(s) in vitro. In some embodiments, a C9orf72
oligonucleotide has an IC50 of no more than about 0.001 nM in a
cell(s) in vitro.
[0932] In some embodiments, a provided C9orf72 oligonucleotide
comprises any pattern of stereochemistry described herein. In some
embodiments, a provided C9orf72 oligonucleotide comprises any
pattern of stereochemistry described herein and is capable of
directing RNase H-mediated C9orf72 knockdown. In some embodiments,
a provided C9orf72 oligonucleotide comprises any pattern of
stereochemistry described herein and is capable of directing RNase
H-mediated C9orf72 knockdown.
[0933] In some embodiments, a provided C9orf72 oligonucleotide
comprises any modification or pattern of modification described
herein. In some embodiments, a provided C9orf72 oligonucleotide
comprises any pattern of modification described herein and is
capable of directing RNase H-mediated C9orf72 knockdown. In some
embodiments, a modification or pattern of modification is a
modification or pattern of modifications at the 2' position of a
sugar. In some embodiments, a modification or pattern of
modification is a modification or pattern of modifications at the
2' position of a sugar, including but not limited to, 2'-deoxy,
2'-F, 2'-OMe, 2'-MOE, and 2'-OR1, wherein R is optionally
substituted C1-6 alkyl.
[0934] In some embodiments, the present disclosure demonstrates
that Sp internucleotidic linkages, among other things, at the 5'-
and 3'-ends can improve oligonucleotide stability. In some
embodiments, the present disclosure demonstrates that, among other
things, natural phosphate linkages and/or Rp internucleotidic
linkages can improve removal of oligonucleotides from a system. As
appreciated by a person having ordinary skill in the art, various
assays known in the art can be utilized to assess such properties
in accordance with the present disclosure.
[0935] In some embodiments, provided C9orf72 oligonucleotides
capable of directing C9orf72 knockdown comprise one or more
modified sugar moieties. In some embodiments, a modified sugar
moiety comprises a 2'-modification. In some embodiments, a modified
sugar moiety comprises a 2'-modification. In some embodiments, a
2'-modification is 2'-OR.sup.1. In some embodiments, a
2'-modification is a 2'-OMe. In some embodiments, a 2'-modification
is a 2'-MOE. In some embodiments, a 2'-modification is an LNA sugar
modification. In some embodiments, a 2'-modification is 2'-F. In
some embodiments, each sugar modification is independently a
2'-modification. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F. In some embodiments, each sugar
modification is independently 2'-OR.sup.1 or 2'-F, wherein R is
optionally substituted C.sub.1-6 alkyl. In some embodiments, each
sugar modification is independently 2'-OR.sup.1 or 2'-F, wherein at
least one is 2'-F. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl, and wherein at least one is 2'-OR. In
some embodiments, each sugar modification is independently
2'-OR.sup.1 or 2'-F, wherein at least one is 2'-F, and at least one
is 2'-OR. In some embodiments, each sugar modification is
independently 2'-OR.sup.1 or 2'-F, wherein R.sup.1 is optionally
substituted C.sub.1-6 alkyl, and wherein at least one is 2'-F, and
at least one is 2'-OR.sup.1.
[0936] In some embodiments, provided C9orf72 oligonucleotides
capable of directing C9orf72 knockdown comprise one or more 2'-F.
In some embodiments, provided C9orf72 oligonucleotides capable of
directing C9orf72 knockdown comprise at least one 2'-OMe. In some
embodiments, provided C9orf72 oligonucleotides capable of directing
C9orf72 knockdown comprise at least two or more consecutive 2'-F.
In some embodiments, provided C9orf72 oligonucleotides capable of
directing C9orf72 knockdown comprise at least two or more
consecutive 2'-OMe.
[0937] In some embodiments, provided C9orf72 oligonucleotides
capable of directing C9orf72 knockdown comprise alternating 2'-F
modified sugar moieties and 2'-OR.sup.1 modified sugar moieties. In
some embodiments, provided C9orf72 oligonucleotides capable of
directing C9orf72 knockdown comprise alternating 2'-F modified
sugar moieties and 2'-OMe modified sugar moieties, e.g.,
[(2'-F)(2'-OMe)]x, [(2'-OMe)(2'-F)]x, etc., wherein x is 1-50. In
some embodiments, provided C9orf72 oligonucleotides capable of
directing C9orf72 knockdown comprise at least two pairs of
alternating 2'-F and 2'-OMe modifications. In some embodiments,
provided C9orf72 oligonucleotides comprises alternating
phosphodiester and phosphorothioate internucleotidic linkages,
e.g., [(PO)(PS)]x, [(PS)(PO)]x, etc., wherein x is 1-50.
[0938] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides, wherein: [0939] oligonucleotides of the first
plurality have the same base sequence; and [0940] oligonucleotides
of the first plurality comprise one or more modified sugar
moieties, or comprise one or more natural phosphate linkages and
one or more modified internucleotidic linkages.
[0941] In some embodiments, provided C9orf72 oligonucleotides
comprise one or more 2'-F. In some embodiments, in provided C9orf72
oligonucleotides, a nucleoside comprising a 2'-modification is
followed by a modified internucleotidic linkage. In some
embodiments, in provided C9orf72 oligonucleotides, a nucleoside
comprising a 2'-modification is preceded by a modified
internucleotidic linkage. In some embodiments, a modified
internucleotidic linkage is a chiral internucleotidic linkage. In
some embodiments, a modified internucleotidic linkage is a
phosphorothioate. In some embodiments, a chiral internucleotidic
linkage is Sp. In some embodiments, in provided C9orf72
oligonucleotides, a nucleoside comprising a 2'-modification is
followed by an Sp chiral internucleotidic linkage. In some
embodiments, in provided C9orf72 oligonucleotides, a nucleoside
comprising a 2'-F is followed by an Sp chiral internucleotidic
linkage. In some embodiments, in provided C9orf72 oligonucleotides,
a nucleoside comprising a 2'-modification is preceded by an Sp
chiral internucleotidic linkage. In some embodiments, in provided
C9orf72 oligonucleotides, a nucleoside comprising a 2'-F is
preceded by an Sp chiral internucleotidic linkage. In some
embodiments, a chiral internucleotidic linkage is Rp. In some
embodiments, in provided C9orf72 oligonucleotides, a nucleoside
comprising a 2'-modification is followed by an Rp chiral
internucleotidic linkage. In some embodiments, in provided C9orf72
oligonucleotides, a nucleoside comprising a 2'-F is followed by an
Rp chiral internucleotidic linkage. In some embodiments, in
provided C9orf72 oligonucleotides, a nucleoside comprising a
2'-modification is preceded by an Rp chiral internucleotidic
linkage. In some embodiments, in provided C9orf72 oligonucleotides,
a nucleoside comprising a 2'-F is preceded by an Rp chiral
internucleotidic linkage. In some embodiments, provided C9orf72
oligonucleotides are capable of directing a decrease in the
expression, level and/or activity of a C9orf72 target gene or its
gene product. In some embodiments, a C9orf72 target gene comprises
a repeat expansion. In some embodiments, C9orf72 oligonucleotides
of the first plurality comprise one or more natural phosphate
linkages and one or more modified internucleotidic linkages.
[0942] In some embodiments, provided compositions alter transcript
C9orf72 knockdown so that an undesired target and/or biological
function are suppressed. In some embodiments, in such cases
provided composition can also induce cleavage of the transcript
after hybridization.
[0943] In some embodiments, compared to a reference condition,
provided chirally controlled C9orf72 oligonucleotide compositions
are surprisingly effective. In some embodiments, desired biological
effects (e.g., as measured by increased levels of desired mRNA,
proteins, etc., decreased levels of undesired mRNA, proteins, etc.)
can be enhanced by more than 5, 10, 15, 20, 25, 30, 40, 50, or 100
fold. In some embodiments, a change is measured by increase of a
desired mRNA level compared to a reference condition.
[0944] In some embodiments, provided C9orf72 oligonucleotides
contain increased levels of one or more isotopes. In some
embodiments, provided C9orf72 oligonucleotides are labeled, e.g.,
by one or more isotopes of one or more elements, e.g., hydrogen,
carbon, nitrogen, etc. In some embodiments, provided C9orf72
oligonucleotides in provided compositions, e.g., C9orf72
oligonucleotides of a first plurality, comprise base modifications,
sugar modifications, and/or internucleotidic linkage modifications,
wherein the oligonucleotides contain an enriched level of
deuterium. In some embodiments, provided C9orf72 oligonucleotides
are labeled with deuterium (replacing -.sup.1H with --.sup.2H) at
one or more positions. In some embodiments, one or more .sup.1H of
a C9orf72 oligonucleotide or any moiety conjugated to the
oligonucleotide (e.g., a targeting moiety, etc.) is substituted
with .sup.2H. Such oligonucleotides can be used in any composition
or method described herein.
[0945] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides which: [0946] 1) have a common base sequence
complementary to a C9orf72 target sequence in a transcript; and
[0947] 2) comprise one or more modified sugar moieties and modified
internucleotidic linkages.
[0948] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides capable of directing C9orf72 knockdown, wherein a
C9orf72 oligonucleotides type is defined by: [0949] 1) base
sequence; [0950] 2) pattern of backbone linkages; [0951] 3) pattern
of backbone chiral centers; and [0952] 4) pattern of backbone
phosphorus modifications, which composition is chirally controlled
in that it is enriched, relative to a substantially racemic
preparation of oligonucleotides having the same base sequence, for
oligonucleotides of the particular oligonucleotide type, [0953] the
oligonucleotide composition being characterized in that, when it is
contacted with the transcript in a C9orf72 knockdown system,
C9orf72 knockdown-mediated C9orf72 knockdown of the transcript is
improved relative to that observed under reference conditions
selected from the group consisting of absence of the composition,
presence of a reference composition, and combinations thereof.
[0954] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides, wherein: oligonucleotides of the first plurality
have the same base sequence; oligonucleotides of the first
plurality comprise structural elements (a) 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleoside units
comprising 2'-F, 2'-OMe, 2'-deoxy and/or 2'-MOE modified sugar
moieties; (b) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20 or more modified internucleotidic linkages, (c) 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or
more chirally controlled modified internucleotidic linkages, and
(d) 2, 3, 4, 5, 6, 7, 8, 9, 10 or more natural phosphate linkages.
In some embodiments, the oligonucleotides of the first plurality
comprise structural elements (a), (b) and (c). In some embodiments,
the oligonucleotides of the first plurality comprise structural
elements (b), (c) and (d). In some embodiments, the
oligonucleotides of the first plurality comprise structural
elements (a), (b) and (d). In some embodiments, the
oligonucleotides of the first plurality comprise structural
elements (a), (c) and (d). In some embodiments, the
oligonucleotides of the first plurality comprise structural
elements (a) and (b). In some embodiments, the oligonucleotides of
the first plurality comprise structural elements (a) and (c). In
some embodiments, the oligonucleotides of the first plurality
comprise structural elements (a) and (d). In some embodiments, the
oligonucleotides of the first plurality comprise structural
elements (b) and (c). In some embodiments, the oligonucleotides of
the first plurality comprise structural elements (b) and (d). In
some embodiments, the oligonucleotides of the first plurality
comprise structural elements (c) and (d).
[0955] In some embodiments, a modified internucleotidic linkage has
a structure of Formula I. In some embodiments, a modified
internucleotidic linkage has a structure of Formula I-a.
[0956] As demonstrated in the present disclosure, in some
embodiments, a provided C9orf72 oligonucleotide composition is
characterized in that, when it is contacted with the transcript in
a C9orf72 knockdown system, C9orf72 knockdown-mediated C9orf72
knockdown of the transcript is improved relative to that observed
under reference conditions selected from the group consisting of
absence of the composition, presence of a reference composition,
and combinations thereof. In some embodiments, C9orf72 knockdown is
increased 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 fold or more. In
some embodiments, as exemplified in the present disclosure, levels
of the plurality of oligonucleotides, e.g., a first plurality of
oligonucleotides, in provided compositions are pre-determined.
[0957] In some embodiments, a common base sequence and length may
be referred to as a common base sequence. In some embodiments,
C9orf72 oligonucleotides having a common base sequence may have the
same pattern of nucleoside modifications, e.g., sugar
modifications, base modifications, etc. In some embodiments, a
pattern of nucleoside modifications may be represented by a
combination of locations and modifications. In some embodiments, a
pattern of backbone linkages comprises locations and types (e.g.,
phosphate, phosphorothioate, substituted phosphorothioate, etc.) of
each internucleotidic linkages. A pattern of backbone chiral
centers of a C9orf72 oligonucleotide can be designated by a
combination of linkage phosphorus stereochemistry (Rp/Sp) from 5'
to 3'. As exemplified above, locations of non-chiral linkages may
be obtained, for example, from pattern of backbone linkages.
[0958] In some embodiments, the present disclosure provides a
C9orf72 oligonucleotide composition comprising a first plurality of
oligonucleotides capable of directing C9orf72 knockdown, wherein
oligonucleotides are of a particular oligonucleotide type
characterized by: [0959] 1) a common base sequence and length;
[0960] 2) a common pattern of backbone linkages; and [0961] 3) a
common pattern of backbone chiral centers; which composition is
chirally controlled in that it is enriched, relative to a
substantially racemic preparation of oligonucleotides having the
same base sequence and length, for oligonucleotides of the
particular oligonucleotide type.
[0962] As understood by a person having ordinary skill in the art,
a stereorandom or racemic preparation of oligonucleotides is
prepared by non-stereoselective and/or low-stereoselective coupling
of nucleotide monomers, typically without using any chiral
auxiliaries, chiral modification reagents, and/or chiral catalysts.
In some embodiments, in a substantially racemic (or chirally
uncontrolled) preparation of oligonucleotides, all or most coupling
steps are not chirally controlled in that the coupling steps are
not specifically conducted to provide enhanced stereoselectivity.
An example substantially racemic preparation of oligonucleotides is
the preparation of phosphorothioate oligonucleotides through
sulfurizing phosphite triesters from commonly used phosphoramidite
oligonucleotide synthesis with either tetraethylthiuram disulfide
or (TETD) or 3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD), a
well-known process in the art. In some embodiments, substantially
racemic preparation of oligonucleotides provides substantially
racemic oligonucleotide compositions (or chirally uncontrolled
oligonucleotide compositions). In some embodiments, at least one
coupling of a nucleotide monomer has a diastereoselectivity lower
than about 60:40, 70:30, 80:20, 85:15, 90:10, 91:9, 92:8, 97:3,
98:2, or 99:1.
[0963] As understood by a person having ordinary skill in the art,
in some embodiments, diastereoselectivity of a coupling or a
linkage can be assessed through the diastereoselectivity of a dimer
formation under the same or comparable conditions, wherein the
dimer has the same 5'- and 3'-nucleosides and internucleotidic
linkage.
[0964] In some embodiments, C9orf72 oligonucleotides having a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers have a
common pattern of backbone phosphorus modifications and a common
pattern of base modifications. In some embodiments, C9orf72
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers have a common pattern of backbone phosphorus
modifications and a common pattern of nucleoside modifications. In
some embodiments, C9orf72 oligonucleotides having a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers have identical
structures.
[0965] In some embodiments, C9orf72 oligonucleotides of a C9orf72
oligonucleotide type have a common pattern of backbone phosphorus
modifications and a common pattern of sugar modifications. In some
embodiments, C9orf72 oligonucleotides of a C9orf72 oligonucleotide
type have a common pattern of backbone phosphorus modifications and
a common pattern of base modifications. In some embodiments,
C9orf72 oligonucleotides of a C9orf72 oligonucleotide type have a
common pattern of backbone phosphorus modifications and a common
pattern of nucleoside modifications. In some embodiments, C9orf72
oligonucleotides of a C9orf72 oligonucleotide type are
identical.
[0966] In some embodiments, a C9orf72 oligonucleotide is a
substantially pure preparation of a C9orf72 oligonucleotide type in
that oligonucleotides in the composition that are not of the
oligonucleotide type are impurities form the preparation process of
said oligonucleotide type, in some case, after certain purification
procedures.
[0967] In some embodiments, at least about 20% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, at least about 25%
of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments, at
least about 30% of the oligonucleotides in the composition have a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers. In some
embodiments, at least about 35% of the oligonucleotides in the
composition have a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers. In some embodiments, at least about 40% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, at least about 45%
of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments, at
least about 50% of the oligonucleotides in the composition have a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers. In some
embodiments, at least about 55% of the oligonucleotides in the
composition have a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers. In some embodiments, at least about 60% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, at least about 65%
of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments, at
least about 70% of the oligonucleotides in the composition have a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers. In some
embodiments, at least about 75% of the oligonucleotides in the
composition have a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers. In some embodiments, at least about 80% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, at least about 85%
of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments, at
least about 90% of the oligonucleotides in the composition have a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers. In some
embodiments, at least about 92% of the oligonucleotides in the
composition have a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers. In some embodiments, at least about 94% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, at least about 95%
of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments, at
least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the
oligonucleotides in the composition have a common base sequence and
length, a common pattern of backbone linkages, and a common pattern
of backbone chiral centers. In some embodiments, greater than about
99% of the oligonucleotides in the composition have a common base
sequence and length, a common pattern of backbone linkages, and a
common pattern of backbone chiral centers. In some embodiments,
purity of a C9orf72 oligonucleotide of a C9orf72 oligonucleotide
can be expressed as the percentage of oligonucleotides in the
composition that have a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers.
[0968] In some embodiments, C9orf72 oligonucleotides having a
common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers have a
common pattern of backbone phosphorus modifications. In some
embodiments, C9orf72 oligonucleotides having a common base sequence
and length, a common pattern of backbone linkages, and a common
pattern of backbone chiral centers have a common pattern of
backbone phosphorus modifications and a common pattern of
nucleoside modifications. In some embodiments, C9orf72
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers have a common pattern of backbone phosphorus
modifications and a common pattern of sugar modifications. In some
embodiments, C9orf72 oligonucleotides having a common base sequence
and length, a common pattern of backbone linkages, and a common
pattern of backbone chiral centers have a common pattern of
backbone phosphorus modifications and a common pattern of base
modifications. In some embodiments, C9orf72 oligonucleotides having
a common base sequence and length, a common pattern of backbone
linkages, and a common pattern of backbone chiral centers have a
common pattern of backbone phosphorus modifications and a common
pattern of nucleoside modifications. In some embodiments, C9orf72
oligonucleotides having a common base sequence and length, a common
pattern of backbone linkages, and a common pattern of backbone
chiral centers are identical.
[0969] As noted above and understood in the art, in some
embodiments, the base sequence of a C9orf72 oligonucleotide may
refer to the identity and/or modification status of nucleoside
residues (e.g., of sugar and/or base components, relative to
standard naturally occurring nucleotides such as adenine, cytosine,
guanosine, thymine, and uracil) in the oligonucleotide and/or to
the hybridization character (i.e., the ability to hybridize with
particular complementary residues) of such residues.
[0970] In some embodiments, purity of a C9orf72 oligonucleotide can
be controlled by stereoselectivity of each coupling step in its
preparation process. In some embodiments, a coupling step has a
stereoselectivity (e.g., diastereoselectivity) of 60% (60% of the
new internucleotidic linkage formed from the coupling step has the
intended stereochemistry).
[0971] In some embodiments, provided compositions comprise
oligonucleotides containing one or more residues which are modified
at the sugar moiety. In some embodiments, provided compositions
comprise oligonucleotides containing one or more residues which are
modified at the 2' position of the sugar moiety (referred to herein
as a "2'-modification"). Examples of such modifications are
described above and herein and include, but are not limited to,
2'-OMe, 2'-MOE, 2'-LNA, 2'-F, FRNA, FANA, S-cEt, etc. In some
embodiments, provided compositions comprise oligonucleotides
containing one or more residues which are 2'-modified. For example,
in some embodiments, provided C9orf72 oligonucleotides contain one
or more residues which are 2'-O-methoxyethyl (2'-MOE)-modified
residues. In some embodiments, provided compositions comprise
oligonucleotides which do not contain any 2'-modifications. In some
embodiments, provided compositions are oligonucleotides which do
not contain any 2'-MOE residues. That is, in some embodiments,
provided C9orf72 oligonucleotides are not MOE-modified. Additional
example sugar modifications are described in the present
disclosure.
[0972] In some embodiments, a sugar moiety without a
2'-modification is a sugar moiety found in a natural DNA
nucleoside.
[0973] A person of ordinary skill in the art understands that
various regions of a C9orf72 target transcript can be targeted by
provided compositions and methods. In some embodiments, a base
sequence of provided C9orf72 oligonucleotides comprises an intron
sequence. In some embodiments, a base sequence of provided C9orf72
oligonucleotides comprises an exon sequence. In some embodiments, a
base sequence of provided C9orf72 oligonucleotides comprises an
intron and an exon sequence.
[0974] As understood by a person having ordinary skill in the art,
provided C9orf72 oligonucleotides and compositions, among other
things, can target a great number of nucleic acid polymers. For
instance, in some embodiments, provided C9orf72 oligonucleotides
and compositions may target a transcript of a nucleic acid
sequence, wherein a common base sequence of oligonucleotides (e.g.,
a base sequence of a C9orf72 oligonucleotide type) comprises or is
a sequence complementary to a sequence of the transcript.
[0975] In some embodiments, as described in this disclosure,
provided C9orf72 oligonucleotides and compositions may provide new
cleavage patterns, higher cleavage rate, higher cleavage degree,
higher cleavage selectivity, etc. In some embodiments, provided
compositions can selectively suppress (e.g., cleave) a transcript
from a C9orf72 target nucleic acid sequence which has one or more
similar sequences exist within a subject or a population, each of
the target and its similar sequences contains a specific
nucleotidic characteristic sequence element that defines the target
sequence relative to the similar sequences.
[0976] In some embodiments, a similar sequence has greater than
60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99% sequence identity with a C9orf72 target sequence.
In some embodiments, a C9orf72 target sequence is a disease-causing
copy of a nucleic acid sequence comprising one or more mutations,
and a similar sequence is a copy not causing the disease (wild
type). In some embodiments, a C9orf72 target sequence comprises a
mutation, wherein a similar sequence is the corresponding wild-type
sequence. In some embodiments, a C9orf72 target sequence is a
mutant allele, while a similar sequence is a wild-type allele. In
some embodiments, a C9orf72 target sequence is in an intron
comprising a hexanucleotide repeat expansion. In some embodiments,
the region of a C9orf72 target sequence that is complementary to a
common base sequence of a provided C9orf72 oligonucleotide
composition differs from the corresponding region of a similar
sequence at less than 5, less than 4, less than 3, less than 2, or
only 1 base pairs.
[0977] In some embodiments, a common base sequence comprises or is
a sequence complementary to a characteristic sequence element. In
some embodiments, a common base sequence comprises a sequence
complementary to a characteristic sequence element. In some
embodiments, a common base sequence is a sequence complementary to
a characteristic sequence element. In some embodiments, a common
base sequence comprises or is a sequence 100% complementary to a
characteristic sequence element. In some embodiments, a common base
sequence comprises a sequence 100% complementary to a
characteristic sequence element. In some embodiments, a common base
sequence is a sequence 100% complementary to a characteristic
sequence element.
[0978] Among other things, the present disclosure recognizes that a
base sequence may have impact on oligonucleotide properties. In
some embodiments, a base sequence may have impact on cleavage
pattern of a C9orf72 target when oligonucleotides having the base
sequence are utilized for suppressing a C9orf72 target, e.g.,
through a pathway involving RNase H: for example, structurally
similar (all phosphorothioate linkages, all stereorandom)
oligonucleotides have different sequences may have different
cleavage patterns.
[0979] As a person having ordinary skill in the art understands,
provided C9orf72 oligonucleotide compositions and methods have
various uses as known by a person having ordinary skill in the art.
Methods for assessing provided compositions, and properties and
uses thereof, are also widely known and practiced by a person
having ordinary skill in the art. Example properties, uses, and/or
methods include but are not limited to those described in
WO/2014/012081 and WO/2015/107425.
[0980] In some embodiments, a chiral internucleotidic linkage has
the structure of Formula I. In some embodiments, a chiral
internucleotidic linkage is phosphorothioate. In some embodiments,
each chiral internucleotidic linkage in a single oligonucleotide of
a provided composition independently has the structure of Formula
I. In some embodiments, each chiral internucleotidic linkage in a
single oligonucleotide of a provided composition is a
phosphorothioate.
[0981] In some embodiments, C9orf72 oligonucleotides of the present
disclosure comprise one or more modified sugar moieties. In some
embodiments, C9orf72 oligonucleotides of the present disclosure
comprise one or more modified base moieties. As known by a person
of ordinary skill in the art and described in the disclosure,
various modifications can be introduced to a sugar and/or moiety.
For example, in some embodiments, a modification is a modification
described in U.S. Pat. No. 9,006,198, WO2014/012081 and
WO/2015/107425, the sugar and base modifications of each of which
are incorporated herein by reference.
[0982] In some embodiments, a sugar modification is a
2'-modification. Commonly used 2'-modifications include but are not
limited to 2'-OR.sup.1, wherein R.sup.1 is not hydrogen. In some
embodiments, a modification is 2'-OR, wherein R is optionally
substituted aliphatic. In some embodiments, a modification is
2'-OMe. In some embodiments, a modification is 2'-O-MOE. In some
embodiments, the present disclosure demonstrates that inclusion
and/or location of particular chirally pure internucleotidic
linkages can provide stability improvements comparable to or better
than those achieved through use of modified backbone linkages,
bases, and/or sugars. In some embodiments, a provided single
oligonucleotide of a provided composition has no modifications on
the sugars. In some embodiments, a provided single oligonucleotide
of a provided composition has no modifications on 2'-positions of
the sugars (i.e., the two groups at the 2'-position are either
--H/--H or -H/--OH). In some embodiments, a provided single
oligonucleotide of a provided composition does not have any 2'-MOE
modifications.
[0983] In some embodiments, a 2'-modification is --O-L- or -L-
which connects the 2'-carbon of a sugar moiety to another carbon of
a sugar moiety. In some embodiments, a 2'-modification is --O-L- or
-L- which connects the 2'-carbon of a sugar moiety to the 4'-carbon
of a sugar moiety. In some embodiments, a 2'-modification is S-cEt.
In some embodiments, a modified sugar moiety is an LNA moiety.
[0984] In some embodiments, a locked nucleic acid or LNA or LNA
nucleoside or LNA nucleotide is or comprises a nucleic acid monomer
having a bridge connecting two carbon atoms between the 4' and 2'
position of the nucleoside sugar unit, thereby forming a bicyclic
sugar. Examples of such a bicyclic sugar include but are not
limited to alpha-L-Methyleneoxy (4'-CH.sub.2--O-2') LNA,
beta-D-Methyleneoxy (4'-CH.sub.2--O-2') LNA, Ethyleneoxy
(4'-(CH.sub.2).sub.2--O-2') LNA, Aminooxy (4'-CH.sub.2--O--N(R)-2')
LNA, and Oxyamino (4'-CH.sub.2--N(R)--O-2') LNA. In some
embodiments, R is R.sub.1 or R.sub.2.
[0985] In some embodiments, LNA compounds include, but are not
limited to, compounds having at least one bridge between the 4' and
the 2' position of the sugar wherein each of the bridges
independently comprises 1 or from 2 to 4 linked groups
independently selected from --[C(R.sub.1)(R.sub.2)].sub.n--,
--C(R.sub.1).dbd.C(R.sub.2)--, --C(R.sub.1).dbd.N--,
--C(.dbd.NR)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R).sub.2--, --S(.dbd.O)-- and --N(R.sub.1)--; wherein: x is 0,
1, or 2; n is 1, 2, 3, or 4; each R.sub.1 and R.sub.2 is,
independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.20 aryl, a heterocycle radical, a
substituted heterocycle radical, heteroaryl, substituted
heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted
C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H),
substituted acyl, a heterocycle radical, a substituted heterocycle
radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12
aminoalkyl or a protecting group. Non-limiting examples of 4'-2'
bridging groups encompassed within the definition of LNA include,
but are not limited to one of formulae:
--[C(R.sub.1)(R.sub.2)].sub.n--,
--[C(R.sub.1)(R.sub.2)].sub.n--O--,
--C(R.sub.1R.sub.2)--N(R.sub.1)--O-- or
C(R.sub.1R.sub.2)--O--N(R.sub.1)--. Furthermore, other bridging
groups encompassed with the definition of LNA are 4'-CH.sub.2-2',
4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2',
4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R.sub.1)-2' and
4'-CH.sub.2--N(R.sub.1)--O-2'-bridges, wherein each R.sub.1 and
R.sub.2 is, independently, H, a protecting group or
C.sub.1-C.sub.12 alkyl. Also included within the definition of LNA
are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring
is connected to the 4' carbon atom of the sugar ring, thereby
forming a methyleneoxy (4'-CH.sub.2--O-2') bridge to form the
bicyclic sugar moiety. The bridge can also be a methylene
(--CH.sub.2--) group connecting the 2' oxygen atom and the 4'
carbon atom, for which the term methyleneoxy (4'-CH.sub.2--O-2')
LNA is used. In some embodiments, in the case of the bicylic sugar
moiety having an ethylene bridging group in this position, the term
ethyleneoxy (4'-CH.sub.2CH.sub.2--O-2') LNA is used.
alpha-L-methyleneoxy (4'-CH.sub.2--O-2'), an isomer of methyleneoxy
(4'-CH.sub.2--O-2') LNA, is also encompassed within the definition
of LNA, as used herein.
[0986] In some embodiments, a 2'-modification is --F. In some
embodiments, a 2'-modification is FANA. In some embodiments, a
2'-modification is FRNA.
[0987] In some embodiments, a sugar modification is a
5'-modification, e.g., R-5'-Me, S-5'-Me, etc.
[0988] In some embodiments, a sugar modification changes the size
of the sugar ring. In some embodiments, a sugar modification is the
sugar moiety in FHNA.
[0989] In some embodiments, a sugar modification replaces a sugar
moiety with another cyclic or acyclic moiety. Examples of such
moieties are widely known in the art, including but not limited to
those used in morpholino (optionally with its phosphorodiamidate
linkage), glycol nucleic acids, etc.
[0990] In some embodiments, a C9orf72 oligonucleotide is selected
from the group consisting of the C9orf72 oligonucleotides disclosed
herein, and any C9orf72 oligonucleotide of any format described
herein. Those skilled in the art, reading the present
specification, will appreciate that the present disclosure
specifically does not exclude the possibility that any
oligonucleotide described herein which is labeled as a C9orf72
oligonucleotide may also or alternatively operate through another
mechanism (e.g., as an antisense oligonucleotide; mediating
knock-down via a RNase H mechanism; sterically hindering
translation; or any other biochemical mechanism).
[0991] In some embodiments, an antisense oligonucleotide (ASO) is
or comprises a C9orf72 oligonucleotide selected from the group
consisting of any C9orf72 oligonucleotide disclosed herein, and any
oligonucleotide of any format described herein. Those skilled in
the art, reading the present specification, will appreciate that
the present disclosure specifically does not exclude the
possibility that any oligonucleotide described herein which is
labeled as an antisense oligonucleotide (ASO) may also or
alternatively operate through another mechanism (e.g., as a C9orf72
knockdown utilizing RISC); the disclosure also notes that various
oligonucleotides may operate via different mechanisms (utilizing
RNase H, sterically blocking translation or other
post-transcriptional processes, changing the conformation of a
C9orf72 target nucleic acid, etc.).
Chirally Controlled Oligonucleotides and Chirally Controlled
Oligonucleotide Compositions
[0992] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, a C9orf72 target gene comprises a repeat expansion. In
some embodiments, a C9orf72 target gene comprises a hexanucleotide
repeat expansion.
[0993] The present disclosure provides chirally controlled C9orf72
oligonucleotides, and chirally controlled C9orf72 oligonucleotide
compositions which are of high crude purity and of high
diastereomeric purity. In some embodiments, the present disclosure
provides chirally controlled C9orf72 oligonucleotides, and chirally
controlled C9orf72 oligonucleotide compositions which are of high
crude purity. In some embodiments, the present disclosure provides
chirally controlled C9orf72 oligonucleotides, and chirally
controlled C9orf72 oligonucleotide compositions which are of high
diastereomeric purity.
[0994] In some embodiments, a C9orf72 oligonucleotide is a
substantially pure preparation of a C9orf72 oligonucleotide type in
that oligonucleotides in the composition that are not of the
oligonucleotide type are impurities form the preparation process of
said oligonucleotide type, in some case, after certain purification
procedures.
[0995] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different stereochemistry and/or different
P-modifications relative to one another. In certain embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide, wherein at least two individual internucleotidic
linkages within the oligonucleotide have different P-modifications
relative to one another. In certain embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide,
wherein at least two of the individual internucleotidic linkages
within the oligonucleotide have different P-modifications relative
to one another, and wherein the chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage. In certain embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide,
wherein at least two of the individual internucleotidic linkages
within the oligonucleotide have different P-modifications relative
to one another, and wherein the chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least one phosphorothioate diester
internucleotidic linkage. In certain embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide,
wherein at least two of the individual internucleotidic linkages
within the oligonucleotide have different P-modifications relative
to one another, and wherein the chirally controlled C9orf72
oligonucleotide comprises at least one phosphorothioate triester
internucleotidic linkage. In certain embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide,
wherein at least two of the individual internucleotidic linkages
within the oligonucleotide have different P-modifications relative
to one another, and wherein the chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least one phosphorothioate triester
internucleotidic linkage.
[0996] Internucleotidic Linkages
[0997] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, a C9orf72 target gene comprises a repeat expansion. In
some embodiments, provided C9orf72 oligonucleotides comprise any
internucleotidic linkage described herein or known in the art.
[0998] In some embodiments, a C9orf72 oligonucleotide can comprise
any internucleotidic linkage described herein or known in the
art.
[0999] A non-limiting example of an internucleotidic linkage or
unmodified internucleotidic linkage is a phosphodiester;
non-limiting examples of modified internucleotidic linkages include
those in which one or more oxygen of a phosphodiester has been
replaced by, as non-limiting examples, sulfur (as in a
phosphorothioate), H, alkyl, or another moiety or element which is
not oxygen. A non-limiting example of an internucleotidic linkage
is a moiety which does not a comprise a phosphorus but serves to
link two sugars. A non-limiting example of an internucleotidic
linkage is a moiety which does not a comprise a phosphorus but
serves to link two sugars in the backbone of a C9orf72
oligonucleotide. Disclosed herein are additional non-limiting
examples of nucleotides, modified nucleotides, nucleotide analogs,
internucleotidic linkages, modified internucleotidic linkages,
bases, modified bases, and base analogs, sugars, modified sugars,
and sugar analogs, and nucleosides, modified nucleosides, and
nucleoside analogs.
[1000] In certain embodiments, a internucleotidic linkage has the
structure of Formula I
##STR00178##
wherein each variable is as defined and described below. In some
embodiments, a linkage of Formula I is chiral. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide comprising one or more modified internucleotidic
linkages of Formula I. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising
one or more modified internucleotidic linkages of Formula I, and
wherein individual internucleotidic linkages of Formula I within
the oligonucleotide have different P-modifications relative to one
another. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising one or more
modified internucleotidic linkages of Formula I, and wherein
individual internucleotidic linkages of Formula I within the
oligonucleotide have different --X-L-R.sup.1 relative to one
another. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising one or more
modified internucleotidic linkages of Formula I, and wherein
individual internucleotidic linkages of Formula I within the
oligonucleotide have different X relative to one another. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising one or more modified
internucleotidic linkages of Formula I, and wherein individual
internucleotidic linkages of Formula I within the oligonucleotide
have different -L-R.sup.1 relative to one another.
[1001] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different stereochemistry and/or different
P-modifications relative to one another. In some embodiments, the
present disclosure provides a chirally controlled C9orf72
oligonucleotide, wherein at least two of the individual
internucleotidic linkages within the oligonucleotide have different
stereochemistry relative to one another, and wherein at least a
portion of the structure of the chirally controlled C9orf72
oligonucleotide is characterized by a repeating pattern of
alternating stereochemistry.
[1002] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide, wherein at least two
of the individual internucleotidic linkages within the
oligonucleotide have different P-modifications relative to one
another, in that they have different X atoms in their --XLR.sup.1
moieties, and/or in that they have different L groups in their
--XLR.sup.1 moieties, and/or that they have different R.sup.1 atoms
in their --XLR.sup.1 moieties, wherein XLR.sup.1 is equivalent to
X-L-R.sup.1 and X, L, and R.sup.1 are as defined in Formula I,
disclosed herein.
[1003] In some embodiments, L is a covalent bond or an optionally
substituted, linear or branched C.sub.1-C.sub.10 alkylene, wherein
one or more methylene units of L are optionally and independently
replaced by an optionally substituted C.sub.1-C.sub.6 alkylene,
C.sub.1-C.sub.6 alkenylene, --C.ident.C--, --C(R').sub.2--, --Cy-,
--O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R')--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--N(R')S(O).sub.2--, --SC(O)--, --C(O)S--, --OC(O)--, or
--C(O)O--;
R.sup.1 is halogen, R, or an optionally substituted
C.sub.1-C.sub.50 aliphatic wherein one or more methylene units are
optionally and independently replaced by an optionally substituted
C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6 alkenylene,
--C.ident.C--, --C(R').sub.2--, --Cy-, --O--, --S--, --S--S--,
--N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--SC(O)--, --C(O)S--, --OC(O)--, or --C(O)O--; each R' is
independently --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R, or: two R'
on the same nitrogen are taken together with their intervening
atoms to form an optionally substituted heterocyclic or heteroaryl
ring, or two R' on the same carbon are taken together with their
intervening atoms to form an optionally substituted aryl,
carbocyclic, heterocyclic, or heteroaryl ring; --Cy- is an
optionally substituted bivalent ring selected from phenylene,
carbocyclylene, arylene, heteroarylene, and heterocyclylene; each R
is independently hydrogen, or an optionally substituted group
selected from C.sub.1-C.sub.6 aliphatic, phenyl, carbocyclyl, aryl,
heteroaryl, and heterocyclyl; and each
##STR00179##
independently represents a connection to a nucleoside.
[1004] In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises one or more modified internucleotidic
phosphorus linkages. Examples of such modified internucleotidic
phosphorus linkages are described further herein.
[1005] In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises different internucleotidic phosphorus
linkages. In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least one modified internucleotidic
linkage. Examples of such modified internucleotidic phosphorus
linkages are described further herein.
[1006] In some embodiments, a phosphorothioate triester linkage
comprises a chiral auxiliary, which, for example, is used to
control the stereoselectivity of a reaction. In some embodiments, a
phosphorothioate triester linkage does not comprise a chiral
auxiliary. In some embodiments, a phosphorothioate triester linkage
is intentionally maintained until and/or during the administration
to a subject.
[1007] In some embodiments, a chirally controlled C9orf72
oligonucleotide is linked to a solid support. In some embodiments,
a chirally controlled C9orf72 oligonucleotide is cleaved from a
solid support.
[1008] In some embodiments, a chirally controlled C9orf72
oligonucleotide comprises at least one phosphate diester
internucleotidic linkage and at least two consecutive modified
internucleotidic linkages. In some embodiments, a chirally
controlled C9orf72 oligonucleotide comprises at least one phosphate
diester internucleotidic linkage and at least two consecutive
phosphorothioate triester internucleotidic linkages.
[1009] In some embodiments, the present disclosure provides
compositions comprising or consisting of a plurality of provided
C9orf72 oligonucleotides (e.g., chirally controlled C9orf72
oligonucleotide compositions). In some embodiments, all such
provided C9orf72 oligonucleotides are of the same type, i.e., all
have the same base sequence, pattern of backbone linkages (i.e.,
pattern of internucleotidic linkage types, for example, phosphate,
phosphorothioate, etc), pattern of backbone chiral centers (i.e.
pattern of linkage phosphorus stereochemistry (Rp/Sp)), and pattern
of backbone phosphorus modifications (e.g., pattern of "-XLR.sup.1"
groups in Formula I, disclosed herein). In some embodiments, all
oligonucleotides of the same type are identical. In many
embodiments, however, provided compositions comprise a plurality of
oligonucleotides types, typically in pre-determined relative
amounts.
[1010] In some embodiments, a C9orf72 oligonucleotide can comprise
any internucleotidic linkage described herein or known in the art.
In some embodiments, a C9orf72 oligonucleotide can comprise any
internucleotidic linkage described herein or known in the art in
combination with any other structural element or modification
described herein, including but not limited to, base sequence or
portion thereof, sugar, base (nucleobase); stereochemistry or
pattern thereof, additional chemical moiety, including but not
limited to, a targeting moiety, a carbohydrate moiety, etc.;
additional chemical moiety, including but not limited to, a
targeting moiety, etc.; format or any structural element thereof,
and/or any other structural element or modification described
herein; and in some embodiments, the present disclosure pertains to
multimers of any such oligonucleotides.
[1011] In some embodiments, the present disclosure provides C9orf72
oligonucleotides comprising one or more modified internucleotidic
linkages independently having the structure of Formula I, disclosed
herein.
[1012] In some embodiments of Formula I, P in T.sup.LD is P*. In
some embodiments, P* is an asymmetric phosphorus atom and is either
Rp or Sp. In some embodiments, P* is Rp. In other embodiments, P*
is Sp. In some embodiments, a C9orf72 oligonucleotide comprises one
or more internucleotidic linkages of Formula I wherein each P* is
independently Rp or Sp. In some embodiments, a C9orf72
oligonucleotide comprises one or more internucleotidic linkages of
Formula I wherein each P* is Rp. In some embodiments, a C9orf72
oligonucleotide comprises one or more internucleotidic linkages of
Formula I wherein each P* is Sp. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein P* is Rp. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein P* is Sp. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein P* is Rp, and at least one internucleotidic
linkage of Formula I wherein P* is Sp.
[1013] In some embodiments of Formula I, W is O, S, or Se. In some
embodiments, W is O. In some embodiments, W is S. In some
embodiments, W is Se. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein W is O. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein W is S. In some embodiments, a C9orf72
oligonucleotide comprises at least one internucleotidic linkage of
Formula I wherein W is Se.
[1014] In some embodiments of Formula I, a C9orf72 oligonucleotide
comprises at least one internucleotidic linkage of Formula I
wherein W is O. In some embodiments, a C9orf72 oligonucleotide
comprises at least one internucleotidic linkage of Formula I
wherein W is S.
[1015] In some embodiments, each R is independently hydrogen, or an
optionally substituted group selected from C.sub.1-C.sub.6
aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, and
heterocyclyl.
[1016] In some embodiments, R is hydrogen. In some embodiments, R
is an optionally substituted group selected from C.sub.1-C.sub.6
aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, and
heterocyclyl.
[1017] In some embodiments, R is an optionally substituted
C.sub.1-C.sub.6 aliphatic. In some embodiments, R is an optionally
substituted C.sub.1-C.sub.6 alkyl. In some embodiments, R is
optionally substituted, linear or branched hexyl. In some
embodiments, R is optionally substituted, linear or branched
pentyl. In some embodiments, R is optionally substituted, linear or
branched butyl. In some embodiments, R is optionally substituted,
linear or branched propyl. In some embodiments, R is optionally
substituted ethyl. In some embodiments, R is optionally substituted
methyl.
[1018] In some embodiments, R is optionally substituted phenyl. In
some embodiments, R is substituted phenyl. In some embodiments, R
is phenyl.
[1019] In some embodiments, R is optionally substituted
carbocyclyl. In some embodiments, R is optionally substituted
C.sub.3-C.sub.10 carbocyclyl. In some embodiments, R is optionally
substituted monocyclic carbocyclyl. In some embodiments, R is
optionally substituted cycloheptyl. In some embodiments, R is
optionally substituted cyclohexyl. In some embodiments, R is
optionally substituted cyclopentyl. In some embodiments, R is
optionally substituted cyclobutyl. In some embodiments, R is an
optionally substituted cyclopropyl. In some embodiments, R is
optionally substituted bicyclic carbocyclyl.
[1020] In some embodiments, R is an optionally substituted aryl. In
some embodiments, R is an optionally substituted bicyclic aryl
ring.
[1021] In some embodiments, R is an optionally substituted
heteroaryl. In some embodiments, R is an optionally substituted 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, sulfur, and oxygen. In some
embodiments, R is a substituted 5-6 membered monocyclic heteroaryl
ring having 1-3 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an unsubstituted 5-6
membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, sulfur, and oxygen.
[1022] In some embodiments, R is an optionally substituted
5-membered monocyclic heteroaryl ring having 1-3 heteroatoms
independently selected from nitrogen, sulfur, and oxygen. In some
embodiments, R is an optionally substituted 6 membered monocyclic
heteroaryl ring having 1-3 heteroatoms independently selected from
nitrogen, oxygen, and sulfur.
[1023] In some embodiments, R is an optionally substituted
5-membered monocyclic heteroaryl ring having 1 heteroatom selected
from nitrogen, oxygen, and sulfur. In some embodiments, R is
selected from pyrrolyl, furanyl, and thienyl.
[1024] In some embodiments, R is an optionally substituted
5-membered heteroaryl ring having 2 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In certain embodiments,
R is an optionally substituted 5-membered heteroaryl ring having 1
nitrogen atom, and an additional heteroatom selected from sulfur
and oxygen. Example R groups include optionally substituted
pyrazolyl, imidazolyl, thiazolyl, isothiazolyl, oxazolyl or
isoxazolyl.
[1025] In some embodiments, R is a 6-membered heteroaryl ring
having 1-3 nitrogen atoms. In other embodiments, R is an optionally
substituted 6-membered heteroaryl ring having 1-2 nitrogen atoms.
In some embodiments, R is an optionally substituted 6-membered
heteroaryl ring having 2 nitrogen atoms. In certain embodiments, R
is an optionally substituted 6-membered heteroaryl ring having 1
nitrogen. Example R groups include optionally substituted
pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, or
tetrazinyl.
[1026] In certain embodiments, R is an optionally substituted 8-10
membered bicyclic heteroaryl ring having 1-4 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In some
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having 1-4 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In other embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In
certain embodiments, R is an optionally substituted 5,6-fused
heteroaryl ring having 1 heteroatom independently selected from
nitrogen, oxygen, and sulfur. In some embodiments, R is an
optionally substituted indolyl. In some embodiments, R is an
optionally substituted azabicyclo[3.2.1]octanyl. In certain
embodiments, R is an optionally substituted 5,6-fused heteroaryl
ring having 2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted azaindolyl. In some embodiments, R is an optionally
substituted benzimidazolyl. In some embodiments, R is an optionally
substituted benzothiazolyl. In some embodiments, R is an optionally
substituted benzoxazolyl. In some embodiments, R is an optionally
substituted indazolyl. In certain embodiments, R is an optionally
substituted 5,6-fused heteroaryl ring having 3 heteroatoms
independently selected from nitrogen, oxygen, and sulfur.
[1027] In certain embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having 1-4 heteroatoms independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted 6,6-fused heteroaryl ring having 1-2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In other embodiments, R is an optionally substituted
6,6-fused heteroaryl ring having 1 heteroatom independently
selected from nitrogen, oxygen, and sulfur. In some embodiments, R
is an optionally substituted quinolinyl. In some embodiments, R is
an optionally substituted isoquinolinyl. According to one aspect, R
is an optionally substituted 6,6-fused heteroaryl ring having 2
heteroatoms independently selected from nitrogen, oxygen, and
sulfur. In some embodiments, R is a quinazoline or a
quinoxaline.
[1028] In some embodiments, R is an optionally substituted
heterocyclyl. In some embodiments, R is an optionally substituted
3-7 membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is a substituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an unsubstituted 3-7
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur.
[1029] In some embodiments, R is an optionally substituted
heterocyclyl. In some embodiments, R is an optionally substituted 6
membered saturated or partially unsaturated heterocyclic ring
having 1-2 heteroatoms independently selected from nitrogen,
oxygen, and sulfur. In some embodiments, R is an optionally
substituted 6 membered partially unsaturated heterocyclic ring
having 2 heteroatoms independently selected from nitrogen, oxygen,
and sulfur. In some embodiments, R is an optionally substituted 6
membered partially unsaturated heterocyclic ring having 2 oxygen
atom.
[1030] In certain embodiments, R is a 3-7 membered saturated or
partially unsaturated heterocyclic ring having 1-2 heteroatoms
independently selected from nitrogen, oxygen, and sulfur. In
certain embodiments, R is oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, oxepaneyl, aziridineyl, azetidineyl,
pyrrolidinyl, piperidinyl, azepanyl, thiiranyl, thietanyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, thiepanyl, dioxolanyl,
oxathiolanyl, oxazolidinyl, imidazolidinyl, thiazolidinyl,
dithiolanyl, dioxanyl, morpholinyl, oxathianyl, piperazinyl,
thiomorpholinyl, dithianyl, dioxepanyl, oxazepanyl, oxathiepanyl,
dithiepanyl, diazepanyl, dihydrofuranonyl, tetrahydropyranonyl,
oxepanonyl, pyrolidinonyl, piperidinonyl, azepanonyl,
dihydrothiophenonyl, tetrahydrothiopyranonyl, thiepanonyl,
oxazolidinonyl, oxazinanonyl, oxazepanonyl, dioxolanonyl,
dioxanonyl, dioxepanonyl, oxathiolinonyl, oxathianonyl,
oxathiepanonyl, thiazolidinonyl, thiazinanonyl, thiazepanonyl,
imidazolidinonyl, tetrahydropyrimidinonyl, diazepanonyl,
imidazolidinedionyl, oxazolidinedionyl, thiazolidinedionyl,
dioxolanedionyl, oxathiolanedionyl, piperazinedionyl,
morpholinedionyl, thiomorpholinedionyl, tetrahydropyranyl,
tetrahydrofuranyl, morpholinyl, thiomorpholinyl, piperidinyl,
piperazinyl, pyrrolidinyl, tetrahydrothiophenyl, or
tetrahydrothiopyranyl. In some embodiments, R is an optionally
substituted 5-membered saturated or partially unsaturated
heterocyclic ring having 1-2 heteroatoms independently selected
from nitrogen, oxygen, and sulfur.
[1031] In some embodiments, a structure of Formula I is a structure
of Formula I as described in WO2017/210647. In some embodiments,
the internucleotidic linkage of Formula I has the structure of
Formula I-a:
##STR00180##
wherein each variable is independently described in the present
disclosure, as in Formula I.
[1032] In some embodiments, the internucleotidic linkage of Formula
I has the structure of Formula I-b:
##STR00181##
wherein each variable is independently described in the present
disclosure, as in Formula I.
[1033] In some embodiments, the internucleotidic linkage of Formula
I is an phosphorothioate triester linkage having the structure of
Formula I-c:
##STR00182##
wherein: P* is an asymmetric phosphorus atom and is either Rp or
Sp; L is a covalent bond or an optionally substituted, linear or
branched C.sub.1-C.sub.10 alkylene, wherein one or more methylene
units of L are optionally and independently replaced by an
optionally substituted C.sub.1-C.sub.6 alkylene, C.sub.1-C.sub.6
alkenylene, --C.ident.C--, --C(R').sub.2--, --Cy-, --O--, --S--,
--S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--, --C(O)N(R')--,
--N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--, --OC(O)N(R')--,
--S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--, --N(R')S(O).sub.2--,
--SC(O)--, --C(O)S--, --OC(O)--, or --C(O)O--; R.sup.1 is halogen,
R, or an optionally substituted C.sub.1-C.sub.50 aliphatic wherein
one or more methylene units are optionally and independently
replaced by an optionally substituted C.sub.1-C.sub.6 alkylene,
C.sub.1-C.sub.6 alkenylene, --C.ident.C--, --C(R').sub.2--, --Cy-,
--O--, --S--, --S--S--, --N(R')--, --C(O)--, --C(S)--, --C(NR')--,
--C(O)N(R')--, --N(R')C(O)N(R')--, --N(R')C(O)--, --N(R')C(O)O--,
--OC(O)N(R')--, --S(O)--, --S(O).sub.2--, --S(O).sub.2N(R')--,
--N(R')S(O).sub.2--, --SC(O)--, --C(O)S--, --OC(O)--, or --C(O)O--;
each R' is independently --R, --C(O)R, --CO.sub.2R, or --SO.sub.2R,
or: two R' on the same nitrogen are taken together with their
intervening atoms to form an optionally substituted heterocyclic or
heteroaryl ring, or two R' on the same carbon are taken together
with their intervening atoms to form an optionally substituted
aryl, carbocyclic, heterocyclic, or heteroaryl ring; --Cy- is an
optionally substituted bivalent ring selected from phenylene,
carbocyclylene, arylene, heteroarylene, and heterocyclylene; each R
is independently hydrogen, or an optionally substituted group
selected from C.sub.1-C.sub.6 aliphatic, phenyl, carbocyclyl, aryl,
heteroaryl, and heterocyclyl; each
##STR00183##
independently represents a connection to a nucleoside; and R.sup.1
is not --H when L is a covalent bond.
[1034] In some embodiments, the internucleotidic linkage having the
structure of Formula I is
##STR00184##
or an internucleotidic linkage as shown in the art, e.g.,
WO2017/210647.
[1035] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising one or more
phosphate diester linkages, and one or more modified
internucleotide linkages having the formula of I-a, I-b, or
I-c.
[1036] In some embodiments, a modified internucleotidic linkage has
the structure of I. In some embodiments, a modified
internucleotidic linkage has the structure of I-a. In some
embodiments, a modified internucleotidic linkage has the structure
of I-b. In some embodiments, a modified internucleotidic linkage
has the structure of I-c.
[1037] In some embodiments, a modified internucleotidic linkage is
phosphorothioate. Examples of internucleotidic linkages having the
structure of Formula I are widely known in the art, including but
not limited to those described in US 20110294124, US 20120316224,
US 20140194610, US 20150211006, US 20150197540, WO 2015107425,
PCT/US2016/043542, and PCT/US2016/043598, each of which is
incorporated herein by reference. In some embodiments, a modified
internucleotidic linkage is a vinylphosphonate. Whittaker et al.
2008 Tetrahedron Letters 49: 6984-6987.
[1038] Non-limiting examples of internucleotidic linkages also
include those described in the art, including, but not limited to,
those described in any of: Gryaznov, S.; Chen, J.-K. J. Am. Chem.
Soc. 1994, 116, 3143, Jones et al. J. Org. Chem. 1993, 58, 2983,
Koshkin et al. 1998 Tetrahedron 54: 3607-3630, Lauritsen et al.
2002 Chem. Comm. 5: 530-531, Lauritsen et al. 2003 Bioo. Med. Chem.
Lett. 13: 253-256, Mesmaeker et al. Angew. Chem., Int. Ed. Engl.
1994, 33, 226, Petersen et al. 2003 TRENDS Biotech. 21: 74-81,
Schultz et al. 1996 Nucleic Acids Res. 24: 2966, Ts'o et al. Ann.
N. Y. Acad. Sci. 1988, 507, 220, and Vasseur et al. J. Am. Chem.
Soc. 1992, 114, 4006.
[1039] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising at least one
phosphate diester internucleotidic linkage and at least one
phosphorothioate triester linkage having the structure of Formula
I-c. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising at least one
phosphate diester internucleotidic linkage and at least two
phosphorothioate triester linkages having the structure of Formula
I-c. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising at least one
phosphate diester internucleotidic linkage and at least three
phosphorothioate triester linkages having the structure of Formula
I-c. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising at least one
phosphate diester internucleotidic linkage and at least four
phosphorothioate triester linkages having the structure of Formula
I-c. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising at least one
phosphate diester internucleotidic linkage and at least five
phosphorothioate triester linkages having the structure of Formula
I-c.
[1040] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising a sequence
found in any oligonucleotide disclosed herein. In some embodiments,
the present disclosure provides a chirally controlled C9orf72
oligonucleotide comprising a sequence found in any oligonucleotide
disclosed herein, wherein one or more U is replaced with T or vice
versa. In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising a sequence
found in any oligonucleotide disclosed herein, wherein the said
sequence has over 50% identity with the sequence of any
oligonucleotide disclosed herein. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
comprising a sequence found in any oligonucleotide disclosed
herein, wherein the said sequence has over 60% identity with the
sequence of any oligonucleotide disclosed herein. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising a sequence found in any
oligonucleotide disclosed herein, wherein the said sequence has
over 70% identity with the sequence of any oligonucleotide
disclosed herein. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising a
sequence found in any oligonucleotide disclosed herein, wherein the
said sequence has over 80% identity with the sequence of any
oligonucleotide disclosed herein. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
comprising a sequence found in any oligonucleotide disclosed
herein, wherein the said sequence has over 90% identity with the
sequence of any oligonucleotide disclosed herein. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising a sequence found in any
oligonucleotide disclosed herein, wherein the said sequence has
over 95% identity with the sequence of any oligonucleotide
disclosed herein. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising
the sequence of any oligonucleotide disclosed herein. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide having the sequence of any oligonucleotide
disclosed herein. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising a
sequence found in any oligonucleotide disclosed herein, wherein the
oligonucleotides have a pattern of backbone linkages, pattern of
backbone chiral centers, and/or pattern of backbone phosphorus
modifications described herein.
[1041] In some embodiments, the present disclosure provides a
chirally controlled C9orf72 oligonucleotide comprising a sequence
(or a portion of at least 10 contiguous bases thereof) found in any
oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage has a chiral linkage phosphorus. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising a sequence found in any
oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage has the structure of Formula I. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising a sequence (or a portion of at
least 10 contiguous bases thereof) found in any oligonucleotide
disclosed herein, wherein each internucleotidic linkage has the
structure of Formula I. In some embodiments, the present disclosure
provides a chirally controlled C9orf72 oligonucleotide comprising a
sequence (or a portion of at least 10 contiguous bases thereof)
found in any oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage has the structure of Formula I-c. In some
embodiments, the present disclosure provides a chirally controlled
C9orf72 oligonucleotide comprising a sequence (or a portion of at
least 10 contiguous bases thereof) found in any oligonucleotide
disclosed herein, wherein each internucleotidic linkage has the
structure of Formula I-c. In some embodiments, the present
disclosure provides a chirally controlled C9orf72 oligonucleotide
comprising a sequence (or a portion of at least 10 contiguous bases
thereof) found in any oligonucleotide disclosed herein, wherein at
least one internucleotidic linkage is
##STR00185##
In some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide comprising a sequence (or a
portion of at least 10 contiguous bases thereof) found in any
oligonucleotide disclosed herein, wherein each internucleotidic
linkage is
##STR00186##
In some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide comprising a sequence (or a
portion of at least 10 contiguous bases thereof) found in any
oligonucleotide disclosed herein, wherein at least one
internucleotidic linkage is
##STR00187##
In some embodiments, the present disclosure provides a chirally
controlled C9orf72 oligonucleotide comprising a sequence (or a
portion of at least 10 contiguous bases thereof) found in any
oligonucleotide disclosed herein, wherein each internucleotidic
linkage is
##STR00188##
[1042] In some embodiments, a modification at a linkage phosphorus
is characterized by its ability to be transformed to a phosphate
diester, such as those present in naturally occurring DNA and RNA,
by one or more esterases, nucleases, and/or cytochrome P450
enzymes, including but not limited to: CYP1A1, CYP1A2, CYP1B1
(Family: CYP1); CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9,
CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP2J2, CYP2R1, CYP2S1,
CYP2U1, CYP2W1 (CYP2); CYP3A4, CYP3A5, CYP3A7, CYP3A43 (CYP3);
CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4F8, CYP4F11, CYP4F12,
CYP4F22, CYP4V2, CYP4X1, CYP4Z1 (CYP4); CYP5A1 (CYP5); CYP7A1,
CYP7B1 (CYP7); CYP8A1 (prostacyclin synthase), CYP8B1 (bile acid
biosynthesis) (CYP8); CYP11A1, CYP11B1, CYP11B2 (CYP11); CYP17A1
(CYP17); CYP19A1 (CYP19); CYP20A1 (CYP20); CYP21A2 (CYP21); CYP24A1
(CYP24); CYP26A1, CYP2XXX1, CYP26C1 (CYP26); CYP27A1 (bile acid
biosynthesis), CYP27B1 (vitamin D31-alpha hydroxylase, activates
vitamin D3), CYP27C1 (unknown function) (CYP27); CYP39A1 (CYP39);
CYP46A1 (CYP46); or CYP51A1 (lanosterol 14-alpha demethylase)
(CYP51).
[1043] In some embodiments, a modification at phosphorus results in
a P-modification moiety characterized in that it acts as a
pro-drug, e.g., the P-modification moiety facilitates delivery of a
C9orf72 oligonucleotide to a desired location prior to removal. For
instance, in some embodiments, a P-modification moiety results from
PEGylation at the linkage phosphorus. One of skill in the relevant
arts will appreciate that various PEG chain lengths are useful and
that the selection of chain length will be determined in part by
the result that is sought to be achieved by PEGylation. For
instance, in some embodiments, PEGylation is effected in order to
reduce RES uptake and extend in vivo circulation lifetime of a
C9orf72 oligonucleotide.
[1044] In some embodiments, a PEGylation reagent for use in
accordance with the present disclosure is of a molecular weight of
about 300 g/mol to about 100,000 g/mol. In some embodiments, a
PEGylation reagent is of a molecular weight of about 300 g/mol to
about 10,000 g/mol. In some embodiments, a PEGylation reagent is of
a molecular weight of about 300 g/mol to about 5,000 g/mol. In some
embodiments, a PEGylation reagent is of a molecular weight of about
500 g/mol. In some embodiments, a PEGylation reagent of a molecular
weight of about 1000 g/mol. In some embodiments, a PEGylation
reagent is of a molecular weight of about 3000 g/mol. In some
embodiments, a PEGylation reagent is of a molecular weight of about
5000 g/mol.
[1045] In certain embodiments, a PEGylation reagent is PEG500. In
certain embodiments, a PEGylation reagent is PEG1000. In certain
embodiments, a PEGylation reagent is PEG3000. In certain
embodiments, a PEGylation reagent is PEG5000.
[1046] In some embodiments, a P-modification moiety is
characterized in that it acts as an agent which promotes cell entry
and/or endosomal escape, such as a membrane-disruptive lipid or
peptide.
[1047] In some embodiments, a P-modification moiety is
characterized in that it acts as a targeting agent. In some
embodiments, a P-modification moiety is or comprises a targeting
agent. The phrase "targeting agent," as used herein, is an entity
that is associates with a payload of interest (e.g., with a C9orf72
oligonucleotide or oligonucleotide composition) and also interacts
with a C9orf72 target site of interest so that the payload of
interest is targeted to the target site of interest when associated
with the targeting agent to a materially greater extent than is
observed under otherwise comparable conditions when the payload of
interest is not associated with the targeting agent. A targeting
agent may be, or comprise, any of a variety of chemical moieties,
including, for example, small molecule moieties, nucleic acids,
polypeptides, carbohydrates, etc. Targeting agents are described
further by Adarsh et al., "Organelle Specific Targeted Drug
Delivery--A Review," International Journal of Research in
Pharmaceutical and Biomedical Sciences, 2011, p. 895.
[1048] Examples of such targeting agents include, but are not
limited to, proteins (e.g. Transferrin), C9orf72 oligopeptides
(e.g., cyclic and acyclic RGD-containing oligopedptides),
antibodies (monoclonal and polyclonal antibodies, e.g. IgG, IgA,
IgM, IgD, IgE antibodies), sugars/carbohydrates (e.g.,
monosaccharides and/or oligosaccharides (mannose,
mannose-6-phosphate, galactose, and the like)), vitamins (e.g.,
folate), or other small biomolecules. In some embodiments, a
targeting moiety is a steroid molecule (e.g., bile acids including
cholic acid, deoxycholic acid, dehydrocholic acid; cortisone;
digoxigenin; testosterone; cholesterol; cationic steroids such as
cortisone having a trimethylaminomethyl hydrazide group attached
via a double bond at the 3-position of the cortisone ring, etc.).
In some embodiments, a targeting moiety is a lipophilic molecule
(e.g., alicyclic hydrocarbons, saturated and unsaturated fatty
acids, waxes, terpenes, and polyalicyclic hydrocarbons such as
adamantine and buckminsterfullerenes). In some embodiments, a
lipophilic molecule is a terpenoid such as vitamin A, retinoic
acid, retinal, or dehydroretinal. In some embodiments, a targeting
moiety is a peptide.
[1049] In some embodiments, a P-modification moiety is a targeting
agent of formula --X-L-R.sup.1 wherein each of X, L, and R are as
defined in Formula I, disclosed herein.
[1050] In some embodiments, a P-modification moiety is
characterized in that it facilitates cell specific delivery.
[1051] In some embodiments, a P-modification moiety is
characterized in that it falls into one or more of the
above-described categories. For instance, in some embodiments, a
P-modification moiety acts as a PK enhancer and a targeting ligand.
In some embodiments, a P-modification moiety acts as a pro-drug and
an endosomal escape agent. One of skill in the relevant arts would
recognize that numerous other such combinations are possible and
are contemplated by the present disclosure.
[1052] In some embodiments, a carbocyclyl, aryl, heteroaryl, or
heterocyclyl group, or a bivalent or polyvalent group thereof, is a
C.sub.3-C.sub.30 carbocyclyl, aryl, heteroaryl, or heterocyclyl
group, or a bivalent and/or polyvalent group thereof.
Bases (Nucleobases)
[1053] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, a C9orf72 target gene comprises a repeat expansion. In
some embodiments, provided C9orf72 oligonucleotides comprise any
nucleobase described herein or known in the art.
[1054] In some embodiments, a nucleobase present in a provided
C9orf72 oligonucleotide is a natural nucleobase or a modified
nucleobase derived from a natural nucleobase. Examples include, but
are not limited to, uracil, thymine, adenine, cytosine, and guanine
having their respective amino groups protected by acyl protecting
groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil,
5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs
such as pseudoisocytosine and pseudouracil and other modified
nucleobases such as 8-substituted purines, xanthine, or
hypoxanthine (the latter two being the natural degradation
products). Example modified nucleobases are disclosed in Chiu and
Rana, R N A, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids
Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive
Natural Products Chemistry, vol. 7, 313. In some embodiments, a
modified nucleobase is substituted uracil, thymine, adenine,
cytosine, or guanine. In some embodiments, a modified nucleobase is
a functional replacement, e.g., in terms of hydrogen bonding and/or
base pairing, of uracil, thymine, adenine, cytosine, or guanine. In
some embodiments, a nucleobase is optionally substituted uracil,
thymine, adenine, cytosine, 5-methylcytosine, or guanine. In some
embodiments, a nucleobase is uracil, thymine, adenine, cytosine,
5-methylcytosine, or guanine.
[1055] In some embodiments, a modified base is optionally
substituted adenine, cytosine, guanine, thymine, or uracil. In some
embodiments, a modified nucleobase is independently adenine,
cytosine, guanine, thymine or uracil, modified by one or more
modifications by which: [1056] (1) a nucleobase is modified by one
or more optionally substituted groups independently selected from
acyl, halogen, amino, azide, alkyl, alkenyl, alkynyl, aryl,
heteroalkyl, heteroalkenyl, heteroalkynyl, heterocyclyl,
heteroaryl, carboxyl, hydroxyl, biotin, avidin, streptavidin,
substituted silyl, and combinations thereof, [1057] (2) one or more
atoms of a nucleobase are independently replaced with a different
atom selected from carbon, nitrogen and sulfur; [1058] (3) one or
more double bonds in a nucleobase are independently hydrogenated;
or [1059] (4) one or more aryl or heteroaryl rings are
independently inserted into a nucleobase.
[1060] In some embodiments, a modified nucleobase is a modified
nucleobase as shown in the art, e.g., WO2017/210647. Modified
nucleobases also include expanded-size nucleobases in which one or
more aryl rings, such as phenyl rings, have been added. Nucleic
base replacements described in the Glen Research catalog (Glen
Research, Sterling, Va.); Krueger A T et al, Acc. Chem. Res., 2007,
40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner
S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F.
E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I.,
Curr. Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as
useful for the synthesis of the nucleic acids described herein. In
some embodiments, an expanded-size nucleobase is an expanded-size
nucleobase as shown in the art, e.g., WO2017/210647 Herein,
modified nucleobases also encompass structures that are not
considered nucleobases but are other moieties such as, but not
limited to, corrin- or porphyrin-derived rings. Porphyrin-derived
base replacements have been described in Morales-Rojas, H and Kool,
E T, Org. Lett., 2002, 4, 4377-4380. In some embodiments, a
porphyrin-derived ring is a porphyrin-derived ring as shown in the
art, e.g., WO2017/219647 In some embodiments, a modified nucleobase
is a modified nucleobase as shown in the art, e.g., WO2017/219647
In some embodiments, a modified nucleobase is fluorescent. Examples
of such fluorescent modified nucleobases include phenanthrene,
pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl,
terthiophene, benzoterthiophene, coumarin, lumazine, tethered
stillbene, benzo-uracil, and naphtho-uracil, as shown in the art,
e.g., WO2017/210647 In some embodiments, a nucleobase or modified
nucleobase is selected from: C5-propyne T, C5-propyne C,
C5-Thiazole, Phenoxazine, 2-Thio-thymine,
5-Triazolylphenyl-thymine, Diaminopurine, and
N2-Aminopropylguanine.
[1061] In some embodiments, a modified nucleobase is selected from:
5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl
substituted pyrimidines, alkyl substituted purines, and N-2, N-6
and 0-6 substituted purines. In certain embodiments, modified
nucleobases are selected from: 2-aminopropyladenine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-N-methylguanine, 6-N-methyladenine, 2-propyladenine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl
(--C.ident.C--CH.sub.3) uracil, 5-propynylcytosine, 6-azouracil,
6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl,
8-aza and other 8-substituted purines, 5-halo, particularly
5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine,
7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine,
7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine,
6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine,
4-N-benzoyluracil, 5-methyl 4-N-benzoylcytosine, 5-methyl
4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous
bases, size-expanded bases, and fluorinated bases. Further modified
nucleobases include tricyclic pyrimidines, such as
1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and
9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified
nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example,
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, Kroschwitz, J. I., Ed., John Wiley &
Sons, 1990, 858-859; Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, Crooke, S. T. and Lebleu, B.,
Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6
and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press,
2008, 163-166 and 442-443.
[1062] Example United States patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include without limitation, US2003/0158403,
U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187; 5,459,255;
5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594, 121;
5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588;
5,830,653; and 6,005,096.
[1063] In some embodiments, a modified nucleobase is unsubstituted.
In some embodiments, a modified nucleobase is substituted. In some
embodiments, a modified nucleobase is substituted such that it
contains, e.g., heteroatoms, alkyl groups, or linking moieties
connected to fluorescent moieties, biotin or avidin moieties, or
other protein or peptides. In some embodiments, a modified
nucleobase is a "universal base" that is not a nucleobase in the
most classical sense, but that functions similarly to a nucleobase.
One representative example of such a universal base is
3-nitropyrrole.
[1064] In some embodiments, other nucleosides can also be used in
the process disclosed herein and include nucleosides that
incorporate modified nucleobases, or nucleobases covalently bound
to modified sugars. Some examples of nucleosides that incorporate
modified nucleobases include 4-acetylcytidine;
5-(carboxyhydroxylmethyl)uridine; 2''-O-methylcytidine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyluridine; dihydrouridine;
2''-O-methylpseudouridine; beta,D-galactosylqueosine;
2''-O-methylguanosine; N.sup.6-isopentenyladenosine;
1-methyladenosine; 1-methylpseudouridine; 1-methylguanosine;
1-methylinosine; 2,2-dimethylguanosine; 2-methyladenosine;
2-methylguanosine; N.sup.7-methylguanosine; 3-methyl-cytidine;
5-methylcytidine; 5-hydroxymethylcytidine; 5-formylcytosine;
5-carboxylcytosine; N.sup.6-methyladenosine; 7-methylguanosine;
5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine;
beta,D-mannosylqueosine; 5-methoxycarbonylmethyluridine;
5-methoxyuridine; 2-methylthio-N.sup.6-isopentenyladenosine;
N-((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine;
N-((9-beta,D-ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine;
uridine-5-oxyacetic acid methylester; uridine-5-oxyacetic acid (v);
pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine;
2-thiouridine; 4-thiouridine; 5-methyluridine;
2''-O-methyl-5-methyluridine; and2''--O-methyluridine.
[1065] In some embodiments, nucleosides include 6-modified bicyclic
nucleosides that have either (R) or (S)-chirality at the 6-position
and include the analogs described in U.S. Pat. No. 7,399,845. In
other embodiments, nucleosides include 5''-modified bicyclic
nucleosides that have either (R) or (S)-chirality at the 5-position
and include the analogs described in US Patent Application
Publication No. 20070287831.
[1066] In some embodiments, a nucleobase or modified nucleobase
comprises one or more biomolecule binding moieties such as e.g.,
antibodies, antibody fragments, biotin, avidin, streptavidin,
receptor ligands, or chelating moieties. In other embodiments, a
nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil,
or 2,6-diaminopurine. In some embodiments, a nucleobase or modified
nucleobase is modified by substitution with a fluorescent or
biomolecule binding moiety. In some embodiments, the substituent on
a nucleobase or modified nucleobase is a fluorescent moiety. In
some embodiments, the substituent on a nucleobase or modified
nucleobase is biotin or avidin.
[1067] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,457,191; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692;
6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368;
6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and
7,495,088, the modified nucleobases, sugars, and internucleotidic
linkages of each of which are incorporated by reference.
[1068] In some embodiments, a base is optionally substituted A, T,
C, G or U, wherein one or more --NH.sub.2 are independently and
optionally replaced with --C(-L-R.sup.1).sub.3, one or more --NH--
are independently and optionally replaced with
--C(-L-R.sup.1).sub.2--, one or more .dbd.N-- are independently and
optionally replaced with --C(-L-R.sup.1)--, one or more .dbd.CH--
are independently and optionally replaced with .dbd.N--, and one or
more .dbd.O are independently and optionally replaced with .dbd.S,
.dbd.N(-L-R.sup.1), or .dbd.C(-L-R.sup.1).sub.2, wherein two or
more --L-R.sup.1 are optionally taken together with their
intervening atoms to form a 3-30 membered bicyclic or polycyclic
ring having 0-10 heteroatom ring atoms. In some embodiments, a
modified base is optionally substituted A, T, C, G or U, wherein
one or more --NH.sub.2 are independently and optionally replaced
with --C(-L-R.sup.1).sub.3, one or more --NH-- are independently
and optionally replaced with --C(-L-R.sup.1).sub.2--, one or more
.dbd.N-- are independently and optionally replaced with
--C(-L-R.sup.1)--, one or more .dbd.CH-- are independently and
optionally replaced with .dbd.N--, and one or more .dbd.O are
independently and optionally replaced with .dbd.S,
.dbd.N(-L-R.sup.1), or .dbd.C(-L-R.sup.1).sub.2, wherein two or
more -L-R.sup.1 are optionally taken together with their
intervening atoms to form a 3-30 membered bicyclic or polycyclic
ring having 0-10 heteroatom ring atoms, wherein the modified base
is different than the natural A, T, C, G and U. In some
embodiments, a base is optionally substituted A, T, C, G or U. In
some embodiments, a modified base is substituted A, T, C, G or U,
wherein the modified base is different than the natural A, T, C, G
and U.
[1069] In some embodiments, a nucleoside is any described in any
of: Gryaznov, S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143;
Hendrix et al. 1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg.
Med. Chem. 4: 5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et
al. J. Org. Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids
Res. 12: 3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630;
Kumar et al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et
al. 2002 Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med.
Chem. Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed.
Engl. 1994, 33, 226; Morita et al. 2001 Nucl. Acids Res. Supp. 1:
241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76;
Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al.
1997 Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins
Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50):
8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan
et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS
Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396;
Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al. 2009
J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53:
8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et
al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol.
Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew;
Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha
P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat,
Balkrishen; et al. From Nucleic Acids Symposium Series (2008),
52(1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et
al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org.
Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131; Ts'o et
al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995
Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem.
Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO
2016/079181.
[1070] Example nucleobases are also described in US 20110294124, US
20120316224, US 20140194610, US 20150211006, US 20150197540, WO
2015107425, PCT/US2016/043542, and PCT/US2016/043598, each of which
is incorporated herein by reference.
[1071] In some embodiments, a C9orf72 oligonucleotides comprises a
nucleobase, synthetic or modified nucleobase, nucleoside or
nucleotide, or modified nucleoside or modified nucleotide described
in Feldman et al. 2017 J. Am. Chem. Soc. 139: 11427-11433, Feldman
et al. 2017 Proc. Natl. Acad. Sci. USA 114: E6478-E6479, Hwang et
al. 2009 Nucl. Acids Res. 37: 4757-4763, Hwang et al. 2008 J. Am.
Chem. Soc. 130: 14872-14882, Lavergne et al. 2012 Chem. Eur. J. 18:
1231-1239, Lavergne et al. 2013 J. Am. Chem. Soc. 135: 5408-5419,
Ledbetter et al. 2018 J. Am. Chem. Soc. 140: 758-765, Malyshev et
al. 2009 J. Am. Chem. Soc. 131: 14620-14621, Seo et al. 2009
ChemBioChem 10: 2394-2400, including, but not limited to: d3FB,
d2Py analogs, d2Py, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy, d45DMPy,
d5FM, d5PrM, d5SICS, dFEMO, dMMO2, dNaM, dNM01, dTPT3, nucleotides
with 2'-azido, 2'-chloro, 2'-amino or arabinose sugars,
isocarbostiryl-, napthyl- and azaindole-nucleotides, and
modifications and derivatives and functionalized versions thereof,
including but not limited to those in which the sugar comprises a
2'-modification and/or other modification, and dMMO2 derivatives
with meta-chlorine, -bromine, -iodine, -methyl, or -propinyl
substituents.
[1072] Sugars
[1073] In some embodiments, provided C9orf72 oligonucleotides are
capable of directing a decrease in the expression, level and/or
activity of a C9orf72 target gene or its gene product. In some
embodiments, a C9orf72 target gene comprises a repeat expansion. In
some embodiments, provided C9orf72 oligonucleotides comprise any
sugar described herein or known in the art.
[1074] In some embodiments, provided C9orf72 oligonucleotides
capable of directing C9orf72 knockdown comprise one or more
modified sugar moieties beside the natural sugar moieties.
[1075] The most common naturally occurring nucleotides are
comprised of ribose sugars linked to the nucleobases adenosine (A),
cytosine (C), guanine (G), and thymine (T) or uracil (U). Also
contemplated are modified nucleotides wherein a phosphate group or
linkage phosphorus in the nucleotides can be linked to various
positions of a sugar or modified sugar. As non-limiting examples,
the phosphate group or linkage phosphorus can be linked to the 2'',
3'', 4'' or 5'' hydroxyl moiety of a sugar or modified sugar.
Nucleotides that incorporate modified nucleobases as described
herein are also contemplated in this context. In some embodiments,
nucleotides or modified nucleotides comprising an unprotected --OH
moiety are used in accordance with methods of the present
disclosure.
[1076] In some embodiments, a C9orf72 oligonucleotide can comprise
any base (nucleobase), modified base or base analog described
herein or known in the art. In some embodiments, a C9orf72
oligonucleotide can comprise any base described herein or known in
the art in combination with any other structural element or
modification described herein, including but not limited to, base
sequence or portion thereof, sugar; internucleotidic linkage;
stereochemistry or pattern thereof, additional chemical moiety,
including but not limited to, a targeting moiety, etc.; pattern of
modifications of sugars, bases or internucleotidic linkages; format
or any structural element thereof, and/or any other structural
element or modification described herein; and in some embodiments,
the present disclosure pertains to multimers of any such
oligonucleotides.
[1077] In some embodiments, a C9orf72 oligonucleotide can comprise
any sugar.
[1078] In some embodiments, a sugar has a structure of:
##STR00189##
[1079] Modified sugars can be incorporated into a provided C9orf72
oligonucleotide. In some embodiments, a modified sugar contains one
or more substituents at the 2'' position including one of the
following: --F; --CF.sub.3, --CN, --N.sub.3, --NO, --NO.sub.2,
--OR', --SR', or --N(R').sub.2, wherein each R' is independently
described in the present disclosure; --O--(C.sub.1-C.sub.10 alkyl),
--S--(C.sub.1-C.sub.10 alkyl), --NH--(C.sub.1-C.sub.10 alkyl), or
--N(C.sub.1-C.sub.10 alkyl).sub.2; --O--(C.sub.2-C.sub.10 alkenyl),
--S--(C.sub.2-C.sub.10 alkenyl), --NH--(C.sub.2-C.sub.10 alkenyl),
or --N(C.sub.2-C.sub.10 alkenyl).sub.2; --O--(C.sub.2-C.sub.10
alkynyl), --S--(C.sub.2-C.sub.10 alkynyl), --NH--(C.sub.2-C.sub.10
alkynyl), or --N(C.sub.2-C.sub.10 alkynyl).sub.2; or
--O--(C.sub.1-C.sub.10 alkylene)-O--(C.sub.1-C.sub.10 alkyl),
--O--(C.sub.2-C.sub.10 alkylene)-NH--(C.sub.1-C.sub.10 alkyl) or
--O--(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl).sub.2,
--NH--(C.sub.1-C.sub.10 alkylene)-O--(C.sub.1-C.sub.10 alkyl), or
--N(C.sub.1-C.sub.10 alkyl)-(C.sub.1-C.sub.10
alkylene)-O--(C.sub.1-C.sub.10 alkyl), wherein the alkyl, alkylene,
alkenyl and alkynyl may be substituted or unsubstituted. Examples
of substituents include, and are not limited to,
--O(CH.sub.2).sub.nOCH.sub.3, and --O(CH.sub.2).sub.nNH.sub.2,
wherein n is from 1 to about 10, MOE, DMAOE, DMAEOE. Also
contemplated herein are modified sugars described in WO
2001/088198; and Martin et al., Helv. Chim. Acta, 1995, 78,
486-504. In some embodiments, a modified sugar comprises one or
more groups selected from a substituted silyl group, an RNA
cleaving group, a reporter group, a fluorescent label, an
intercalator, a group for improving the pharmacokinetic properties
of a nucleic acid, a group for improving the pharmacodynamic
properties of a nucleic acid, or other substituents having similar
properties. In some embodiments, modifications are made at one or
more of the 2', 3', 4', 5', or 6' positions of the sugar or
modified sugar, including the 3' position of the sugar on the
3'-terminal nucleotide or in the 5' position of the 5'-terminal
nucleotide.
[1080] In some embodiments, a 2'-modification is 2'-F.
[1081] In some embodiments, the 2'-OH of a ribose is replaced with
a substituent including one of the following: --H, --F; --CF.sub.3,
--CN, --N.sub.3, --NO, --NO.sub.2, --OR', --SR', or --N(R').sub.2,
wherein each R' is independently described in the present
disclosure; --O--(C.sub.2-C.sub.10 alkyl), --S--(C.sub.1-C.sub.10
alkyl), --NH--(C.sub.1-C.sub.10 alkyl), or --N(C.sub.1-C.sub.10
alkyl).sub.2; --O--(C.sub.2-C.sub.10 alkenyl),
--S--(C.sub.2-C.sub.10 alkenyl), --NH--(C.sub.2-C.sub.10 alkenyl),
or --N(C.sub.2-C.sub.10 alkenyl).sub.2; --O--(C.sub.2-C.sub.10
alkynyl), --S--(C.sub.2-C.sub.10 alkynyl), --NH--(C.sub.2-C.sub.10
alkynyl), or --N(C.sub.2-C.sub.10 alkynyl).sub.2; or
--O--(C.sub.1-C.sub.10 alkylene)-O--(C.sub.1-C.sub.10 alkyl),
--O--(C.sub.1-C.sub.10 alkylene)-NH--(C.sub.1-C.sub.10 alkyl) or
--O--(C.sub.1-C.sub.10 alkylene)-NH(C.sub.1-C.sub.10 alkyl).sub.2,
--NH--(C.sub.1-C.sub.10 alkylene)-O--(C.sub.1-C.sub.10 alkyl), or
--N(C.sub.1-C.sub.10 alkyl)-(C.sub.1-C.sub.10
alkylene)-O--(C.sub.1-C.sub.10 alkyl), wherein the alkyl, alkylene,
alkenyl and alkynyl may be substituted or unsubstituted. In some
embodiments, the 2'-OH is replaced with --H (deoxyribose). In some
embodiments, the 2'-OH is replaced with --F. In some embodiments,
the 2'-OH is replaced with --OR'. In some embodiments, the 2'-OH is
replaced with --OMe. In some embodiments, the 2'-OH is replaced
with --OCH.sub.2CH.sub.2OMe.
[1082] Modified sugars also include locked nucleic acids (LNAs). In
some embodiments, two substituents on sugar carbon atoms are taken
together to form a bivalent moiety. In some embodiments, two
substituents are on two different sugar carbon atoms. In some
embodiments, a formed bivalent moiety has the structure of -L-as
defined herein. In some embodiments, -L- is --O--CH.sub.2--,
wherein --CH.sub.2-- is optionally substituted. In some
embodiments, -L- is --O--CH.sub.2--. In some embodiments, -L- is
--O--CH(Et)-. In some embodiments, -L- is between C2 and C4 of a
sugar moiety. In some embodiments, a locked nucleic acid has the
structure indicated below. A locked nucleic acid of the structure
below is indicated, wherein B represents a nucleobase or modified
nucleobase as described herein, and wherein, e.g., R.sup.2s and
R.sup.4s are R taken together with their intervening atoms to form
a ring. In some embodiments, a modified nucleoside has a structure
of:
##STR00190##
wherein B is a base.
[1083] In some embodiments, a modified sugar is an ENA such as
those described in, e.g., Seth et al., J Am Chem Soc. 2010 October
27; 132(42): 14942-14950. In some embodiments, a modified sugar is
any of those found in an XNA (xenonucleic acid), for instance,
arabinose, anhydrohexitol, threose, 2'fluoroarabinose, or
cyclohexene.
[1084] Modified sugars include cyclobutyl or cyclopentyl moieties
in place of the pentofuranosyl sugar. Representative United States
patents that teach the preparation of such modified sugar
structures include, but are not limited to, U.S. Pat. Nos.
4,981,957; 5,118,800; 5,319,080; and 5,359,044. Some modified
sugars that are contemplated include sugars in which the oxygen
atom within the ribose ring is replaced by nitrogen, sulfur,
selenium, or carbon. In some embodiments, a modified sugar is a
modified ribose wherein the oxygen atom within the ribose ring is
replaced with nitrogen, and wherein the nitrogen is optionally
substituted with an alkyl group (e.g., methyl, ethyl, isopropyl,
etc).
[1085] Non-limiting examples of modified sugars include glycerol,
which form glycerol nucleic acid (GNA). One example of a GNA is
shown below and is described in Zhang, R et al., J Am. Chem. Soc.,
2008, 130, 5846-5847; Zhang L, et al., J Am. Chem. Soc., 2005, 127,
4174-4175 and Tsai C H et al., PNAS, 2007, 14598-14603. In some
embodiments, a nucleoside has a structure of:
##STR00191##
Wherein B is a base.
[1086] A flexible nucleic acid (FNA) based on the mixed acetal
aminal of formyl glycerol, is described in Joyce G F et al., PNAS,
1987, 84, 4398-4402 and Heuberger B D and Switzer C, J. Am. Chem.
Soc., 2008, 130, 412-413. In some embodiments, a nucleoside has a
structure of
##STR00192##
Wherein B is a base.
[1087] Additional non-limiting examples of modified sugars and/or
modified nucleosides and/or modified nucleotides include
hexopyranosyl (6' to 4'), pentopyranosyl (4' to 2'), pentopyranosyl
(4' to 3'), 5'-deoxy-5'-C-malonyl, squaryldiamide, and
tetrofuranosyl (3' to 2') sugars. In some embodiments, a modified
nucleoside comprises a hexopyranosyl (6' to 4') sugar and has the
structure of any one in the following formulae:
##STR00193##
wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein wherein XLR.sup.1 is equivalent to
X-L-R.sup.1 and X, L, and R.sup.1 are as defined in Formula I,
disclosed herein, and B is a base.
[1088] In some embodiments, a modified nucleotide comprises a
pentopyranosyl (4' to 2') sugar and has a structure of any one in
the following formulae:
##STR00194##
wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein, wherein XLR.sup.1 is equivalent to
X-L-R.sup.1 and X, L, and R.sup.1 are as defined in Formula I,
disclosed herein, and B is a base.
[1089] In some embodiments, a modified nucleotide comprises a
pentopyranosyl (4' to 3') sugar and is of any one in the following
formulae:
##STR00195##
wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein, wherein XLR.sup.1 is equivalent to
X-L-R and X, L, and R.sup.1 are as defined in Formula I, disclosed
herein, and B is a base.
[1090] In some embodiments, a modified nucleotide comprises a
tetrofuranosyl (3' to 2') sugar and is of either in the following
formulae:
##STR00196##
wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein, wherein XLR.sup.1 is equivalent to
X-L-R.sup.1 and X, L, and R.sup.1 are as defined in Formula I,
disclosed herein, and B is a base.
[1091] In some embodiments, a modified nucleotide comprises a
modified sugar and is of any one in the following formulae:
##STR00197##
wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein, wherein XLR.sup.1 is equivalent to
X-L-R and X, L, and R.sup.1 are as defined in Formula I, disclosed
herein, and B is a base.
[1092] In some embodiments, one or more hydroxyl group in a sugar
moiety is optionally and independently replaced with halogen,
R'--N(R').sub.2, --OR', or --SR', wherein each R' is independently
described in the present disclosure.
[1093] In some embodiments, a modified nucleotide is as illustrated
below, wherein X.sup.s corresponds to the P-modification group
"--XLR.sup.1" described herein, wherein XLR.sup.1 is equivalent to
X-L-R and X, L, and R.sup.1 are as defined in Formula I, disclosed
herein, B is a base, and X is selected from --S--, --Se--,
--CH.sub.2--, --NMe-, -NEt- and --NiPr--
##STR00198## ##STR00199## ##STR00200##
[1094] Modified sugars can be prepared by methods known in the art,
including, but not limited to: A. Eschenmoser, Science (1999),
284:2118; M. Bohringer et al, Helv. Chim. Acta (1992),
75:1416-1477; M. Egli et al, J. Am. Chem. Soc. (2006),
128(33):10847-56; A. Eschenmoser i nChemical Synthesis: Gnosis to
Prognosis, C. Chatgilialoglu and V. Sniekus, Ed., (Kluwer Academic,
Netherlands, 1996), p. 293; K.-U. Schoning et al, Science (2000),
290:1347-1351; A. Eschenmoser et al, Helv. Chim. Acta (1992),
75:218; J. Hunziker et al, Helv. Chim. Acta (1993), 76:259; G.
Otting et al, Helv. Chim. Acta (1993), 76:2701; K. Groebke et al,
Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser, Science
(1999), 284:2118. Modifications to the 2' modifications can be
found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and
all references therein. Specific modifications to the ribose can be
found in the following references: 2'-fluoro (Kawasaki et. al., J.
Med. Chem., 1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta
1996, 79, 1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310). In some embodiments, a modified sugar is any of those
described in PCT Publication No. WO2012/030683, incorporated herein
by reference, and/or depicted herein. In some embodiments, a
modified sugar is any modified sugar described in any of: Gryaznov,
S; Chen, J.-K. J. Am. Chem. Soc. 1994, 116, 3143; Hendrix et al.
1997 Chem. Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4:
5; Jepsen et al. 2004 Oligo. 14: 130-146; Jones et al. J. Org.
Chem. 1993, 58, 2983; Koizumi et al. 2003 Nuc. Acids Res. 12:
3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et
al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002
Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem.
Lett. 13: 253-256; Mesmaeker et al. Angew. Chem., Int. Ed. Engl.
1994, 33, 226; Morita et al. 2001 Nucl. Acids Res. Supp. 1:
241-242; Morita et al. 2002 Bioo. Med. Chem. Lett. 12: 73-76;
Morita et al. 2003 Bioo. Med. Chem. Lett. 2211-2226; Nielsen et al.
1997 Chem. Soc. Rev. 73; Nielsen et al. 1997 J. Chem. Soc. Perkins
Transl. 1: 3423-3433; Obika et al. 1997 Tetrahedron Lett. 38 (50):
8735-8; Obika et al. 1998 Tetrahedron Lett. 39: 5401-5404; Pallan
et al. 2012 Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS
Biotech. 21: 74-81; Rajwanshi et al. 1999 Chem. Commun. 1395-1396;
Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et al. 2009
J. Med. Chem. 52: 10-13; Seth et al. 2010 J. Med. Chem. 53:
8309-8318; Seth et al. 2010 J. Org. Chem. 75: 1569-1581; Seth et
al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol.
Ther-Nuc. Acids. 1, e47; Seth, Punit P; Siwkowski, Andrew;
Allerson, Charles R; Vasquez, Guillermo; Lee, Sam; Prakash, Thazha
P; Kinberger, Garth; Migawa, Michael T; Gaus, Hans; Bhat,
Balkrishen; et al. From Nucleic Acids Symposium Series (2008),
52(1), 553-554; Singh et al. 1998 Chem. Comm. 1247-1248; Singh et
al. 1998 J. Org. Chem. 63: 10035-39; Singh et al. 1998 J. Org.
Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm. 2130-2131; Ts'o et
al. Ann. N. Y. Acad. Sci. 1988, 507, 220; Van Aerschot et al. 1995
Angew. Chem. Int. Ed. Engl. 34: 1338; Vasseur et al. J. Am. Chem.
Soc. 1992, 114, 4006; WO 20070900071; WO 20070900071; or WO
2016/079181.
[1095] In some embodiments, a modified sugar moiety is an
optionally substituted pentose or hexose moiety. In some
embodiments, a modified sugar moiety is an optionally substituted
pentose moiety. In some embodiments, a modified sugar moiety is an
optionally substituted hexose moiety. In some embodiments, a
modified sugar moiety is an optionally substituted ribose or
hexitol moiety. In some embodiments, a modified sugar moiety is an
optionally substituted ribose moiety. In some embodiments, a
modified sugar moiety is an optionally substituted hexitol
moiety.
[1096] In some embodiments, an example modified nucleotide is
selected from:
##STR00201##
In some embodiments, a nucleotide has a structure selected from any
of:
##STR00202## ##STR00203##
In some embodiments, a modified nucleoside has a structure selected
from:
##STR00204##
Wherein R.sup.1 and R are independently --H, --F, --OMe-MOE or
substituted or unsubstituted C.sub.1-6 alkyl;
##STR00205##
where R.sup.e is substituted or unsubstituted C.sub.1-6 alkyl or
H
##STR00206## ##STR00207##
Additional chemically modified sugars are described in WO
2008/101157, WO 2007/134181, WO 2016/167780, and published US
Patent Application US2005-0130923. In some embodiments, a
nucleotide and adjacent nucleoside have the structure of:
##STR00208##
[1097] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl group (R or S), 4'-S, 2'-F, 2'-OCH.sub.3,
2'-OCH.sub.2CH.sub.3, 2'--OCH.sub.2CH.sub.2F and
2'-O(CH.sub.2).sub.20CH.sub.3 substituent groups. The substituent
at the 2' position can also be selected from allyl, amino, azido,
thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3, OCH.sub.2F,
O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n),
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.1)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)-
, where each R.sub.1, R.sub.m and R is, independently, H or
substituted or unsubstituted C.sub.1-C.sub.10 alkyl.
[1098] In some embodiments, a bicyclic nucleoside includes any
modified nucleoside comprising a bicyclic sugar moiety. Examples of
bicyclic nucleic acids (BNAs) include without limitation
nucleosides comprising a bridge between the 4' and the 2' ribosyl
ring atoms. In some embodiments, antisense compounds provided
herein include one or more BNA nucleosides wherein the bridge
comprises one of the formulas: 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2)--S-2'; 4, --(CH.sub.2).sub.2--O-2' (ENA);
4'-CH(CH.sub.3)-0-2' and 4'-CH(CH.sub.2OCH.sub.3)--O-2' (and
analogs thereof, see U.S. Pat. No. 7,399,845);
4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof, see
PCT/US2008/068922 published as WO/2009/006478);
4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof, see
PCT/US2008/064591 published as WO/2008/150729);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent
Application US2004-0171570); 4'-CH.sub.2--N(R)--O-2', wherein R is
H, C.sub.1-C.sub.12 alkyl, or a protecting group (see U.S. Pat. No.
7,427,672); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Chattopadhyaya et
al, J. Org. Chem., 2009, 74, 118-134); and 4,
--CH.sub.2--C(.dbd.CH.sub.2)-2' (and analogs thereof, see
PCT/US2008/066154 published as WO 2008/154401).
[1099] Further bicyclic nucleosides have been reported in the
literature (see for example: Srivastava et al, J. Am. Chem. Soc.,
2007, 129(26) 8362-8379; Frieden et al, Nucleic Acids Research,
2003, 21, 6365-6372; Elayadi et al, Curr. Opinion Inverts. Drugs,
2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7; Oram et
al, Curr. Opinion Mol Ther., 2001, 3, 239-243; Wahlestedt et al,
Proc. Natl Acad. Sci. U.S.A, 2000, 97, 5633-5638; Singh et al,
Chem. Commun., 1998, 4, 455-456; Koshkin et al, Tetrahedron, 1998,
54, 3607-3630; Kumar et al, Bioorg. Med. Chem. Lett., 1998, 8,
2219-2222; Singh et al, J. Org. Chem., 1998, 63, 10035-10039; U.S.
Pat. Nos. 7,399,845; 7,053,207; 7,034,133; 6,794,499; 6,770,748;
6,670,461; 6,525,191; 6,268,490; U.S. Patent Publication Nos.:
US2008-0039618; US2007-0287831; US2004-0171570; U.S. Patent
Applications, Ser. Nos. 12/129,154; 61/099,844; 61/097,787;
61/086,231; 61/056,564; 61/026,998; 61/026,995; 60/989,574;
International applications WO 2007/134181; WO 2005/021570; WO
2004/106356; and PCT International Applications Nos.:
PCT/US2008/068922; PCT/US2008/066154; and PCT/US2008/064591).
[1100] In some embodiments, a bicyclic nucleoside can be prepared
having one or more stereochemical sugar configurations including
for example alpha-L-ribofuranose and beta-D-ribofuranose (see PCT
international application PCT/DK98/00393, published as WO
99/14226). In some embodiments, a monocyclic nucleosides is a
nucleoside comprising a modified sugar moiety that is not a
bicyclic sugar moiety. In some embodiments, the sugar moiety, or
sugar moiety analogue, of a nucleoside may be modified or
substituted at any position. In some embodiments, a 4'-2' bicyclic
nucleoside or 4' to 2' bicyclic nucleoside is a bicyclic nucleoside
comprising a furanose ring comprising a bridge connecting two
carbon atoms of the furanose ring connects the 2' carbon atom and
the 4' carbon atom of the sugar ring. In some embodiments, bicyclic
sugar moieties of BNA nucleosides include, but are not limited to,
compounds having at least one bridge between the 4' and the 2'
carbon atoms of the pentofuranosyl sugar moiety including without
limitation, bridges comprising 1 or from 1 to 4 linked groups
independently selected from --[C(R.sub.a)(R.sub.b)].sub.n,
--C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--,
--C(.dbd.NR.sub.a)--, --C(.dbd.O)--, --C(.dbd.S)--, --O--,
--Si(R.sub.a).sub.2--, --S(.dbd.O)x-, and --N(R)--; wherein: x is
0, 1, or 2; n is 1, 2, 3, or 4; each R.sub.a and R.sub.b is,
independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl,
C.sub.5-C.sub.2Oaryl, substituted C.sub.5-C.sub.20 aryl,
heterocycle radical, substituted heterocycle radical, heteroaryl,
substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical,
substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1,
NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl
(C(.dbd.O)--H), substituted acyl, CN, sulfonyl
(S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and
each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12
alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12
alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12
alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20
aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H),
substituted acyl, a heterocycle radical, a substituted heterocycle
radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12
aminoalkyl or a protecting group.
[1101] In some embodiments, the bridge of a bicyclic sugar moiety
is --[C(R.sub.a)(R.sub.b)].sub.n,
--[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)-N(R)--O--
or --C(R.sub.aR.sub.b)--O--N(R)--. In some embodiments, the bridge
is 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2',
4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2',
4'-CH.sub.2--O--N(R)-2' and 4'-CH.sub.2--N(R)--O-2'-- wherein each
R is, independently, H, a protecting group or C.sub.1-C.sub.12
alkyl.
[1102] In some embodiments, bicyclic nucleosides are further
defined by isomeric configuration. For example, a nucleoside
comprising a 4'-(CH.sub.2)--O-2' bridge, may be in the alpha-L
configuration or in the beta-D configuration. alpha-L-methyleneoxy
(4'-CH.sub.2--O-2') BNA's have been incorporated into antisense
oligonucleotides that showed antisense activity (Frieden et al.,
Nucleic Acids Research, 2003, 21, 6365-6372).
[1103] In some embodiments, bicyclic nucleosides include those
having a 4' to 2' bridge wherein such bridges include without
limitation, a-L-4'-(CH.sub.2)--O-2', .beta.-D-4'-CH.sub.2--O-2',
4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R)-2',
4'-CH.sub.2--N(R)--O-2', 4'-CH(CH.sub.3)--O-2', 4'-CH.sub.2--S-2',
4'-CH.sub.2--N(R)-2', 4'-CH.sub.2--CH(CH.sub.3)-2', and
4'-(CH.sub.2).sub.3-2', wherein R is H, a protecting group or
C.sub.1-C.sub.12 alkyl.
[1104] Analogs of various bicyclic nucleosides that have 4' to 2'
bridging groups such as 4'-CH.sub.2-0-2' and 4'-CH.sub.2--S-2',
have also been prepared (Kumar et al, Bioorg. Med. Chem. Lett.,
1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide
duplexes comprising bicyclic nucleosides for use as substrates for
nucleic acid polymerases has also been described (Wengel et al, WO
99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel
conformationally restricted high-affinity oligonucleotide analog
has been described in the art (Singh et al, J. Org. Chem., 1998,
63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's
have been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[1105] One carbocyclic bicyclic nucleoside having a
4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge
4'-CH.dbd.CH--CH.sub.2-2' have been described (Frier et al.,
Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al.,
J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation
of carbocyclic bicyclic nucleosides along with their
oligomerization and biochemical studies have also been described
(Srivastava et al, J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
[1106] In some embodiments, bicyclic nucleosides include, but are
not limited to, alpha-L-methyleneoxy (4'-CH.sub.2--O-2') BNA,
beta-D-methyleneoxy (4'-CH.sub.2--O-2') BNA, ethyleneoxy
(4'-(CH.sub.2).sub.2--O-2') BNA, aminooxy (4'-CH.sub.2--O--N(R)-2')
BNA, oxyamino (4'-CH.sub.2--N(R)--O-2') BNA, methyl(methyleneoxy)
(4'-CH(CH.sub.3)--O-2') BNA (also referred to as constrained ethyl
or cEt), methylene-thio (4'-CH.sub.2--S-2') BNA, methylene-amino
(4'-CH.sub.2--N(R)-2') BNA, methyl carbocyclic
(4'-CH.sub.2--CH(CH.sub.3)-2') BNA, propylene carbocyclic
(4'-(CH.sub.2).sub.3-2') BNA, and vinyl BNA.
[1107] In some embodiments, a modified tetrahydropyran nucleoside
or modified THP nucleoside is a nucleoside having a six-membered
tetrahydropyran "sugar" substituted for the pentofuranosyl residue
in normal nucleosides and can be referred to as a sugar surrogate.
Modified THP nucleosides include, but are not limited to, what is
referred to in the art as hexitol nucleic acid (HNA), anitol
nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann,
Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having
a tetrahydropyranyl ring system as illustrated below.
[1108] In some embodiments, sugar surrogates comprise rings having
more than 5 atoms and more than one heteroatom. For example
nucleosides comprising morpholino sugar moieties and their use in
oligomeric compounds has been reported (see for example: Braasch et
al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.
5,698,685; 5,166,315; 5,185,444; and 5,034,506).
[1109] Combinations of modifications are also provided without
limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT
International Application WO 2008/101157 for other disclosed
5',2'-bis substituted nucleosides) and replacement of the ribosyl
ring oxygen atom with S and further substitution at the 2'-position
(see published U.S. Patent Application US2005-0130923) or
alternatively 5'-substitution of a bicyclic nucleic acid (see PCT
International Application WO 2007/134181, wherein a
4'-CH.sub.2--O-2' bicyclic nucleoside is further substituted at the
5' position with a 5'-methyl or a 5'-vinyl group). The synthesis
and preparation of carbocyclic bicyclic nucleosides along with
their oligomerization and biochemical studies have also been
described (see, e.g., Srivastava et al, J. Am. Chem. Soc. 2007,
129(26), 8362-8379).
[1110] In some embodiments, antisense compounds comprise one or
more modified cyclohexenyl nucleosides, which is a nucleoside
having a six-membered cyclohexenyl in place of the pentofuranosyl
residue in naturally occurring nucleosides. Modified cyclohexenyl
nucleosides include, but are not limited to those described in the
art (see for example commonly owned, published PCT Application WO
2010/036696, Robeyns et al, J. Am. Chem. Soc., 2008, 130(6),
1979-1984; Horvath et al, Tetrahedron Letters, 2007, 48, 3621-3623;
Nauwelaerts et al, J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu
et al., Nucleosides, Nucleotides & Nucleic Acids, 2005,
24(5-7), 993-998; Nauwelaerts et al, Nucleic Acids Research, 2005,
33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section
F: Structural Biology and Crystallization Communications, 2005,
F61(6), 585-586; Gu et al, Tetrahedron, 2004, 60(9), 2111-2123; Gu
et al, Oligonucleotides, 2003, 13(6), 479-489; Wang et al, J. Org.
Chem., 2003, 68, 4499-4505; Verbeure et al, Nucleic Acids Research,
2001, 29(24), 4941-4947; Wang et al, J. Org. Chem., 2001, 66,
8478-82; Wang et al, Nucleosides, Nucleotides & Nucleic Acids,
2001, 20(4-7), 785-788; Wang et al, J. Am. Chem., 2000, 122,
8595-8602; Published PCT application, WO 06/047842; and Published
PCT Application WO 1/049687.
[1111] Many other monocyclic, bicyclic and tricyclic ring systems
are known in the art and are suitable as sugar surrogates that can
be used to modify nucleosides for incorporation into oligomeric
compounds as provided herein (see for example review article:
Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854).
Such ring systems can undergo various additional substitutions to
further enhance their activity. In some embodiments, a 2'-modified
sugar is a furanosyl sugar modified at the 2' position. In some
embodiments, such modifications include substituents selected from:
a halide, including, but not limited to substituted and
unsubstituted alkoxy, substituted and unsubstituted thioalkyl,
substituted and unsubstituted amino alkyl, substituted and
unsubstituted alkyl, substituted and unsubstituted allyl, and
substituted and unsubstituted alkynyl. In some embodiments, 2'
modifications are selected from substituents including, but not
limited to: O[(CH.sub.2).sub.nO].sub.mCH, O(CH.sub.2)NH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF,
O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m
are from 1 to about 10. Other 2'- substituent groups can also be
selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl,
alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an R A cleaving group, a
reporter group, an intercalator, a group for improving
pharmacokinetic properties, or a group for improving the
pharmacodynamic properties of an antisense compound, and other
substituents having similar properties. In some embodiments,
modified nucleosides comprise a 2'-MOE side chain (Baker et al, J.
Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have
been described as having improved binding affinity compared to
unmodified nucleosides and to other modified nucleosides, such as
2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having
the 2'-MOE substituent also have been shown to be antisense
inhibitors of gene expression with promising features for in vivo
use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al.,
Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans.,
1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides,
1997, 16, 917-926).
[1112] In some embodiments, a 2'-modified" or 2'-substituted
nucleoside is a nucleoside comprising a sugar comprising a
substituent at the 2' position other than H or OH. In some
embodiments, 2'-modified nucleosides, include, but are not limited
to, bicyclic nucleosides wherein the bridge connecting two carbon
atoms of the sugar ring connects the 2' carbon and another carbon
of the sugar ring; and nucleosides with non-bridging 2'
substituents, such as allyl, amino, azido, thio, O-allyl,
O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3,
O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R,), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C1-C.sub.10 alkyl.
[1113] Methods for the preparations of modified sugars are well
known to those skilled in the art. Some representative U.S. patents
that teach the preparation of such modified sugars include without
limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and
International Application PCT/US2005/019219 published as WO
2005/121371.
[1114] In some embodiments, R.sup.1 is R as defined and described.
In some embodiments, R.sup.2 is R. In some embodiments, R.sup.e is
R. In some embodiments, R.sup.e is H, CH.sub.3, Bn, COCF.sub.3,
benzoyl, benzyl, pyren-1-ylcarbonyl, pyren-1-ylmethyl,
2-aminoethyl. In some embodiments, a non-limiting example
internucleotidic linkage or sugar is or comprises a component of
any of: N-methanocarba, C3-amide, Formacetal, Thioformacetal, MMI,
PMO (phosphorodiamidate linked morpholino), PNA (peptide nucleic
acid), LNA, cMOE BNA, cEt BNA, .alpha.-L-NA or a related analog,
HNA, Me-ANA, MOE-ANA, Ara-FHNA, FHNA, R-6'-Me-FHNA, S-6'-Me-FHNA,
ENA, or c-ANA. In some embodiments, a non-limiting example
internucleotidic linkage or sugar is or comprises a component of
any of those described in Allerson et al. 2005 J. Med. Chem. 48:
901-4; BMCL 201121: 1122; BMCL 2011 21: 588; BMCL 2012 22: 296;
Chattopadhyaya et al. 2007 J. Am. Chem. Soc. 129: 8362; Chem. Bio.
Chem. 2013 14: 58; Curr. Prot. Nucl. Acids Chem. 2011 1.24.1; Egli
et al. 2011 J. Am. Chem. Soc. 133: 16642; Hendrix et al. 1997 Chem.
Eur. J. 3: 110; Hyrup et al. 1996 Bioorg. Med. Chem. 4: 5; Imanishi
1997 Tet. Lett. 38: 8735; J. Am. Chem. Soc. 1994, 116, 3143; J.
Med. Chem. 2009 52: 10; J. Org. Chem. 2010 75: 1589; Jepsen et al.
2004 Oligo. 14: 130-146; Jones et al. J. Org. Chem. 1993, 58, 2983;
Jung et al. 2014 ACIEE 53: 9893; Kodama et al. 2014 AGDS; Koizumi
2003 BMC 11: 2211; Koizumi et al. 2003 Nuc. Acids Res. 12:
3267-3273; Koshkin et al. 1998 Tetrahedron 54: 3607-3630; Kumar et
al. 1998 Bioo. Med. Chem. Let. 8: 2219-2222; Lauritsen et al. 2002
Chem. Comm. 5: 530-531; Lauritsen et al. 2003 Bioo. Med. Chem.
Lett. 13: 253-256; Lima et al. 2012 Cell 150: 883-894; Mesmaeker et
al. Angew. Chem., Int. Ed. Engl. 1994, 33, 226; Migawa et al. 2013
Org. Lett. 15: 4316; Mol. Ther. Nucl. Acids 2012 1: e47; Morita et
al. 2001 Nucl. Acids Res. Supp. 1: 241-242; Morita et al. 2002
Bioo. Med. Chem. Lett. 12: 73-76; Morita et al. 2003 Bioo. Med.
Chem. Lett. 2211-2226; Murray et al. 2012 Nucl. Acids Res. 40:
6135; Nielsen et al. 1997 Chem. Soc. Rev. 73; Nielsen et al. 1997
J. Chem. Soc. Perkins Transl. 1: 3423-3433; Obika et al. 1997
Tetrahedron Lett. 38 (50): 8735-8; Obika et al. 1998 Tetrahedron
Lett. 39: 5401-5404; Obika et al. 2008 J. Am. Chem. Soc. 130: 4886;
Obika et al. 2011 Org. Lett. 13: 6050; Oestergaard et al. 2014 JOC
79: 8877; Pallan et al. 2012 Biochem. 51: 7; Pallan et al. 2012
Chem. Comm. 48: 8195-8197; Petersen et al. 2003 TRENDS Biotech. 21:
74-81; Prakash et al. 2010 J. Med. Chem. 53: 1636; Prakash et al.
2015 Nucl. Acids Res. 43: 2993-3011; Prakash et al. 2016 Bioorg.
Med. Chem. Lett. 26: 2817-2820; Rajwanshi et al. 1999 Chem. Commun.
1395-1396; Schultz et al. 1996 Nucleic Acids Res. 24: 2966; Seth et
al. 2008 Nucl. Acid Sym. Ser. 52: 553; Seth et al. 2009 J. Med.
Chem. 52: 10-13; Seth et al. 2010 J. Am. Chem. Soc. 132: 14942;
Seth et al. 2010 J. Med. Chem. 53: 8309-8318; Seth et al. 2010 J.
Org. Chem. 75: 1569-1581; Seth et al. 2011 BMCL 21: 4690; Seth et
al. 2012 Bioo. Med. Chem. Lett. 22: 296-299; Seth et al. 2012 Mol.
Ther-Nuc. Acids. 1, e47; Seth et al., Nucleic Acids Symposium
Series (2008), 52(1), 553-554; Singh et al. 1998 Chem. Comm.
1247-1248; Singh et al. 1998 J. Org. Chem. 63: 10035-39; Singh et
al. 1998 J. Org. Chem. 63: 6078-6079; Sorensen 2003 Chem. Comm.
2130-2131; Starrup et al. 2010 Nucl. Acids Res. 38: 7100; Swayze et
al. 2007 Nucl. Acids Res. 35: 687; Ts'o et al. Ann. N. Y. Acad.
Sci. 1988, 507, 220; Van Aerschot et al. 1995 Angew. Chem. Int. Ed.
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WO 20070900071; WO 2016/079181; U.S. Pat. Nos. 6,326,199;
6,066,500; and 6,440,739, the base and sugar modifications of each
of which is herein incorporated by reference.
[1115] In some embodiments, a C9orf72 oligonucleotide can comprise
any sugar described herein or known in the art. In some
embodiments, a C9orf72 oligonucleotide can comprise any sugar
described herein or known in the art in combination with any other
structural element or modification described herein, including but
not limited to, base sequence or portion thereof, base;
internucleotidic linkage; stereochemistry or pattern thereof,
additional chemical moiety, including but not limited to, a
targeting moiety, etc.; pattern of modifications of sugars, bases
or internucleotidic linkages; format or any structural element
thereof, and/or any other structural element or modification
described herein; and in some embodiments, the present disclosure
pertains to multimers of any such oligonucleotides.
Biological Applications
[1116] As described herein, provided compositions and methods are
capable of improving knockdown of RNA, including knockdown of
C9orf72 RNA transcripts. In some embodiments, provided compositions
and methods provide improved knockdown of C9orf72 transcripts
(including but not limited to those comprising a repeat expansion)
compared to a reference condition selected from the group
consisting of absence of the composition, presence of a reference
composition, and combinations thereof.
[1117] In some embodiment, a C9orf72 oligonucleotide is capable of
preferentially decreasing (knocking down) the expression, level
and/or activity of a mutant or repeat expansion-containing C9orf72
gene or gene product (e.g., one comprising a hexanucleotide repeat
expansion) relative to that of a wild-type or non-repeat
expansion-containing C9orf72 gene or gene product (e.g., one
lacking a hexanucleotide repeat expansion).
[1118] Preferential knockdown of repeat expansion-containing
C9orf72 oligonucleotides is illustrated, for example, in FIGS. 4A
and B. C9orf72 oligonucleotides WV-3688, WV-6408, WV-7658, WV-7659,
WV-8011 and WV-8012 were all able to preferentially knock down the
level of repeat expansion-containing C9orf72 RNA transcripts
relative to the level of non-repeat expansion-containing C9orf72
RNA transcripts (e.g., total transcripts, most of which are normal
transcripts which do not comprise a repeat expansion).
[1119] WV-3688, WV-6408, WV-7658, WV-7659, WV-8011, and WV-8012 all
have the base sequence of CCUCACTCACCCACTCGCCA (for WV-3688) or
CCTCACTCACCCACTCGCCA (the remainder), and have a sequence of:
mC*mCmUmCmA*C*T*C*A*C*C*C*A*C*T*mCmGmCmC*mA,
m5Ceo*m5CeoTeom5CeoAeo*C*T*C*A*C*C*C*A*C*T*m5CeoGeom5Ceom5Ceo*Aeo,
m5Ceo*Rm5CeoTeom5CeoAeo*RC*ST*SC*RA*SC*SC*RC*SA*SC*ST*Rm5CeoGeom5Ceom5Ceo
*RAeo,
m5Ceo*Rm5CeoTeom5CeoAeo*RC*ST*SC*RA*SC*SC*SC*SA*SC*ST*Rm5CeoGeom5C-
eom5Ceo*RAeo,
mC*Sm5CeoTeom5CeomA*SC*ST*SC*RA*SC*SC*SC*SA*SC*ST*SmC*SmG*SmC*SmC*SmA,
mC*Sm5CeoTeom5CeomA*SC*ST*SC*RA*SC*SC*RC*SA*SC*ST*SmC*SmG*SmC*SmC*SmA,
respectively. Total transcripts include V2, V3 and V1, both normal
(healthy, without repeat expansions) and mutant (pathological,
comprising a repeat expansion). Various transcripts are diagrammed
in FIG. 1. V1 is reportedly transcribed at very low levels (around
1% of the total C9orf72 transcripts) and does not contribute
significantly to the levels of transcripts comprising
hexanucleotide repeat expansions or to the levels of transcripts
detected in assays for V3 transcripts.
[1120] V1, V2 and V3 are naturally produced pre-mRNA variants of
the C9orf72 transcript produced by alternative pre-mRNA splicing.
DeJesus-Hernandez et al. 2011. In variants 1 and 3 the expanded
GGGGCC repeat is located in an intron between two alternatively
spliced exons, whereas in variant 2 the repeat is located in the
promoter region and thus not present in the transcript. V1 is
C9orf72 Variant 1 transcript, which represents the shortest
transcript and encodes the shorter C9orf72 protein (isoform b), see
NM_145005.5. V2 is C9orf72 Variant 2 transcript, which differs in
the 5' UTR and 3' coding region and UTR compared to variant 1. The
resulting C9orf72 protein (isoform a) is longer compared to isoform
1. Variants 2 and 3 encode the same C9orf72 protein; see
NM_018325.3. V3 is C9orf72 Variant 3 transcript, which differs in
the 5' UTR and 3' coding region and UTR compared to variant 1. The
resulting C9orf72 protein (isoform a) is longer compared to isoform
1; Variants 2 and 3 encode the same protein, see NM_001256054.1.
Transcript variants 1 and 3 are predicted to encode for a 481 amino
acid long protein encoded by C90RF72 exons 2-11 (NP_060795.1;
isoform a), whereas variant 2 is predicted to encode a shorter 222
amino acid protein encoded by exons 2-5 (NP_659442.2; isoform b).
It is noted that, according to some reports, the V1, V2 and V3
transcripts are not equally abundant; reportedly, V2 is the major
transcript, representing 90% of total transcripts, V3 representing
9%, and V1 representing 1%. Therefore, without being bound by any
particular theory, this disclosure suggests that a decrease in
total transcripts mediated by some C9orf72 oligonucleotides
includes representation of knockdown of repeat expansion-containing
transcripts. The data show that many C9orf72 oligonucleotides were
thus capable of mediating preferential knockdown of repeat
expansion-containing C9orf72 transcripts relative to non-repeat
expansion-containing C9orf72 transcripts. For example, WV-6408
achieved 80%: 35% knockdown of repeat associated transcripts (V3):
total (mostly normal) C9 mRNA. WV-3537 and WV-3174 were also
capable of mediating some preferential knockdown of repeat
expansion-containing transcripts. In contrast, C9orf72
oligonucleotides WV-3662 and WV-3536, representing the sequences of
SEQ ID NO: 0553 of WO2015054676 and the complement of SEQ ID NO:
0057 of WO2016168592, representatively, were not capable of
mediating preferential knockdown of repeat expansion-containing
C9orf72 transcripts relative to non-repeat expansion-containing
C9orf72 transcripts (FIGS. 4A and B).
[1121] In these experiments, patient derived ALS neurons (detailed
in Example 9) were used for screening. Negative control
oligonucleotide WV-2376 does not target C9orf72. Control
oligonucleotide WV-3542 is described in Table 1A. In FIGS. 4C and
4D, oligonucleotides were tested at 1 and 10 .mu.M.
[1122] FIGS. 5 and 6 present example data demonstrating the in vivo
capability of C9orf72 oligonucleotides to mediate preferential
knockdown of repeat expansion-containing C9orf72 transcripts in the
C9-BAC mouse spinal cord and cortex, respectively. Presented data
were those of: WV-6408, WV-8009, WV-8010, WV-8011, and WV-8012.
FIGS. 5A and 6A show knockdown of total transcripts (including
repeat expansion-containing and non-repeat expansion-containing
transcripts). FIGS. 5B and 6B show knockdown of V3 (repeat
expansion-containing) transcripts. FIGS. 5C and 6C show knockdown
of Intron/AS transcripts (with probes targeting a region 3' to the
repeat transcript expansion, the detected area includes both sense
and antisense transcripts of the intronic region). Additional
experimental details are provided in Example 9. Additional
information related to preferential knockdown of repeat
expansion-containing C9orf72 transcripts is presented herein.
[1123] In some embodiments, a C9orf72 oligonucleotide can
preferentially knockdown or decrease the expression, level and/or
activity of mutant (e.g., repeat expansion containing) V3 C9orf72
transcripts relative to the total C9orf72 transcripts.
[1124] In some embodiments, a C9orf72 oligonucleotide is capable of
mediating a decrease in the expression, activity and/or level of a
DPR protein translated from a repeat expansion.
[1125] In some embodiments, a C9orf72 oligonucleotide is capable of
mediating a decrease in the expression, activity and/or level of a
C9orf72 gene product. In some embodiments, a C9orf72 gene product
is a protein, such as a dipeptide repeat (DPR) protein. In some
embodiments, DPRs can be produced by RAN translation in any of the
six reading frames of a repeat-containing C9orf72 transcript. In
some embodiments, a dipeptide repeat protein is produced via RNA
(repeat-associated and non-ATG-dependent translation) of either the
sense or the antisense strand of a hexanucleotide repeat region.
DPR proteins are described, for example, in Zu et al. 2011 Proc.
Natl. Acad. Sci. USA 108: 260-265; Zu et al. Proc. Natl. Acad. Sci.
USA. 2013 Dec. 17; 110(51):E4968-77; Lopez-Gonzalez et al., 2016,
Neuron 92, 1-9; May et al. Acta Neuropathol (2014) 128:485-503; and
Freibaum et al. 2017 Front. Mol. Neurosci. 10, Article 35; and
Westergard et al., 2016, Cell Reports 17, 645-652. In some
embodiments, a C9orf72 dipeptide repeat is or comprises any of:
poly-(proline-alanine) (poly-PA or) or poly-(alanine-proline) or
(poly-AP); poly-(proline-arginine) (poly-PR) or
poly-(arginine-proline) (poly-RP); or poly-(proline-glycine)
(poly-PG) or poly-(glycine-proline (poly-GP). Poly-GA is reportedly
abundantly expressed in the C9orf72 brains, followed by poly-GP and
poly-GR, while poly-PA and poly-PR resulting from translation of
the antisense transcript are rare. Reportedly, Poly-GA and the
other DPR species are transmitted between cells and how DPR uptake
affects the receiving cells. Zhou et al. detected cell-to-cell
transmission of all hydrophobic DPR species and show that poly-GA
boosts repeat RNA levels and DPR expression, suggesting DPR
transmission may trigger a vicious cycle; treating cells with
anti-GA antibodies reduced intracellular aggregation of DPRs. Zhou
et al. 2017. EMBO Mol. Med. 9(5):687-702. Chang et al. reported
that Glycine-Alanine Dipeptide Repeat proteins form toxic amyloids
possessing cell-to-cell transmission properties. Chang et al. 2016.
J. Biol. Chem. 291: 4903-4911.
[1126] In some embodiments, a DPR protein is a polyGP. As
non-limiting examples, the amino acid sequence of a DPR protein is
or comprises any of:
TABLE-US-00003 GAGAGAGAGAGAGAGAGAGAWSGRARGRARGGAAVAVPAPA-
AAEAQAVASG, GPGPGPGPGPGPGPGPGPGRGRGGPGGGPGAGLRLRCLRPRR RRRRR-WRVGE,
or GRGRGRGRGRGRGRGRGRGVVGAGPGAGPGRGCGCGACARGG
GGAGG-GEWVSEEAASWRVAVWGSAAGKRRG (from a sense frame); or
PRPRPRPRPR-PRPRPRPRPLARDS, GPGPGPGPGPGPGPGPGP, or
PAPAPAPAPAPAPAPAPAPSARLLSS- RACYRLRLFPSLFSSG (from an antisense
frame).
[1127] As shown in FIG. 10 and detailed in Example 13, C9orf72
oligonucleotides WV-6408, WV-8009, WV-8010, WV-8011, and WV-8012
all reduced the level of polyGP (pGP, a dipeptide repeat protein)
in the hippocampus of C9-BAC mice. In addition, C9orf72
oligonucleotides WV-8549 and WV-8551 also reduced polyGP levels in
the mouse hippocampus (data not shown).
[1128] C9orf72 gene products also include foci, which comprise a
complex of a C9orf72 RNA or a portion thereof (e.g., an excised
intron) bound by multiple RNA-binding proteins. Foci are described
in, for example, Mori et al. 2013 Acta Neuropath. 125: 413-423. In
some embodiments, a C9orf72 oligonucleotide is capable of mediating
a decrease in the number of cells comprising a focus, and/or the
number of foci per cell.
[1129] As non-limiting example data, administration of C9orf72
oligonucleotides WV-7658 and WV-7659 in mouse demonstrated a 51.8%
and 62.2% decrease in the number of foci counted per 100 motor
neuron nuclei [compared to PBS (negative control)] in the spinal
cord anterior horn (location of the lower motor neurons); and 58.3%
and 70.9% decrease, respectively, in the number of cells with more
than 5 foci/cell; and a 49.1% and 55.0% decrease, respectively, in
the number of foci per 100 motor neurons.
[1130] Without wishing to be bound by any particular theory, the
present disclosure suggests that a significant knockdown of V3
C9orf72 transcript and/or decrease in the expression, activity
and/or level of a DPR protein and/or a decrease in the number of
cells comprising a focus, and/or the number of foci per cell can
lead to or be associated with a significant inhibition of cellular
pathology, with the underlying biology rationale that the expanded
hexanucleotide repeat allele leads to longer resident time of the
pre-spliced C9orf72 transcripts and the spliced intron, which makes
them more vulnerable to intronic targeting oligonucleotides.
Without wishing to be bound by any particular theory, the present
disclosure suggests that an about 50% knockdown of V3 C9orf72
transcript can lead to or be associated with an about 90%
inhibition of cellular pathology.
[1131] An improvement mediated by a C9orf72 oligonucleotide can be
an improvement of any desired biological functions, including but
not limited to treatment and/or prevention of a C9orf72-related
disorder or a symptom thereof. In some embodiments, a
C9orf72-related disorder is amyotrophic lateral sclerosis (ALS),
frontotemporal dementia (FTD), corticobasal degeneration syndrome
(CBD), atypical Parkinsonian syndrome, olivopontocerebellar
degeneration (OPCD), primary lateral sclerosis (PLS), progressive
muscular atrophy (PMA), Huntington's disease (HD) phenocopy,
Alzheimer's disease (AD), bipolar disorder, schizophrenia, or other
non-motor disorders. In some embodiments, a symptom of a
C9orf72-related disorder is selected from: agitation, anxiety,
blunted emotions, changes in food preference, decreased energy
and/or motivation, dementia, depression, difficulty in breathing,
difficulty in swallowing, difficulty in projecting the voice,
difficulty with respiration, distractibility, fasciculation and/or
cramping of muscles, impaired balance, impaired motor function,
inappropriate social behavior, lack of empathy, loss of memory,
mood swings, muscle twitching, muscle weakness, neglect of personal
hygiene, repetitive or compulsive behavior, shortness of breath,
slurring of speech, unsteady gait, vision abnormality, weakness in
the extremities.
[1132] In some embodiments, a symptom of a C9orf72-related disorder
is semantic dementia, decrease in language comprehension, or
difficulty in using correct or precise language. In some
embodiments, a c9orf72-related disorder or a symptom thereof is
corticobasal degeneration syndrome (CBD), shakiness, lack of
coordination, muscle rigidity and/or spasm, progressive
supranuclear palsy (PSP), a walking and/or balance problem,
frequent falls, muscle stiffness, muscle stiffness in the neck
and/or upper body, loss of physical function, and/or abnormal eye
movement.
[1133] In some embodiments, FTD is behavioral variant
frontotemporal dementia (bvFTD). In some embodiments, in bvFTD,
reportedly, the most significant initial symptoms are associated
with personality and behavior. In some embodiments, a c9orf72
oligonucleotide is capable of reducing the extent or rate at which
a subject experiences disinhibition, which presents as a loss of
restraint in personal relations and social life, as assessed
according to methods well-known in the art.
[1134] In some embodiments, the present disclosure provides a
method of treating a disease by administering a composition
comprising a first plurality of oligonucleotides sharing a common
base sequence comprising a common base sequence, which nucleotide
sequence is complementary to a target sequence in the target
C9orf72 transcript, [1135] the improvement that comprises using as
the oligonucleotide composition a stereocontrolled oligonucleotide
composition characterized in that, when it is contacted with the
C9orf72 transcript in an oligonucleotide or a knockdown system,
RNase H-mediated knockdown of the C9orf72 transcript is improved
relative to that observed under a reference condition selected from
the group consisting of absence of the composition, presence of a
reference composition, and combinations thereof.
[1136] Evaluation and Testing of Efficacy of C9orf72
Oligonucleotides
[1137] Various techniques and tools, including but not limited to
many known in the art, can be used for evaluation and testing of
C9orf72 oligonucleotides.
[1138] In some embodiments, evaluation and testing of efficacy of
C9orf72 oligonucleotides can be performed by quantifying a change
or improvement in the level, activity, expression, allele-specific
expression and/or intracellular distribution of a C9orf72 target
nucleic acid or a corresponding gene product following delivery of
a C9orf72 oligonucleotide. In some embodiments, delivery can be via
a transfection agent or without a transfection agent (e.g.,
gymnotic).
[1139] In some embodiments, evaluation and testing of efficacy of
C9orf72 oligonucleotides can be performed by quantifying a change
in the level, activity, expression and/or intracellular of a
C9orf72 gene product (including but not limited to a transcript,
DPR or focus) following introduction of a C9orf72 oligonucleotide.
C9orf72 gene products include RNA produced from a C9orf72 gene or
locus.
[1140] In some embodiments, the present disclosure provides a
method of identifying and/or characterizing an oligonucleotide
composition, the method comprising steps of: providing at least one
composition comprising a first plurality of oligonucleotides; and
assessing delivery relative to a reference composition.
[1141] In some embodiments, the present disclosure provides a
method of identifying and/or characterizing an oligonucleotide
composition, the method comprising steps of: [1142] providing at
least one composition comprising a first plurality of
oligonucleotides; and [1143] assessing cellular uptake relative to
a reference composition.
[1144] In some embodiments, properties of a provided
oligonucleotide compositions are compared to a reference
oligonucleotide composition.
[1145] In some embodiments, a reference oligonucleotide composition
is a stereorandom oligonucleotide composition. In some embodiments,
a reference oligonucleotide composition is a stereorandom
composition of oligonucleotides of which all internucleotidic
linkages are phosphorothioate. In some embodiments, a reference
oligonucleotide composition is a DNA oligonucleotide composition
with all phosphate linkages.
[1146] In some embodiments, a reference composition is a
composition of oligonucleotides having the same base sequence and
the same chemical modifications. In some embodiments, a reference
composition is a composition of oligonucleotides having the same
base sequence and the same pattern of chemical modifications. In
some embodiments, a reference composition is a chirally
un-controlled (or stereorandom) composition of oligonucleotides
having the same base sequence and chemical modifications.
[1147] In some embodiments, a reference composition is a
composition of oligonucleotides having the same base sequence but
different chemical modifications, including but not limited to
chemical modifications described herein. In some embodiments, a
reference composition is a composition of oligonucleotides having
the same base sequence but different patterns of internucleotidic
linkages and/or stereochemistry of internucleotidic linkages and/or
chemical modifications.
[1148] Various methods are known in the art for the detection of
C9orf72 gene products, the expression, level and/or activity of
which might be altered after introduction or administration of a
C9orf72 oligonucleotide. As non-limiting examples: C9orf72
transcripts and their knockdown can be quantified with qPCR,
C9orf72 protein levels can be determined via Western blot, RNA foci
by FISH (fluorescence in situ hybridization), DPRs by Western blot,
ELISA, or mass spectrometry. Commercially available C9orf72
antibodies include anti-C9orf72 antibody GT779 (1:2000; GeneTex,
Irvine, Calif.). In addition, functional assays can be performed on
motor neurons (MN) expressing wild-type and/or mutant C9orf72 by
Electrophysiology and NMJ formation.
[1149] In some embodiments, evaluation and testing of efficacy of
C9orf72 oligonucleotides can be performed in vitro in a cell. In
some embodiments, the cell is a cell which expresses C9orf72. In
some embodiments, a cell is a SH-SY5Y (human neuroblastoma) cell
engineered to express C9orf72. In some embodiments, a cell is a
SH-SY5Y cell engineering to express C9orf72, as described in WO
2016/167780. In some embodiments, a cell is a patient-derived cell,
patient-derived fibroblast, iPSC or iPSN. In some embodiments, a
cell is an iPSC derived neuron or motor neuron. Various cells
suitable for testing of a C9orf72 oligonucleotide include
patient-derived fibroblasts, iPSCs and iPSNs and described in, for
example, Donelly et al. 2013 Neuron 80, 415-428; Sareen et al. 2013
Sci. Trans. Med. 5: 208ra149; Swartz et al. STEM CELLS
TRANSLATIONAL MEDICINE 2016; 5:1-12; and Almeida et al. 2013 Acta
Neuropathol. 126: 385-399. In some embodiments, a cell is a BAC
transgenic mouse-derived cell, including without limitation, a
mouse embryonic fibroblast or cortical primary neuron. In some
embodiments, evaluation and testing involves a population of cells.
In some embodiments, a population of cells is a population of iCell
Neurons (also referenced as iNeurons), an iPS cell-derived mixed
population of human cerebral cortical neurons that exhibit native
electrical and biochemical activity, commercially available from
Cellular Dynamics International, Madison, Wis. Additional cells,
including Spinal Cord Motor Neurons, Midbrain, Dopaminergic
Neurons, Glutamatergic Neurons, GABAergic Neurons, Mixed Cortical
Neurons, Medium Spiny Striatal GABAergic Neurons,
Parvalbumin-Enriched Cortical GABAergic Neurons, Layer V Cortical
Glutamatergic Neurons, are commercially available from BrainXell,
Madison, Wis.
[1150] In some embodiments, evaluation of a C9orf72 oligonucleotide
can be performed in an animal. In some embodiments, an animal is a
mouse. C9orf72 mouse models and experimental procedures using them
are described in Hukema et al. 2014 Acta Neuropath. Comm. 2: 166;
Ferguson et al. 2016 J. Anat. 226: 871-891; Lagier-Tourenne et al.
Proc. Natl. Acad. Sci. USA. 2013 Nov. 19; 110(47):E4530-9; Koppers
et al. Ann. Neurol. 2015; 78:426-438; Kramer et al. 2016 Science
353: 708; Liu et al., 2016, Neuron 90, 521-534; Peters et al.,
2015, Neuron 88, 902-909; Picher-Martel et al. Acta
Neuropathologica Communications (2016) 4:70. A C9-BAC mouse model
is described herein (see Example 9).
[1151] In some embodiments, target nucleic acid levels can be
quantitated by any method known in the art, many of which can be
accomplished with kits and materials which are commercially
available, and which methods are well known and routine in the art.
Such methods include, e.g., Northern blot analysis, competitive
polymerase chain reaction (PCR), or quantitative real-time PCR. RNA
analysis can be performed on total cellular RNA or poly(A)+ mRNA.
Probes and primers are designed to hybridize to a C9orf72 nucleic
acid. Methods for designing real-time PCR probes and primers are
well known in the art.
[1152] In some embodiments, evaluation and testing of efficacy of
C9orf72 oligonucleotides can be performed using a luciferase assay.
A non-limiting example of such an assay is detailed in Example 3,
below. In some embodiments, a luciferase assay employs a construct
comprising the luciferase gene (or an efficacious portion thereof)
linked to a portion of the sense C9orf72 transcript, such as nt
1-374 or nt 158-900 (both of which comprise a hexanucleotide repeat
expansion). In some embodiments, nt 1-374 comprises exon 1a and the
intron between exons 1a and 1b. In some embodiments, a luciferase
assay employs a construct comprising the luciferase gene (or an
efficacious portion thereof) linked to a portion of the antisense
C9orf72 transcript, such as nt 900 to 1 (which comprises a
hexanucleotide repeat expansion). In some embodiments, a luciferase
assay is performed in a transfect COS-7 cell.
[1153] In some embodiments, a C9orf72 protein level can be
evaluated or quantitated in any method known in the art, including,
but not limited to, enzyme-linked immunosorbent assay (ELISA),
Western blot analysis (immunoblotting), immunocytochemistry,
fluorescence-activated cell sorting (FACS), immunohistochemistry,
immunoprecipitation, protein activity assays (for example, caspase
activity assays), and quantitative protein assays. Antibodies
useful for the detection of mouse, rat, monkey, and human C9orf72
are commercially available; additional antibodies to C9orf72 can be
generated via methods known in the art.
[1154] An assay for detecting levels of an oligonucleotide or other
nucleic acid is described herein (e.g., in Example 14). This assay
can be used to detect, as non-limiting examples, a C9orf72
oligonucleotide or any other nucleic acid of interest, including
nucleic acids or other oligonucleotides which do not target C9orf72
and nucleic acids.
[1155] Evaluation and testing of efficacy of C9orf72
oligonucleotides can be performed in vitro or in vivo by
determining the change in number of repeat RNA foci (or RNA foci)
in cells following delivery of the C9orf72 oligonucleotide. A
repeat RNA focus is a structure formed when a RNA comprising a
hexanucleotide repeat sequesters RNA-binding proteins, and is a
measure and/or cause of RNA-mediated toxicity. In some embodiments,
a RNA focus can be a sense or an antisense RNA focus. When a
C9orf72 oligonucleotide is administered in vivo to an animal, the
presence and/or number of RNA foci can be determined or examined in
the brain of the animal, or a portion thereof, such as, without
limitation, the cerebellum, cerebral cortex, hippocampus, thalamus,
medulla, or any other portion of the brain. The number of foci per
cell (e.g., up to 5 or greater than 5) or average thereof and/or
the number of cells comprising a focus can be determined after
delivery of a C9orf72 oligonucleotide. A decrease in any or all of
these numbers indicates the efficacy of a C9orf72 oligonucleotide.
RNA foci can be detected by an method known in the art, including,
but not limited to FISH (fluorescence in situ hybridization); a
non-limiting example of FISH is presented in Example 14.
[1156] Evaluation and testing of efficacy of C9orf72
oligonucleotides can be performed in vitro by determining the
change in haploinsufficiency in cells following delivery of the
C9orf72 oligonucleotide. Haploinsufficiency occurs, for example,
when a hexanucleotide repeat RNA acts as a negative effector on
C9orf72 transcription and/or expression of a C9orf72 gene, thus
decreasing the overall amount of C9orf72 transcript or gene
product. A decrease in haploinsufficiency indicates the efficacy of
a C9orf72 oligonucleotide.
[1157] In some embodiments, a C9orf72 oligonucleotide does not
significantly decrease the expression, activity and/or level of the
C9orf72 protein. In some embodiments, a C9orf72 oligonucleotide
decreases the expression, activity and/or level of a C9orf72 repeat
expansion or a gene product thereof, but does not significantly
decrease the expression, activity and/or level of the C9orf72
protein.
[1158] In some embodiments, a C9orf72 oligonucleotide (a) decreases
the expression, activity and/or level of a C9orf72 repeat expansion
or a gene product thereof, and (b) does not decrease the
expression, activity and/or level of C9orf72 to a degree sufficient
to cause a disease condition. Various disease conditions related to
insufficient production of C9orf72 include improper endosomal
trafficking, a robust immune phenotype characterized by myeloid
expansion, T cell activation, increased plasma cells, elevated
autoantibodies, immune-mediated glomerulonephropathy, and/or an
auto-immune response, as described in, for example, Farg et al.
2014 Human Mol. Gen. 23: 3579-3595; and Atanasio et al. Sci Rep.
2016 Mar. 16; 6:23204. doi: 10.1038/srep23204.
[1159] Evaluation and testing of efficacy of C9orf72
oligonucleotides can be performed in vivo. In some embodiments,
C9orf72 oligonucleotides can be evaluated and/or tested in animals.
In some embodiments, C9orf72 oligos can be evaluated and/or tested
in humans and/or other animals to mediate a change or improvement
in the level, activity, expression, allele-specific expression
and/or intracellular distribution and/or to prevent, treat,
ameliorate or slow the progress of a C9orf72-related disorder or at
least one symptom of a C9orf72-related disorder. In some
embodiments, such in vivo evaluation and/or testing can determine,
after introduction of a C9orf72 oligonucleotide, phenotypic
changes, such as, improved motor function and respiration. In some
embodiments, a motor function can be measured by a determination of
changes in any of various tests known in the art including: balance
beam, grip strength, hindpaw footprint testing (e.g., in an
animal), open field performance, pole climb, and rotarod. In some
embodiments, respiration can measured by a determination of changes
in any of various tests known in the art including: compliance
measurements, invasive resistance, and whole body
plethysmograph.
[1160] In some embodiments, the testing of the efficacy of a
C9orf72 oligonucleotide be accomplished by contacting a motor
neuron cell from a subject with a neurological disease with the
C9orf72 oligonucleotide and determining whether the motor neuron
cell degenerates. If the motor neuron cell does not degenerate, the
C9orf72 oligonucleotide may be capable of reducing or inhibiting
motor neuron degeneration. The motor neuron cell may be derived
from a pluripotent stem cell. The pluripotent stem cell may have
been reprogrammed from a cell from the subject. The cell from the
subject may be a somatic cell, for example. The somatic cell may be
a fibroblast, a lymphocyte, or a keratinocyte, for example. The
assessment of whether a motor neuron cell degenerates or not may be
based on a comparison to a control. In some embodiments, the
control level may be a predetermined or reference value, which is
employed as a benchmark against which to assess the measured and/or
visual result. The predetermined or reference value may be a level
in a sample (e.g. motor neuron cell) from a subject not suffering
from a neurological disease or from a sample from a subject
suffering from a neurological disease but wherein the motor neuron
cell is not contacted with the C9orf72 oligonucleotide. The
predetermined or reference value may be a level in a sample from a
subject suffering from a neurological disease. In any of these
screening methods, the cell from the subject having the
neurological disease may comprise the (GGGGCC)n hexanucleotide
expansion in C9orf72.
[1161] The efficacy of C9orf72 can also be tested in suitable test
animals, such as those described in, as non-limiting examples:
Peters et al. 2015 Neuron. 88(5):902-9; O'Rourke et al. 2015
Neuron. 88(5): 892-901; and Liu et al. 2016 Neuron. 90(3):521-34.
In some embodiments, a test animal is a C9-BAC mouse. The efficacy
of C9orf72 can also be tested in C9-BAC transgenic mice with 450
repeat expansions, which were also described in Jiang et al. 2016
Neuron 90, 1-16.
[1162] In some embodiments, in a test animal, levels of various
C9orf72 transcripts can be determined, as can be C9orf72 protein
level, RNA foci, and levels of DPRs (dipeptide repeat proteins).
Tests can be performed on C9orf72 oligonucleotides and in
comparison with reference oligonucleotides. Several C9orf72
oligonucleotides disclosed herein are capable of reducing the
percentage of cells comprising RNAi foci and the average number of
foci per cell (data shown below and data not shown). Several
C9orf72 oligonucleotides disclosed herein are capable of reducing
the level of DPRs such as polyGP. As shown in FIG. 10, C9orf72
oligonucleotides WV-6408, WV-8009, WV-8010, WV-8011, and WV-8012
all reduced the level of polyGP (pGP, a dipeptide repeat protein)
in the hippocampus of C9-BAC mice. In addition, C9orf72
oligonucleotides WV-8549 and WV-8551 also reduced polyGP levels in
the mouse hippocampus (data not shown).
[1163] In some embodiments, a c9orf72 oligonucleotide is capable of
reducing the extent or rate of neurodegeneration caused by ALS, FTD
or other c9orf72-related disorder. In some embodiments, in addition
to an improvement, or at least reduction in the extent or rate of
deterioration of any nervous system tissue, in behavioral symptoms,
therapeutic efficacy of a c9orf72 oligonucleotide in a subject or
other animal can also be monitored with brain scans, e.g., CAT
scan, functional MRI, or PET scan, or other methods known in the
art.
[1164] Various assays for analysis of C9orf72 oligonucleotides are
described herein, for example in Example 9, 13, and 14, and
include, inter alia, Reporter assay (Luciferase Assay), e.g.,
performed in an ALS neuron, and measuring, for example, analysis of
V3/intron expression, activity and/or level; stability assay; TLR9
assay; Complement assay; PD (Pharmacodynamics) (C9-BAC, icv or
Intracerebroventricular injection), e.g., PD and/or efficacy tested
in C9orf72-BAC (C9-BAC) mouse model; in vivo procedures, including
but not limited to injection into a lateral ventricle or other
areas of the central nervous system (including but not limited to
cortex and spinal cord) of a test animal, such as a mouse; analysis
of number of foci and/or number of cells comprising foci: PolyGP
(or pGP or DPR assay).
[1165] In some embodiments, selection criteria are used to evaluate
the data resulting from the various assays and to select
particularly desirable C9orf72 oligonucleotides. In some
embodiments, at least one selection criterion is used. In some
embodiments, two or more selection criteria are used. In some
embodiments, selection criteria for a Luciferase assay (e.g.,
V3/intron knockdown) is at least partial knockdown of the V3
introns and/or at least partial knockdown of the intron transcript.
In some embodiments, selection criteria for a Luciferase assay
(e.g., V3/intron knockdown) is 50% KD (knockdown) of the V3 introns
and 50% KD of the intron transcript. In some embodiments, selection
criteria include a determination of IC.sub.50. In some embodiments,
selection criteria include an IC.sub.50 of less than about 10 nM,
less than about 5 nM or less than about 1 nM. In some embodiments,
selection criteria for a stability assay is at least 50% stability
[a level of at least 50% of the oligonucleotide is still remaining
and/or detectable] at Day 1. In some embodiments, selection
criteria for a stability assay is at least 50% stability at Day 2.
In some embodiments, selection criteria for a stability assay is at
least 50% stability at Day 3. In some embodiments, selection
criteria for a stability assay is at least 50% stability at Day 4.
In some embodiments, selection criteria for a stability assay is at
least 50% stability at Day 5. In some embodiments, selection
criteria for a stability assay is 80% [at least 80% of the
oligonucleotide remains] at Day 5. In some embodiments, selection
criteria is at least partial knockdown in number of foci and/or
number of cells comprising foci. In some embodiments, selection
criteria is at least 50% KD (knockdown) in number of foci and/or
number of cells comprising foci. In some embodiments, selection
criteria include lack of activation in a TLR9 assay. In some
embodiments, selection criteria include lack of activation in a
complement assay. In some embodiments, selection criteria include
knockdown in a lateral ventricle or other area of the central
nervous system (including but not limited to cortex and spinal
cord) of a test animal, such as a mouse. In some embodiments,
selection criteria include knockdown by at least 50% in a lateral
ventricle or other area of the central nervous system (including
but not limited to cortex and spinal cord) of a test animal, such
as a mouse. In some embodiments, selection criteria include a
knockdown in the expression, activity and/or level of DPR protein.
In some embodiments, selection criteria include a knockdown in the
expression, activity and/or level of DPR protein. In some
embodiments, selection criteria include a knockdown in the
expression, activity and/or level of DPR protein by at least 50%.
In some embodiments, selection criteria include a knockdown in the
expression, activity and/or level of the DPR protein PolyGP by at
least 50%.
[1166] Oligonucleotides which have been evaluated and tested for
efficacy in knocking down C9orf72 have various uses, including
administration for use in treatment or prevention of a
C9orf72-related disorder or a symptom thereof.
[1167] Assay for Detecting Target Nucleic Acids of Interest
[1168] In some embodiments, the present disclosure pertains to a
hybridization assay for detecting and/or quantifying a target
nucleic acid (e.g., a target oligonucleotide), wherein the assay
utilizes a capture probe, which is at least partially complementary
to the target nucleic acid, and a detection probe; wherein the
detection probe or a complex comprising the capture probe, the
detection probe and the target nucleic acid is capable of being
detected. Such an assay can be used to detect a C9orf72
oligonucleotide (e.g., in a tissue or fluid sample), or used to
detect any target nucleic acid (to any target or sequence) in any
sample. In some embodiments, the capture probe comprises a primary
amine, which is capable of reacting to an amino-reactive solid
support, thereby immobilizing the probe on the solid support. In
some embodiments, the amino-reactive solid support comprises maleic
anhydride. Immobilization of the probe can be performed with click
chemistry using an alkyne and an azide moiety on the probe and the
solid support. For click chemistry, the alkyne or azide can be, for
example, at the 5' or 3' end of the probe, and can optionally be
attached via a linker. For the click chemistry, the solid support,
for example, comprises an alkyne or an azide moiety. In some
embodiments, click chemistry includes that described in, as a
non-limiting example, Kolb et al. 2011 Angew. Chem. Int. Ed. 40:
2004-2021.
[1169] In some embodiments, a probe or complex which is capable of
being detected directly or indirectly is involved in producing a
detectable signal. In some embodiments, a probe or complex is (a)
capable of producing a detectable signal in the absence of another
chemical component (as a non-limiting example, having a moiety
capable of producing a detectable signal, such as a fluorescent dye
or radiolabel), or (b) comprises a ligand, label or other component
which, when bound by an appropriate second moiety, is capable of
producing a detectable signal. In some embodiments, a probe or
complex of type (b) comprises a label such as biotin, digoxigenin,
hapten, ligand, etc., which can be bound by an appropriate second
chemical entity such as an antibody which, when bound to the label,
is capable of producing a signal, e.g., via a radiolabel,
chemiluminesce, dye, alkaline phosphatase signal, peroxidase
signal, etc.
[1170] In some embodiments, the capture probe is immobilized on a
solid support. In some embodiments, the capture probe is
hybridized, bound or ligated to the target nucleic acid, and the
detection probe is also hybridized, bound or ligated to the target
nucleic acid, and the complex is capable of being detected. Many
variants of hybridization assays are known in the art. In some
embodiments, in a hybridization assay, the capture and the
detection probe are the same probe, and a single-stranded nuclease
is used to degrade probe which is not bound (or not fully bound) to
a target nucleic acid.
[1171] In some embodiments, the present disclosure pertains to a
hybridization assay for detecting and/or quantifying a target
nucleic acid (e.g., a target oligonucleotide), wherein a probe
(e.g., a capture probe) is at least partially complementary to the
target nucleic acid and comprises a primary amine, wherein the
primary amine is capable of reacting to an amino-reactive solid
support, thereby immobilizing the probe on the solid support. The
primary amine can be, for example, at the 5' or 3' end of the
probe, and can optionally be attached via a linker. In some
embodiments, the amino-reactive solid support comprises maleic
anhydride.
[1172] The target oligonucleotide can be, for example, a C9orf72
oligonucleotide or an oligonucleotide to any target of
interest.
[1173] In some embodiments, the assay is a hybridization assay,
sandwich hybridization assay, competitive hybridization assay, dual
ligation hybridization assay, nuclease hybridization assay, or
electrochemical or electrochemical hybridization assay.
[1174] In some embodiments, the assay is a sandwich hybridization
assay, wherein a capture probe is bound to a solid support and is
capable of annealing to a portion of the target oligonucleotide;
wherein a detection probe is capable of being detected and is
capable of annealing to another portion of the target
oligonucleotide; and wherein the hybridization of both the capture
probe and the detection probe to the target oligonucleotide
produces a complex which is capable of being detected.
[1175] In some embodiments, the assay is a nuclease hybridization
assay and the capture probe is a cutting probe fully complementary
to the target oligonucleotide, wherein a cutting probe which is
bound by full-length target oligonucleotides is capable of being
detected; and wherein a cutting probe which is free (not bound to a
target oligonucleotide) or which is bound to a shortmer, metabolite
or degradation product of a target oligonucleotide is degraded by S
nuclease treatment and therefore does not produce a detectable
signal.
[1176] In some embodiments, the assay is a hybridization-ligation
assay, wherein the capture probe is a template probe, which is
fully complementary to the target oligonucleotide and is intended
to serve as a substrate for ligase-mediated ligation of the target
oligonucleotide and a detection probe.
[1177] In some embodiments, the present disclosure pertains to a
method of detecting and/or quantifying a target nucleic acid (e.g.,
a target oligonucleotide), for example, in a sample, e.g., a tissue
or fluid, comprising the steps of (1) providing a capture probe,
wherein the capture probe is at least partially complementary to
the target nucleic acid and comprises a primary amine, wherein the
primary amine is capable of being bound by an amino-reactive solid
support, thereby immobilizing the probe on the solid support; (2)
immobilizing the capture probe to the solid support; (3) providing
a detection probe, wherein the detection probe is at least
partially complementary to the target nucleic acid (e.g., in a
region of the target nucleic acid different from the region to
which the capture probe binds) and is capable of directly or
indirectly producing a signal; wherein steps (2) and (3) can be
performed in either order; (4) bringing the tissue or fluid in
contact with the capture probe and detection probe under conditions
suitable for hybridization of the probes to the target nucleic
acid; (5) removing detection probe not hybridized to the target
nucleic acid; and (6) detecting for the signal directly or
indirectly produced by the detection probe, wherein detection of
the signal indicates the detection and/or quantification of the
target nucleic acid.
[1178] In some embodiments, the target oligonucleotide is a C9orf72
oligonucleotide. In some embodiments, the target oligonucleotide is
not a C9orf72 oligonucleotide. In some embodiments, a target
nucleic acid is an oligonucleotide, an antisense oligonucleotide, a
siRNA agent, a double-stranded siRNA agent, a single-stranded siRNA
agent, or a nucleic acid associated with a disease (e.g., a gene or
gene product which is expressed or over-expressed in a disease
state, such as a transcript whose abundance is increased in cancer
cells, or which nucleic acid comprises a mutation associated with a
disease or disorder).
[1179] In some embodiments, the amino-reactive solid support
comprises maleic anhydride.
[1180] FIG. 11. FIG. 11A shows an example hybridization ELISA assay
for measuring target oligonucleotide (e.g., ASO) levels, e.g., in
tissues and fluids, including but not limited to animal biopsies.
FIG. 11B shows example chemistry for binding a primary
amine-labeled capture probe to an amino-reactive solid support,
such as a plate comprising maleic anhydride.
[1181] The target oligonucleotide is reannealed to the detection
probe, and then combined with the capture probe, which is attached
to an amino-reactive plate via a primary amine label. Dual
hybridization (e.g., sandwich hybridization) occurs between the
capture probe, detection probe and the target oligonucleotide; a
gap (not shown in FIG. 11A) is allowable between the capture probe
and detection probe, leaving a single-stranded portion of the
target oligonucleotide not bound to the capture or detection probe.
The solid support (e.g., a plate surface) comprises maleic
anhydride (e.g., a maleic anhydride activated plate), which
spontaneously reacts with the primary amine label on the end of a
capture probe (e.g., at pH 8 to 9), immobilizing the probe to the
solid support. In some embodiments, a solid support is a plate,
tube, filter, bead, polymeric bead, gold, particle, well, or
multiwell plate.
[1182] As a non-limiting example, the following conditions can be
used:
Coating: 500 nM in 2.5% Na2CO3 pH9.0 50 ul/well, 37 C, 2 hr
Sample/Detection probe: 300 nM Detect probe as diluent, 4 C, O/N
Streptavidin-AP: 1:2000 in PBST 50 ul/well, RT, 1-2 hr Substrate
AttoPhos: 100 ul/well, RT, 5 min read
[1183] For example: The target nucleic acid is preannealed to the
detection probe, and then combined with the capture probe, which is
attached to a plate via a click chemistry using an alkyne (azide)
moiety on the probe and the solid support. Dual hybridization
(e.g., sandwich hybridization) occurs between the capture probe,
detection probe and the target nucleic acid; a gap is allowable
between the capture probe and detection probe, leaving a
single-stranded portion of the target oligonucleotide not bound to
the capture or detection probe. The solid support (e.g., a plate
surface) comprises alkyne (or azide) moiety, which reacts with the
azide (or alkyne) moiety label on the end of a capture probe with
click chemistry, immobilizing the probe to the solid support. In
some embodiments, a solid support is a plate, tube, filter, bead,
polymeric bead, gold, particle, well, or multiwell plate.
[1184] A non-limiting example of an assay is provided below:
[1185] Hybridization ELISA assay to measure target oligonucleotide
level in tissues, including animal biopsies:
[1186] The reverse complement sequence of the target
oligonucleotide can be divided into 2 segments, each represented by
a capture or detection probe. The 5'- sequence (of the target
oligonucleotide) can be 5-15 nt; the 3' sequence can be 5-15 nt.
However, the 5'-probe sequence (hybridizing to the 3'-portion of
the target oligonucleotide) should not overlap the 3' probe
sequence when they are both hybridized to the target
oligonucleotide. A gap between 5'- probe and 3'-probe is allowable.
Each probe should have a melting temperature (Tm) at least 25 C,
preferably >45 C, even more preferably >50 C. To achieve high
Tm, modified nucleotides can be used, such as Locked Nucleic Acids
(LNA) or Peptide Nucleic Acids (PNA). Other nucleotides in the
probe can be either DNA or RNA nucleotides or any other forms of
modified nucleotides, such as those having a 2'-OMe, 2'-F, or
2'-MOE modification.
[1187] The 5'-probe can also be labeled with a detection moiety
with a linker at the 5'-position. This probe is the Detection
Probe.
[1188] The 5'-probe (hybridizing to the 3'-portion of the target
oligonucleotide) can be labeled with a primary amine with a linker
at the 5'-position. This probe is the Capture Probe. The linker is
used to link the primary amine to the probe nucleotides. The linker
can be a C6-, C12- linker, PEG, TEG or any nucleotide sequence not
related to the oligonucleotide (such as oligo dT). A 5'-primary
amine with a linker can be put on during synthesis or post
synthesis.
[1189] The 3'-probe can also be labeled with primary amine with a
linker sequences at 3'-position. This probe is the Capture
Probe.
[1190] The 3'-probe (hybridizing to the 5'-portion of the target
oligonucleotide) can be labeled with a detection moiety with a
linker at the 3'-position. This probe is the Detection Probe. The
detection moiety can be biotin, digoxigenin, HaloTag.RTM. ligand
(Promega, Madison, Wis.), or any other hapten. The detection moiety
can also be Sulfo-Tag (Meso Scale Diagnostics, Rockville, Md.). The
linker is used to link the detection moiety with the probe
nucleotides. The linker can be a C6-, C12-linker, PEG, TEG or any
nucleotide sequence not related to oligonucleotide (such as oligo
dT). A 3'-detection moiety with a linker can be put on during
synthesis or post synthesis.
[1191] The Capture Probes (with a primary amine either at the 5'-
or 3'- end of probe) can be immobilized on a solid surface
activated to react with a primary amine, such as Maleic Anhydride
Activated Plates (Pierce; available from ThermoFisher, Waltham,
Mass.) or N-oxysuccinimide (NOS) activated DNA-BIND plate (Corning
Life Sciences, Tewksbury, Mass.). The plate can also be other kind
of plates activated for amine conjugation, such as MSD plate (Meso
Scale Diagnostics, Rockville, Md.). The surface can be a solid
support such as beads, gold particles, carboxylated polystyrene
microparticles (MagPlex Microspheres, Luminex Corporation;
available from ThermoFisher, Waltham, Mass.), or Dynabeads (Thermo
Fisher Scientific, Waltham, Mass.), so that flow based assay
platform can be used, such as Luminex or bead-array platform
(BD.TM. Cytometric Bead Array--CBA, BD Biosciences, San Jose,
Calif.).
[1192] The biological samples containing the target
oligonucleotide, such as tissue lysates or liquid biological fluids
(plasma, blood, serum, CSF, urine, or other tissue or fluid), are
mixed with the detection probe at a proper concentration of the
oligonucleotide and detection probe, heat-denatured then put on
surfaces coated with Capture Probes (plates or microparticles) to
promote sequence specific hybridization either at room temperature
or 4 C for a period of time (hybridization), in an appropriate
hybridization buffer. Excessive detection probes are removed by
washing the surfaces (plates or beads). Then the surface is
incubated with reagents which recognize the detection moieties,
such as avidin/streptavidin for biotin, antibodies to DIG or
haptens, or HaloTag to its ligand.
[1193] The detection reagents are usually labeled with an enzyme,
such as horseradish peroxidase (HRP) or alkaline phosphatase (AP),
or fluorophores or Sulfo-Tag. After extensive washes, enzyme
labeled detection reagents are detected by adding respective
substrates, such as TMB for HRP or AttoPhos for AP, and plates are
read by plate reader in absorbance mode or fluorescence mode
(fluorescent substrates). In some embodiments, a label comprises
Fluorescein, B-Phycoerythrin, Rhodamine, Cyanine Dye,
Allophycocyanin or a variant or derivative thereof.
[1194] Fluorophore labeled detection reagents can be used for
flow-based detection platform, such as Luminex or Bead-array
platform.
[1195] Sulfo-Tagged detection reagents can be read by MSD reader
(Meso Scale Discovery) directly.
[1196] The oligonucleotide amount can be calculated using a
standard curve of serial dilution of test articles run in the same
assay.
[1197] Another non-limiting example of a hybridization assay is
provided in Example 14.
[1198] Various assays for utility of oligonucleotides (including
but not limited to C9orf72 oligonucleotides) are described herein
and/or known in the art.
[1199] Administration of Provided Oligonucleotides and Compositions
Thereof
[1200] In some embodiments, provided oligonucleotides are capable
of directing a decrease in the expression and/or level of a target
gene or its gene product.
[1201] In some embodiments, a target gene is a C9orf72 comprising a
hexanucleotide repeat expansion.
[1202] In some embodiments, a provided oligonucleotide composition
is administered at a dose and/or frequency lower than that of an
otherwise comparable reference oligonucleotide composition with
comparable effect in improving the knockdown of a target,
including, as a non-limiting example, a C9orf72 transcript. In some
embodiments, a stereocontrolled oligonucleotide composition is
administered at a dose and/or frequency lower than that of an
otherwise comparable stereorandom reference oligonucleotide
composition with comparable effect in improving the knockdown of
the target C9orf72 transcript.
[1203] In some embodiments, the present disclosure recognizes that
properties, e.g., improved knockdown activity, etc. of
oligonucleotides and compositions thereof can be optimized by
chemical modifications and/or stereochemistry. In some embodiments,
the present disclosure provides methods for optimizing
oligonucleotide properties through chemical modifications and
stereochemistry.
[1204] In some embodiments, the present disclosure provides a
method of administering a oligonucleotide composition comprising a
first plurality of oligonucleotides and having a common nucleotide
sequence, the improvement that comprises: [1205] administering an
oligonucleotide comprising a first plurality of oligonucleotides
that is characterized by improved delivery relative to a reference
oligonucleotide composition of the same common nucleotide
sequence.
[1206] In some embodiments, provided C9orf72 oligonucleotides,
compositions and methods provide improved delivery. In some
embodiments, provided oligonucleotides, compositions and methods
provide improved cytoplasmatic delivery. In some embodiments,
improved delivery is to a population of cells. In some embodiments,
improved delivery is to a tissue. In some embodiments, improved
delivery is to an organ. In some embodiments, improved delivery is
to the central nervous system or a portion thereof, e.g., CNS. In
some embodiments, improved delivery is to an organism. Example
structural elements (e.g., chemical modifications, stereochemistry,
combinations thereof, etc.), oligonucleotides, compositions and
methods that provide improved delivery are extensively described in
this disclosure.
[1207] Various dosing regimens can be utilized to administer
provided chirally controlled oligonucleotide compositions. In some
embodiments, multiple unit doses are administered, separated by
periods of time. In some embodiments, a given composition has a
recommended dosing regimen, which may involve one or more doses. In
some embodiments, a dosing regimen comprises a plurality of doses
each of which are separated from one another by a time period of
the same length; in some embodiments, a dosing regimen comprises a
plurality of doses and at least two different time periods
separating individual doses. In some embodiments, all doses within
a dosing regimen are of the same unit dose amount. In some
embodiments, different doses within a dosing regimen are of
different amounts. In some embodiments, a dosing regimen comprises
a first dose in a first dose amount, followed by one or more
additional doses in a second dose amount different from the first
dose amount. In some embodiments, a dosing regimen comprises a
first dose in a first dose amount, followed by one or more
additional doses in a second (or subsequent) dose amount that is
same as or different from the first dose (or another prior dose)
amount. In some embodiments, a dosing regimen comprises
administering at least one unit dose for at least one day. In some
embodiments, a dosing regimen comprises administering more than one
dose over a time period of at least one day, and sometimes more
than one day. In some embodiments, a dosing regimen comprises
administering multiple doses over a time period of at least week.
In some embodiments, the time period is at least 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or
more (e.g., about 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100
or more) weeks. In some embodiments, a dosing regimen comprises
administering one dose per week f or more than one week. In some
embodiments, a dosing regimen comprises administering one dose per
week for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100 or more) weeks. In some embodiments, a
dosing regimen comprises administering one dose every two weeks f
or more than two week period. In some embodiments, a dosing regimen
comprises administering one dose every two weeks over a time period
of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40 or more (e.g., about 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100 or more) weeks. In some embodiments, a dosing
regimen comprises administering one dose per month for one month.
In some embodiments, a dosing regimen comprises administering one
dose per month f or more than one month. In some embodiments, a
dosing regimen comprises administering one dose per month for 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, or more months. In some embodiments,
a dosing regimen comprises administering one dose per week for
about 10 weeks. In some embodiments, a dosing regimen comprises
administering one dose per week for about 20 weeks. In some
embodiments, a dosing regimen comprises administering one dose per
week for about 30 weeks. In some embodiments, a dosing regimen
comprises administering one dose per week for 26 weeks. In some
embodiments, an oligonucleotide is administered according to a
dosing regimen that differs from that utilized for a chirally
uncontrolled (e.g., stereorandom) oligonucleotide composition of
the same sequence, and/or of a different chirally controlled
oligonucleotide composition of the same sequence. In some
embodiments, an oligonucleotide is administered according to a
dosing regimen that is reduced as compared with that of a chirally
uncontrolled (e.g., stereorandom) oligonucleotide composition of
the same sequence in that it achieves a lower level of total
exposure over a given unit of time, involves one or more lower unit
doses, and/or includes a smaller number of doses over a given unit
of time. In some embodiments, an oligonucleotide is administered
according to a dosing regimen that extends for a longer period of
time than does that of a chirally uncontrolled (e.g., stereorandom)
oligonucleotide composition of the same sequence Without wishing to
be limited by theory, Applicant notes that in some embodiments, the
shorter dosing regimen, and/or longer time periods between doses,
may be due to the improved stability, bioavailability, and/or
efficacy of a chirally controlled oligonucleotide composition. In
some embodiments, an oligonucleotide has a longer dosing regimen
compared to the corresponding chirally uncontrolled oligonucleotide
composition. In some embodiments, an oligonucleotide has a shorter
time period between at least two doses compared to the
corresponding chirally uncontrolled oligonucleotide composition.
Without wishing to be limited by theory, Applicant notes that in
some embodiments longer dosing regimen, and/or shorter time periods
between doses, may be due to the improved safety of a chirally
controlled oligonucleotide composition.
[1208] In some embodiments, with their improved delivery (and other
properties), provided compositions can be administered in lower
dosages and/or with lower frequency to achieve biological effects,
for example, clinical efficacy.
[1209] A single dose can contain various amounts of
oligonucleotides. In some embodiments, a single dose can contain
various amounts of a type of chirally controlled oligonucleotide,
as desired suitable by the application. In some embodiments, a
single dose contains about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300 or more (e.g., about
350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,
1000 or more) mg of a type of chirally controlled oligonucleotide.
In some embodiments, a single dose contains about 1 mg of a type of
chirally controlled oligonucleotide. In some embodiments, a single
dose contains about 5 mg of a type of chirally controlled
oligonucleotide. In some embodiments, a single dose contains about
10 mg of a type of chirally controlled oligonucleotide. In some
embodiments, a single dose contains about 15 mg of a type of
chirally controlled oligonucleotide. In some embodiments, a single
dose contains about 20 mg of a type of chirally controlled
oligonucleotide. In some embodiments, a single dose contains about
50 mg of a type of chirally controlled oligonucleotide. In some
embodiments, a single dose contains about 100 mg of a type of
chirally controlled oligonucleotide. In some embodiments, a single
dose contains about 150 mg of a type of chirally controlled
oligonucleotide. In some embodiments, a single dose contains about
200 mg of a type of chirally controlled oligonucleotide. In some
embodiments, a single dose contains about 250 mg of a type of
chirally controlled oligonucleotide. In some embodiments, a single
dose contains about 300 mg of a type of chirally controlled
oligonucleotide. In some embodiments, a chirally controlled
oligonucleotide is administered at a lower amount in a single dose,
and/or in total dose, than a chirally uncontrolled oligonucleotide.
In some embodiments, a chirally controlled oligonucleotide is
administered at a lower amount in a single dose, and/or in total
dose, than a chirally uncontrolled oligonucleotide due to improved
efficacy. In some embodiments, a chirally controlled
oligonucleotide is administered at a higher amount in a single
dose, and/or in total dose, than a chirally uncontrolled
oligonucleotide. In some embodiments, a chirally controlled
oligonucleotide is administered at a higher amount in a single
dose, and/or in total dose, than a chirally uncontrolled
oligonucleotide due to improved safety.
[1210] Treatment of C9orf72-Related Disorders or a Symptom
Thereof
[1211] In some embodiments, provided oligonucleotides are capable
of directing a decrease in the expression, level and/or activity of
a C9orf72 target gene or a gene product thereof. In some
embodiments, an C9orf72-related disorder is a disorder related to,
caused and/or associated with abnormal or excessive activity, level
and/or expression of, a deleterious mutation in, or abnormal tissue
or inter- or intracellular distribution of an C9orf72 gene or a
gene product thereof. In some embodiments, a C9orf72-related
disorder is amyotrophic lateral sclerosis (ALS), frontotemporal
dementia (FTD), corticobasal degeneration syndrome (CBD), atypical
Parkinsonian syndrome, olivopontocerebellar degeneration (OPCD),
primary lateral sclerosis (PLS), progressive muscular atrophy
(PMA), Huntington's disease (HD) phenocopy, Alzheimer's disease
(AD), bipolar disorder, schizophrenia, or other non-motor
disorders. Symptoms of a C9orf72-related disorder include those
described herein and known in the art.
[1212] Without wishing to be bound by any particular theory or
terminology, the present specification notes that, with the
understanding of C9orf72-related diseases constantly evolving, the
exact labeling of various c9orf72-related diseases is also
reportedly evolving. In some embodiments, c9orf72 oligonucleotides
are useful for decreasing levels of hexanucleotide
repeat-containing mutant alleles of C9orf72 (at the protein and/or
mRNA level) and/or decrease the level of dipeptide repeat proteins
produced from hexanucleotide-repeat-containing mutant C9orf72 mRNA,
wherein the oliognucleotides are useful for treating a C9orf72
related disease.
[1213] In some embodiments, a c9orf72-related disorder is FTD. In
some embodiments, FTD is an abbreviation for frontotemporal
dementia or frontotemporal degeneration. In some embodiments,
frontotemporal degeneration (FTD) is a disease process that affects
the frontal and temporal lobes of the brain. It causes a group of
disorders characterized by changes in behavior, personality,
language, and/or movement. Clinical diagnoses of FTD include any
one or more of: behavioral variant FTD (bvFTD), primary progressive
aphasia (PPA), and the movement disorders progressive supranuclear
palsy (PSP) and corticobasal degeneration (CBD). In some
embodiments, a patient suffering from or susceptible to PPA, PSP or
CBD does not exhibit or identify with dementia. In some
embodiments, frontotemporal dementia is equivalent to or
characterized by the symptoms of bvFTD.
[1214] The present disclosure pertains to methods of using
oligonucleotides disclosed herein which are capable of targeting
C9orf72 and useful for treating and/or manufacturing a treatment
for a C9orf72-related disorder. In some embodiments, a base
sequence of an oligonucleotide can comprise or consist of a base
sequence which has a specified maximum number of mismatches from a
specified base sequence.
[1215] In some embodiments, the present disclosure pertains to the
use of a composition of comprising a C9orf72 oligonucleotide for
the manufacture of a medicament for treating a neurodegenerative
disease.
[1216] In some embodiments, the present disclosure pertains to a
method of treating or ameliorating an C9orf72-related disorder in a
patient thereof, the method comprising the step of administering to
the patient a therapeutically effective amount of an
oligonucleotide to C9orf72.
[1217] In some embodiments, the present disclosure pertains to a
method comprising administering to an animal a composition
comprising a C9orf72 oligonucleotide.
[1218] In some embodiments, the animal is a subject, e.g., a
human.
[1219] In some embodiments, a subject or patient suitable for
treatment of a C9orf72-related disorder, such as administration of
a C9orf72 oligonucleotide, can be identified or diagnosed by a
health care professional. A C9orf72-related disease is one of
several neurological diseases. In some embodiments, a diagnose of a
subject as having a neurological disease can be performed by the
assessment of one or more symptoms, e.g., a symptom of motor neuron
degeneration. In some embodiments, to diagnose a neurological
disease, a physical exam may be followed by a thorough neurological
exam. In some embodiments, the neurological exam may assess motor
and sensory skills, nerve function, hearing and speech, vision,
coordination and balance, mental status, and changes in mood or
behavior. Non-limiting symptoms of a disease associated with a
neurological disease may be weakness in the arms, legs, feet, or
ankles; slurring of speech; difficulty lifting the front part of
the foot and toes; hand weakness or clumsiness; muscle paralysis;
rigid muscles; involuntary jerking or writing movements (chorea);
involuntary, sustained contracture of muscles (dystonia);
bradykinesia; loss of automatic movements; impaired posture and
balance; lack of flexibility; tingling parts in the body; electric
shock sensations that occur with movement of the head; twitching in
arm, shoulders, and tongue; difficulty swallowing; difficulty
breathing; difficulty chewing; partial or complete loss of vision;
double vision; slow or abnormal eye movements; tremor; unsteady
gait; fatigue; loss of memory; dizziness; difficulty thinking or
concentrating; difficulty reading or writing; misinterpretation of
spatial relationships; disorientation; depression; anxiety;
difficulty making decisions and judgments; loss of impulse control;
difficulty in planning and performing familiar tasks;
aggressiveness; irritability; social withdrawal; mood swings;
dementia; change in sleeping habits; wandering; change in
appetite.
[1220] In some embodiments, the composition prevents, treats,
ameliorates, or slows progression of at least one symptom of a
C9orf72-related disorder.
[1221] In some embodiments, an animal or human is suffering from a
symptom of a C9orf72-related disorder.
[1222] In some embodiments, the present disclosure pertains to a
method for introducing an oligonucleotide that decreases C9orf72
gene expression into a cell, the method comprising: contacting the
cell with an oligonucleotide or a C9orf72 oligonucleotides.
[1223] In some embodiments, the present disclosure pertains to a
method for decreasing C9orf72 gene expression in a mammal in need
thereof, the method comprising: administering to the mammal a
nucleic acid-lipid particle comprising an oligonucleotide to
C9orf72.
[1224] In some embodiments, the present disclosure pertains to a
method for the in vivo delivery of an oligonucleotide that targets
C9orf72 gene expression, the method comprising: administering to a
mammal an oligonucleotide to C9orf72.
[1225] In some embodiments, the present disclosure pertains to a
method for treating and/or ameliorating one or more symptoms
associated with a C9orf72-related disorder in a mammal in need
thereof, the method comprising: administering to the mammal a
therapeutically effective amount of a nucleic acid-lipid particle
comprising an oligonucleotide to C9orf72.
[1226] In some embodiments, the present disclosure pertains to a
method of inhibiting C9orf72 expression in a cell, the method
comprising: (a) contacting the cell with an oligonucleotide to
C9orf72; and (b) maintaining the cell produced in step (a) for a
time sufficient to obtain degradation of the mRNA transcript of an
C9orf72 gene, thereby inhibiting expression of the C9orf72 gene in
the cell.
[1227] In some embodiments, C9orf72 expression is inhibited by at
least 30%.
[1228] In some embodiments, the present disclosure pertains to a
method of treating a disorder mediated by C9orf72 expression
comprising administering to a human in need of such treatment a
therapeutically effective amount of an oligonucleotide to
C9orf72.
[1229] In some embodiments, administration causes a decrease in the
expression, activity and/or level of a C9orf72 transcript
containing a repeat expansion or a gene product thereof.
[1230] In some embodiments, the present disclosure pertains to a
method of treatment of a C9orf72-related disorder.
[1231] In some embodiments, the present disclosure pertains to a
method comprising the steps of: Providing a system comprising two
or more different splicing products of the same mRNA, wherein at
least one splicing product is disease-associated and at least one
splicing product is non-disease-associated; introducing into a
system an oligonucleotide, wherein the oligonucleotide is
complementary to a sequence which is present in the at least one
disease-associated splicing product, but not present in the at
least one non-disease-associated splicing product, wherein the
oligonucleotide is capable of reducing the expression, level and/or
activity of the disease-associated splicing product relative to the
expression, level and/or activity of the non-disease-associated
splicing product.
[1232] In some embodiments of the method, the oligonucleotide is
complementary to an intron-exon junction present on the
disease-associated splicing product but not present on the
non-disease-associated splicing product.
[1233] In some embodiments of the method, the oligonucleotide
comprises at least one chirally controlled internucleotidic
linkage.
[1234] In some embodiments of the method, the oligonucleotide is a
c9orf72 oligonucleotide and the system is a subject suffering from
and/or susceptible a c9orf2-related disorder.
[1235] In some embodiments, a subject is administered a second
therapeutic agent or method.
[1236] In some embodiments, a subject is administered a c9orf72
oligonucleotide and one or more second therapeutic agent or
method.
[1237] In some embodiments, a second therapeutic agent or method is
capable of preventing, treating, ameliorating or slowing the
progress of a neurological disease.
[1238] In some embodiments, a second therapeutic agent or method is
capable of preventing, treating, ameliorating or slowing the
progress of a C9orf72-related disorder.
[1239] In some embodiments, a second therapeutic agent or method is
capable of preventing, treating, ameliorating or slowing the
progress of a neurological disease selected from: an endosomal
and/or lysosomal trafficking modulator, a glutamate receptor
inhibitor, a PIKFYVE kinase inhibitor, and a potassium channel
activator.
[1240] In some embodiments a second therapeutic agent or method
comprises an antibody to a dipeptide repeat protein or an agent
(e.g., an antibody or small molecule) which disrupts the formation
of or decreases the abundance or number of RNA foci.
[1241] In some embodiments, a second therapeutic agent or method
indirectly decreases the expression, activity and/or level of
C9orf72, as non-limiting examples, by knocking down a gene or gene
product which increases the expression, activity and/or level of
C9orf72. In some embodiments, a second therapeutic agent or method
knocks down SUPT4H1, the human Spt4 ortholog, knockdown of which
decreased production of sense and antisense C9orf72 RNA foci, as
well as DPR proteins. Kramer et al. 2016 Science 353: 708. In some
embodiments, a second therapeutic agent or method is a nucleic
acid, small molecule, gene therapy or other agent or method
described in the literature, including, as a non-limiting example,
Mis et al. Mol Neurobiol. 2017 August; 54(6):4466-4476.
[1242] In some embodiments, a second therapeutic agent is
physically conjugated to a C9orf72 oligonucleotide. In some
embodiments, a C9orf72 oligonucleotide is physically conjugated to
a second oligonucleotide which decreases (directly or indirectly)
the expression, activity and/or level of C9orf72, or which is
useful for treating a symptom of a C9orf72-related disorder. In
some embodiments, a first C9orf72 oligonucleotide is physically
conjugated to a second C9orf72 oligonucleotide, which can be
identical to the first C9orf72 oligonucleotide or not identical,
and which can target a different or the same or an overlapping
sequence as the first C9orf72 oligonucleotide. In some embodiments,
a C9orf72 oligonucleotide is conjugated or co-administered or
incorporated into the same treatment regime as an oligonucleotide
which knocks down SUPT4H1. In some embodiments, a C9orf72
oligonucleotide is conjugated or co-administered or incorporated
into the same treatment regime as a second therapeutic agent which
improves the expression, activity and/or level of another
(non-C9orf72) gene or gene product which is associated with a
C9orf72-related disorder such as ALS or FTD, such as: SOD1, TARDBP,
FUS/TLS, MAPT, TDP-43, SUPT4H1, or FUS/TLS.
[1243] In some embodiments, improving the expression, activity
and/or level of such a gene or gene product includes, inter alia:
decreasing the expression, activity and/or level of such a gene or
gene product is such is too high in the disease state; increasing
the expression, activity and/or level or such a gene or gene
product is such is too low in the disease state; and/or decreasing
the expression, activity and/or level of a mutant and/or
disease-associated variant of such a gene or gene product. In some
embodiments, a second therapeutic agent is an oligonucleotide. In
some embodiments, a second therapeutic agent is an oligonucleotide
physically conjugated to a C9orf72 oligonucleotide. In some
embodiments, a second therapeutic agent comprises monomethyl
fumarate (MMF), which reportedly activates Nrf2, and/or an omega-3
fatty acid. In some embodiments, a second therapeutic agent
comprises monomethyl fumarate (MMF) and/or the omega-3 fatty acid,
docosahexaenoic acid (DHA), which reportedly inhibits NF-.kappa.B.
In some embodiments, a second therapeutic agent comprises a
conjugate of monomethyl fumarate (MMF) and the omega-3 fatty acid,
docosahexaenoic acid (DHA). In some embodiments, a second
therapeutic agent is CAT-4001 (Catabasis Pharmaceuticals,
Cambridge, Mass., US).
[1244] In some embodiments, a second therapeutic agent is capable
of preventing, treating, ameliorating or slowing the progress of a
neurological disease selected from: an endosomal and/or lysosomal
trafficking modulator, a glutamate receptor inhibitor, a PIKFYVE
kinase inhibitor, and a potassium channel activator described in
WO2016/210372. In some embodiments, a potassium channel activator
is retigabine. In some embodiments, a glutamate receptor is on a
motor neuron (MN) or spinal motor neuron. In some embodiments, a
glutamate receptor is NMDA, AMPA, or kainite. In some embodiments,
a glutamate receptor inhibitor is AP5
((2R)-amino-5-phosphonovaleric acid;
(2R)-amino-5-phosphonopentanoate), CNQX
(6-cyano-7-nitroquinoxaline-2,3-dione), or NBQX
(2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione).
[1245] In some embodiments, a second therapeutic agent is capable
of decreasing the expression, level and/or activity of a gene (or a
gene product thereof) associated with a c9orf72-related disorder,
such as SOD1, TARDBP, FUS/TLS, MAPT, TDP-43, SUPT4H1, or FUS/TLS.
In some embodiments, a second therapeutic agent is an agent which
decreases the expression, level and/or activity of a gene (or a
gene product thereof) associated with amyotrophic lateral sclerosis
(ALS) or frontotemporal dementia (FTD), such as SOD1, TARDBP,
FUS/TLS, MAPT, TDP-43, SUPT4H1, or FUS/TLS. In some embodiments, a
second therapeutic agent is capable of controlling excessive
oxidative stress. In some embodiments, a second therapeutic agent
is Radicava.RTM. (edaravone). In some embodiments, a second
therapeutic agent is ursodeoxycholic acid (UDCA). In some
embodiments, a second therapeutic agent is capable of affecting
neurons by reducing their activity through blocking Na+ entrance
into the neurons, and blocking the release of the chemicals that
cause the activity of the motor neurons. In some embodiments, a
second therapeutic agent is riluzole. In some embodiments, a second
therapeutic agent is capable of: reducing fatigue, easing muscle
cramps, controlling spasticity, and/or reducing excess saliva and
phlegm. In some embodiments, a second therapeutic agent is capable
of reducing pain. In some embodiments, a second therapeutic agent
is a nonsteroidal and/or anti-inflammatory drug and/or opioid. In
some embodiments, a second therapeutic agent is capable of reducing
depression, sleep disturbance, dysphagia, spasticity, difficulty
swallowing saliva, and/or constipation. In some embodiments, a
second therapeutic agent is baclofen or diazepam. In some
embodiments, a second therapeutic agent is or comprises
trihexyphenidyl, amitriptyline and/or glycopyrrolate. In some
embodiments, a second therapeutic agent is a dsRNA or siRNA which
comprises a strand which has a sequence which comprises at least 15
contiguous nt of the sequence of any oligonucleotide disclosed
herein.
Pharmaceutical Compositions
[1246] In some embodiments, the present disclosure provides
pharmaceutical compositions comprising a provided compound, e.g., a
provided oligonucleotide, or a pharmaceutically acceptable salt
thereof, and a pharmaceutical carrier. In some embodiments, an
oligonucleotide is a C9orf72 oligonucleotide.
[1247] When used as therapeutics, a provided oligonucleotide or
oligonucleotide composition described herein is administered as a
pharmaceutical composition. In some embodiments, the pharmaceutical
composition is suitable for administration of an oligonucleotide to
an area of the body affected by a disorder, including but not
limited to the central nervous system. In some embodiments, the
pharmaceutical composition comprises a therapeutically effective
amount of a provided oligonucleotides, or a pharmaceutically
acceptable salt thereof, and at least one pharmaceutically
acceptable inactive ingredient selected from pharmaceutically
acceptable diluents, pharmaceutically acceptable excipients, and
pharmaceutically acceptable carriers.
[1248] In some embodiments, a provided C9orf72 is conjugated to an
additional chemical moiety suitable for use in delivery to the
central nervous system, selected from: glucose, GluNAc (N-acetyl
amine glucosamine) and anisamide, and a molecule of any of the
structures of:
##STR00209## ##STR00210##
which are described in more detail in Examples 1 and 2.
[1249] In some embodiments, an additional chemical moiety
conjugated to an oligonucleotide is capable of targeting the
oligonucleotide to a cell in the nervous system.
[1250] In some embodiments, an additional chemical moiety
conjugated to a provided oligonucleotide comprises anisamide or a
derivative or analog thereof and is capable of targeting the
provided oligonucleotide to a cell expressing a particular
receptor, such as the sigma 1 receptor.
[1251] In some embodiments, a provided oligonucleotide is
formulated for administration to a body cell and/or tissue
expressing its target.
[1252] In some embodiments, an additional chemical moiety
conjugated to a C9orf72 oligonucleotide is capable of targeting the
C9orf72 oligonucleotide to a cell in the nervous system.
[1253] In some embodiments, an additional chemical moiety
conjugated to a C9orf72 oligonucleotide comprises anisamide or a
derivative or analog thereof and is capable of targeting the
C9orf72 oligonucleotide to a cell expressing a particular receptor,
such as the sigma 1 receptor.
[1254] In some embodiments, a provided C9orf72 oligonucleotide is
formulated for administration to a body cell and/or tissue
expressing C9orf72. In some embodiments, such a body cell and/or
tissue is a neuron or a cell and/or tissue of the central nervous
system. In some embodiments, broad distribution of oligonucleotides
and compositions, described herein, within the central nervous
system may be achieved with intraparenchymal administration,
intrathecal administration, or intracerebroventricular
administration.
[1255] In some embodiments, the pharmaceutical composition is
formulated for intravenous injection, oral administration, buccal
administration, inhalation, nasal administration, topical
administration, ophthalmic administration or otic administration.
In some embodiments, the pharmaceutical composition is a tablet, a
pill, a capsule, a liquid, an inhalant, a nasal spray solution, a
suppository, a suspension, a gel, a colloid, a dispersion, a
suspension, a solution, an emulsion, an ointment, a lotion, an eye
drop or an ear drop.
[1256] In some embodiments, the present disclosure provides a
pharmaceutical composition comprising chirally controlled
oligonucleotide, or composition thereof, in admixture with a
pharmaceutically acceptable excipient. One of skill in the art will
recognize that the pharmaceutical compositions include the
pharmaceutically acceptable salts of the chirally controlled
oligonucleotide, or composition thereof, described above.
[1257] A variety of supramolecular nanocarriers can be used to
deliver nucleic acids. Example nanocarriers include, but are not
limited to liposomes, cationic polymer complexes and various
polymeric. Complexation of nucleic acids with various polycations
is another approach for intracellular delivery; this includes use
of PEGlyated polycations, polyethyleneamine (PEI) complexes,
cationic block co-polymers, and dendrimers. Several cationic
nanocarriers, including PEI and polyamidoamine dendrimers help to
release contents from endosomes. Other approaches include use of
polymeric nanoparticles, microspheres, liposomes, dendrimers,
biodegradable polymers, conjugates, prodrugs, inorganic colloids
such as sulfur or iron, antibodies, implants, biodegradable
implants, biodegradable microspheres, osmotically controlled
implants, lipid nanoparticles, emulsions, oily solutions, aqueous
solutions, biodegradable polymers, poly(lactide-coglycolic acid),
poly(lactic acid), liquid depot, polymer micelles, quantum dots and
lipoplexes. In some embodiments, an oligonucleotide is conjugated
to another molecular.
[1258] Additional nucleic acid delivery strategies are known in
addition to the example delivery strategies described herein.
[1259] In therapeutic and/or diagnostic applications, the compounds
of the disclosure can be formulated for a variety of modes of
administration, including systemic and topical or localized
administration. Techniques and formulations generally may be found
in Remington, The Science and Practice of Pharmacy, (20th ed.
2000).
[1260] Provided oligonucleotides, and compositions thereof, are
effective over a wide dosage range. For example, in the treatment
of adult humans, dosages from about 0.01 to about 1000 mg, from
about 0.5 to about 100 mg, from about 1 to about 50 mg per day, and
from about 5 to about 100 mg per day are examples of dosages that
may be used. The exact dosage will depend upon the route of
administration, the form in which the compound is administered, the
subject to be treated, the body weight of the subject to be
treated, and the preference and experience of the attending
physician.
[1261] Pharmaceutically acceptable salts are generally well known
to those of ordinary skill in the art, and may include, by way of
example but not limitation, acetate, benzenesulfonate, besylate,
benzoate, bicarbonate, bitartrate, bromide, calcium edetate,
camsylate, carbonate, citrate, edetate, edisylate, estolate,
esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate,
lactobionate, malate, maleate, mandelate, mesylate, mucate,
napsylate, nitrate, pamoate (embonate), pantothenate,
phosphate/diphosphate, polygalacturonate, salicylate, stearate,
subacetate, succinate, sulfate, tannate, tartrate, or teoclate.
Other pharmaceutically acceptable salts may be found in, for
example, Remington, The Science and Practice of Pharmacy (20th ed.
2000). Preferred pharmaceutically acceptable salts include, for
example, acetate, benzoate, bromide, carbonate, citrate, gluconate,
hydrobromide, hydrochloride, maleate, mesylate, napsylate, pamoate
(embonate), phosphate, salicylate, succinate, sulfate, or
tartrate.
[1262] In some embodiments, a provided C9orf72 oligonucleotides is
formulated in a pharmaceutical composition described in U.S.
Applications No. 61/774,759; 61/918,175, filed Dec. 19, 2013;
61/918,927; 61/918,182; 61/918,941; 62/025,224; 62/046,487; or
International Applications No. PCT/US04/042911; PCT/EP2010/070412;
or PCT/I B2014/059503.
[1263] Depending on the specific conditions being treated, such
agents may be formulated into liquid or solid dosage forms and
administered systemically or locally. The agents may be delivered,
for example, in a timed- or sustained-low release form as is known
to those skilled in the art. Techniques for formulation and
administration may be found in Remington, The Science and Practice
of Pharmacy (20th ed. 2000). Suitable routes may include oral,
buccal, by inhalation spray, sublingual, rectal, transdermal,
vaginal, transmucosal, nasal or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intra-articullar, intra-sternal,
intra-synovial, intra-hepatic, intralesional, intracranial,
intraperitoneal, intranasal, or intraocular injections or other
modes of delivery.
[1264] For injection, the agents of the disclosure may be
formulated and diluted in aqueous solutions, such as in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological saline buffer. For such
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art.
[1265] Use of pharmaceutically acceptable inert carriers to
formulate the compounds herein disclosed for the practice of the
disclosure into dosages suitable for systemic administration is
within the scope of the disclosure. With proper choice of carrier
and suitable manufacturing practice, the compositions of the
present disclosure, in particular, those formulated as solutions,
may be administered parenterally, such as by intravenous
injection.
[1266] The compounds can be formulated readily using
pharmaceutically acceptable carriers well known in the art into
dosages suitable for oral administration. Such carriers enable the
compounds of the disclosure to be formulated as tablets, pills,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a subject (e.g., patient) to be
treated.
[1267] For nasal or inhalation delivery, the agents of the
disclosure may also be formulated by methods known to those of
skill in the art, and may include, for example, but not limited to,
examples of solubilizing, diluting, or dispersing substances such
as, saline, preservatives, such as benzyl alcohol, absorption
promoters, and fluorocarbons.
[1268] In certain embodiments, oligonucleotides and compositions
are delivered to the CNS. In certain embodiments, oligonucleotides
and compositions are delivered to the cerebrospinal fluid. In
certain embodiments, oligonucleotides and compositions are
administered to the brain parenchyma. In certain embodiments,
oligonucleotides and compositions are delivered to an
animal/subject by intrathecal administration, or
intracerebroventricular administration. Broad distribution of
oligonucleotides and compositions, described herein, within the
central nervous system may be achieved with intraparenchymal
administration, intrathecal administration, or
intracerebroventricular administration.
[1269] In certain embodiments, parenteral administration is by
injection, by, e.g., a syringe, a pump, etc. In certain
embodiments, the injection is a bolus injection. In certain
embodiments, the injection is administered directly to a tissue,
such as striatum, caudate, cortex, hippocampus and cerebellum.
[1270] In certain embodiments, methods of specifically localizing a
pharmaceutical agent, such as by bolus injection, decreases median
effective concentration (EC50) by a factor of 20, 25, 30, 35, 40,
45 or 50. In certain embodiments, the pharmaceutical agent in an
antisense compound as further described herein. In certain
embodiments, the targeted tissue is brain tissue. In certain
embodiments the targeted tissue is striatal tissue. In certain
embodiments, decreasing EC50 is desirable because it reduces the
dose required to achieve a pharmacological result in a patient in
need thereof.
[1271] In certain embodiments, an antisense oligonucleotide is
delivered by injection or infusion once every month, every two
months, every 90 days, every 3 months, every 6 months, twice a year
or once a year.
[1272] Pharmaceutical compositions suitable for use in the present
disclosure include compositions wherein the active ingredients are
contained in an effective amount to achieve its intended purpose.
Determination of the effective amounts is well within the
capability of those skilled in the art, especially in light of the
detailed disclosure provided herein.
[1273] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of an active compound into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, dragees, capsules, or
solutions.
[1274] Pharmaceutical preparations for oral use can be obtained by
combining an active compound with solid excipients, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethyl-cellulose (CMC), and/or polyvinylpyrrolidone (PVP:
povidone). If desired, disintegrating agents may be added, such as
the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a
salt thereof such as sodium alginate.
[1275] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol
gel, polyethylene glycol (PEG), and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dye-stuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[1276] Pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin, and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, an active compound may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols (PEGs). In
addition, stabilizers may be added.
[1277] A composition can be obtained by combining an active
compound with a lipid. In some embodiments, the lipid is conjugated
to an active compound. In some embodiments, the lipid is not
conjugated to an active compound. In some embodiments, a lipid
comprises a C.sub.10-C.sub.40 linear, saturated or partially
unsaturated, aliphatic chain. In some embodiments, a lipid
comprises a C.sub.10-C.sub.40 linear, saturated or partially
unsaturated, aliphatic chain, optionally substituted with one or
more C.sub.1-4 aliphatic group. In some embodiments, the lipid is
selected from the group consisting of: lauric acid, myristic acid,
palmitic acid, stearic acid, oleic acid, linoleic acid,
alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid
(cis-DHA), turbinaric acid and dilinoleyl. In some embodiments, an
active compound is any oligonucleotide or other nucleic acid
described herein. In some embodiments, an active compound is a
nucleic acid of a sequence comprising or consisting of any sequence
of any nucleic acid listed in Table 1A. In some embodiments, a
composition comprises a lipid and an an active compound, and
further comprises another component selected from: another lipid,
and a targeting compound or moiety. In some embodiments, a lipid
includes, without limitation: an amino lipid; an amphipathic lipid;
an anionic lipid; an apolipoprotein; a cationic lipid; a low
molecular weight cationic lipid; a cationic lipid such as CLinDMA
and DLinDMA; an ionizable cationic lipid; a cloaking component; a
helper lipid; a lipopeptide; a neutral lipid; a neutral
zwitterionic lipid; a hydrophobic small molecule; a hydrophobic
vitamin; a PEG-lipid; an uncharged lipid modified with one or more
hydrophilic polymers; phospholipid; a phospholipid such as
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; a stealth lipid; a
sterol; a cholesterol; and a targeting lipid; and any other lipid
described herein or reported in the art. In some embodiments, a
composition comprises a lipid and a portion of another lipid
capable of mediating at least one function of another lipid. In
some embodiments, a targeting compound or moiety is capable of
targeting a compound (e.g., a composition comprising a lipid and a
active compound) to a particular cell or tissue or subset of cells
or tissues. In some embodiments, a targeting moiety is designed to
take advantage of cell- or tissue-specific expression of particular
targets, receptors, proteins, or other subcellular components; In
some embodiments, a targeting moiety is a ligand (e.g., a small
molecule, antibody, peptide, protein, carbohydrate, aptamer, etc.)
that targets a composition to a cell or tissue, and/or binds to a
target, receptor, protein, or other subcellular component.
[1278] Certain example lipids for use in preparation of a
composition for delivery of an active compound allow (e.g., do not
prevent or interfere with) the function of an active compound.
Non-limiting example lipids include: lauric acid, myristic acid,
palmitic acid, stearic acid, oleic acid, linoleic acid,
alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid
(cis-DHA), turbinaric acid and dilinoleyl.
[1279] As described in the present disclosure, lipid conjugation,
such as conjugation with fatty acids, may improve one or more
properties of oligonucleotides.
[1280] In some embodiments, a composition for delivery of an active
compound is capable of targeting an active compound to particular
cells or tissues, as desired. In some embodiments, a composition
for delivery of an active compound is capable of targeting an
active compound to a muscle cell or tissue. In some embodiments,
the present disclosure pertains to compositions and methods related
to delivery of active compounds, wherein the compositions comprise
an active compound a lipid. In various embodiments to a muscle cell
or tissue, the lipid is selected from: lauric acid, myristic acid,
palmitic acid, stearic acid, oleic acid, linoleic acid,
alpha-linolenic acid, gamma-linolenic acid, docosahexaenoic acid
(cis-DHA), turbinaric acid and dilinoleyl.
[1281] Depending upon the particular disorder to be treated or
prevented, additional therapeutic agents, which are normally
administered to treat or prevent that condition, may be
administered together with C9orf oligonucleotides of this
disclosure.
[1282] In some embodiments, a second therapeutic agent administered
with a first C9orf72 oligonucleotide is a second, different,
C9orf72 oligonucleotide.
[1283] In some embodiments, C9orf72 oligonucleotides disclosed
herein can be used for a method for the prevention and/or treatment
of a C9orf72-related disorder or a symptom thereof, or for the
manufacture of medicament for use in such a method.
EXEMPLIFICATION
[1284] Certain examples of provided technologies (compounds
(oligonucleotides, reagents, etc.), compositions, methods (methods
of preparation, use, assessment, etc.)) were presented below.
[1285] Various technologies for preparing oligonucleotides and
oligonucleotide compositions (both stereorandom and chirally
controlled) are known and can be utilized in accordance with the
present disclosure, including, for example, those in
WO/2010/064146, WO/2011/005761, WO/2013/012758, WO/2014/010250,
US2013/0178612, WO/2014/012081, WO/2015/107425, WO/2017/015555, and
WO/2017/062862, the methods and reagents of each of which are
incorporated herein by reference.
Example 1
Conjugation of Oligonucleotides
[1286] In some embodiments, the present disclosure provides methods
for conjugation of oligonucleotides, for example, for better
delivery to CNS. Examples 1 and 2 show conjugation of
oligonucleotides for CNS delivery.
[1287] In some embodiments, provided oligonucleotides comprise
chemical moieties connected to the 5'-end optionally through linker
moieties. In some embodiments, provided oligonucleotides comprises
chemical moieties connected to the 5'-end --OH optionally through a
linker. In some embodiments, the present disclosure provides the
following 5' c Conjugation strategies:
##STR00211##
[1288] In some embodiments, provided oligonucleotides comprise
chemical moieties connected to the 5'-end optionally through linker
moieties. In some embodiments, the present disclosure provides the
following 3' cConjugation strategies:
##STR00212##
[1289] Various chemical moieties, e.g., ligand for cell receptors,
can be utilized in accordance with the present disclosure, for
example, those described in Juliano et al., J. Am. Chem. Soc. 2010,
132, 8848; Banerjee R et al., Int J Cancer. 2004, 112, 693; J. Med.
Chem., 2017, 60 (10), pp 4161-4172; etc. In some embodiments, a
chemical moiety is selected from:
##STR00213## ##STR00214##
Conjugates of Various Oligonucleotides
##STR00215## ##STR00216##
[1290] Synthesis of
4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(be-
nzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)pr-
opyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-(-
(benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenic-
osan-21-oic Acid
##STR00217## ##STR00218## ##STR00219##
[1292] Step 1: A solution of di-tert-butyl
3,3'-((2-amino-2-((3-(tert-butoxy)-3-oxopropoxy)methyl)propane-1,3-diyl)b-
is(oxy))dipropanoate 1(4.0 g, 7.91 mmol) and
dihydro-2H-pyran-2,6(3H)-dione (0.903 g, 7.91 mmol) in THF (40 mL)
was stirred at 50 C for 3 hrs and at rt for 3 hrs. LC-MS showed
desired product. Solvent was evaporated to give acid 2, which was
directly used for next step without purification.
[1293] Step 2: To a solution of
5-((9-((3-(tert-butoxy)-3-oxopropoxy)methyl)-2,2,16,16-tetramethyl-4,14-d-
ioxo-3,7,11,15-tetraoxaheptadecan-9-yl)amino)-5-oxopentanoic acid 2
(4.90 g, 7.91 mmol) and (bromomethyl)benzene(1.623-g, 9.49 mmol) in
DMF was added anhydrous K.sub.2CO.sub.3 (3.27 g, 23.73 mmol). The
mixture was stirred at 40.degree. C. for 4 hrs and at room
temperature for overnight. Solvent was evaporated under reduced
pressure. The reaction mixture was diluted with EtOAc, washed with
water, dried over anhydrous sodium sulfate, concentrated under
reduced pressure to give a residue, which was purified by ISCO
eluting with 1000EtOAc in hexane to 50% EtOAc in hexane to give
di-tert-butyl
3,3'-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropox-
y)methyl)propane-1,3-diyl)bis(oxy))dipropanoate 3 (5.43 g, 7.65
mmol, 9700 yield) as a colorless oil. .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 7.36-7.28 (i, 5H), 6.10 (s, 1H), 5.12 (s,
2H), 3.70 (s, 6H), 3.64 (t, J=8.0 Hz, 6H), 2.50-2.38 (m, 8H), 2.22
(t, J=7.3 Hz, 2H), 1.95 (p, J=7.4 Hz, 2H), 1.45 (s, 27H); MS, 710.5
(M+H)+.
[1294] Step 3: A solution of di-tert-butyl
3,3'-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((3-(tert-butoxy)-3-oxopropox-
y)methyl)propane-, 3-diyl)bis(oxy))dipropanoate 3(5.43 g, 7.65
mmol) in formic acid (50 mL) was stirred at room temperature for 48
hrs. LC-MS showed the reaction was not complete. Solvent was
evaporated under reduced pressure. The crude product was
re-dissolved in formic acid (50 mL) and was stirred at room
temperature for 6 hrs. LC-MS showed the reaction was complete.
Solvent was evaporated under reduced pressure, co-evaporated with
toluene (3.times.) under reduced pressure, and dried under vacuum
to give
3,3'-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)prop-
ane-1,3-diyl)bis(oxy))dipropanoic acid 4 (4.22 g, 7.79 mmol, 102%
yield) as a white solid. .sup.1H NMR (500 MHz, DMSO-d.sub.6)
.delta. 12.11 (s, 3H), 7.41-7.27 (m, 5H), 6.97 (s, 1H), 5.07 (s,
2H), 3.55 (t, J=6.4 Hz, 6H), 3.53 (s, 6H), 2.40 (t, J=6.3 Hz, 6H),
2.37-2.26 (m, 2H), 2.08 (t, J=7.3 Hz, 2H), 1.70 (p, J=7.4 Hz, 2H);
MS, 542.3 (M+H)+.
[1295] Step 4: A solution of
3,3'-((2-(5-(benzyloxy)-5-oxopentanamido)-2-((2-carboxyethoxy)methyl)prop-
ane-1,3-diyl)bis(oxy))dipropanoic acid 4 (4.10 g, 7.57 mmol) and
HOBt (4.60 g, 34.1 mmol) in DCM (60 mL) and DMF (15 mL) at
0.degree. C. was added tert-butyl (3-aminopropyl)carbamate (5.94 g,
34.1 mmol), EDAC HCl salt (6.53 g, 34.1 mmol) and DIPEA (10.55 ml,
60.6 mmol). The reaction mixture was stirred at 0.degree. C. for 15
minutes and at room temperature for 20 hrs. LC-MS showed the
reaction was not complete. EDAC HCl salt (2.0 g) and tert-butyl
(3-aminopropyl)carbamate (1.0 g) was added into the reaction
mixture. The reaction mixture was stirred at room temperature for 4
hrs. Solvent was evaporated to give a residue, which was dissolved
in EtOAc (300 mL), washed with water (1.times.), saturated sodium
bicarbonate (2.times.), 10% citric acid (2.times.) and water, dried
over sodium sulfate, and concentrated to give a residue which was
purified by ISCO (80 g gold cartridge) eluting with DCM to 30% MeOH
in DCM to give benzyl
15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2--
dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate 5
(6.99 g, 6.92 mmol, 91% yield) as a white solid. .sup.1H NMR (500
MHz, Chloroform-d) .delta. 7.38-7.33 (m, 5H), 6.89 (brs, 3H), 6.44
(s, 1H), 5.23 (brs, 3H), 5.12 (s, 2H), 3.71-3.62 (m, 12H), 3.29 (q,
J=6.2 Hz, 6H), 3.14 (q, J=6.5 Hz, 6H), 2.43 (dt, J=27.0, 6.7 Hz,
8H), 2.24 (t, J=7.2 Hz, 2H), 1.96 (p, J=7.5 Hz, 2H), 1.64-1.59 (m,
6H), 1.43 (d, J=5.8 Hz, 27H); MS (ESI): 1011.5 (M+H)+.
[1296] Step 5: To a solution of benzyl
15,15-bis(13,13-dimethyl-5,11-dioxo-2,12-dioxa-6,10-diazatetradecyl)-2,2--
dimethyl-4,10,17-trioxo-3,13-dioxa-5,9,16-triazahenicosan-21-oate
(0.326 g, 0.46 mmol) in DCM (5 mL) was added TFA (2 mL). The
reaction mixture was stirred at room temperature for 4 hrs. LC-MS
showed the reaction was completed. Solvent was evaporated under
reduced pressure to give benzyl
5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15--
dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate
as a colorless oil. Directly use for next step without
purification.
[1297] Step 6: To a solution of
5-(((2R,3R,4R,5R,6R)-3-acetamido-4,5-diacetoxy-6-(acetoxymethyl)tetrahydr-
o-2H-pyran-2-yl)oxy)pentanoic acid (1.10 g, 1.61 mmol), HBTU (0.558
g, 1.47 mmol), HOBT (0.062 g, 0.46 mmol) and DIPEA (1.2 mL, 6.9
mmol) in DCM (6 mL) followed by benzyl
5-((1,19-diamino-10-((3-((3-aminopropyl)amino)-3-oxopropoxy)methyl)-5,15--
dioxo-8,12-dioxa-4,16-diazanonadecan-10-yl)amino)-5-oxopentanoate
in acetonitrile (5 mL). The mixture was stirred at room temperature
for 3 hrs. Solvent was evaporated under reduced pressure to give a
residue, which was purified by ISCO (40 g gold column) eluting with
DCM to 20% MeOH in DCM to give
4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(be-
nzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)pr-
opyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-(-
(benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenic-
osan-21-oic benzyl ester (1.14 g, 92% yield) as a white solid. MS
(ESI): 1353.7 (M/2+H).sup.+.
[1298] Step 7: To a solution of
4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(be-
nzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)pr-
opyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-(-
(benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenic-
osan-21-oic benzyl ester (1.09 g, 0.400 mmol) in EtOAc (50 mL) was
added 10% Pd--C(200 mg) and methanol (2 mL). The reaction mixture
was stirred at room temperature for 3 hrs. LC-MS showed the
reaction was complete, diluted with EtOAc, and filtered through
celite, washed with 20% MeOH in EtOAc, concentrated under reduced
pressure to give
4,10,17-trioxo-15,15-bis((3-oxo-3-((3-(4-(((2R,3R,4S,5R,6R)-3,4,5-tris(be-
nzoyloxy)-6-((benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)butanamido)pr-
opyl)amino)propoxy)methyl)-1-(((2R,3R,4S,5R,6R)-3,4,5-tris(benzoyloxy)-6-(-
(benzoyloxy)methyl)tetrahydro-2H-pyran-2-yl)oxy)-13-oxa-5,9,16-triazahenic-
osan-21-oic acid (1.06 g, 100%) as a white solid. MS (ESI): 1308.1
(M+H).sup.+.
Example 2
Synthesis of 4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic Acid
##STR00220##
[1300] To solid reagents 4-(2-aminoethyl)benzenesulfonamide (2.00
g, 9.99 mmol) and dihydrofuran-2,5-dione (0.999 g, 9.99 mmol) was
added THF (30 mL). The reaction mixture was stirred at 60.degree.
C. for 7 hrs. Solvent was evaporated under reduced pressure to give
4-oxo-4-((4-sulfamoylphenethyl)amino)butanoic acid (3.00 g, 9.99
mmol, 100% yield) as a white solid. .sup.1H NMR (400 MHz, DMSO-d6)
.delta. 12.09 (s, 1H), 7.96 (t, J=5.6 Hz, 1H), 7.72 (d, J=8.1 Hz,
2H), 7.38 (d, J=8.1 Hz, 2H), 7.29 (s, 2H), 3.26 (q, J=6.8 Hz, 2H),
2.76 (t, J=7.2 Hz, 2H), 2.40 (t, J=6.9 Hz, 2H), 2.27 (t, J=6.9 Hz,
2H); MS (ESI), 301.1 (M+H).sup.+.
General Procedure for Conjugation of Sulfonamides with WV-7557
Synthesis of WV-7558 and 7559
[1301] Procedure: Synthesis of WV-7558 and WV-7559 follows same
procedure as described below. To a solution of sulfonamide (5
equivalents), in 2 ml DMF was added HATU (4.5 equivalents) and
DIPEA (25 equivalents). This mixture was stirred well for 2 minutes
(Scheme 1 and 2).
##STR00221##
[1302] To this solution was added, a solution of WV-7557 (1
equivalent) in water and shaken well for 60 minutes. The solvent
was removed under vacuum and crude product was purified by RP
column (C18) chromatography to obtain the product. The purified
product was desalted over a C-18 cartridge using sodium acetate
solution.
Synthesis of WV-7558
[1303] Following the general procedure outlined above, 4-sulfamoyl
benzoic acid (11 mg, 54.5 .mu.mol), HATU (18.6 mg, 49 .mu.mol) and
DIPEA (35 mg, 272 .mu.mol) were stirred for 2 minutes in 2 ml DMF
(Scheme 1). This activated HATU intermediate was added into a
solution of WV-7557 (75 mg, 10.9 .mu.mol) in 0.75 ml water. The
reaction vial was shaken for 60 minutes. Solvent was removed under
reduced pressure, purification and desalting was performed as
described above. Amount of product obtained is 20 mg. Molecular
weight of the product calculated: 7063; Deconvoluted mass obtained:
7065
##STR00222##
Synthesis of WV-7559
[1304] Following the general procedure outlined above, 4-sulfamoyl
benzoic acid (16.3 mg, 54.5 .mu.mol), HATU (18.6 mg, 49 .mu.mol)
and DIPEA (35 mg, 272 .mu.mol) were stirred for 2 minutes in 2 ml
DMF (Scheme 2). This activated HATU intermediate was added into a
solution of WV-7557 (75 mg, 10.9 .mu.mol) in 0.75 ml water. The
reaction vial was shaken for 60 minutes. Solvent was removed under
reduced pressure, purification and desalting was performed as
described above. Amount of product obtained is 13 mg. Molecular
weight of the product calculated: 7162; Deconvoluted mass obtained:
7165.
##STR00223##
General Procedure for Conjugation of Tri Antennary Anisamide with
WV-7557 and WV 8444: Synthesis of WV-7560 and WV 8447
##STR00224##
[1306] General Procedure:
[1307] To a solution of triantennary anisamide (2 equivalents), in
2 ml DMF was added HATU (1.8 equivalents) and DIPEA (10
equivalents). This mixture was stirred well for 2 minutes. To this
solution was added a solution of WV-7557 (1 equivalent) in water
and shaken well for 60 minutes. The solvent was removed under
vacuum and crude product was purified by RP column (C8)
chromatography to obtain the product. The purified product was
desalted over a C-18 cartridge using sodium acetate solution.
Synthesis of WV-7560
[1308] To a solution of triantennary anisamide (11 mg, 9.8
.mu.mol), in 2 ml DMF was added HATU (3.34 mg, 8.82 .mu.mol) and
DIPEA (6.3 mg, 9 .mu.l, 49 .mu.mol). This mixture was stirred well
for 2 minutes (Scheme 3). To this solution was added a solution of
WV-7557 (33.7 mg, 4.9 .mu.mol) in 0.88 ml water and shaken well for
60 minutes. The solvent was removed under vacuum and crude product
was purified by RP column (C8) chromatography to obtain the product
WV-7560 (25 mg). The purified product was desalted over a C-18
cartridge using sodium acetate solution. Molecular weight of
product calculated: 7982; De-convoluted mass obtained: 7987.
##STR00225##
Synthesis of WV-8447
[1309] To a solution of triantennary anisamide (13 mg, 11.6
.mu.mol), in 2 ml DMF was added HATU (4 mg, 10.4 .mu.mol) and DIPEA
(7.5 mg, 10.3 .mu.l, 58 .mu.mol). This mixture was stirred well for
2 minutes (Scheme 4). To this solution was added a solution of
WV-8444 (40 mg, 5.8 .mu.mol) in 1 ml water and shaken well for 60
minutes. The solvent was removed under vacuum and the crude product
was purified by RP column (C8) chromatography to obtain the product
WV-8447. The purified product was desalted over a C-18 cartridge
using sodium acetate solution. Molecular weight of product
calculated: 7970; De-convoluted mass obtained: 7975.
##STR00226##
General Procedure for Conjugation of Triantennary
Glucosamine/Glucose Derivative with WV-7557 or WV-8444
[1310] To a solution of triantennary glucosamine or glucose
derivative (2 equivalents), in 2 ml DMF was added HATU (1.8
equivalents) and DIPEA (10 equivalents). This mixture was stirred
well for 2 minutes. To this solution was added a solution of
WV-7557 or WV-8444 (1 equivalent) in water and shaken well for 60
minutes. The solvent was removed under vacuum and crude product was
treated with 30% NH.sub.4OH solution at room temperature for 24
hours. The solvent was removed under vacuum and the crude product
was purified by RP column (C8) chromatography to obtain the
product. The purified product was desalted over a C-18 cartridge
using sodium acetate solution.
##STR00227##
Synthesis of WV-8896
[1311] Following the general procedure shown above, Glucosamine
derivative (23.3 mg, 11.6 .mu.mol), HATU (4 mg, 10.44 .mu.mol) and
DIPEA (7.5 mg, 58 .mu.mol) was stirred in 2 ml DMF (Scheme 5). To
this solution was added 40 mg (5.8 .mu.mol) of WV-7557 in 1 ml
water. Reaction mixture was stirred for 60 minutes to obtain the
desired product. This product was treated with NH.sub.4OH as
described above. Amount of product obtained is 20 mg. Molecular
weight calculated: 8496; Deconvoluted mass obtained: 8494
##STR00228##
Synthesis of WV-8448
[1312] Following the general procedure shown above, Glucose
derivative (57 mg, 21.8 .mu.mol), HATU (7.5 mg, 19.6 .mu.mol) and
DIPEA (14.6 mg, 109 .mu.mol) was stirred in 2 ml DMF (Scheme 6). To
this solution was added 75 mg (10.9 .mu.mol) of WV-7557 in 1 ml
water. Reaction mixture was stirred for 60 minutes to obtain the
desired product. This product was heated at 40.degree. C. with
NH.sub.4OH as described above to obtain the product. Molecular
weight calculated: 8227; Deconvoluted mass obtained: 8233.
##STR00229##
Synthesis of WV-8446
[1313] Following the general procedure shown above, Glucose
derivative (30 mg, 11.6 mol), HATU (4 mg, 10.4 mol) and DIPEA (7.5
mg, 58 mol) was stirred in 2 ml DMF (Scheme 7). To this solution
was added 40 mg (5.8 .mu.mol) of WV 8444 in 1 ml water. Reaction
mixture was stirred for 60 minutes to obtain the desired product.
This product was heated at 40.degree. C. with NH.sub.4OH as
described above to obtain the product. Molecular weight calculated:
8214; Deconvoluted mass obtained: 8218.
##STR00230##
Synthesis of WV-8445
[1314] Following the general procedure shown above, Glucosamine
derivative (24 mg, 12 .mu.mol), HATU (4 mg, 10.4 .mu.mol) and DIPEA
(7.5 mg, 58 .mu.mol) was stirred in 2 ml DMF (Scheme 8). To this
solution was added 40 mg (5.8 .mu.mol) of WV 8444 in 1 ml water.
Reaction mixture was stirred for 60 minutes to obtain the desired
product. This product was heated at 40.degree. C. with NH.sub.4OH
as described above to obtain the product. Molecular weight
calculated: 8477; Deconvoluted mass obtained: 8484.
##STR00231##
Synthesis of GlucNAc Linker
##STR00232##
[1316] GlucNAc acid.sup.1 1 (1.88 g, 4.2 mmol) and HOBT (0.73 g,
5.4 mmol) was stirred in anhydrous DMF-DCM mixture (11+15 ml) under
nitrogen at room temperature for 10 minutes. HBTU (2.05 g, 5.4
mmol) was added followed by DIPEA (2.17 g, 16.8 mmol) at 10.degree.
C. To this solution was added tri-amine salt.sup.2 2 (1.38 g, 1.2
mmol) and stirred overnight. Solvent was removed under vacuum and
the residue was dissolved in ethyl acetate (200 ml). To this
solution was added 100 ml of a mixture of sat. ammonium chloride,
sat. sodium chloride, sat. sodium bicarbonate and water (1:1:1:1).
The ethyl acetate layer was turbid initially. After thoroughly
shaking the layers got separated. Aqueous layer was extracted with
ethyl acetate (.times.2). Combined organic fractions were washed
with brine and dried over anhydrous sodium sulfate. Solvent removal
under reduced pressure afforded 490 mg of crude product. This
product was purified by CC on an ISCO machine. The eluent was
DCM-Methanol (0-20% methanol in DCM). Amount of product obtained
was 1.26 g (50%). LC-MS (+ mode): 1768 (M-1GlucNAc), 1438 (M-2
GlucNAc), 1108 (M-3 GlucNAc), 1049 (M/2+1).
##STR00233##
[1317] Procedure:
[1318] To a solution of benzyl ester 4 (0.25 g, 0.119 mmol) in 7 ml
dry methanol, under an atmosphere of argon, was added 1.5 ml (9.4
mmol) Triethylsilane (TES) drop wise. A vigorous reaction sets in
and the RM was stirred for 3 hours. LC-MS analysis of the product
indicates completion of reaction. The RM was filtered over celite
and solvent was removed under vacuum. The crude product was
triturated (.times.3) with ether-methanol (3:1) mixture and dried
under vacuum. This product 5 was used for next reaction without
further purification. 1H NMR (500 MHz, DMSO-D6): .delta. 7.90 (3H,
d, J=10 Hz), 7.80 (t, 3H), 7.70 (t, 3H), 5.03 (t, 3H), 4.77 (t,
3H), 4.54 (3H, d, J=10 Hz), 4.14 (3H, dd, J.sub.1=9 Hz, J.sub.2=5
Hz), 3.97-3.93 (m, 3H), 3.79-3.74 (m, 3H), 3.69-3.61 (m, 6H),
3.51-3.47 (m, 3H), 3.40-3.35 (m, 3H), 3.31 (d, 3H, J=9 Hz), 2.98
(m, 12H), 2.23 (t, 3H), 2.13 (t, 3H), 2.01-1.99 (m, 3H), 1.97 (s,
9H), 1.92 (s, 9H), 1.86 (s, 9H), 1.71 (s, 9H), 1.49-1.32 (m, 22H),
1.18 (br s, 12H).
[1319] Ref 1 and 2: US Patent WO 2014/025805 A1; dated 13 Feb.
2014.
REFERENCES
[1320] Juliano et al. J. Am. Chem. Soc. 2010, 132, 8848 [1321]
Banerjee R et al. Int. J. Cancer. 2004, 112, 693 [1322] He et al.
J. Med. Chem., 2017, 60 (10), pp 4161-4172
General Procedure for the Deprotection of Amine
##STR00234##
[1324] 15.2 g of NHBoc amine was dissolved in dry DCM (100 ml) then
TFA (50 ml) was added dropwise at RT. Reaction mixture was stirred
at RT overnight. Solvents were removed under reduced pressure then
co-evaporated with toluene (2.times.50 mL) then used for the next
step without any further purification. NMR in CD.sub.3OD confirmed
the NHBoc deprotection.
General Procedure for the Anisamide Formation
##STR00235##
[1326] Procedure-A:
[1327] The crude amine from the previous step was dissolved in a
mixture of DCM (100 ml) and Et.sub.3N (10 equ.) at RT. During this
process, the reaction mixture was cooled with a water bath. Then
4-Methoxybenzoyl chloride (4 equ) was added dropwise to the
reaction mixture under argon atmosphere at RT, stirring continued
for 3 h. Reaction mixture was diluted with water and extracted with
DCM. Organic layer was extracted with aq. NaHCO.sub.3, 1N HCl,
brine then dried with magnesium sulfate evaporated to dryness. The
crude product was purified by silica column chromatography using
DCM-MeOH as eluent.
[1328] Procedure-B:
[1329] The crude amine (0.27 equ), acid and HOBt (1 equ) were
dissolved in a mixture of DCM and DMF (2:1) in an appropriate sized
RBF under argon. EDAC.HCl (1.25 equ) was added portion wise to the
reaction mixture under constant stirring. After 15 mins, the
reaction mixture was cooled to 10.degree. C. then DIEA (2.7 equ)
was added over a period of 5 mins. Slowly warmed the reaction
mixture to ambient temperature and stirred under argon for
overnight. TLC indicated completion of the reaction TLC condition,
DCM: MeOH (9.5:0.5). Solvents were removed under reduced pressure,
then water was added to the residue, and a gummy solid separated
out. The clear solution was decanted, and the solid residue was
dissolved in EtOAc and washed successively with water, 10% aqueous
citric acid, aq. NaHCO.sub.3, followed by saturated brine. The
organic layer was separated and dried over magnesium sulfate.
Solvent was removed under reduced pressure then the crude product
was purified with silica column to get the pure product.
[1330] Anisamide:
##STR00236##
[1331] Anizamide was obtained from the amine in 32% yield over 2
steps using the above procedure-B: 1H NMR (CDCl.sub.3):
.delta.=7.74 (d, 6H), 7.44 (t, 2H), 7.34 (t, 1H), 7.26 (m, 5H),
7.05 (m, 3H), 6.83 (d, 6H), 6.46 (s, 1H), 5.01 (s, 2H), 3.75 (s,
9H), 3.57 (m, 12H), 3.37 (m, 6H), 3.25 (m, 6H), 2.31 (m, 8H), 2.11
(m, 2H), 1.84 (m, 2H), 1.62 (m, 6H) ppm.
[1332] Anisamide:
##STR00237##
[1333] Anizamide was obtained from the amine in 57% yield over 2
steps using the above procedure-A: 1H NMR (CDCl.sub.3):
.delta.=7.75 (m, 3H), 7.73 (d, 6H), 7.43 (t, 3H), 7.25 (m, 5H),
6.80 (d, 6H), 6.51 (brs, 1H), 5.01 (s, 2H), 3.72 (s, 9H), 3.58 (m,
6H), 3.21 (m, 12H), 2.33 (t, 3H), 2.25 (t, 2H), 2.02 (t, 2H), 1.64
(q, 6H), 1.52 (p, 2H), 1.41 (q, 2H), 1.12 (m, 12H) ppm.
General Procedure for the Debenzylation
##STR00238##
[1335] The benzyl ester (10 g) was dissolved in a mixture of ethyl
acetate (100 ml) and methanol (25 ml) then Pd/C, 1 g (10% palladium
content) was added under argon atmosphere then the reaction mixture
was vacuumed and flushed with hydrogen and stirred at RT under H2
atmosphere for 3 h. TLC indicated completion of the reaction,
filtered through pad of celite and washed with methanol, evaporated
to dryness to yield a foamy white solid.
[1336] Acid:
##STR00239##
[1337] Yield 98%, 1H NMR (CD.sub.3OD): .delta.=8.35 (t, 1H), 8.01
(t, 1H), 7.82 (d, 6H), 7.27 (d, 1H), 6.99 (d, 6H), 3.85 (s, 9H),
3.68 (m, 12H), 3.41 (m 6H), 3.29 (m, 6H), 2.42 (m, 6H), 2.31 (q,
2H), 2.21 (td, 2H), 1.80 (m, 8H) ppm.
[1338] Acid:
##STR00240##
[1339] Yield 94%, 1H NMR (CD.sub.3OD): .delta.=8.36 (t, 2H), 8.02
(t, 2H), 7.82 (d, 6H), 7.23 (d, 1H), 6.98 (d, 6H), 3.85 (s, 9H),
3.70 (s, 6H), 3.67 (t, 6H), 3.41 (q, 4H), 3.28 (m, 8H), 2.42 (t,
6H), 2.27 (t, 2H), 2.13 (t, 2H), 1.79 (p, 6H), 1.54 (dp, 4H), 1.25
(m, 12H) ppm.
[1340] Additional compositions, including oligonucleotides
comprising analogues of anisamide, are presented below:
##STR00241## ##STR00242##
Example 3
Activity of Various C9orf72 Oligonucleotides in Dual-Luciferase
Reporter Assay
[1341] Tables 2A to 2C present data pertaining to the activities of
various C9orf72 oligonucleotides in a Dual-luciferase reporter
assay
[1342] Each data point in Tables 2A to C is % of Renilla signal
compared to WV-993, a control oligonucleotide which does not target
C9orf72. In these tables, an ASO is a C9orf72 oligonucleotide.
[1343] Numbers are replicates performed simultaneously. The numbers
represent the amount of residual gene expression. For example,
WV-3677 replicate 1 (in the first row in Table 2A) shows 1.522608%
residual gene expression, representing 98.477392% knockdown.
[1344] Constructs: C9orf72 nt 1-374 (Table 2A), C9orf72 nt 158-900
(Table 2B) or C9orf72 nt 900-1 (Table 2C) sequences were separately
cloned into the NotI site of the psiCHECK-2 vector (Promega
Corporation, Madison, Wis.), which is in the middle of the 3'UTR of
the hRluc gene. The new construct thus comprises a portion of the
wild-type C9orf72 which does not contain a hexanucleotide repeat
expansion.
[1345] Sequences used for making these constructs are presented
below:
TABLE-US-00004 C9, 1-374 (Exon 1a and part of intron 1)
ACGTAACCTACGGTGTCCCGCTAGGAAAGAGAGGTGCGTCAAACAGCGA
CAAGTTCCGCCCACGTAAAAGATGACGCTTGGTGTGTCAGCCGTCCCTG
CTGCCCGGTTGCTTCTCTTTTGGGGGCGGGGTCTAGCAAGAGCAGGTGT
GGGTTTAGGAGGTGTGTGTTTTTGTTTTTCCCACCCTCTCTCCCCACTA
CTTGCTCTCACAGTACTCGCTGAGGGTGAACAAGAAAAGACCTGATAAA
GATTAACCAGAAGAAAACAAGGAGGGAAACAACCGCAGCCTGTAGCAAG
CTCTGGAACTCAGGAGTCGCGCGCTAGGGGCCGGGGCCGGGGCCGGGGC
GTGGTCGGGGCGGGCCCGGGGGCGGGCCCGG C9, 158-900 (intron 1)
GTGTGTGTTTTTGTTTTTCCCACCCTCTCTCCCCACTACTTGCTCTCAC
AGTACTCGCTGAGGGTGAACAAGAAAAGACCTGATAAAGATTAACCAGA
AGAAAACAAGGAGGGAAACAACCGCAGCCTGTAGCAAGCTCTGGAACTC
AGGAGTCGCGCGCTAGGGGCCGGGGCCGGGGCCGGGGCGTGGTCGGGGC
GGGCCCGGGGGCGGGCCCGGGGCGGGGCTGCGGTTGCGGTGCCTGCGCC
CGCGGCGGCGGAGGCGCAGGCGGTGGCGAGTGGGTGAGTGAGGAGGCGG
CATCCTGGCGGGTGGCTGTTTGGGGTTCGGCTGCCGGGAAGAGGCGCGG
GTAGAAGCGGGGGCTCTCCTCAGAGCTCGACGCATTTTTACTTTCCCTC
TCATTTCTCTGACCGAAGCTGGGTGTCGGGCTTTCGCCTCTAGCGACTG
GTGGAATTGCCTGCATCCGGGCCCCGGGCTTCCCGGCGGCGGCGGCGGC
GGCGGCGGCGCAGGGACAAGGGATGGGGATCTGGCCTCTTCCTTGCTTT
CCCGCCCTCAGTACCCGAGCTGTCTCCTTCCCGGGGACCCGCTGGGAGC
GCTGCCGCTGCGGGCTCGAGAAAAGGGAGCCTCGGGTACTGAGAGGCCT
CGCCTGGGGGAAGGCCGGAGGGTGGGCGGCGCGCGGCTTCTGCGGACCA
AGTCGGGGTTCGCTAGGAACCCGAGACGGTCCCTGCCGGCGAGGAGATC ATGCGGG C9, 900-1
(antisense RNA) CCCGCATGATCTCCTCGCCGGCAGGGACCGTCTCGGGTTCCTAGCGAAC
CCCGACTTGGTCCGCAGAAGCCGCGCGCCGCCCACCCTCCGGCCTTCCC
CCAGGCGAGGCCTCTCAGTACCCGAGGCTCCCTTTTCTCGAGCCCGCAG
CGGCAGCGCTCCCAGCGGGTCCCCGGGAAGGAGACAGCTCGGGTACTGA
GGGCGGGAAAGCAAGGAAGAGGCCAGATCCCCATCCCTTGTCCCTGCGC
CGCCGCCGCCGCCGCCGCCGCCGGGAAGCCCGGGGCCCGGATGCAGGCA
ATTCCACCAGTCGCTAGAGGCGAAAGCCCGACACCCAGCTTCGGTCAGA
GAAATGAGAGGGAAAGTAAAAATGCGTCGAGCTCTGAGGAGAGCCCCCG
CTTCTACCCGCGCCTCTTCCCGGCAGCCGAACCCCAAACAGCCACCCGC
CAGGATGCCGCCTCCTCACTCACCCACTCGCCACCGCCTGCGCCTCCGC
CGCCGCGGGCGCAGGCACCGCAACCGCAGCCCCGCCCCGGGCCCGCCCC
CGGGCCCGCCCCGACCACGCCCCGGCCCCGGCCCCGGCCCCTAGCGCGC
GACTCCTGAGTTCCAGAGCTTGCTACAGGCTGCGGTTGTTTCCCTCCTT
GTTTTCTTCTGGTTAATCTTTATCAGGTCTTTTCTTGTTCACCCTCAGC
GAGTACTGTGAGAGCAAGTAGTGGGGAGAGAGGGTGGGAAAAACAAAAA
CACACACCTCCTAAACCCACACCTGCTCTTGCTAGACCCCGCCCCCAAA
AGAGAAGCAACCGGGCAGCAGGGACGGCTGACACACCAAGCGTCATCTT
TTACGTGGGCGGAACTTGTCGCTGTTTGACGCACCTCTCTTTCCTAGCG
GGACACCGTAGGTTACGT
[1346] Each of the constructs expresses two Luminescent proteins:
Firefly luciferase from hluc gene and Renilla luciferase from hRluc
gene.
[1347] The construct(20 ng) and tested oligonucleotides(different
doses) were cotransfected with Lipofectamine 2000 into Cos 7 cells
or 48 hours and the Firefly and Renilla signal were read with a
plate reader.
[1348] An efficacious C9orf72 oligonucleotide targeting the
inserted sequences should decrease the Renilla signal without
affecting the Firefly signal. The data analysis normalizes the
Renilla with Firefly signal and compares the efficacy of the tested
oligonucleotide to the control oligonucleotide. In Tables 2A and
21B, the numbers represent the percentage of remaining Renilla
signal. For example, for WV-3677, 1.5 residual level was detected
in a replicate and 2.4 in another replicate at 30 nM representing
98.5 and 97.6 knockdown respectively).
TABLE-US-00005 TABLE 2A Exon 1a targeting C9orf72 oligonucleotides
(tested using the construct comprising C9orf72 1-374)
oligonucleotide oligonucleotide oligonucleotide ID (30 nM) (15 nM)
WV-3677 1.5 2.4 2.8 2.8 WV-3678 8.0 11.4 13.5 15.9 WV-3679 5.3 6.8
9.2 7.0 WV-3680 26.2 24.6 26.4 27.4 WV-3681 16.2 17.0 19.3 17.3
WV-3682 26.6 31.3 23.6 27.1 WV-3683 12.0 18.5 6.5 7.2 WV-3684 30.9
45.1 23.2 23.0 WV-3685 9.4 41.5 9.9 9.8 WV-3686 19.7 22.9 24.0 15.4
WV-6928 7.1 6.1 10.3 11.3 WV-6929 13.9 19.2 21.7 26.8 WV-6930 5.5
6.6 9.4 10.3 WV-6931 4.1 6.2 7.3 7.3 WV-6932 12.1 10.4 9.4 12.2
WV-6933 6.5 8.1 12.9 14.8 WV-6934 12.6 13.8 25.6 26.9 WV-6935 7.0
11.5 16.0 17.2 WV-6936 22.3 20.7 16.9 16.4 WV-6937 6.6 8.1 8.3 9.8
WV-6938 6.6 8.5 21.5 21.8 WV-6939 10.4 14.4 28.3 25.4 WV-6940 5.7
5.1 6.5 6.5 WV-6941 9.4 14.1 19.5 24.6 WV-6942 10.1 12.6 18.0 20.9
WV-6943 20.6 25.9 32.0 39.2 WV-6944 24.2 23.2 24.6 29.0 WV-6945
24.6 26.0 43.3 41.3 WV-6946 25.4 33.9 40.7 49.2 WV-6947 20.5 24.0
43.7 44.7 WV-6948 30.7 25.7 38.9 43.3 WV-6949 18.1 21.0 34.9 43.6
WV-3677 2.8 3.4 3.8 3.3
TABLE-US-00006 TABLE 2B Intron1 Targeting C9orf72 oligonucleotides
(using construct comprising C9orf72 158-900) oligonucleotide
oligonucleotide oligonucleotide ID (30 nM) (15 nM) WV-3687 10.3
15.5 33.1 19.5 WV-3688 11.7 11.1 10.2 11.8 WV-3689 3.6 12.6 8.6 8.2
WV-3690 4.7 9.3 5.4 4.8 WV-3691 25.0 24.3 29.3 29.3 WV-3692 20.9
22.9 27.7 25.5 WV-3693 50.6 36.0 31.0 29.3 WV-3694 18.9 35.3 45.7
45.5 WV-3695 29.2 63.7 55.9 55.2 WV-3696 32.0 73.0 32.3 27.9
WV-3697 18.5 81.8 36.4 38.2 WV-3698 9.5 28.1 33.4 31.1 WV-3699 9.0
40.5 27.8 20.7 WV-3700 37.2 57.3 44.0 35.6 WV-3701 42.6 39.8 44.9
31.7 WV-3702 45.3 14.4 38.5 30.0 WV-3703 48.2 29.8 20.5 15.0
WV-3704 10.4 47.6 12.3 9.5 WV-3705 23.8 21.8 47.1 50.0 WV-3706 35.9
32.5 45.5 39.1 WV-3707 60.9 86.4 73.3 74.1 WV-3708 68.4 76.7 96.9
79.3 WV-3709 89.3 107.7 138.6 113.7 WV-3710 107.8 96.3 121.8 117.5
WV-6471 9.6 14.6 46.5 89.5 WV-6472 8.9 10.0 25.5 77.0 WV-6473 8.6
9.2 24.8 60.3 WV-6474 13.6 8.1 12.8 11.8 WV-6475 25.4 17.2 33.0
26.0 WV-6476 47.8 30.9 24.5 17.9 WV-6477 55.9 93.4 20.9 21.4
WV-6478 40.2 79.3 35.7 33.1 WV-6479 35.5 68.7 27.2 20.6 WV-6480
40.2 59.6 22.2 13.4 WV-6481 40.7 65.7 27.9 26.5 WV-6482 18.9 36.4
39.0 16.0 WV-6483 17.9 23.6 29.4 20.8 WV-6484 21.1 22.6 27.6 18.6
WV-6485 19.0 30.7 32.2 32.7 WV-6486 23.6 15.5 17.6 15.3 WV-6487
24.0 22.5 18.6 20.3 WV-6488 16.1 20.2 18.3 13.5 WV-6489 21.6 34.8
17.4 15.1 WV-3688 16.9 15.3 13.5 11.8 WV-3687 15.5 15.8 24.8 24.1
WV-3536 47.3 57.0 60.2 85.7 WV-6408 3.5 5.7 7.7 7.6 WV-6950 10.3
15.4 23.7 18.7 WV-6951 11.1 9.5 12.0 12.6 WV-6952 7.0 6.2 9.8 11.2
WV-6953 23.2 27.2 41.3 42.2 WV-6954 20.8 19.2 34.8 28.0 WV-6955 9.6
11.0 14.2 11.8 WV-6956 14.4 14.4 21.1 22.0 WV-6957 27.6 26.5 45.9
36.5 WV-6958 23.4 26.4 22.4 22.4 WV-6959 12.7 13.9 17.8 18.0
WV-6960 10.0 10.7 9.8 8.7 WV-6961 22.9 23.4 19.3 18.4 WV-6962 20.1
20.9 17.3 16.0 WV-6963 17.2 21.0 24.7 22.6 WV-6964 24.3 25.6 19.6
19.0 WV-6965 18.6 18.4 20.0 16.4 WV-6966 22.0 22.0 28.7 30.1
WV-6967 36.9 33.0 19.1 19.5 WV-6968 31.4 31.1 18.6 19.7 WV-6969 8.9
9.1 4.7 5.1 WV-6970 19.0 19.0 8.4 7.8 WV-6971 15.8 16.6 16.6 14.0
WV-6972 13.7 15.8 14.5 12.7 WV-6973 39.0 43.9 26.9 20.7 WV-6974
12.8 16.3 9.8 9.6 WV-6975 22.3 23.6 17.1 15.9 WV-6976 9.3 11.7 7.4
7.9 WV-6977 26.3 33.9 13.3 12.6 WV-6978 20.1 20.0 11.0 8.7 WV-6979
22.5 19.9 15.8 14.4 WV-6980 20.0 24.3 17.4 17.2 WV-6981 22.7 22.9
22.2 20.3 WV-6982 13.3 17.9 14.7 13.1 WV-6983 35.9 35.2 17.2 18.2
WV-6984 47.4 67.2 34.1 34.8 WV-6985 13.0 13.8 15.3 15.9 WV-6986
13.8 14.2 15.0 14.8 WV-6987 19.2 19.9 22.5 22.6 WV-6988 18.3 20.4
16.2 17.2 WV-6989 12.5 12.5 12.0 9.4 WV-6990 16.7 26.1 30.0 25.9
WV-6991 26.4 24.2 28.0 29.2 WV-6992 34.7 34.6 44.2 44.6 WV-6993
26.8 29.9 38.1 38.8 WV-6994 14.3 16.2 17.2 16.7 WV-6995 11.5 13.7
13.1 13.9 WV-6996 11.3 9.4 14.8 13.5 WV-6997 8.4 9.9 16.7 20.1
WV-6998 14.4 16.9 25.4 24.0 WV-6999 21.2 23.9 29.8 28.0 WV-7000
23.4 19.1 26.5 27.2 WV-7001 9.3 10.0 9.2 10.0 WV-7002 7.1 9.5 7.0
8.8 WV-7003 28.5 29.0 14.2 13.1 WV-7004 11.3 13.1 11.9 11.3 WV-7005
20.3 19.6 15.3 13.5 WV-7006 16.1 20.3 13.2 12.3 WV-7007 11.5 12.0
10.8 11.3 WV-7008 24.3 21.6 34.7 34.2 WV-7009 18.7 21.6 30.6 32.3
WV-7010 25.1 27.9 29.3 26.2 WV-7011 19.8 23.5 22.6 23.6 WV-7012
31.4 38.5 26.4 23.7 WV-6408 5.2 4.6 7.6 10.5
TABLE-US-00007 TABLE 2C Antisense Transcript Targeting C9orf72
oligonucleotides (tested using the construct with C9orf72 900-1)
oligonucleotide oligonucleotide oligonucleotide ID (30 nM) (15 nM)
WV-3719 31.1 25.4 13.9 14.7 WV-3720 41.9 46.3 24.8 21.4 WV-3721
30.2 36.3 22.6 23.7 WV-3722 20.3 26.0 19.3 16.6 WV-3723 14.1 13.7
9.8 10.0 WV-3724 31.6 33.5 18.6 19.0 WV-3725 64.1 89.0 27.5 32.6
WV-3726 206.7 49.4 50.0 48.5 WV-3727 58.2 47.2 50.2 46.3 WV-3728
39.6 33.7 69.2 58.6 WV-3729 34.1 25.6 49.1 50.2 WV-3730 14.0 20.5
17.6 14.2 WV-3731 21.5 27.8 21.2 32.3 WV-3732 13.1 16.1 22.1 16.8
WV-3733 13.7 20.0 24.1 19.7 WV-3734 9.0 14.7 17.7 25.4 WV-3735 21.2
28.5 47.7 21.2 WV-3736 10.0 17.1 17.7 48.4 WV-3737 11.9 8.4 13.3
14.2 WV-3738 42.0 36.6 39.8 65.7 WV-3739 16.6 12.5 16.1 20.5
WV-3740 43.9 40.0 58.2 67.3 WV-3741 30.6 46.4 23.6 32.1 WV-3742
38.1 38.4 50.2 60.1 WV-3743 23.4 25.2 19.1 16.9 WV-3744 42.6 44.2
66.4 69.3 WV-3745 13.4 10.8 15.0 21.1 WV-3746 183.3 142.7 92.3
106.2 WV-3747 148.5 135.5 93.0 80.1 WV-3748 88.1 78.2 73.0 64.4
WV-3749 90.8 99.5 72.3 62.7 WV-3750 102.3 105.0 77.1 77.6 WV-3751
169.9 150.3 88.4 76.0 WV-3752 115.1 80.1 74.2 53.5
Example 4
Ability of C9orf72 Oligonucleotides to Knock Down C9orf72
Transcripts In Vitro
[1349] Various C9orf72 oligonucleotides were tested for their
ability to knockdown C9orf72 transcripts in vitro. The tested
oligonucleotides had any of 20 different sequences (seq 1 to 20),
and each sequence was tested in each of three different formats
(e.g., 2'-O-Methyl full PS, 2'-O-Methyl PS/PO, or MOE full PS). The
exact sequences and modifications for each C9orf72 oligonucleotide
is presented in Table 1A.
TABLE-US-00008 TABLE 3A C9orf72 oligonucleotides tested in this
experiment Group A Group B Group C 2'-O-Methyl full PS 2'-O-Methyl
PS/PO MOE full PS mRNA seq 1 WV-3561 WV-3657 WV-5905 mRNA seq 2
WV-3562 WV-3658 WV-5906 mRNA seq 3 WV-3563 WV-3659 WV-5907 mRNA seq
4 WV-3564 WV-3660 WV-5908 mRNA seq 5 WV-3565 WV-3661 WV-5909 mRNA
seq 6 WV-3566 WV-3662 WV-5910 mRNA seq 7 WV-3567 WV-3663 WV-5911
mRNA seq 8 WV-3568 WV-3664 WV-5912 mRNA seq 9 WV-3569 WV-3665
WV-5913 mRNA seq 10 WV-3570 WV-3666 WV-5914 mRNA seq 11 WV-3571
WV-3667 WV-5915 mRNA seq 12 WV-3572 WV-3668 WV-5916 mRNA seq 13
WV-3573 WV-3669 WV-5917 mRNA seq 14 WV-3574 WV-3670 WV-5918 mRNA
seq 15 WV-3575 WV-3671 WV-5919 mRNA seq 16 WV-3576 WV-3672 WV-5920
mRNA seq 17 WV-3577 WV-3673 WV-5921 mRNA seq 18 WV-3578 WV-3674
WV-5922 mRNA seq 19 WV-3579 WV-3675 WV-5923 mRNA seq 20 WV-3580
WV-3676 WV-5924
[1350] C9orf72oligonucleotides were tested in ell neurons, at 48
hours, at concentration of 10 uM. Results are presented below.
Numbers represent amount of residual C9orf72 transcripts (measured
were a total of all C9orf72 transcripts) remaining after gymnotic
introduction of the oligonucleotides or controls into the cells.
For example, for Water, in Group A, 0.992 indicates 99.2% retention
of C9orf72 transcript level, or essentially no knockdown relative
to the control. For WV-3675, representing mRNA sequence 19 Group B,
0.316 indicates 31.6% residual C9orf72 transcript level, or 68.4%
knockdown. Unless noted otherwise, other data representing residual
transcript level is presented in this same or a similar format.
TABLE-US-00009 TABLE 3B Activity of C9orf72 oligonucleotides tested
in this experiment Group A Group B Group C 2'-O-Methyl full PS
2'-O-Methyl PS/PO MOE full PS mRNA seq 1 1.169 0.648 1.087 mRNA seq
2 0.782 1.121 0.877 mRNA seq 3 0.597 0.886 0.514 mRNA seq 4 0.572
0.710 0.448 mRNA seq 5 0.593 0.898 0.497 mRNA seq 6 0.478 0.311
0.228 mRNA seq 7 0.402 0.305 0.247 mRNA seq 8 0.850 1.017 0.624
mRNA seq 9 0.923 1.061 0.607 mRNA seq 10 0.963 1.025 0.717 mRNA seq
11 0.963 0.481 0.747 mRNA seq 12 1.068 1.210 0.865 mRNA seq 13
0.886 0.425 0.678 mRNA seq 14 0.976 0.943 0.742 mRNA seq 15 0.886
0.963 0.732 mRNA seq 16 0.892 0.963 0.563 mRNA seq 17 0.782 0.963
0.463 mRNA seq 18 0.473 0.747 0.624 mRNA seq 19 0.660 0.316 0.669
mRNA seq 20 0.678 0.651 0.763 control 1.032 1.017 0.774 Water 0.992
0.985 1.026 WV-3537 0.624
Example 5
Activities of Various C9orf72 Oligonucleotides in Various
Assays
[1351] Tables 4A to D show activity of C9orf72 oligonucleotides in
knocking down C9orf72 transcripts (Table 4A, all transcripts; Table
4B, only V3 transcripts; Table 4C, Intron/AS transcripts; and Table
4D, only Exon 1a transcripts) in vitro in iPSC neurons. Table 4C
shows knockdown of Intron/AS transcripts (with probes targeting a
region 3' to the repeat transcript expansion, the detected area
includes both sense and antisense transcripts of the intronic
region). Relative-fold change in C9orf72/HPRT1 is shown. Three
replicate experiments are shown for the various C9orf72
oligonucleotides, at a concentration of 1 .mu.M (Column A) or 10
.mu.M (Column B). Numbers represent residual transcript level (all
C9orf72 transcripts). For example, with WV-7601, three replicates
were done at a concentration of 1 .mu.M (Group A), showing 82.6%,
86.8% and 77.6% residual C9orf72 transcript level (all C9orf72
transcripts), corresponding to 17.4%, 13.2% and 22.3% knockdown,
respectively. For WV-7601, three replicates were also performed at
a concentration of 10 .mu.M (Group B), showing 76.0%, 68.5%, and
75.0% residual C9orf72 transcript level (all C9orf72 transcripts),
corresponding to 24.0%, 31.5%, and 25.0% knockdown, respectively.
Delivery of oligonucleotides was gymnotic and cells were tested
after 1 week. Controls used included WV-5302 and WV-6493, which
target Malat1. Malat1 and C9orf72 oligonucleotides were also tested
against Malat1; Malat1 oligonucleotides were efficacious in
knocking down Malat1, but C9orf72 oligonucleotides were not
efficacious in knocking down Malat1 (data not shown). Controls also
include WV-2549 and WV-6028, which target a gene target which is
not C9orf72.
TABLE-US-00010 TABLE 4A Activity of C9orf72 oligonucleotides
(residual level of all C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-7601 0.826 0.760 0.868 0.685 0.776 0.750 WV-7657 0.832
0.622 0.844 0.676 0.886 0.719 WV-7658 0.917 0.798 0.850 0.676 0.880
0.704 WV-7659 0.882 0.740 0.946 0.631 0.852 0.626 WV-8005 0.795
0.622 0.768 0.568 0.763 0.609 WV-8006 0.952 0.681 0.835 0.662 0.774
0.700 WV-8007 0.727 0.605 0.697 0.568 0.702 0.545 WV-8008 0.747
0.502 0.637 0.601 0.717 0.584 WV-8009 0.722 0.593 0.732 0.605 0.779
0.553 WV-8010 0.688 0.572 0.742 0.626 0.835 0.622 WV-8011 0.650
0.486 0.702 0.486 0.655 0.483 WV-8012 0.707 0.489 0.687 0.496 0.655
0.496 WV-2549 0.939 0.900 0.920 0.888 0.907 WV-6028 0.972 1.006
0.992 0.932 0.972 0.985 WV-3688 0.852 0.731 0.840 0.711 0.876 0.806
WV-6408 0.773 0.624 0.835 0.641 0.945 0.558 WV-3662 0.423 0.429
0.109 0.086 WV-7118 0.405 0.240 0.380 0.240 0.380 0.237 WV-6936
0.937 1.044 0.862 0.974 0.924 0.915 WV-7027 0.963 0.928 0.868 0.981
0.937 0.994 WV-5302 0.880 0.947 0.874 1.029 0.937 1.022 WV-6493
0.990 0.981 0.833 1.001 0.990 1.044 WV-2376 1.018 0.987 0.911 0.693
0.970 0.764 WV-3542 0.892 0.994 0.892 0.967 1.004 1.022
TABLE-US-00011 TABLE 4B Activity of C9orf72 oligonucleotides
(residual level of V3 C9orf72 transcripts) A (1 .mu.M) B (10 .mu.M)
WV-7603 0.631 0.455 0.725 0.442 0.740 0.445 WV-7604 0.572 0.436
0.622 0.407 0.601 0.362 WV-7605 0.667 0.340 0.695 0.354 0.648 0.374
WV-7606 0.676 0.298 0.495 0.286 0.576 0.247 WV-7601 0.475 0.286
0.557 0.278 0.530 0.247 WV-7657 0.618 0.424 0.676 0.364 0.549 0.407
WV-7658 0.568 0.326 0.542 0.321 0.572 0.304 WV-7659 0.558 0.333
0.539 0.315 0.582 0.296 WV-8005 0.366 0.123 0.327 0.124 0.392 0.147
WV-8006 0.409 0.158 0.438 0.171 0.473 0.157 WV-8007 0.182 0.056
0.196 0.056 0.238 0.050 WV-8008 0.197 0.048 0.183 0.045 0.172 0.043
WV-8009 0.412 0.150 0.379 0.129 0.406 0.110 WV-8010 0.339 0.137
0.344 0.138 0.368 0.128 WV-8011 0.229 0.059 0.244 0.067 0.263 0.055
WV-8012 0.212 0.046 0.244 0.050 0.217 0.057 WV-2549 0.827 0.821
0.936 0.905 0.983 WV-6028 0.943 1.018 0.990 0.983 0.905 1.011
WV-3688 0.735 0.502 0.730 0.472 0.715 0.538 WV-6408 0.505 0.341
0.557 0.343 0.644 0.343 WV-3662 0.357 0.408 0.071 0.028 WV-7118
0.369 0.153 0.404 0.159 0.352 0.148 WV-6936 0.843 0.562 0.792 0.649
0.808 0.589 WV-7027 0.792 0.602 0.819 0.731 0.941 0.778 WV-5302
1.066 1.062 1.059 1.055 1.066 1.077 WV-6493 1.044 1.026 1.030 1.085
0.995 1.115 WV-2376 0.981 1.108 0.968 0.887 0.995 0.828 WV-3542
1.030 1.041 1.009 0.991 1.016 1.070
TABLE-US-00012 TABLE 4C Activity of C9orf72 oligonucleotides
(residual level of Intron/AS C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-7603 0.557 0.654 0.767 0.705 0.799 0.654 WV-7604 0.386
0.375 0.538 0.329 0.535 0.299 WV-7605 0.851 0.585 0.845 0.561 0.663
0.610 WV-7606 0.783 0.408 0.178 0.623 0.343 0.520 WV-7601 0.303
0.260 0.260 0.271 0.265 0.311 WV-7657 0.715 0.606 0.756 0.513 0.405
0.434 WV-7658 0.520 0.345 0.502 0.277 0.677 0.370 WV-7659 0.372
0.417 0.458 0.397 0.359 0.479 WV-8005 0.471 0.346 0.613 0.425 0.626
0.654 WV-8006 0.410 0.355 0.474 0.663 0.471 0.411 WV-8007 0.621
0.531 0.512 0.475 0.548 0.307 WV-8008 0.439 0.645 0.311 0.485 0.564
0.495 WV-8009 0.580 0.593 0.685 0.479 0.592 0.706 WV-8010 0.461
0.394 0.252 0.431 0.407 0.341 WV-8011 0.514 0.415 0.594 0.774 0.972
0.774 WV-8012 0.594 1.050 0.650 0.633 0.606 0.651 WV-2549 0.435
1.198 1.282 1.174 1.318 WV-6028 1.715 2.001 1.049 2.604 0.846 1.058
WV-3688 0.795 0.703 0.687 0.836 0.554 0.764 WV-6408 1.071 1.029
0.741 1.036 0.789 0.940 WV-3662 1.273 1.180 0.802 1.376 WV-7118
1.356 1.094 0.712 1.248 1.156 0.876 WV-6936 1.291 1.375 1.064 1.310
1.443 1.944 WV-7027 0.507 0.727 0.992 1.494 0.768 1.777 WV-5302
1.230 2.157 0.737 0.795 1.101 0.840 WV-6493 0.562 1.463 0.586 0.727
0.536 0.784 WV-2376 0.784 1.985 1.579 0.387 0.594 0.426 WV-3542
1.494 1.515 1.283 1.944 1.704 2.361
TABLE-US-00013 TABLE 4D Activity of C9orf72 oligonucleotides
(residual level of Exon 1a C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-7603 1.006 1.127 1.042 1.051 0.965 0.981 WV-7604 0.823
1.059 0.823 0.848 0.737 0.738 WV-7605 1.282 1.059 1.205 1.023 1.049
1.096 WV-7606 0.907 0.995 0.524 1.008 0.687 1.044 WV-7601 0.707
1.044 0.795 0.909 0.726 0.848 WV-7657 0.985 0.854 0.888 0.728 0.551
0.733 WV-7658 0.979 1.104 0.829 0.786 1.124 1.183 WV-7659 1.160
1.582 1.090 1.119 0.904 1.088 WV-8005 0.923 1.199 0.996 1.119 0.936
1.330 WV-8006 1.121 1.088 1.010 1.216 0.792 0.981 WV-8007 1.168
1.582 0.904 1.358 0.873 1.058 WV-8008 1.090 1.755 0.820 1.560 1.136
1.684 WV-8009 0.892 1.233 0.917 1.001 0.843 0.884 WV-8010 0.755
0.896 0.666 1.111 1.010 1.037 WV-8011 1.028 1.084 1.049 1.153 1.086
1.138 WV-8012 0.986 1.298 0.933 1.138 0.926 1.254 WV-2549 0.946
1.084 1.132 1.047 1.071 WV-6028 1.197 1.194 0.959 1.334 1.086 1.054
WV-3688 1.013 1.122 0.852 0.977 0.795 0.943 WV-6408 1.101 1.254
1.049 1.316 1.172 1.245 WV-3662 0.939 1.028 0.886 1.271 WV-7118
1.070 1.171 1.026 1.020 1.077 1.013 WV-6936 1.077 0.408 0.945 0.773
1.115 0.677 WV-7027 1.123 0.978 1.221 1.204 1.246 1.171 WV-5302
1.281 1.524 1.026 1.116 1.034 0.965 WV-6493 1.100 1.255 0.971 1.282
0.912 1.238 WV-2376 1.171 1.462 1.255 0.747 0.951 0.817 WV-3542
1.383 1.657 1.412 1.680 1.588 2.011
Example 6
Activities of Various C9orf72 Oligonucleotides in Various
Assays
[1352] Tables 5A to D show activity of various C9orf72
oligonucleotides in knocking down C9orf72 transcripts (Table 5A,
all transcripts; Table 5B, only V3 transcripts; Table 5C,
Intron/AS; and Table 5D, only Exon 1a transcripts). Relative-fold
change in C9orf72/HPRT1 is shown. Three replicate experiments are
shown for the various C9orf72 oligonucleotides, at a concentration
of 1 .mu.M (Column A) or 10 M (Column B). As with Tables 5A to D,
numbers represent residual transcript level. Delivery of
oligonucleotides was gymnotic and cells were tested after 1
week.
TABLE-US-00014 TABLE 5A Activity of C9orf72 oligonucleotides
(residual level of all C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-8122 1.031 0.928 0.975 0.802 0.942 0.718 WV-8311 1.090
0.915 0.948 0.744 0.962 0.819 WV-8315 0.923 0.600 0.935 0.596 1.097
0.471 WV-8312 1.164 1.210 1.034 1.003 1.006 0.969 WV-8313 1.201
1.550 1.082 1.277 1.024 1.268 WV-8314 1.105 1.044 1.176 1.052 1.351
1.044 WV-8316 0.926 0.930 0.789 0.873 0.846 0.898 WV-8317 1.013
0.996 0.882 0.886 0.876 0.861 WV-8318 1.078 1.136 0.919 0.969 0.972
1.010 WV-2549 0.885 0.903 0.897 0.915 0.989 0.922 WV-6028 0.840
0.855 0.876 0.879 1.006 0.976 WV-6936 0.958 0.969 0.999 0.892 1.140
1.046 WV-7027 0.752 0.873
TABLE-US-00015 TABLE 5B Activity of C9orf72 oligonucleotides
(residual level of V3 C9orf72 transcripts) A (1 .mu.M) B (10 .mu.M)
WV-8114 0.880 0.372 0.904 0.608 0.826 0.704 WV-8122 0.936 0.708
1.003 0.689 0.936 0.596 WV-8311 0.917 0.377 0.898 0.364 0.930 0.377
WV-8315 1.018 0.552 1.039 0.508 0.997 0.313 WV-8312 0.803 0.655
0.803 0.683 0.855 0.651 WV-8313 0.793 0.501 0.862 0.544 0.832 0.511
WV-8314 0.593 0.335 0.564 0.364 0.576 0.313 WV-8316 0.891 0.801
0.843 0.707 0.787 0.818 WV-8317 0.648 0.497 0.671 0.467 0.699 0.518
WV-8318 0.360 0.235 0.372 0.283 0.388 0.276 WV-2549 1.076 1.052
1.076 1.002 1.053 1.044 WV-6028 0.955 1.065 0.975 1.133 0.996 1.133
WV-6936 0.891 0.722 0.873 0.665 0.982 0.717 WV-7027 0.680 0.655
0.719 0.624 0.676 0.587
TABLE-US-00016 TABLE 5C Activity of C9orf72 oligonucleotides
(residual level of Intron/AS C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-8114 1.960 0.449 1.906 1.090 1.742 1.399 WV-8122 1.284
0.734 1.517 0.416 1.008 0.317 WV-8311 1.987 1.193 1.485 1.306 1.766
1.500 WV-8315 1.396 0.370 0.934 0.298 1.126 0.294 WV-8312 2.898
2.346 3.305 1.602 1.965 0.940 WV-8313 2.072 5.115 1.302 3.282 1.506
3.305 WV-8314 2.464 1.664 2.696 1.585 2.380 1.333 WV-8316 1.965
2.028 1.630 0.835 1.279 1.879 WV-8317 1.687 2.337 1.028 1.872 1.117
WV-8318 2.354 1.898 1.569 1.500 2.000 WV-2549 1.718 1.185 1.455
1.046 1.581 1.244 WV-6028 2.063 1.214 1.821 1.248 2.437 2.099
WV-6936 2.593 1.454 2.471 1.050 3.398 2.144 WV-7027 1.270 1.705
1.075 0.742 1.024 0.521
TABLE-US-00017 TABLE 5D Activity of C9orf72 oligonucleotides
(residual level of Exon 1a C9orf72 transcripts) A (1 .mu.M) B (10
.mu.M) WV-8114 1.422 0.339 1.462 0.713 1.402 0.974 WV-8122 1.212
0.665 1.163 0.480 1.063 0.401 WV-8311 1.392 0.222 1.123 0.194 1.229
0.157 WV-8315 1.070 0.377 0.919 0.347 0.365 0.119 WV-8312 1.407
1.605 1.304 1.081 1.030 0.713 WV-8313 1.667 1.103 1.255 0.819 1.308
0.796 WV-8314 1.373 1.043 1.392 0.980 1.611 0.994 WV-8316 0.948
1.200 0.797 0.744 0.797 1.096 WV-8317 0.941 0.941 0.837 0.808 0.872
WV-8318 0.903 0.866 0.948 0.825 1.002 WV-2549 1.255 0.954 0.971
0.859 1.432 0.974 WV-6028 0.872 0.961 0.819 0.954 0.941 1.388
WV-6936 1.059 0.749 1.216 0.878 1.216 0.890 WV-7027 0.713 1.089
0.770 0.770 0.791 0.872
Example 7
Activities of Various C9orf72 Oligonucleotides in Various
Assays
[1353] Tables 6A and B show activity of various C9orf72
oligonucleotides in knocking down C9orf72 transcripts (Table 6A,
all transcripts; and Table 6B, only V3 transcripts). Relative-fold
change in C9orf72/HPRT1 is shown. Three replicate experiments are
shown for the various C9orf72 oligonucleotides, at a concentration
of 10 .mu.M. As with Tables 3A to D, numbers represent residual
transcript level relative to HPRT1. Delivery of oligonucleotides
was gymnotic and cells were tested after 1 week. As a control,
C9orf72 oligonucleotides were tested and found not to be
efficacious in knocking down Malat1 (data not shown). C9orf72
oligonucleotides were also found not to be efficacious against
another target, PFN1 (data not shown).
TABLE-US-00018 TABLE 6A Activity of C9orf72 oligonucleotides
(residual level of all C9orf72 transcripts) Replicate experiments
(10 .mu.M) WV-8008 0.592 0.564 0.608 WV-8548 0.625 0.634 0.630
WV-8010 0.639 0.497 0.579 WV-8549 0.680 0.643 0.621 WV-8012 0.579
0.445 0.617 WV-8550 0.634 0.580 0.608 WV-8454 0.527 0.405 0.489
WV-8455 0.440 0.381 0.437 WV-8551 0.640 0.649 0.691 WV-6408 0.687
0.687 0.762 WV-3662 0.148 0.153 0.157 WV-6936 0.951 0.875 1.255
WV-5302 0.979 0.945 WV-2376 0.926 0.972 Water (negative control)
1.013 0.932 1.056
TABLE-US-00019 TABLE 6B Activity of C9orf72 oligonucleotides
(residual level of V3 C9orf72 transcripts) Replicate experiments
(10 .mu.M) WV-8008 0.104 0.100 0.121 WV-8548 0.313 0.318 0.294
WV-8010 0.222 0.229 0.229 WV-8549 0.347 0.367 0.280 WV-8012 0.135
0.107 0.117 WV-8550 0.313 0.302 0.290 WV-8454 0.161 0.131 0.137
WV-8455 0.087 0.082 0.109 WV-8551 0.316 0.308 0.293 WV-6408 0.546
0.499 0.562 WV-3662 0.121 0.121 0.132 WV-6936 0.845 0.554 WV-5302
0.926 0.907 WV-2376 0.876 0.907 Water (negative control) 1.055
0.945
[1354] Table 6C, below, shows the IC50 of various C9orf72
oligonucleotides tested in a full dose-response assay in ALS MN
(motor neurons), delivered gymnotically and evaluated after 1 week.
10, 2.5, 0.625, 0.16, 0.04 and 0.001 .mu.M concentrations were
tested.
TABLE-US-00020 TABLE 6C IC50 of some C9orf72 oligonucleotides IC50
(.mu.M) WV-8011 0.9119 WV-8012 0.5319 WV-8454 0.5982 WV-8455 0.5803
WV-8551 1.47 WV-8550 0.7681
Example 8
In Vitro Screening Protocol
[1355] This example describes an in vitro screening protocol for
C9orf72 oligonucleotides. oligonucleotides were delivered
gymnotically to ALS neurons for 48 hours in 24-well plates.
[1356] RNA Extraction
[1357] RNA extraction with RNeasy Plus 96 kit (Qiagen, Waltham,
Mass.) following protocol: Purification of Total RNA from Cells
Using Vacuum/Spin Technology. (gDNA removal is critical.)
[1358] For each well, total RNA was eluted in 60 ul of RNase-free
water.
[1359] Reverse Transcription
[1360] Reverse transcription with High-Capacity RNA-to-cDNA.TM. Kit
(Applied Biosystems; available from ThermoFisher, Waltham,
Mass.)
TABLE-US-00021 2X RT Buffer Mix 9 ul RNA sample 13.5 ul
[1361] Heat denaturation at 72.degree. C. for 5 mins, Cool down the
plate on ice for at least 2 mins.
[1362] To each well of heat denatured RNA, add:
TABLE-US-00022 2X RT Buffer Mix 6 20X RT Enzyme Mix 1.5 ul
[1363] The final volume of the cDNA is 30 ul.
[1364] Real-Time PCR
[1365] Tagman Probes:
[1366] C9orf72 all variants: Hs00376619_ml (FAM), Catalog #4351368
(ThermoFisher, Waltham, Mass.)
[1367] C9orf72 V3: Hs00948764_ml(FAM), Catalog #4351368
(ThermoFisher, Waltham, Mass.)
[1368] C9orf72 Exon 1a:
TABLE-US-00023 Forward primer AGATGACGCTTGGTGTGTC Reverse primer
TAAACCCACACCTGCTCTTG probe CTGCTGCCCGGTTGCTTCTCTTT
[1369] C9orf72 antisense RNA/intron:
TABLE-US-00024 Forward primer GGTCAGAGAAATGAGAGGGAAAG Reverse
primer CGAGTGGGTGAGTGAGGA probe AAATGCGTCGAGCTCTGAGGAGAG
[1370] Internal control: Human HPRT1 (VIC)
[1371] Hs02800695_ml, Catalog #4448486 (ThermoFisher, Waltham,
Mass.)
[1372] PCR Reaction:
TABLE-US-00025 Lightcycler 480 master mix 10 ul C9 probe (FAM) 0.5
ul HPRT 1 (VIC) 0.5 ul cDNA* up to 9 ul Nuclease-free H2O to 20 ul
*2 ul of cDNA for all variants probe. 9 ul of cDNA for other C9
probes.
[1373] Real-time PCR using Bio-rad CFX96 Touch
[1374] Run information:
195.0 C for 3:00
2 95.0 C for 0:10
360.0 C for 0:30
[1375] + Plate Read 4 GOTO 2, 39 more times [1376] END
Example 9
Activities of Various C9orf72 Oligonucleotides in Various
Assays
[1377] Tables 7A to 7C, below, present the activities of various
C9orf72 oligonucleotides tested in various assays.
[1378] Brief Description of Various Assays Performed:
[1379] Reporter:
[1380] Luciferase assay, as described herein. For some
oligonucleotides, two numbers are given (e.g., 1.32/2.63 for
WV-6408); these indicate replicate experiments.
[1381] ALS Neurons:
[1382] Neuronal differentiation of iPSCs: iPSCs derived from
fibroblasts from a C9orf72-associated ALS patient (female, 64 years
old) were obtained from RUCDR Infinite Biologics. iPSCs were
maintained as colonies on Corning Matrigel matrix (Sigma-Aldrich,
St. Louis, Mo.) in mTeSR1 medium (STEMCELL Technologies, Vancouver,
BC). Neural progenitors were produced using the STEMdiff Neural
System (STEMCELL Technologies, Vancouver, BC). iPSCs were suspended
in an AggreWell800 plate and grown as embryoid bodies in STEMdiff
Neural Induction Medium for 5 days, with daily 75% medium changes.
Embryoid bodies were harvested with a 37 .mu.m cell strainer and
plated onto Matrigel-coated plates in STEMdiff Neural Induction
Medium. Medium was changed daily for 7 days, with 85-95% of
embryoid bodies exhibiting neural rosettes 2 days post-plating.
Rosettes were picked manually and transferred to plates coated with
poly-L-ornithine and laminin in STEMdiff Neural Induction Medium
(STEMCELL Technologies, Vancouver, BC). Medium was changed daily
for 7 days, until cells reached 90% confluence and were considered
neural progenitor cells (NPCs). NPCs were dissociated with TrypLE
(Gibco, available through ThermoFisher, Waltham, Mass.) and
passaged at a ratio of 1:2 or 1:3 on poly-L-ornithine/laminin
plates in a neural maintenance medium (NMM, 70% DMEM, 30% Ham's
F12, 1.times.B27 supplement) supplemented with growth factors (20
ng/ml FGF2, 20 ng/ml EGF, 5 .mu.g/ml heparin). For maturation into
neurons, NPCs were maintained and expanded for fewer than five
passages, and at >90% confluence were passaged 1:4 onto
poly-L-orinithine/laminin-coated plates in NMM supplemented with
growth factors. The next day, Day 0 of differentiation, medium was
changed to fresh NMM without growth factors. Differentiating
neurons were maintained in NMM for 4 or more weeks, with twice
weekly 50% medium changes. Cells were re-plated with TrypLE at a
density of 125,000 cells/cm.sup.2 as needed.
[1383] V3/Intron:
[1384] Knockdown (KD) of V3 RNA transcript and intron RNA
transcript were measured in ALS neurons. V3 transcripts knocked
down are both wild-type and repeat-containing (indicated as
"Healthy allele" V3 and "Pathological allele" V3 in FIG. 1). Note,
however, that, while the present disclosure is not bound by any
particular theory, the repeat-containing transcript may have a
longer retention time in the nucleus and thus may be preferentially
knocked down. Intron transcript is indicated by the backwards AS
arrow in FIG. 1. Two numbers indicate the V3 and intron knockdown;
for example, for WV-6408, V3 was knocked down by 59% and intron by
65%.
[1385] Stability:
[1386] Stability was assayed in vitro using Mouse (Ms) brain
homogenates.
[1387] TLR9:
[1388] TLR9 Reporter Assay Protocol: Induction of NF-.kappa.B
(NF-.kappa.B inducible SEAP) activity was analyzed using a human
TLR9 or mouse TLR9 reporter assay (HEK-Blue.TM. TLR9 cells,
InvivoGen, San Diego, Calif.). Oligonucleotides at a concentration
of 50 .mu.M (330 .mu.g/mL) and 2-fold serial dilution were plated
into 96-well-plates in the final volume of 20 .mu.L in water.
HEK-Blue.TM. TLR9 cells were added to each well at a density of
7.2.times.10.sup.4 cells in a volume of 180 .mu.L of HEK Blue.TM.
detection medium. Final working concentration of oligonucleotides
in the wells was 5, 2.5, 1.25, 0.625, 0.312, 0.156, 0.078, and
0.0375 .mu.M. HEK-Blue.TM. TLR9 cells were incubated with
oligonucleotides for 16 hours at 37.degree. C. and 5% CO.sub.2. At
the end of the incubation, absorbance at 655 nM was measured by
Spectramax. Water was a negative control. Positive controls were
WV-2021 and ODN 2359, a CpG oligonucleotide. The results are
expressed as fold change in NF-.kappa.B activation over vehicle
control-treated cells. Reference: Human TLR9 Agonist Kit
(InvivoGen, San Diego, Calif.). In this assay, an oligonucleotide
is considered "Clean" if no or essential no activity was detected.
In some experiments, WV-8005, WV-8006, WV-8007, WV-8008, WV-8009,
WV-8010, WV-8011, WV-8012 and WV-8321 showed no appreciable hTLR9
activity, though some showed small activity in mTRL9.
[1389] Complement
[1390] In some embodiments, complement is assessed in a cynomolgus
monkey serum complement activation ex vivo assay. The effects of
oligonucleotides on complement activation were measured in
cynomolgus monkey serum ex vivo. Serum samples from 3 individual
male cynomolgus monkeys were pooled and the pool was used for the
studies.
[1391] The time course of C3a production was measured by incubating
oligonucleotides at a final concentration of 330 .mu.g/mL or the
water control at 37.degree. C. in freshly thawed cynomolgus monkey
serum (1:30 ratio, v/v). Specifically, 9.24 .mu.L of 10 mg/mL stock
of oligonucleotide in vehicle or vehicle alone was added to 270.76
.mu.L of pooled serum, and the resulting mixtures were incubated at
37.degree. C. At 0, 5, 10, and 30 minutes, 20-.mu.L aliquots were
collected and the reaction was terminated immediately by addition
of 2.2 .mu.L of 18 mg/mL EDTA.
[1392] C3a concentrations were measured using MicroVue C3a Plus
Enzyme Immunoassays at a 1:3000 dilution. The results were
presented as the complement split product concentration increase
upon the treatment of pooled serum with oligonucleotides compared
with the treatment with the vehicle control.
PD (Pharmacodynamics) (C9-BAC, icv or Intracerebroventricular
Injection)
PD and Efficacy were Tested in: C9orf72-BAC (C9-BAC) Mouse
Model
[1393] The transgenic mice used for in vivo pharmacological studies
have been described in O'Rourke et al. 2015 Neuron. 88(5): 892-901.
Briefly, the transgenic construct was designed using a bacterial
artificial chromosome (BAC) clone derived from fibroblasts of a
patient with amyotrophic lateral sclerosis (ALS), carrying the
human chromosome 9 open reading frame 72 gene (C9orf72) with a
hexanucleotide repeat expansion (GGGGCC) in the intron between the
alternatively-spliced non-coding first exons 1a and 1b (variant 3).
The BAC isolated a .about.166 kbp sequence (.about.36 kbp human
C9orf72 genomic sequence, with .about.110 kbp upstream and
.about.20 kbp downstream sequences). Upon amplification of
different BAC subclones, one subclone with a limited contraction to
100-1000 GGGGCC repeats was used. The Tg(C9orf72_3) line 112 mice
(JAX Stock No. 023099, Jackson Laboratories, Bar Harbor, Me.) have
several tandem copies of the C9orf72_3 transgene, with each copy
having between 100-1000 repeats ([GGGGCC]100-1000). However, only
mice expressing 500 or more repeats were selected for in vivo
studies used herein.
[1394] In Vivo Procedures:
[1395] For injections of oligonucleotides into the lateral
ventricle, mice were anesthetized and placed on a rodent
stereotaxic apparatus; they were then implanted with a
stainless-steel guide cannula in one of their lateral ventricles
(coordinates: -0.3 mm posterior, +1.0 mm lateral and -2.2 mm
vertically from bregma), which was secured in place using dental
cement. Mice were allowed a one-week recovery period prior to the
injection of compounds. Typical pharmacological studies involved
the injection of up to 50 .mu.g oligonucleotide in a volume of 2.5
.mu.l on day 1, which was followed by another injection of the same
amount and volume on day 8. Euthanasia was performed on day 15; the
mice were deeply anesthetized with avertin and transcardiacally
perfused with saline. Brains were rapidly removed from the skull,
one hemisphere was processed for histological analyses, the other
hemisphere dissected and frozen on dry ice for biochemical
analyses. Similarly, spinal cord was dissected and frozen on dry
ice (lumbar) or processed for histological analyses
(cervical/thoracic).
[1396] Efficacy (C9-BAC): Foci:
[1397] Tissue Preparation and Histological Analyses
[1398] Hemibrains and spinal cord were drop-fixed in 4%
paraformaldehyde for 24 hours, then transferred to 30% sucrose for
24-48 hours and frozen in liquid nitrogen. Serial sagittal 20-.mu.m
thick sections were cut at -18.degree. C. in a cryostat and placed
on Superfrost slides.
[1399] Efficacy (C9-BAC): PolyGP (DPR Assay):
[1400] Tissue preparation for protein and PolyGP
quantification:
[1401] Brain and spinal cord samples were processed using a 2-step
extraction procedure; each step was followed by centrifugation at
10,000 rpm for 10 minutes at 4.degree. C. The first step consisted
of homogenizing samples in RIPA (50 mM Tris, 150 mM NaCl, 0.5% DOC,
1% NP40, 0.1% SDS and Complete.TM., pH 8.0). The second step
consisted of re-suspension of the pellet in 5M guanidine-HCl.
[1402] PolyGP's were quantified in each pool using a
Mesoscale-based assay. Briefly, the polyclonal antibody AB1358
(Millipore, available from Millipore Sigma, Billerica, Ma.) was
used as both capture and detection antibody. MULTI-ARRAY 96 Sm Spot
Plate Pack, SECTOR Plate was coated with 1 .mu.l of 10 ug/ml
purified anti-polyGP antibody (Millipore, AB1358, available from
Millipore Sigma, Billerica, Ma.) in PBS directly on small spot
overnight at 4 C. After washing 3 times with PBST (0.05% Tween-20
in PBS), the plates were blocked with MSD Blocker A Kit (R93AA-2)
or 10% FBS/PBS, at room temperature for 1 hour. Poly-GP purified
from HEK-293 cells (by anti-FLAG affinity purification after
plasmids transfection, Genescript custom made) were serial diluted
with 10% FBS/PBS and used as standard. 25 of standard poly-GP and
samples (diluted or non-diluted) were added to each well, incubated
at room temperature for 1-2 hours. After 3 washes with PBST,
sulfo-tagged anti-GP (AB1358) were added 25 per well, and incubated
at room temperature for another hour. The plates were then washed 3
times, 150 .mu.l/well of MSD Read Buffer T (lx) (R92TC-2, MSD) was
added to each well and read by MSD (MESO QUICKPLEX SQ 120)
according to manufacturer's default setting.
[1403] Expression of C9orf72 protein was determined by western
blotting. Briefly, proteins from RIPA extracts were size
fractionated by 4-12% SDS-PAGE (Criterion gel, Bio-Rad) and
transferred onto PVDF membrane. To detect C9orf72, the membrane was
immunoblotted using the mouse monoclonal anti-C9orf72 antibody
GT779 (1:2000; GeneTex, Irvine, Calif.), followed by secondary
DyLight conjugated antibody. Visualization was conducted using the
Odyssey/Li-Cor imaging system.
Some Additional Abbreviations
[1404] Cx: Cortex [1405] HP: Hippocampus [1406] KD: knockdown
[1407] SC: Spinal Cord [1408] Str: Striatum
TABLE-US-00026 [1408] TABLE 7A.1 Activity of various C9orf72
oligonucleotides. End- Selection Assay points Criteria WV-6408
WV-3688 WV-7121 WV-7122 WV-7123 WV-7124 WV-7125 WV-7126 Reporter
IC.sub.50 (nM) <5 nM 1.32/2.63 5.56/8.65 3.11 2.08 3.16 6.95
4.39 3.481 ALS V3/intron 50% KD 66/0 50/23 38%/44% 63%/88% 47%/42%
55%/75% 31%/60% 35%/64% neurons (Foci KD) Stability Ms brain 80% at
100 63.2 1.81 0.95 2.23 2.63 90.3 92.5 Day 5 TLR9 Human Clean clean
clean clean clean clean 1.3 fold clean 1.3 fold Complement panel
Clean PD (C9- V3/intron 50% KD in KD in Trends in N/A N/A N/A KD in
N/A BAC, icv) relevant HP, Str HP, Str. SC, not brain and SC and SC
cortex regions
TABLE-US-00027 TABLE 7A.2 Activity of various C9orf72
oligonucleotides. End- Selection Assay points Criteria WV-6408
WV-3688 WV-7127 WV-7128 WV-7129 WV-7130 WV-7131 WV-7132 Reporter
IC.sub.50 (nM) <5 nM 1.32/2.63 5.56/8.65 2.45 2.71 2.27 2.8 2.04
1.91 ALS V3/intron 50% KD 59%/65% 55%/79% 0%/22% 13%/45% 28%/66%
11%/55% 0%/24% 30%/70% neurons Stability Ms brain 80% at 100 63.2
100 100 100 90.7 100 100 Day 5 TLR9 Human Clean clean clean clean
clean clean clean clean 1.3 fold Complement panel Clean PD (C9-
V3/intron 50% KD in KD in Trends in N/A N/A KD in N/A N/A BAC, icv)
relevant HP, Str HP, Str. SC, not brain and SC and SC cortex
regions Efficacy foci 50% KD YES YES (C9-BAC) Poly GP 50% KD
TABLE-US-00028 TABLE 7B Activity of various C9orf72
oligonucleotides. End- Assay points WV-7603 WV-7604 WV-7605 WV-7606
WV-7601 WV-7657 WV-7658 WV-7659 WV-7774 WV-7775 Reporter IC.sub.50
(nM) 1.8 1.6 1.3 1.2 2.4 1.3 0.28 0.20 1.1 1.6 ALS V3/intron 55/33
60/67 64/41 72/48 73/72 60/48 68/67 69/57 neurons Stability Ms
brain 100 100 100 100 88 72 82 100 100 100 TLR9 Human clean clean
clean clean clean clean clean clean 5-fold clean PD (C9- V3/intron
N/A N/A N/A Trend Trend Trend Trend N/A BAC, icv) for KD in for KD
in for KD in for KD in Cx, KD in Cx, KD in Cx, KD in Cx, KD in
Spinal Spinal Spinal Spinal Cord Cord Cord Cord
TABLE-US-00029 TABLE 7C Activity of various C9orf72
oligonucleotides. Assay Endpoints WV-6408 WV-3688 WV-8005 WV-8006
WV-8007 WV-8008 WV-8009 WV-8010 WV-8011 WV-8012 Reporter IC.sub.50
(nM) 1.32/2.63 5.56/8.65 0.5 0.5 0.4 0.3 0.5 0.4 0.4 0.2 ALS
V3/intron 66/0 50/23 87/53 84/52 94/52 95/46 87/41 87/61 94/35
95/22 neurons Stability Ms brain 100 63.2 72 88 77 77 93 92 22? 77
TLR9 Human clean clean clean clean clean clean clean clean clean
clean PD (C9- V3/intron 20/30 Trends in 38/39 28/43 61/77 59/73
BAC, icv) HP, Str. and SC Efficacy foci TBD TBD TBD TBD TBD
(C9-BAC) DPR 25% 37% 11% 56% 69% TBD, to be determined.
TABLE-US-00030 TABLE 7D Activity of various C9orf72
oligonucleotides. Assay Endpoints WV-6408 WV-3688 WV-8321 WV-8322
WV-8329 WV-8454 WV-8455 Reporter IC.sub.50 (nM) 1.32/2.63 5.56/8.65
ALS V3/intron 66/0 50/23 neurons Stability Ms brain 100 63.2 100
100 TLR9 Human clean clean PD (C9- V3/intron KD in HP, Trends in
BAC, icv) Str and SC HP, Str. and SC
Table 8. Activity of Various c9orf72 Oligonucleotides
[1409] In Table 8A to 8X, various c9orf72 oligonucleotides were
tested at 10 .mu.M in ALS motor neurons (MN). The oligonucleotides
differ, inter alia, in base sequence, chemistry pattern (e.g.,
pattern of 2' sugar modifications), backbone internucleotidic
linkage pattern and/or pattern of stereochemistry. In Tables 8A to
8X, shown are residual levels of various c9orf72 transcripts (e.g.,
all transcripts, or only V3, V1, intron 1, etc.) relative to HPRT1,
after treatment with c9orf72 oligonucleotides, wherein 1.000 would
represent 100% relative transcript level (no knockdown) and 0.000
would represent 0% relative transcript level (e.g., 100%
knockdown). In Tables 8A to 8X, results from replicate experiments
are shown.
TABLE-US-00031 TABLE 8A Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-3688
0.619 0.817 0.806 WV-7124 0.800 0.641 0.711 WV-6408 0.646 0.574
0.582 WV-7130 0.344 0.321 1.070 WV-8550 0.310 0.253 0.316 WV-8011
0.113 0.144 0.111 WV-8012 0.157 0.185 0.153 WV-2376 1.188 1.108
1.180 WV-9491 1.034 1.027 1.108 WV-5302 1.140 1.101 1.078 WV-6493
1.056 1.049 1.063 WV-8552 1.300 1.140 0.932 water 0.834 1.041
0.985
TABLE-US-00032 TABLE 8B Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-3688 0.845 0.881 0.862 WV-7124 0.799 0.845 0.893 WV-6408 0.810
0.751 0.767 WV-7130 0.788 0.542 WV-8550 0.686 0.538 0.667 WV-8011
0.440 0.446 0.495 WV-8012 0.597 0.509 0.565 WV-2376 1.092 1.012
0.944 WV-9491 1.245 1.146 1.069 WV-5302 1.170 0.839 1.077 WV-6493
1.115 0.868 0.991 WV-8552 1.092 0.875 1.122 water 1.122 0.950
1.122
TABLE-US-00033 TABLE 8C Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-3688
0.901 0.829 0.655 WV-7124 0.594 0.829 0.702 WV-6408 0.784 0.732
0.888 WV-7130 0.476 0.539 0.972 WV-8550 0.379 0.341 0.466 WV-8011
0.207 0.279 0.216 WV-8012 0.250 0.241 0.291 WV-2376 0.993 0.864
0.920 WV-9491 1.156 0.946 1.049 WV-5302 0.920 1.101 0.933 WV-6493
1.056 0.858 1.071 WV-8552 0.901 1.148 1.140 Water 1.197 0.846
0.999
TABLE-US-00034 TABLE 8D Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-3688 0.538 0.685 WV-7124 0.681 0.538 WV-6408 0.516 0.408 0.509
WV-7130 0.399 0.523 WV-8550 0.443 0.350 0.298 WV-8011 0.336 0.378
0.434 WV-8012 0.446 0.446 0.475 WV-2376 0.685 0.681 0.714 WV-9491
0.880 0.923 1.261 WV-5302 0.745 1.510 1.091 WV-6493 0.826 0.997
1.017 WV-8552 1.210 0.963 1.010 Water 1.315 1.193 0.990
TABLE-US-00035 TABLE 8E Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-3688
0.619 0.817 0.806 WV-6408 0.646 0.574 0.582 WV-8550 0.310 0.253
0.316 WV-3662 0.105 0.121 0.119 WV-7188 0.065 0.074 0.062 WV-9494
0.009 0.006 0.009 WV-6936 0.795 0.972 0.800 WV-7027 0.741 0.882
0.900 WV-8595 0.926 0.741 0.919 WV-2376 1.188 1.108 1.180 WV-9491
1.034 1.027 1.108 WV-5302 1.140 1.101 1.078 Water 0.834 1.041
0.985
TABLE-US-00036 TABLE 8F Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-3688 0.845 0.881 0.862 WV-6408 0.810 0.751 0.767 WV-8550 0.686
0.538 0.667 WV-3662 0.160 0.155 0.145 WV-7188 0.116 0.116 0.108
WV-9494 0.013 0.010 0.012 WV-6936 1.099 1.084 0.957 WV-7027 1.040
0.991 0.931 WV-8595 1.280 1.005 1.186 WV-2376 1.092 1.012 0.944
WV-9491 1.245 1.146 1.069 WV-5302 1.170 0.839 1.077 water 1.122
0.950 1.122
TABLE-US-00037 TABLE 8G Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-3688
0.901 0.829 0.655 WV-6408 0.784 0.732 0.888 WV-8550 0.379 0.341
0.466 WV-3662 0.185 0.099 0.182 WV-7188 0.114 0.128 0.106 WV-9494
0.023 0.018 0.026 WV-6936 0.913 0.939 0.907 WV-7027 0.702 0.757
0.926 WV-8595 0.952 0.959 0.959 WV-2376 0.993 0.864 0.920 WV-9491
1.156 0.946 1.049 WV-5302 0.920 1.101 0.933 Water 1.197 0.846
0.999
TABLE-US-00038 TABLE 8H Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-3688 0.538 0.685 WV-6408 0.516 0.408 0.509 WV-8550 0.443 0.350
0.298 WV-3662 0.700 0.576 WV-7188 0.787 0.455 0.527 WV-9494 0.534
0.302 0.512 WV-6936 0.676 0.815 0.930 WV-7027 1.500 0.936 0.976
WV-8595 0.983 1.361 0.930 WV-2376 0.685 0.681 0.714 WV-9491 0.880
0.923 1.261 WV-5302 0.745 1.510 1.091 Water 1.315 1.193 0.990
TABLE-US-00039 TABLE 8I Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-8550
0.310 0.253 0.316 WV-8011 0.113 0.144 0.111 WV-8012 0.157 0.185
0.153 WV-9493 1.013 0.978 1.034 WV-9492 0.784 0.811 0.741 WV-3536
0.789 0.510 0.678 WV-2376 1.188 1.108 1.180 Water 0.834 1.041
0.985
TABLE-US-00040 TABLE 8J Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-8550 0.686 0.538 0.667 WV-8011 0.440 0.446 0.495 WV-8012 0.597
0.509 0.565 WV-9493 1.122 1.084 1.069 WV-9492 1.107 0.816 0.924
WV-3536 0.991 0.783 0.977 WV-2376 1.092 1.012 0.944 water 1.122
0.950 1.122
TABLE-US-00041 TABLE 8K Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-8550
0.379 0.341 0.466 WV-8011 0.207 0.279 0.216 WV-8012 0.250 0.241
0.291 WV-9493 0.933 0.979 0.952 WV-9492 0.712 0.737 0.858 WV-3536
0.687 0.493 0.598 WV-2376 0.993 0.864 0.920 Water 1.197 0.846
0.999
TABLE-US-00042 TABLE 8L Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-8550 0.443 0.350 0.298 WV-8011 0.336 0.378 0.434 WV-8012 0.446
0.446 0.475 WV-9493 0.917 0.838 0.917 WV-9492 1.075 0.714 WV-3536
0.710 0.969 1.061 WV-2376 0.685 0.681 0.714 Water 1.315 1.193
0.990
TABLE-US-00043 TABLE 8M Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-3688
0.751 0.677 0.573 WV-7124 0.546 0.482 0.799 WV-6408 0.649 0.593
0.573 WV-7130 0.365 0.343 0.389 WV-8550 0.297 0.286 0.260 WV-8011
0.135 0.123 0.097 WV-8012 0.111 0.162 0.106 WV-2376 0.833 1.033
1.092 WV-3542 0.977 1.069 0.970 WV-9491 1.047 0.899 1.011 WV-5302
1.011 0.944 1.162 WV-6493 0.984 1.099 1.502 WV-8552 1.146 1.077
0.991 water 0.899 1.122 1.122
TABLE-US-00044 TABLE 8N Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-3688 0.940 0.847 0.813 WV-7124 0.737 0.764 1.022 WV-6408 0.774
0.717 0.646 WV-7130 0.591 0.559 0.525 WV-8550 0.567 0.536 0.555
WV-8011 0.451 0.421 0.421 WV-8012 0.451 0.429 0.470 WV-2376 1.182
1.029 1.058 WV-3542 0.966 0.902 0.871 WV-9491 1.087 0.973 0.933
WV-5302 0.902 0.966 0.980 WV-6493 1.043 0.966 0.947 WV-8552 1.149
1.087 0.947 water 0.895 1.029 0.987
TABLE-US-00045 TABLE 8O Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-3688
0.846 0.920 0.858 WV-7124 0.829 0.779 1.064 WV-6408 0.946 0.801
0.790 WV-7130 0.758 0.664 0.582 WV-8550 0.562 0.426 0.384 WV-8011
0.213 0.235 0.272 WV-8012 0.368 0.283 0.351 WV-2376 1.086 0.835
0.858 WV-3542 0.846 1.101 0.972 WV-9491 0.939 1.140 0.779 WV-5302
0.979 1.035 1.274 WV-6493 1.181 1.035 0.993 WV-8552 1.214 0.966
0.926 water 1.079 0.870 0.889
TABLE-US-00046 TABLE 8P Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-3688 0.324 0.481 0.626 WV-7124 0.734 0.354 0.181 WV-6408 0.261
0.340 0.548 WV-7130 0.452 0.288 0.449 WV-8550 0.484 0.382 0.424
WV-8011 0.391 0.296 WV-8012 0.461 0.508 0.375 WV-2376 1.038 1.269
WV-3542 1.184 0.879 0.600 WV-9491 1.060 1.023 1.674 WV-5302 1.217
1.295 1.097 WV-6493 1.136 1.418 WV-8552 1.128 1.332 0.776 water
0.968 0.903 0.685
TABLE-US-00047 TABLE 8Q Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-3688
0.751 0.677 0.573 WV-6408 0.649 0.593 0.573 WV-8550 0.297 0.286
0.260 WV-3662 0.267 0.216 0.248 WV-7118 0.311 0.219 0.249 WV-9494
0.031 0.043 0.042 WV-6936 0.827 0.874 0.667 WV-7027 0.868 0.788
0.874 WV-8595 0.725 0.681 0.822 WV-2376 0.833 1.033 1.092 WV-3542
0.977 1.069 0.970 WV-9491 1.047 0.899 1.011 water 0.899 1.122
1.122
TABLE-US-00048 TABLE 8R Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-3688 0.940 0.847 0.813 WV-6408 0.774 0.717 0.646 WV-8550 0.567
0.536 0.555 WV-3662 0.261 0.235 0.238 WV-7118 0.276 0.263 0.291
WV-9494 0.046 0.043 0.047 WV-6936 1.014 1.007 1.007 WV-7027 1.065
0.966 0.947 WV-8595 0.994 0.818 0.830 WV-2376 1.182 1.029 1.058
WV-3542 0.966 0.902 0.871 WV-9491 1.087 0.973 0.933 water 0.895
1.029 0.987
TABLE-US-00049 TABLE 8S Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-3688
0.846 0.920 0.858 WV-6408 0.946 0.801 0.790 WV-8550 0.562 0.426
0.384 WV-3662 0.299 0.272 0.381 WV-7118 0.387 0.358 0.325 WV-9494
0.065 0.050 0.063 WV-6936 0.712 0.966 1.035 WV-7027 0.959 0.952
1.049 WV-8595 0.742 0.790 0.841 WV-2376 1.086 0.835 0.858 WV-3542
0.846 1.101 0.972 WV-9491 0.939 1.140 0.779 water 1.079 0.870
0.889
TABLE-US-00050 TABLE 8T Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-3688 0.324 0.481 0.626 WV-6408 0.261 0.340 0.548 WV-8550 0.484
0.382 0.424 WV-3662 0.995 0.831 0.891 WV-7118 0.596 0.724 0.584
WV-9494 0.699 0.455 0.556 WV-6936 1.144 0.948 WV-7027 0.729 1.176
1.260 WV-8595 1.045 0.837 1.209 WV-2376 1.038 1.269 WV-3542 1.184
0.879 0.600 WV-9491 1.060 1.023 water 0.968 0.903 0.685
TABLE-US-00051 TABLE 8U Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-8550
0.297 0.286 0.260 WV-8011 0.135 0.123 0.097 WV-8012 0.111 0.162
0.106 WV-9493 0.761 0.705 0.649 WV-9492 0.506 0.520 0.478 WV-3536
0.663 0.606 0.805 WV-2376 0.833 1.033 1.092 water 0.899 1.122
1.122
TABLE-US-00052 TABLE 8V Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-8550 0.567 0.536 0.555 WV-8011 0.451 0.421 0.421 WV-8012 0.451
0.429 0.470 WV-9493 1.014 0.824 0.807 WV-9492 0.859 0.818 0.801
WV-3536 0.830 0.790 1.126 WV-2376 1.182 1.029 1.058 water 0.895
1.029 0.987
TABLE-US-00053 TABLE 8W Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-8550
0.562 0.426 0.384 WV-8011 0.213 0.235 0.272 WV-8012 0.368 0.283
0.351 WV-9493 1.049 0.870 0.586 WV-9492 0.993 0.795 0.758 WV-3536
0.683 0.697 1.021 WV-2376 1.086 0.835 0.858 water 1.079 0.870
0.889
TABLE-US-00054 TABLE 8X Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-8550 0.484 0.382 0.424 WV-8011 0.391 0.296 0.781 WV-8012 0.461
0.508 0.375 WV-9493 0.391 0.942 0.724 WV-9492 0.481 0.989 0.942
WV-3536 0.729 0.948 0.580 WV-2376 1.038 1.269 water 0.968 0.903
0.685
Table 9. Activity of Various c9orf72 Oligonucleotides
[1410] In Tables 9A to 9D, various c9orf72 oligonucleotides were
tested at 1 .mu.M in ALS motor neurons (MN). The oligonucleotides
differ, inter alia, in base sequence, chemistry pattern (e.g.,
pattern of 2' sugar modifications), backbone internucleotidic
linkage pattern and/or pattern of stereochemistry. In Tables 9A to
9D, shown are residual levels of various c9orf72 transcripts (e.g.,
all transcripts, or only V3, V1, intron 1, etc.) relative to HPRT1,
after treatment with c9orf72 oligonucleotides, wherein 1.000 would
represent 100% relative transcript level (no knockdown) and 0.000
would represent 0% relative transcript level (e.g., 100%
knockdown). In Tables 9A to 9D, results from replicate experiments
are shown.
TABLE-US-00055 TABLE 9A Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-8550
0.557 0.672 WV-8011 0.389 0.417 WV-9505 0.370 0.378 WV-9506 0.465
0.446 WV-9507 0.799 0.822 WV-9508 0.502 0.478 WV-9509 0.428 0.397
WV-9510 0.589 0.478 WV-2376 1.047 1.018 water 0.899 1.122
TABLE-US-00056 TABLE 9B Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-8550 0.683 0.790 WV-8011 0.571 0.567 WV-9505 0.651 0.651 WV-9506
0.824 0.743 WV-9507 0.835 0.847 WV-9508 0.717 0.679 WV-9509 0.703
0.688 WV-9510 0.801 0.830 WV-2376 1.198 1.149 water 0.895 1.029
TABLE-US-00057 TABLE 9C Activity of various c9orf72
oligonucleotides (residual level of V1 C9orf72 transcripts) WV-8550
0.758 0.979 WV-8011 1.000 0.818 WV-9505 0.702 0.603 WV-9506 0.476
0.972 WV-9507 0.993 1.265 WV-9508 0.870 0.926 WV-9509 0.907 0.806
WV-9510 1.109 1.049 WV-2376 1.301 1.310 water 1.079 0.870
TABLE-US-00058 TABLE 9D Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts)
WV-8550 0.781 0.533 WV-8011 1.002 0.600 WV-9505 1.009 0.916 WV-9506
0.910 0.765 WV-9507 0.634 0.843 WV-9508 0.724 0.657 WV-9509 0.512
0.873 WV-9510 0.245 1.045 WV-2376 1.128 1.226 water 0.968 0.903
Table 10. Activity of Various c9orf72 Oligonucleotides
[1411] In Tables 10A to 10B, various c9orf72 oligonucleotides were
tested at various concentrations from 0.01 to 10 .mu.M in ALS motor
neurons (MN). The oligonucleotides differ, inter alia, in backbone
internucleotidic linkage pattern and/or pattern of stereochemistry.
In the DNA core, various oligonucleotides comprise one or two SSO
[5'-PS (Phosphorothioate) in the Sp configuration, PS in the Sp
configuration, PO (Phophodiester)-3'] or one or two SSR [5'-PS
(Phosphorothioate) in the Sp configuration, PS in the Sp
configuration, PS in the Rp configuration-3']. In Tables 10A to
10B, shown are residual levels of various c9orf72 transcripts
(e.g., all transcripts, or only V3), relative to HPRT1, after
treatment with c9orf72 oligonucleotides, wherein 1.000 would
represent 100% relative transcript level (no knockdown) and 0.000
would represent 0% relative transcript level (e.g., 100%
knockdown). In Tables 10A to 10B, results from replicate
experiments are shown.
TABLE-US-00059 TABLE 10A Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-8011 WV-9394 WV-8012 WV-9395 .sup. 10 uM 0.617 0.621 0.639 0.680
0.613 0.643 0.617 0.760 2.5 uM 0.724 0.724 0.739 0.754 0.699 0.704
0.680 0.849 0.625 uM 0.843 0.855 0.814 0.897 0.792 0.855 0.831
0.897 0.16 uM 0.891 0.897 0.849 0.948 0.982 0.968 0.922 0.879 0.04
uM 1.038 1.097 1.009 0.962 1.082 0.975 0.942 1.082 0.01 uM 1.002
1.024 1.009 1.002 0.935 0.948 0.922 0.955
TABLE-US-00060 TABLE 10B Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-8011
WV-9394 WV-8012 WV-9395 10 uM 0.023 0.042 0.026 0.026 0.033 0.032
0.023 0.025 2.5 uM 0.061 0.072 0.049 0.061 0.050 0.055 0.060 0.625
uM 0.125 0.147 0.133 0.130 0.152 0.146 0.139 0.169 0.16 uM 0.266
0.318 0.227 0.291 0.236 0.310 0.277 0.332 0.04 uM 0.726 0.668 0.578
0.687 0.711 0.628 0.444 0.906 0.01 uM 0.992 0.932 0.817 0.992 0.888
0.978 0.932 0.900
Table 11. Activity of Various c9orf72 Oligonucleotides
[1412] In Tables 11A and 11B, various c9orf72 oligonucleotides were
tested at 10 .mu.M in ALS motor neurons (MN). The oligonucleotides
differ, inter alia, in base sequence, pattern of internucleotidic
linkages, and pattern of chemistry (e.g., pattern of
2'-modifications of sugars), wherein some oligonucleotides have a
symmetric (e.g., Table 11B) and some have an asymmetric format
(e.g., Table 11A). In Tables 11A and 11B, shown are residual levels
of V3c9orf72 transcript relative to HPRT1, after treatment with
c9orf72 oligonucleotides, wherein 1.000 would represent 100%
relative transcript level (no knockdown) and 0.000 would represent
0% relative transcript level (e.g., 100% knockdown). In Tables 11A
and 11B, results from replicate experiments are shown. In this and
other tables, all positive and negative controls performed in
various experiments are not necessarily shown.
TABLE-US-00061 TABLE 11A Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts)
WV-10406 0.250 0.267 0.264 WV-10407 0.301 0.314 0.297 WV-10408
0.201 0.211 0.228 WV-10409 0.301 0.314 0.279 WV-10410 0.301 0.363
0.287 WV-10411 0.381 0.332 0.325 WV-10412 0.368 0.414 0.400
WV-10413 0.492 0.428 0.459 WV-10414 0.341 0.358 0.437 WV-10415
0.160 0.239 0.231 WV-10416 0.239 0.239 0.214 WV-8550 0.173 0.184
0.200 WV-10417 0.309 0.479 0.411 WV-10418 0.198 0.279 0.244
WV-10419 0.314 0.420 0.332 WV-10420 0.453 0.517 0.546 WV-10421
0.447 0.658 0.539 WV-10422 0.485 0.444 0.577 WV-10423 0.573 0.602
0.479 WV-10424 0.711 0.741 0.811 WV-10425 0.558 0.341 0.403 WV-9491
0.984 1.107 1.317 WV-3662 0.047 0.051 0.058 WV-10426 0.531
1.005
TABLE-US-00062 TABLE 11B Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-6936
0.517 0.420 0.502 WV-6989 0.746 0.828 0.767 WV-7002 0.691 0.726
0.598 WV-6474 0.649 0.778 0.716 WV-3688 0.581 0.606 0.593 WV-6969
0.677 0.558 WV-6951 0.672 0.636 0.731 WV-3690 0.767 0.736 0.677
WV-6952 0.857 0.799 0.731 WV-6976 0.658 0.558 0.645 WV-6981 0.686
0.731 0.663 WV-6982 0.863 0.751 0.658 WV-9694 0.610 0.663 0.645
WV-9695 0.663 0.636 0.585 WV-3662 0.043 0.038 0.029 WV-2376 0.899
1.040 0.822
Table 12. Activity of Various c9orf72 Oligonucleotides
[1413] In Tables 12A and 12B, various c9orf72 oligonucleotides were
tested at 2.5 or 10 .mu.M in ALS motor neurons (MN). The
oligonucleotides differ, inter alia, in base sequence, pattern of
internucleotidic linkages, and pattern of chemistry (e.g., pattern
of 2'-modifications of sugars), wherein some oligonucleotides have
a symmetric and some have an asymmetric format. In Tables 12A and
12B, shown are residual levels of V3 or all V c9orf72 transcript
relative to HPRT1, after treatment with c9orf72 oligonucleotides,
wherein 1.000 would represent 100% relative transcript level (no
knockdown) and 0.000 would represent 0% relative transcript level
(e.g., 100% knockdown). In Tables 12A and 12B, results from
replicate experiments are shown. In this and other tables, all
positive and negative controls performed in various experiments are
not necessarily shown.
TABLE-US-00063 TABLE 12A Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) WV-6408
(10 uM) 0.564 0.740 0.714 WV-6408 (2.5 uM) 0.700 0.745 0.657
WV-12480 (10 uM) 0.936 1.024 0.880 WV-12480 (2.5 uM) 0.956 0.873
0.798 WV-12481 (10 uM) 0.541 0.667 0.657 WV-12481 (2.5 uM) 0.676
0.626 0.676 WV-12482 (10 uM) 0.378 0.407 0.357 WV-12482 (2.5 uM)
0.431 0.462 0.475 WV-12483 (10 uM) 0.446 0.458 0.458 WV-12483 (2.5
uM) 0.530 0.478 0.505 WV-12484 (10 uM) 0.580 0.667 0.705 WV-12484
(2.5 uM) 0.530 0.662 0.714 WV-12486 (10 uM) 0.527 0.597 0.657
WV-12486 (2.5 uM) 0.538 0.719 0.667 WV-8548 (10 uM) 0.372 0.383
0.367 WV-8548 (2.5 uM) 0.523 0.509 0.516 WV-12439 (10 uM) 0.419
0.549 0.446 WV-12439 (2.5 uM) 0.755 0.609 0.478 WV-12440 (10 uM)
0.352 0.485 0.462 WV-12440 (2.5 uM) 0.635 0.485 0.588 WV-12441 (10
uM) 0.246 0.261 WV-12441 (2.5 uM) 0.434 0.360 0.357 WV-12442 (10
uM) 0.861 0.505 WV-12442 (2.5 uM) 0.671 0.553 WV-12443 (10 uM)
WV-12443 (2.5 uM) 0.481 0.613 0.315 WV-12444 (10 uM) 0.251 0.391
0.367 WV-12444 (2.5 uM) 0.471 0.561 WV-12446 (10 uM) 0.481 0.495
0.564 WV-12446 (2.5 uM) 0.644 0.850 WV-12445 (10 uM) 0.657 0.605
0.588 WV-12445 (2.5 uM) 0.662 0.880 WV-12447 (10 uM) 0.286 0.491
0.329 WV-12447 (2.5 uM) 0.618 0.564 WV-12448 (10 uM) 0.191 0.320
0.214 WV-12448 (2.5 uM) 0.468 0.440 WV-12449 (10 uM) 0.505 0.465
WV-12449 (2.5 uM) 0.597 0.478 WV-12450 (10 uM) WV-12450 (2.5 uM)
0.491 0.534 0.553 WV-12451 (10 uM) 0.443 0.458 0.462 WV-12451 (2.5
uM) 0.545 0.452 0.502 WV-8550 (10 uM) 0.273 0.298 0.278 WV-8550
(2.5 uM) 0.478 0.488 0.440 WV-9491 (10 uM) 0.635 1.053 1.010
WV-9491 (2.5 uM) 1.106 0.815 0.850 WV-3542 (10 uM) 0.962 1.053
1.061 WV-3542 (2.5 uM) 1.113 0.983 1.039
TABLE-US-00064 TABLE 12B Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
WV-6408 (10 uM) 0.861 0.843 0.861 WV-6408 (2.5 uM) 0.861 0.949
0.861 WV-12480 (10 uM) 0.982 0.982 0.969 WV-12480 (2.5 uM) 0.923
0.910 0.956 WV-12481 (10 uM) 0.838 0.832 0.809 WV-12481 (2.5 uM)
0.838 0.820 0.867 WV-12482 (10 uM) 0.714 0.714 0.653 WV-12482 (2.5
uM) 0.699 0.744 0.734 WV-12483 (10 uM) 0.976 0.936 0.936 WV-12483
(2.5 uM) 0.820 0.861 0.917 WV-12484 (10 uM) 0.996 0.969 0.976
WV-12484 (2.5 uM) 0.929 0.923 0.982 WV-12486 (10 uM) 0.760 0.820
0.724 WV-12486 (2.5 uM) 0.782 0.843 0.832 WV-8548 (10 uM) 0.729
0.760 0.771 WV-8548 (2.5 uM) 0.771 0.826 0.798 WV-12439 (10 uM)
0.898 0.873 0.855 WV-12439 (2.5 uM) 0.949 0.873 0.820 WV-12440 (10
uM) 0.803 0.809 0.771 WV-12440 (2.5 uM) 0.849 0.792 0.771 WV-12441
(10 uM) 0.431 0.657 0.685 WV-12441 (2.5 uM) 0.657 0.719 0.695
WV-12442 (10 uM) 0.976 0.861 0.996 WV-12442 (2.5 uM) 0.929 0.461
0.495 WV-12443 (10 uM) 0.923 0.798 0.996 WV-12443 (2.5 uM) 0.484
0.879 0.601 WV-12444 (10 uM) 0.653 0.680 0.666 WV-12444 (2.5 uM)
0.734 0.792 WV-12446 (10 uM) 0.820 0.849 0.849 WV-12446 (2.5 uM)
0.838 0.898 WV-12445 (10 uM) 0.861 0.849 0.855 WV-12445 (2.5 uM)
0.898 0.898 WV-12447 (10 uM) 0.744 0.755 0.739 WV-12447 (2.5 uM)
0.782 0.798 WV-12448 (10 uM) 0.704 0.699 0.662 WV-12448 (2.5 uM)
0.792 0.776 WV-12449 (10 uM) 1.098 0.676 0.443 WV-12449 (2.5 uM)
0.873 0.861 WV-12450 (10 uM) 0.580 0.695 0.704 WV-12450 (2.5 uM)
0.917 0.923 0.996 WV-12451 (10 uM) 0.832 0.885 0.820 WV-12451 (2.5
uM) 0.861 0.792 0.867 WV-8550 (10 uM) 0.724 0.739 0.771 WV-8550
(2.5 uM) 0.803 0.815 0.873 WV-9491 (10 uM) 0.982 0.969 1.060
WV-9491 (2.5 uM) 0.962 0.996 1.053 WV-3542 (10 uM) 1.017 1.038
1.024 WV-3542 (2.5 uM) 1.031 0.879 0.949
Table 13. Activity of Various c9orf72 Oligonucleotides
[1414] In Tables 13A to 13F, various c9orf72 oligonucleotides were
tested in c9BAC mice; mice were administered c9orf72
oligonucleotides ICV in two doses, each 50 .mu.g, one week apart,
and tissue was collected a week after the second dose. The
oligonucleotides differ, inter alia, in base sequence, pattern of
internucleotidic linkages, and pattern of chemistry (e.g., pattern
of 2'-modifications of sugars), wherein some oligonucleotides have
asymmetric and some have an asymmetric format. In Tables 13A to
13F, shown are residual levels of c9orf72 transcriptions [e.g., all
transcripts (all V) or only V3] relative to HPRT1, after treatment
with c9orf72 oligonucleotides, wherein 1.000 would represent 100%
relative transcript level (no knockdown) and 0.000 would represent
0% relative transcript level (e.g., 100% knockdown). Results from
replicate experiments are shown. Tissues evaluated: SC, spinal
cord; and CX, cerebral cortex.
TABLE-US-00065 TABLE 13A Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 1.011 0.798
0.676 0.735 0.705 0.523 0.862 1.011 0.553 0.787 0.963 0.530 1.032
0.969 0.745 0.725 0.950 0.549 1.091 0.976 0.720 0.777 0.997 0.827
1.039 0.950 0.750 0.844 0.844 0.715 0.997 0.838 0.868 0.740 0.917
0.976 0.856 0.771 0.333 0.761 0.844 0.662 1.114 0.850 0.750 0.671
0.705 0.690
TABLE-US-00066 TABLE 13B Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in CX)
WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 0.935 0.754 0.708
0.684 0.739 0.439 0.897 1.089 0.537 0.643 0.968 0.556 1.002 0.891
0.666 0.670 1.059 0.413 1.009 0.928 0.704 0.781 1.009 0.775 0.968
0.981 0.792 0.497 0.837 0.643 1.167 0.749 0.928 0.568 0.848 1.030
0.872 0.533 0.229 0.814 0.803 0.759 1.151 0.968 0.968 0.694 0.575
0.808
TABLE-US-00067 TABLE 13C Activity of various c9orf72
oligonucleotides (residual level of intron 1/AS C9orf72 transcripts
in CX) WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 0.426 1.124
1.248 0.619 2.712 1.256 0.441 0.840 1.944 2.113 2.344 2.280 0.852
0.846 3.072 0.993 2.377 1.213 0.646 3.137 0.888 1.433 0.325 3.704
1.109 2.693 1.230 1.453 3.247 1.568 1.180 0.673 1.740 1.404 1.827
0.301 1.931 2.218 0.864
TABLE-US-00068 TABLE 13D Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 1.635 0.747
0.692 0.603 0.528 0.747 0.999 1.042 0.747 1.504 0.673 0.507 1.525
0.768 0.692 0.536 0.779 0.659 0.742 0.835 0.721 0.598 0.806 0.727
0.779 0.717 0.603 0.632 0.551 0.712 0.678 1.172 0.615 1.515 0.574
0.517 0.697 0.727 0.795 0.558 0.574 0.586 0.945 0.939 0.578 0.582
0.795 0.558
TABLE-US-00069 TABLE 13E Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in SC)
WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 1.325 0.681 0.686
0.465 0.513 0.805 1.122 1.307 0.735 0.816 0.746 0.355 1.382 0.725
0.827 0.408 0.905 0.725 0.788 0.772 0.799 0.389 0.856 0.475 0.874
0.499 0.672 0.416 0.557 0.833 0.761 0.887 0.527 0.931 0.550 0.309
0.777 0.715 1.069 0.443 0.557 0.301 0.970 0.950 0.431 0.482 0.816
0.499
TABLE-US-00070 TABLE 13F Activity of various c9orf72
oligonucleotides (residual level of intron 1/AS C9orf72 transcripts
in SC) WV- PBS 8548 WV-12482 WV-12483 WV-12444 WV-12448 1.812 0.054
1.070 0.065 0.070 0.869 1.942 1.545 0.998 0.241 0.074 0.055 1.163
0.258 0.131 0.438 0.528 0.075 1.503 0.281 0.072 0.721 0.789 0.149
0.124 0.381 0.099 0.091 0.701 2.293 0.015 0.057 0.058 0.757 0.450
0.206 0.129 0.358 0.016 0.472 0.027 0.287 0.021
Table 14. Activity of Various c9orf72 Oligonucleotides
[1415] In Tables 14A to 14B, various c9orf72 oligonucleotides were
tested in motor neurons, with oligonucleotides delivered
gymnotically at concentrations from 0.003 to 10 .mu.M
(Concentrations are provided as exp10). Tested c9orf72
oligonucleotide WV-11532 comprises three neutral internucleotidic
linkages. In Tables 14A and 14B, shown are residual levels of
c9orf72 transcriptions [e.g., all transcripts (all V) or only V3]
relative to HPRT1, after treatment with c9orf72 oligonucleotides,
wherein 1.000 would represent 100% relative transcript level (no
knockdown) and 0.000 would represent 0% relative transcript level
(e.g., 100% knockdown). Results from replicate experiments are
shown.
TABLE-US-00071 TABLE 14A Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts)
Conc. WV-8008 WV-11532 -2.495 0.999 0.958 0.913 1.006 0.894 0.900
-1.796 0.965 0.864 0.882 0.972 0.829 0.858 -1.097 1.006 0.900 0.932
0.907 0.888 0.858 -0.398 0.800 0.742 0.806 0.795 0.747 0.742 0.301
0.624 0.611 0.687 0.562 0.554 0.554 1 0.524 0.500 0.521 0.409 0.411
0.387
TABLE-US-00072 TABLE 14B Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts) Conc.
WV-8008 WV-11532 -2.495 0.947 0.871 1.014 0.927 0.853 0.908 -1.796
0.877 0.841 0.908 0.836 0.769 0.841 -1.097 0.665 0.743 0.871 0.620
0.633 0.717 -0.398 0.555 0.427 0.707 0.421 0.415 0.427 0.301 0.210
0.178 0.304 0.096 0.105 0.094 1 0.056 0.071 0.083 0.012 0.015
0.015
Table 15. Activity of Various c9orf72 Oligonucleotides
[1416] In Tables 15A to 15H, various c9orf72 oligonucleotides which
target the AS (antisense strand) were tested in c9 BAC mice; mice
were administered c9orf72 oligonucleotides ICV in two doses, each
50 g, one week apart, and tissue was collected a week after the
second dose. In Tables 15A to 15H, shown are residual levels of AS
(antisense strand) c9orf72 transcripts relative to HPRT1, after
treatment with c9orf72 oligonucleotides, wherein 1.000would
represent 100 relative transcript level (no knockdown) and 0.000
would represent 0% relative transcript level (e.g., 100%
knockdown). Results from replicate experiments are shown. Tissues
evaluated: SC, spinal cord; and CX, cerebral cortex.
TABLE-US-00073 TABLE 15A Activity of various c9orf72
oligonucleotides (residual level of AS C9orf72 transcripts in CX)
PBS 1.385 0.973 0.642 WV-3542 0.727 0.901 0.927 1.065 1.729 WV-7117
0.637 0.895 1.000 0.559 0.953 WV-5969 1.973 1.102 1.141 1.506 0.895
WV-5979 1.214 1.094 1.079 1.249 1.506 WV-5980 0.669 1.591 1.206
0.993 1.636 WV-5981 1.320 0.859 0.454 0.769 1.157 WV-5982 1.157
1.189 0.540 0.940 1.206 WV-5985 0.927 1.275 1.537 1.223 0.933
WV-5987 0.616 1.311 1.249 1.117 0.591
TABLE-US-00074 TABLE 15B Activity of various c9orf72
oligonucleotides (residual level of AS C9orf72 transcripts in SC)
PBS 0.932 1.048 1.020 WV-3542 1.238 1.131 0.965 1.155 0.882 WV-7117
0.645 0.472 0.687 0.389 0.363 WV-5969 1.108 0.971 1.213 1.247 1.213
WV-5979 1.264 0.965 1.085 0.846 0.913 WV-5980 0.397 1.070 1.027
0.823 1.355 WV-5981 0.876 0.992 1.085 0.823 1.155 WV-5982 1.238
1.171 0.806 0.811 0.664 WV-5985 0.741 0.817 0.925 0.773 0.789
WV-5987 0.659 0.517 0.602 0.757 0.566
TABLE-US-00075 TABLE 15C Activity of various c9orf72
oligonucleotides (residual level of AS C9orf72 transcripts in CX)
PBS 0.928 0.596 1.388 1.089 WV-3542 0.455 0.744 0.872 1.251 0.537
WV-7117 0.744 0.436 0.814 0.544 1.052 WV-5967 0.604 0.526 0.915
1.175 0.749 WV-5970 0.723 0.961 1.467 1.200 1.104 WV-5971 0.592
1.167 1.167 1.074 1.183 WV-5972 1.331 0.974 1.009 0.890 1.436
WV-5973 0.744 0.638 1.359 0.708 1.009 WV-5974 1.104 0.837 0.802
0.837 1.127 WV-5978 0.703 0.703 0.842 0.575 1.066
TABLE-US-00076 TABLE 15D Activity of various c9orf72
oligonucleotides (residual level of AS C9orf72 transcripts in SC)
PBS 0.843 0.860 1.037 1.260 WV-3542 1.313 1.217 1.304 1.260 1.037
WV-7117 0.781 0.362 0.357 0.458 1.151 WV-5967 1.437 1.097 0.909
1.151 1.175 WV-5970 1.313 1.030 0.808 1.175 1.097 WV-5971 1.628
3.102 1.313 1.417 1.794 WV-5972 1.572 2.757 1.819 1.794 2.060
WV-5973 1.143 0.922 1.192 1.313 1.304 WV-5974 1.467 1.674 1.175
1.037 1.407 WV-5978 0.975 1.104 1.081 0.848 1.030
TABLE-US-00077 TABLE 15E Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) PBS 1.061 0.943 0.997 WV-3542 1.068 1.025 1.061 1.137 1.235
WV-7117 0.443 0.388 0.787 0.509 0.561 WV-5969 1.270 1.083 1.137
1.113 1.046 WV-5979 1.161 1.083 1.010 1.137 1.010 WV-5980 1.046
1.253 1.129 1.053 1.297 WV-5981 0.917 0.983 0.815 0.892 1.053
WV-5982 1.017 1.039 0.886 1.068 1.075 WV-5985 1.075 1.169 1.177
1.161 0.868 WV-5987 0.990 1.032 1.153 1.010 0.868
TABLE-US-00078 TABLE 15F Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) PBS 0.960 0.953 1.087 WV-3542 1.029 1.087 0.933 0.953 0.973
WV-7117 0.268 0.203 0.722 0.190 0.196 WV-5969 0.987 0.921 0.960
1.014 1.014 WV-5979 0.933 0.980 0.987 0.980 0.973 WV-5980 0.774
0.987 0.940 0.980 1.095 WV-5981 0.921 0.940 0.994 0.933 1.036
WV-5982 1.072 1.014 0.927 0.940 0.871 WV-5985 1.036 1.000 0.960
0.927 0.927 WV-5987 0.946 0.824 0.883 0.841 0.883
TABLE-US-00079 TABLE 15G Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) PBS 1.073 0.859 1.095 0.818 0.973 WV-3542 0.813 0.824 0.947
1.149 0.830 WV-7117 0.470 0.274 0.703 0.563 1.001 WV-5967 0.404
0.818 0.973 1.065 0.896 WV-5970 0.987 1.095 0.960 0.973 1.118
WV-5971 0.830 0.953 1.087 0.947 1.134 WV-5972 1.182 0.960 0.987
1.065 1.103 WV-5973 0.973 0.896 0.987 0.902 1.103 WV-5974 1.142
1.058 0.967 0.967 1.043 WV-5978 0.980 0.836 1.001 0.953 0.973
TABLE-US-00080 TABLE 15H Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) PBS 1.062 0.950 0.912 1.018 1.077 WV-3542 0.997 0.924 0.918
0.937 0.887 WV-7117 0.700 0.207 0.256 0.241 0.761 WV-5967 1.099
1.004 1.077 0.977 0.977 WV-5970 1.026 0.931 0.899 0.970 1.004
WV-5971 1.004 1.162 0.931 0.997 1.146 WV-5972 0.977 1.114 0.977
1.033 1.178 WV-5973 0.931 0.964 0.997 1.018 1.033 WV-5974 1.062
1.077 0.905 0.912 0.997 WV-5978 0.991 0.997 1.054 0.984 0.964
[1417] In Table 15I.1 to Table 15I.6 and Table 15J.1 to Table
15J.6, various c9orf72 oligonucleotides were tested in C9BAC mice.
Tested c9orf72 oligonucleotides have different base sequences and
varying numbers and positions of non-negatively charged
internucleotidic linkages. Shown are residual levels of C9orf72
transcriptions [e.g., all transcripts (all V) or only V3] relative
to HPRT1, after treatment with c9orf72 oligonucleotides, wherein
1.000 would represent 100% relative transcript level (no knockdown)
and 0.000 would represent 0% relative transcript level (e.g., 100%
knockdown). In Tables 15I.1 to 15M.3, and various other tables
herein, C9orf72 transcript levels are shown relative to HPRT1, and
data from replicates are shown.
TABLE-US-00081 TABLE 15I.1 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) PBS WV-12441 WV-13803 WV-13804 WV-13805 0.94 0.89 0.70 0.91
0.90 1.02 0.71 0.76 0.71 0.77 0.94 0.77 0.54 0.70 0.72 0.98 0.81
0.60 0.66 0.75 1.09 0.62 0.76 0.66 0.73 1.08 0.75 0.63 0.78 0.68
0.94 0.73 0.39 0.81 0.81 0.58 0.63 0.57 0.80
TABLE-US-00082 TABLE 15I.2 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in CX)
PBS WV-12441 WV-13803 WV-13804 WV-13805 1.12 0.75 0.71 1.11 0.86
1.09 0.66 0.78 0.76 0.76 0.99 0.89 0.48 0.85 0.58 0.94 0.77 0.63
0.65 0.76 0.97 0.61 0.77 0.70 0.78 0.94 0.93 0.75 0.80 0.76 0.95
0.83 0.24 0.91 0.87 0.57 0.56 0.56 0.81
TABLE-US-00083 TABLE 15I.3 Activity of various c9orf72
oligonucleotides (residual level of intron 1 transcripts in CX) PBS
WV-12441 WV-13803 WV-13804 WV-13805 0.45 0.62 0.86 0.29 0.87 1.54
0.61 0.99 0.40 0.84 0.83 0.35 0.31 0.54 0.70 0.90 0.44 0.55 0.68
0.55 0.82 0.18 0.86 0.73 0.31 1.76 0.64 0.67 0.70 0.75 0.70 0.60
0.32 1.15 0.55 0.47 0.36 1.20
TABLE-US-00084 TABLE 15I.4 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) PBS WV-12441 WV-13803 WV-13804 WV-13805 0.67 0.49 0.63 0.71
0.88 0.99 0.44 0.62 0.60 0.94 0.52 0.53 0.51 0.78 1.03 0.91 0.47
0.85 0.64 1.16 0.78 0.59 0.43 0.99 1.03 0.59 0.47 0.74 0.63 0.96
0.72 0.50 0.79 0.64 0.43 0.49 0.51 0.82
TABLE-US-00085 TABLE 15I.5 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in SC)
PBS WV-12441 WV-13803 WV-13804 WV-13805 0.46 0.19 0.40 0.54 0.91
0.58 0.16 0.41 0.45 1.03 0.26 0.18 0.25 0.76 0.96 0.73 0.16 0.24
0.58 1.19 0.54 0.31 0.16 1.21 1.02 0.39 0.21 0.55 0.54 0.89 0.62
0.23 0.39 0.41 0.19 0.17 0.22 0.61
TABLE-US-00086 TABLE 15I.6 Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts in
SC) PBS WV-12441 WV-13803 WV-13804 WV-13805 0.16 0.11 0.13 0.20
0.67 1.40 0.07 0.22 0.16 1.72 0.22 0.04 0.09 0.64 1.10 0.99 0.06
0.22 0.11 1.58 0.09 0.25 0.17 1.33 0.45 0.27 0.08 0.44 0.15 0.48
0.52 0.12 0.60 0.19 0.12 0.25 0.12 0.41
TABLE-US-00087 TABLE 15J.1 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) PBS WV-12483 WV-13806 WV-13807 WV-13808 0.86 0.84 0.80 0.76
0.90 1.03 0.91 0.92 0.60 0.89 0.93 0.79 0.83 0.77 0.98 1.00 0.74
0.96 0.83 0.76 1.05 0.73 0.68 0.71 0.85 1.15 0.76 0.90 0.85 0.78
0.75 0.96 0.98 0.82 0.79 0.79 0.86 0.81
TABLE-US-00088 TABLE 15J.2 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in CX)
PBS WV-12483 WV-13806 WV-13807 WV-13808 0.98 0.71 0.79 0.88 0.94
1.18 0.95 1.08 0.58 0.77 0.91 0.83 0.78 0.92 0.94 1.03 0.82 1.08
0.82 0.73 1.03 0.80 0.64 0.61 1.08 0.87 1.06 1.08 0.74 1.00 0.83
1.19 0.94 1.11 0.89 0.94 0.72 0.78
TABLE-US-00089 TABLE 15J.3 Activity of various c9orf72
oligonucleotides (residual level of intron 1 transcripts in CX) PBS
WV-12483 WV-13806 WV-13807 WV-13808 0.46 0.67 1.31 1.04 1.45 1.50
2.15 1.39 0.42 1.40 0.95 1.13 0.75 0.63 1.98 1.24 1.66 1.51 1.33
1.29 0.78 0.88 0.87 0.89 1.58 1.08 0.45 1.16 0.88 0.96 0.80 0.83
1.56 1.54 1.37 1.37 0.53 1.49
TABLE-US-00090 TABLE 15J.4 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) PBS WV-12483 WV-13806 WV-13807 WV-13808 0.90 1.00 0.83 0.87
0.79 0.99 0.64 0.71 0.70 0.74 1.08 0.68 0.80 0.77 0.73 0.98 0.64
1.13 0.75 0.77 0.99 0.70 0.68 0.67 0.79 1.05 0.72 0.67 0.95 0.68
0.75 0.68 0.87 0.85 0.76 0.76 0.79 0.77
TABLE-US-00091 TABLE 15J.5 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in SC)
PBS WV-12483 WV-13806 WV-13807 WV-13808 0.95 0.77 0.47 0.87 0.51
1.06 0.49 0.49 0.60 0.54 1.10 0.42 0.51 0.75 0.48 0.91 0.37 0.95
0.80 0.54 0.95 0.48 0.40 0.48 0.76 1.04 0.54 0.40 0.54 0.79 0.49
0.52 0.53 1.02 0.56 0.49 0.37 0.79
TABLE-US-00092 TABLE 15J.6 Activity of various c9orf72
oligonucleotides (residual level of intron 1 C9orf72 transcripts in
SC) PBS WV-12483 WV-13806 WV-13807 WV-13808 0.97 0.42 0.67 0.50
0.75 1.81 0.32 0.60 0.15 0.75 1.10 0.35 0.74 0.24 0.59 0.94 0.60
1.10 1.14 0.76 0.77 0.57 0.56 0.68 0.59 0.41 0.12 0.72 0.58 0.39
0.27 0.45 0.58 0.14 2.11 0.37 0.69 0.67
[1418] Tables 15K.1 to 15L.2 show the activity of various C9orf72
oligonucleotides in ALS motor neurons in vitro.
TABLE-US-00093 TABLE 15K.1 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72
transcripts/HPRT1) WV-13312 (1 uM) 0.61 0.69 0.66 WV-13312 (0.2 uM)
0.90 0.97 0.92 WV-8007 (1 uM) 0.69 0.80 0.71 WV-8007 (0.2 uM) 1.05
0.83 0.92 WV-13313 (1 uM) 0.68 0.67 0.64 WV-13313 (0.2 uM) 0.93
0.89 0.90 WV-8008 (1 uM) 0.63 0.76 0.66 WV-8008 (0.2 uM) 0.90 0.99
0.98 WV-13305 (1 uM) 0.63 0.68 0.71 WV-13305 (0.2 uM) 0.72 0.96
0.88 WV-13308 (1 uM) 0.60 0.75 0.62 WV-13308 (0.2 uM) 0.77 0.79
0.90 WV-13309 (1 uM) 0.67 0.63 0.66 WV-13309 (0.2 uM) 0.84 0.77
0.79 WV-14552 (1 uM) 0.70 0.71 0.65 WV-14552 (0.2 uM) 0.79 0.73
0.81 WV-14553 (1 uM) 0.79 0.58 0.62 WV-14553 (0.2 uM) 0.81 0.83
0.75 WV-14554 (1 uM) 0.63 0.64 0.69 WV-14554 (0.2 uM) 0.81 0.64
0.63 WV-14555 (1 uM) 0.70 0.66 0.62 WV-14555 (0.2 uM) 0.65 0.78
0.88 WV-8550 (1 uM) 0.81 0.67 0.76 WV-8550 (0.2 M) 0.86 0.75 0.80
Water 1.17 1.13 1.02
TABLE-US-00094 TABLE 15K.2 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts/HPRT1)
WV-13312 (1 uM) 0.17 0.20 0.21 WV-13312 (0.2 uM) 0.56 0.63 0.59
WV-8007 (1 uM) 0.42 0.42 0.39 WV-8007 (0.2 uM) 0.79 0.76 0.76
WV-13313 (1 uM) 0.24 0.25 0.22 WV-13313 (0.2 uM) 0.67 0.60 0.64
WV-8008 (0.1 uM) 0.29 0.34 0.30 WV-8008 (0.2 uM) 0.64 0.83 0.68
WV-13305 (1 uM) 0.23 0.22 0.21 WV-13305 (0.2 uM) 0.48 0.65 0.60
WV-13308 (1 uM) 0.27 0.31 0.27 WV-13308 (0.2 uM) 0.51 0.63 0.68
WV-13309 (1 uM) 0.17 0.15 0.12 WV-13309 (0.2 uM) 0.52 0.60 0.55
WV-14552 (1 uM) 0.28 0.29 0.24 WV-14552 (0.2 uM) 0.77 0.73 0.79
WV-14553 (1 uM) 0.27 0.20 0.21 WV-14553 (0.2 uM) 0.75 0.74 0.58
WV-14554 (1 uM) 0.24 0.24 0.31 WV-14554 (0.2 uM) 0.64 0.53 0.55
WV-14555 (1 uM) 0.19 0.25 0.20 WV-14555 (0.2 uM) 0.55 0.55 0.65
WV-8550 (1 uM) 0.42 0.38 0.38 WV-8550 (0.2 uM) 0.87 0.73 0.76 Water
1.19 1.21 1.09
[1419] Tables 15L.1 to 15M.3 show activity of various C9orf72
oligonucleotides.
[1420] Shown are residual levels of c9orf72 transcriptions [e.g.,
all transcripts (all V) or only V3] relative to HPRT1, after
treatment with c9orf72 oligonucleotides, wherein 1.000 would
represent 100% relative transcript level (no knockdown) and 0.000
would represent 0% relative transcript level (e.g., 100%
knockdown). In these and other tables, not all controls are
shown.
[1421] In these and various other tables, tested C9orf72
oligonucleotides vary in base sequence, format (e.g., some have an
asymmetrical format), pattern of internucleotidic linkages and/or
in pattern of stereochemistry of internucleotidic linkages.
TABLE-US-00095 TABLE 15L.1 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72
transcripts/HPRT1) WV-12441 WV-13803 WV-12483 WV-13806 (10 uM) (10
uM) (10 uM) (10 uM) Water 0.47 0.10 0.68 0.79 1.17 0.48 0.14 0.89
0.68 1.13
TABLE-US-00096 TABLE 15L.2 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts/HPRT1)
WV-12441 WV-13803 WV-12483 WV-13806 (10 uM) (10 uM) (10 uM) (10 uM)
Water 0.14 0.02 0.17 0.11 1.19 0.13 0.02 0.18 0.07 1.21
TABLE-US-00097 TABLE 15M.1 Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72
transcripts/HPRT1) WV-10406 0.25 0.27 0.26 WV-10407 0.30 0.31 0.30
WV-10408 0.20 0.21 0.23 WV-10409 0.30 0.31 0.28 WV-10410 0.30 0.36
0.29 WV-10411 0.38 0.33 0.32 WV-10412 0.37 0.41 0.40 WV-10413 0.49
0.43 0.46 WV-10414 0.34 0.36 0.44 WV-10415 0.16 0.24 0.23 WV-10416
0.24 0.24 0.21 WV-8550 0.17 0.18 0.20 WV-10417 0.31 0.48 0.41
WV-10418 0.20 0.28 0.24 WV-10419 0.31 0.42 0.33 WV-10420 0.45 0.52
0.55 WV-10421 0.45 0.66 0.54 WV-10422 0.49 0.44 0.58 WV-10423 0.57
0.60 0.48 WV-10424 0.71 0.74 0.81 WV-10425 0.56 0.34 0.40 WV-9491
0.98 1.11 1.32 WV-3662 0.05 0.05 0.06 WV-10426 0.53 1.00
TABLE-US-00098 TABLE 15M.2 Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts/HPRT1)
WV-10406 0.62 0.60 0.62 WV-10407 0.60 0.60 0.67 WV-10408 0.57 0.54
0.60 WV-10409 0.57 0.63 0.52 WV-10410 0.59 0.58 0.52 WV-10411 0.68
0.58 0.56 WV-10412 0.67 0.75 0.67 WV-10413 0.73 0.68 0.67 WV-10414
0.68 0.60 0.66 WV-10415 0.57 0.56 0.58 WV-10416 0.64 0.67 0.61
WV-8550 0.56 0.60 0.62 WV-10417 0.62 0.62 0.60 WV-10418 0.49 0.54
0.58 WV-10419 0.48 0.47 0.44 WV-10420 0.56 0.50 0.53 WV-10421 0.57
0.56 0.60 WV-10422 0.56 0.61 0.59 WV-10423 0.63 0.58 0.59 WV-10424
0.81 0.79 0.76 WV-10425 0.77 0.73 0.76 WV-9491 1.04 1.08 1.05
WV-3662 0.08 0.07 0.08 WV-10426 0.87 0.72 0.97
TABLE-US-00099 TABLE 15M.3 Activity of various c9orf72
oligonucleotides (residual level of intron/AS C9orf72
transcripts/HPRT1) WV-10406 0.39 0.56 0.67 WV-10407 0.57 0.54 0.66
WV-10408 0.65 0.57 0.74 WV-10409 0.39 0.77 0.72 WV-10410 0.55 0.64
0.63 WV-10411 0.77 0.74 0.66 WV-10412 0.50 0.71 0.64 WV-10413 0.71
0.64 0.74 WV-10414 0.72 0.75 0.73 WV-10415 0.40 0.49 0.66 WV-10416
0.60 0.54 0.53 WV-8550 0.45 0.44 0.49 WV-10417 0.26 0.62 0.67
WV-10418 0.44 0.63 0.59 WV-10419 0.59 0.65 0.60 WV-10420 0.65 0.66
0.73 WV-10421 0.48 0.59 0.62 WV-10422 0.52 0.22 0.74 WV-10423 0.73
0.50 0.52 WV-10424 0.40 0.47 0.65 WV-10425 0.52 0.28 0.40 WV-9491
0.64 0.81 1.03 WV-3662 0.53 0.48 0.73 WV-10426 1.85 1.47
Table 16. Activity of Various c9orf72 Oligonucleotides
[1422] In Tables 16A to 16H, combination of various S and AS (sense
and antisense) c9orf72 oligonucleotides were tested in c9 BAC mice;
mice were administered c9orf72 oligonucleotides ICV in two doses
one week apart, and tissue was collected a week after the second
dose. WV-7117 was administered in two doses of 50 .mu.g each;
WV-5987 was administered in two doses of 50 .mu.g each; WV-5987 was
administered in two doses of 100 .mu.g each; and the combination of
WV-7117 (50 .mu.g) and WV-5987 (50 .mu.g) was administered in two
doses. In Tables 16A to 16H, shown are residual levels of c9orf72
transcripts relative to HPRT1, after treatment with c9orf72
oligonucleotides, wherein 1.000 would represent 100% relative
transcript level (no knockdown) and 0.000 would represent 0%
relative transcript level (e.g., 100% knockdown). Results from
replicate experiments are shown. Tissues evaluated: SC, spinal
cord; and CX, cerebral cortex.
TABLE-US-00100 TABLE 16A Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
CX) WV-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50 ug)
(100 ug) (50 ug) 1.111 0.490 1.233 1.001 0.433 1.134 0.451 1.058
1.358 0.608 1.158 0.642 1.312 1.921 0.427 1.103 0.548 2.716 0.439
1.001 0.395 1.073 1.466 0.769 0.836 0.326 1.036 1.436 0.689 0.866
0.290 1.377 1.103 0.390 0.791 0.497 1.780 0.445
TABLE-US-00101 TABLE 16B Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in CX)
WV-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50 ug) (100
ug) (50 ug) 1.155 0.510 0.978 0.971 0.546 1.131 0.417 1.055 1.027
0.650 0.999 0.711 1.070 1.308 0.450 1.147 0.450 1.600 0.506 0.881
0.363 0.840 0.985 0.636 1.027 0.327 0.800 1.108 0.985 0.888 0.318
1.273 1.131 0.339 0.773 0.489 1.318 0.506
TABLE-US-00102 TABLE 16C Activity of various c9orf72
oligonucleotides (residual level of intron/AS C9orf72 transcripts
in CX) WV-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50
ug) (100 ug) (50 ug) 0.178 0.799 1.203 2.080 0.649 1.019 0.307
1.849 3.964 0.828 0.696 0.350 1.523 5.303 0.203 0.517 0.042 4.104
0.550 2.214 0.189 1.667 3.242 1.019 0.887 0.108 1.092 3.829 0.408
1.471 0.293 0.428 1.811 2.184 1.019 0.706 1.115 1.187
TABLE-US-00103 TABLE 16D Activity of various c9orf72
oligonucleotides (residual level of all V C9orf72 transcripts in
SC) WV-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50 ug)
(100 ug) (50 ug) 0.955 0.256 0.916 1.082 0.324 1.017 0.226 0.989
0.929 0.251 1.128 0.201 0.989 1.144 0.244 0.962 0.340 0.942 0.260
1.038 0.370 0.929 1.168 0.355 0.975 0.234 0.982 1.168 0.237 0.982
0.214 0.955 1.053 0.237 0.942 0.221 0.975 0.236
TABLE-US-00104 TABLE 16E Activity of various c9orf72
oligonucleotides (residual level of V3 C9orf72 transcripts in SC)
WV-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50 ug) (100
ug) (50 ug) 0.924 0.213 1.047 0.998 0.262 1.040 0.155 1.069 0.950
0.248 0.984 0.121 1.004 1.335 0.196 0.905 0.234 0.931 0.175 1.033
0.244 0.924 1.325 0.248 1.069 0.136 0.931 1.211 0.180 1.047 0.154
0.937 1.092 0.146 0.998 0.130 1.047 0.163
TABLE-US-00105 TABLE 16F Activity of various c9orf72
oligonucleotides (residual level of AS C9orf72 transcripts in SC)
VW-7117 WV-5987 WV-5987 WV-7117 + WV-5987 PBS (50 ug) (50 ug) (100
ug) (50 ug) 0.484 0.209 1.023 1.159 0.286 0.604 0.223 0.968 0.878
0.168 1.059 0.125 0.734 0.981 0.399 0.734 0.078 1.104 0.209 1.662
0.229 0.728 0.935 0.078 1.192 0.254 0.885 1.508 0.106 1.242 0.377
0.854 1.870 0.232 1.023 0.224 0.670 0.315
Example 10
Activity of Various Oligonucleotides
[1423] The efficacy of various additional chemical moieties, which
can be used in construction of C9orf72 oligonucleotides, were
tested in oligonucleotides which target a different gene target,
Malat1. Data is provided in FIGS. 7A to 7D and described here.
[1424] Single-stranded Malat1 oligonucleotide WV-3174 was
conjugated to any of various conjugates (Mod027, Mod028 or Mod007),
to produce WV-7558, WV-7559, and WV-7560, detailed below in Table
17, and diagrammed in Example 1. WV-3174 is a cross-species
oligonucleotide, as its base sequence has no mismatches with the
corresponding sequence in human, dog (Canis lupis familiaris
(mm10)), mouse (Mus musculus (mm10)), rat (Rattus norvegicus (m6)),
and monkeys Macaca mulatta (rheMac8) and Macaca fascicularis
(macFas5).
[1425] Table 17, below, provides information for some Malat1
oligonucleotides. Included in Table 17 is WV-8448, which is
described elsewhere herein.
TABLE-US-00106 TABLE 17 Malat1 oligonucleotides Base Stereo- Name
Modified Sequence Sequence chemistry WV- mU * mG * mC * mC * mA *
UGCCAGGCTG XXXXXXXXXX 3174 G * G * C * T * G * G * T * GTTATGACUC
XXXXXXXXX T * A * T * mG * mA * mC * mU * mC WV- Mod027L001 mU * mG
* mC * UGCCAGGCTG OXXXXXXXXX 7558 mC * mA * G * G * C * T *
GTTATGACUC XXXXXXXXXX G * G * T * T * A * T * mG * mA * mC * mU *
mC WV- Mod028L001 mU * mG * mC * UGCCAGGCTG OXXXXXXXXX 7559 mC * mA
* G * G * C * T * G * GTTATGACUC XXXXXXXXXX G * T * T * A * T * mG
* mA * mC * mU * mC WV- Mod007L001 mU * mG * mC * UGCCAGGCTG
OXXXXXXXXX 7560 mC * mA * G * G * C * T * G * GTTATGACUC XXXXXXXXXX
G * T * T * A * T * mG * mA * mC * mU * mC WV- Mod059L001mU * mG *
mC * UGCCAGGCTG OXXXXXXXXX 8448 mC * mA * G * G * C * T * G *
GTTATGACUC XXXXXXXXXX G * T * T * A * T * mG * mA * mC * mU * mC
For definitions of various components in Modified Sequence and
Stereochemistry, see descriptions and texts following and related
to Table 1A.
[1426] These experiments demonstrate greater biodistribution and
greater knockdown with sulfonamide- or anisamide-conjugated
WV-3174. Animals tested: Male C57BL/6 mice, 10-12 week-old, 5
groups, 50 mice. ICV cannulation was performed. Phase 1: N.dbd.10;
ICV injections of PBS, 50, 100, 150 or 250 mg ICV (2 mice per
group), Clin Obs for 2 days. Phase 2: N.dbd.40; ICV injection of
PBS or oligonucleotide on Day 1 in awake animals. Necropsy 7 days
after injection. Necropsy: whole body perfusion with PBS.
Procedure: Dissect lumbar spinal cord (PD) and place
thoracic/cervical spinal cord in formalin (histology); dissect one
hemibrain (cortex, hippocampus, striatum, cerebellum), flash freeze
(exposures/transcripts). Second hemibrain post fixed in formalin,
cryoprotected and flash frozen (Malat1 KD/oligonucleotide
visualization).
[1427] The parameters for Phase 2 were as follows:
TABLE-US-00107 TABLE 18 Parameters for Phase 2 Malat1 animal
testing Test Dosing Dose Group Article Dose Regimen Volume # mice 1
PBS NA ICV 2.5 ml 8 2 WV-3174 50 mg ICV 2.5 ml 8 3 WV-7558 50 mg
ICV 2.5 ml 8 4 WV-7559 50 mg ICV 2.5 ml 8 5 WV-7560 50 mg ICV ml
8
[1428] FIGS. 7B to 7D show MALAT1 knockdown in spinal cord.
Triantennary anisamide conjugated Malat1 oligonucleotide (WV-3174)
shows significant knockdown of Malat1 (70%). Triantennary anisamide
conjugated Malat1 oligonucleotide (WV-3174) also shows significant
accumulation in spinal cord.
[1429] FIGS. 7E, 7F and 7G show MALAT1 knockdown in cortex.
Triantennary anisamide conjugated Malat1 oligonucleotide (WV-3174)
shows knockdown of Malat1 (.about.34%). Triantennary anisamide
conjugated Malat1 oligonucleotide (WV-3174) also shows higher
accumulation in Cortex.
Example 11
Effects of C9orf72 Oligonucleotides In Vivo on C9orf72 Transcripts
in C9-BAC Mice
[1430] A pharmacodynamics study was performed to compare the
effects of C9orf72 oligonucleotides on knockdown of C9orf72
transcripts in C9-BAC mice.
[1431] C9orf72 oligonucleotides tested were: WV-6408, WV-8009,
WV-8010, WV-8011, and WV-8012. Negative controls were PBS
(phosphate-buffered saline) and WV-2376, which does not target
C9orf72.
[1432] Animals used: Male and Female C9-BAC mice, 12 week-old, 7
groups, 50 mice.
[1433] ICV cannulation was performed. ICV injection of PBS or 50
.mu.g of oligonucleotide on Day 1 in awake animals. 2nd dose of PBS
or 50 .mu.g of oligonucleotide on Day 8. Dose volume, 2.5 .mu.l.
Necropsy 2 weeks after first injection.
[1434] Necropsy: whole body perfusion with PBS. Dissect lumbar
spinal cord (PD) and place thoracic/cervical spinal cord in
formalin (histology); dissect one hemibrain (cortex, hippocampus,
striatum, cerebellum), flash freeze (exposures/transcripts). Second
hemibrain post fixed in formalin, cryoprotected and flash frozen
(RNA foci/oligonucleotide visualization).
[1435] Results are shown in FIGS. 8A to H.
[1436] Transcripts were analyzed from the cerebral cortex (FIGS. 8A
to D) and spinal cord (FIGS. 8E to H). Transcripts analyzed were:
All transcripts (FIGS. 8A and E); V3 (FIGS. 8B and F); V3 (exon 1a)
(FIGS. 8C and G); and Intron1/AS (FIGS. 8D and H).
[1437] Several C9orf72 oligonucleotides were shown to be capable of
knocking down C9orf72 transcripts, including V3, in the cortex and
spinal cord of C9-BAC mice.
Example 12
Distribution of C9orf72 Oligonucleotides In Vivo in Spinal Cord and
Cerebral Cortex of C9-BAC Mice
[1438] A pharmacokinetics study was performed to examine the
distribution of C9orf72 oligonucleotides in vivo in spinal cord and
cerebral cortex of C9-BAC mice.
[1439] C9orf72 oligonucleotides tested were: WV-6408, WV-8009,
WV-8010, WV-8011, and WV-8012. Negative controls were PBS
(phosphate-buffered saline) and WV-2376, which does not target
C9orf72.
[1440] Results are shown in FIGS. 9A (spinal cord) and 9B (cerebral
cortex). The color red indicates points outside the standard curve
range.
[1441] Several C9orf72 oligonucleotides showed significant
accumulation in spinal cord and cortex.
Example 13
Effect of C9orf72 Oligonucleotides In Vivo on polyGP Levels in
Hippocampus of C9-BAC Mice
[1442] A study was performed to evaluate the effect of C9orf72
oligonucleotides in vivo on polyGP (a dipeptide repeat protein)
levels in hippocampus of C9-BAC mice.
[1443] C9orf72 oligonucleotides tested were: WV-6408, WV-8009,
WV-8010, WV-8011, and WV-8012. Negative controls were PBS
(phosphate-buffered saline) and WV-2376, which does not target
C9orf72. WT is a control.
[1444] Method:
[1445] Tissues were homogenized in RIPA buffer and clarified by
centrifugation. Lysate concentration was determined by Pierce
Protein 660 nm assay (reagent available as Catalog number: 22660
from ThermoFisher, Waltham, Ma.) and normalized in RIPA lysis and
extraction buffer (reagent available as Catalog number: 89900 from
ThermoFisher, Waltham, Ma.). MSD 96-well Small Spot Standard plates
were coated with anti-polyGP (Millipore ABN1358, available from
Millipore Sigma, Billerica, Ma.) overnight at 4C, washed in PBST,
and blocked with PBS containing 10% fetal bovine serum. Lysate
samples were diluted 1:4 in PBS/10% FBS and loaded at 75 ug per
well, and incubated at room temperature. A standard curve was
composed of affinity purified Flag-polyGP (GenScript, Piscataway,
N.J.) diluted into wild-type mouse brain RIPA lysate. Detection was
performed with Sulfo-tag-conjugated anti-polyGP, and read with MSD
Read Buffer T with Surfactant in a MSD QuickPlex SQ 120 (Meso Scale
Diagnostics, Rockville, Md.) instrument.
[1446] The data, shown in FIG. 10, was quantified from a standard
curve of GenScript Flag-polyGP diluted in wild-type mouse brain
lysate.
[1447] The data show that the C9orf72 oligonucleotides were capable
of decreasing the level of polyGP (a dipeptide repeat protein) in
hippocampus in C9-BAC mice.
Example 14
Additional Protocols
[1448] Additional protocols for experiments are presented
below.
[1449] A non-limiting example of a hybridization assay for
detecting a target nucleic acid is described herein. Such an assay
can be used for detecting and/or quantifying a C9orf72
oligonucleotide, or any other nucleic acid or oligonucleotide to
any target, including targets which are not C9orf72.
[1450] Pharmacokinetics Studies:
[1451] Tissue Preparation for Oligonucleotide Quantification and
Transcript Quantification:
[1452] Tissues were dissected and fresh-frozen in the pre-weighted
Eppendorf tubes. Tissue weight were calculated by re-weight the
tubes. 4 volume of Trizol or lysis buffer (4 M Guanidine; 0.33%
N-Lauryl Sarcosine; 25 mM Sodium Citrate; 10 mM DTT) were added to
one unit weight (4 of buffer for 1 mg tissue). Tissue lysis were
done by Precellys Evolution tissue homogenizer (Bertin
Technologies, Montigny-le-Bretonneux, France) until all the tissue
pieces were dissolved at 4 C. 30-50 .mu.l of tissue lysates were
saved in 96 well plate for PK measurement, and rest of lysates were
stored at -80 C (if it is in lysis buffer) or continue with RNA
extraction (if it is in Trizol buffer).
[1453] Transcript Quantification:
[1454] Hybridization probes (IDT-DNA)
TABLE-US-00108 Capture probe: "C9-Intron-Cap" /5AmMC12/TGGCGAGTGG
Detection probe: "C9-Intron-Det": GTGAGTGAGG/3BioTEG/
[1455] 5AmC12 is a 5'-amine with C.sub.12 linker.
[1456] 3BioTEG is a Biotinylated probe.
[1457] Maleic anhydride activated 96 well plate (Pierce 15110) was
coated with 50 .mu.l of capture probe at 500 nM in 2.5%
NaHCO.sub.3(Gibco, 25080-094) for 2 hours at 37 C. The plate then
washed 3 times with PBST (PBS+0.1% Tween-20), blocked with 5% fat
free milk-PBST at 37 C for 1 hour. Payload oligonucleotide was
serial diluted into matrix. This standard together with original
samples were diluted with lysis buffer (4 M Guanidine; 0.33%
N-Lauryl Sarcosine; 25 mM Sodium Citrate; 10 mM DTT) so that
oligonucleotide amount in all samples is less than 50 ng/ml. 20 of
diluted samples were mixed with 180 of 333 nM detection probe
diluted in PBST, then denatured in PCR machine (65 C, 10 min, 95 C,
15 min, 4 C .infin.). 50 of denatured samples were distributed in
blocked ELISA plate in triplicates, and incubated overnight at 4 C.
After 3 washes of PBST, 1:2000 streptavidin-AP (SouthernBiotech,
7100-04) in PBST was added, 50 .mu.l per well and incubated at room
temperature for 1 hour. After extensive wash with PBST, 100 .mu.l
of AttoPhos (Promega S1000) was added, incubated at room
temperature in dark for 10 min and read on plate reader (Molecular
Device, M5) fluorescence channel: Ex435 nm, Em555 nm. The
oligonucleotide in samples were calculated according to standard
curve by 4-parameter regression.
[1458] FISH Protocol for GGGGCC and GGCCCC RNA Foci
[1459] Fixation:
[1460] The slides were dried at room temperature for 30 mins then
fixed in 4% PFA for 20 mins. After fixation, the slides were washed
for 3 times in PBS then stored at 4.degree. C. in 70% prechilled
ethanol for at least 30 min.
[1461] Pre-Hybridization:
[1462] The slides were rehydrated in FISH washing buffer (40%
formamide, 2.times.SSC in DEPC water) for 10 min. Hybridization
buffer (40% Formamide, 2.times.SSC, 0.1 mg/ml BSA, 0.1 g/ml dextran
sulfate, 1% Vanadyl sulfate complex, 0.25 mg/ml tRNA in DEPC water)
was added on slides and incubated at 55.degree. C. for 30 min.
[1463] Preparation of the Probe:
[1464] Cy3-(GGCCCC)3 (detecting sense repeat expansion) and
Cy3-(GGGGCC)3 (detecting antisense repeat expansion) probes were
denatured at 95.degree. C. for 10 mins. After cooling down on ice,
the probes were diluted to 200 ng/ml with cold hybridization
buffer.
[1465] Hybridization:
[1466] The slides were briefly washed with FISH washing buffer and
diluted probes were added on the slides. The slides were incubated
at 55.degree. C. for 3 hours in a hybridization oven. After
hybridization, slides were washed 3 times at 55.degree. C. with
FISH washing buffer, 15 min per wash. Then slides were briefly
washed once with 1.times.PBS.
[1467] Neuronal Nuclei Immunofluorescence Staining:
[1468] The slides were blocked with blocking solution (2% normal
goat serum in PBS) for 1 hour. Anti-NeuN antibody (MAB377,
Millipore) was diluted 1:500 in blocking solution and applied to
the slides at 4.degree. C. over night. The slides were then washed
3 times with PBS and incubate with 1:500 diluted goat anti-mouse
secondary antibody with Alexa Fluor 488(Life technology) at room
temperature for 1 hour. Then the slides were washed 3 times with
PBS. Finally, the sides were mounted with DAPI for imaging.
[1469] Imaging and Foci Quantification:
[1470] The images were taken with RPI spinning disk confocal
microscope (Zeiss) at 40.times. magnification. 488, CY3 and DAPI
channels were collected. RNA foci were quantified with ImageJ
software(NIH).
[1471] While various embodiments have been described and
illustrated herein, those of ordinary skill in the art will readily
envision a variety of other means and/or structures for performing
the functions and/or obtaining the results and/or one or more of
the advantages described in the present disclosure, and each of
such variations and/or modifications is deemed to be included. More
generally, those skilled in the art will readily appreciate that
all parameters, dimensions, materials, and configurations described
herein are meant to be example and that the actual parameters,
dimensions, materials, and/or configurations will depend upon the
specific application or applications for which the teachings of the
present disclosure is/are used. Those skilled in the art will
recognize, or be able to ascertain using no more than routine
experimentation, many equivalents to the specific embodiments of
the disclosure described in the present disclosure. It is,
therefore, to be understood that the foregoing embodiments are
presented by way of example only and that, within the scope of the
appended claims and equivalents thereto, claimed technologies may
be practiced otherwise than as specifically described and claimed.
In addition, any combination of two or more features, systems,
articles, materials, kits, and/or methods, if such features,
systems, articles, materials, kits, and/or methods are not mutually
inconsistent, is included within the scope of the present
disclosure.
* * * * *
References