U.S. patent application number 15/892560 was filed with the patent office on 2019-01-31 for compositions and methods for modulation of smn2 splicing in a subject.
This patent application is currently assigned to Biogen MA Inc.. The applicant listed for this patent is Biogen MA Inc., Cold Spring Harbor Laboratory. Invention is credited to C. Frank Bennett, Seng H. Cheng, Yimin Hua, Gene Hung, Katherine W. Klinger, Adrian R. Krainer, Marco A. Passini, Frank Rigo, Lamya Shihabuddin.
Application Number | 20190030058 15/892560 |
Document ID | / |
Family ID | 43356760 |
Filed Date | 2019-01-31 |
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United States Patent
Application |
20190030058 |
Kind Code |
A1 |
Bennett; C. Frank ; et
al. |
January 31, 2019 |
COMPOSITIONS AND METHODS FOR MODULATION OF SMN2 SPLICING IN A
SUBJECT
Abstract
Disclosed herein are compounds, compositions and methods for
modulating splicing of SMN2 mRNA in a subject. Also provided are
uses of disclosed compounds and compositions in the manufacture of
a medicament for treatment of diseases and disorders, including
spinal muscular atrophy.
Inventors: |
Bennett; C. Frank;
(Carlsbad, CA) ; Hung; Gene; (Carlsbad, CA)
; Rigo; Frank; (Carlsbad, CA) ; Krainer; Adrian
R.; (Huntington Station, NY) ; Hua; Yimin;
(Jericho, NY) ; Passini; Marco A.; (Shrewsbury,
MA) ; Shihabuddin; Lamya; (Brighton, MA) ;
Cheng; Seng H.; (Natick, MA) ; Klinger; Katherine
W.; (Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen MA Inc.
Cold Spring Harbor Laboratory |
Cambridge
Cold Spring Harbor |
MA
NY |
US
US |
|
|
Assignee: |
Biogen MA Inc.
Cambridge
MA
Cold Spring Harbor Laboratory
Cold Spring Harbor
NY
|
Family ID: |
43356760 |
Appl. No.: |
15/892560 |
Filed: |
February 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15630445 |
Jun 22, 2017 |
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15892560 |
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14617388 |
Feb 9, 2015 |
9717750 |
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15630445 |
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13380021 |
Apr 12, 2012 |
8980853 |
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PCT/US2010/039077 |
Jun 17, 2010 |
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14617388 |
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61218031 |
Jun 17, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 11/00 20180101; C12N 2310/315 20130101; C12N 2310/321
20130101; A61P 25/00 20180101; C12N 2320/33 20130101; A61K 31/7088
20130101; C12N 2310/3525 20130101; A61P 25/02 20180101; A61P 25/28
20180101; A61P 21/02 20180101; C12N 2310/11 20130101; A61K 31/712
20130101; A61P 21/00 20180101; C12N 2320/32 20130101; C12N 15/113
20130101 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C12N 15/113 20100101 C12N015/113 |
Claims
1. A method comprising administering to a subject an antisense
compound comprising an antisense oligonucleotide complementary to
intron 7 of a nucleic acid encoding human SMN2 pre-mRNA, wherein
the antisense compound is administered into the cerebrospinal
fluid.
2. The method of claim 1, wherein the administration is: (i) into
the intrathecal space; (ii) into the cerebrospinal fluid in the
brain.
3. (canceled)
4. The method of claim 1, wherein the administration comprises a
bolus injection.
5.-14. (canceled)
15. The method of claim 1 comprising administering at least one
induction dose during an induction phase and administering at least
one maintenance dose during a maintenance phase.
16.-32. (canceled)
33. The method of claim 1, comprising co-administration of the
antisense compound and at least one other therapy.
34.-41. (canceled)
42. The method of claim 1, wherein (i) inclusion of exon 7 of SMN2
mRNA in a motoneuron in the subject is increased; or (ii) inclusion
of exon 7 amino acids in SMN2 polypeptide in a motoneuron in the
subject is increased.
43. (canceled)
44. A method of increasing (i) inclusion of exon 7 of SMN2 mRNA in
a motoneuron in a subject or (ii) increasing inclusion of exon 7
amino acids in SMN2 polypeptide in a motoneuron in a subject, the
method comprising administering to the subject an antisense
compound comprising an antisense oligonucleotide complementary to
intron 7 of a nucleic acid encoding human SMN2 and thereby
increasing inclusion of exon 7 of SMN2 mRNA in the motoneuron in
the subject or increasing inclusion of exon 7 amino acids in SMN2
polypeptide in a motoneuron in the subject.
45. (canceled)
46. The method of claim 44, wherein the subject has SMA.
47. The method of claim 44, wherein the subject has type I SMA,
type II SMA, or type III SMA.
48.-49. (canceled)
50. The method of claim 44, wherein a first dose is administered in
utero.
51. The method of claim 50, wherein the first dose is administered
prior to complete formation of the blood-brain-barrier.
52.-60. (canceled)
61. The method of claim 1, further comprising identifying a subject
having SMA.
62.-84. (canceled)
85. The method of claim 1, wherein each nucleoside of the antisense
oligonucleotide comprises a 2'-methoxyethyl sugar moiety.
86. (canceled)
87. The method of claim 1, wherein each internucleoside linkage of
the antisense oligonucleotide is a phosphorothioate internucleoside
linkage.
88. The method of claim 1, wherein the antisense oligonucleotide
consists of 10 to 25 linked nucleosides.
89.-93. (canceled)
94. The method of claim 1, wherein the oligonucleotide has a
nucleobase sequence comprising at least 10 contiguous nucleobases
of the nucleobase sequence SEQ ID NO: 1.
95. The method of claim 94, wherein the oligonucleotide has a
nucleobase sequence comprising at least 15 contiguous nucleobases
of the nucleobase sequence SEQ ID NO: 1.
96.-103. (canceled)
104. An antisense compound comprising an antisense oligonucleotide
complementary to intron 7 of a nucleic acid encoding human
SMN2.
105.-107. (canceled)
108. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier, the antisense compound of claim 104, and one or
more of: valproic acid, riluzole, hydroxyurea, a butyrate, and
trichostatin-A.
Description
SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled 20100617_CORE0086WOSEQ.txt, created Jun. 17, 2010,
which is 5 Kb in size. The information in the electronic format of
the sequence listing is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0002] Newly synthesized eukaryotic mRNA molecules, known as
primary transcripts or pre-mRNA are processed before translation.
Processing of the pre-mRNAs includes addition of a 5' methylated
cap and an approximately 200-250 base poly(A) tail to the 3' end of
the transcript. Processing of mRNA from pre-mRNA also frequently
involves splicing of the pre-mRNA, which occurs in the maturation
of 90-95% of mammalian mRNAs. Introns (or intervening sequences)
are regions of a pre-mRNA (or the DNA encoding it) that are not
included in the coding sequence of the mature mRNA. Exons are
regions of a primary transcript that remain in the mature mRNA. The
exons are spliced together to form the mature mRNA sequence. Splice
junctions are also referred to as splice sites with the 5' side of
the junction often called the "5' splice site," or "splice donor
site" and the 3' side the "3' splice site" or "splice acceptor
site." In splicing, the 3' end of an upstream exon is joined to the
5' end of the downstream exon. Thus the unspliced pre-mRNA has an
exon/intron junction at the 5' end of an intron and an intron/exon
junction at the 3' end of an intron. After the intron is removed,
the exons are contiguous at what is sometimes referred to as the
exon/exon junction or boundary in the mature mRNA. Cryptic splice
sites are those which are less often used but may be used when the
usual splice site is blocked or unavailable. Alternative splicing,
defined as the splicing together of different combinations of
exons, often results in multiple mRNA transcripts from a single
gene.
[0003] Up to 50% of human genetic diseases resulting from a point
mutation result in aberrant pre-mRNA processing. Such point
mutations can either disrupt a current splice site or create a new
splice site, resulting in mRNA transcripts comprised of a different
combination of exons or with deletions in exons. Point mutations
also can result in activation of a cryptic splice site or disrupt
regulatory cis elements (i.e. splicing enhancers or silencers)
(Cartegni et al., Nat. Rev. Genet., 2002, 3, 285-298; Drawczak et
al., Hum. Genet., 1992, 90, 41-54). Antisense oligonucleotides have
been used to target mutations that lead to aberrant splicing in
several genetic diseases in order to redirect splicing to give a
desired splice product (Kole, Acta Biochimica Polonica, 1997, 44,
231-238).
[0004] Antisense compounds have also been used to alter the ratio
of naturally occurring alternate splice variants such as the long
and short forms of Bcl-x pre-mRNA (U.S. Pat. No. 6,172,216; U.S.
Pat. No. 6,214,986; Taylor et al., Nat. Biotechnol. 1999, 17,
1097-1100) or to force skipping of specific exons containing
premature termination codons (Wilton et al., Neuromuscul. Disord.,
1999, 9, 330-338). U.S. Pat. No. 5,627,274 and WO 94/26887 disclose
compositions and methods for combating aberrant splicing in a
pre-mRNA molecule containing a mutation using antisense
oligonucleotides which do not activate RNAse H.
[0005] Proximal spinal muscular atrophy (SMA) is a genetic,
neurodegenerative disorder characterized by the loss of spinal
motor neurons. SMA is an autosomal recessive disease of early onset
and is currently the leading cause of death among infants. The
severity of SMA varies among patients and has thus been classified
into three types. Type I SMA is the most severe form with onset at
birth or within 6 months and typically results in death within 2
years. Children with type I SMA are unable to sit or walk. Type II
SMA is the intermediate form and patients are able to sit, but
cannot stand or walk. Patients with type III SMA, a chronic form of
the disease, typically develop SMA after 18 months of age (Lefebvre
et al., Hum. Mol. Genet., 1998, 7, 1531-1536).
[0006] The molecular basis of SMA is caused by the loss of both
copies of survival motor neuron gene 1 (SMN1), which may also be
known as SMN Telomeric, a protein that is part of a multi-protein
complex thought to be involved in snRNP biogenesis and recycling. A
nearly identical gene, SMN2, which may also be known as SMN
Centromeric, exists in a duplicated region on chromosome 5q13 and
modulates disease severity. Expression of the normal SMN1 gene
results solely in expression of survival motor neuron (SMN)
protein. Although SMN1 and SMN2 have the potential to code for the
same protein, SMN2 contains a translationally silent mutation at
position +6 of exon 7, which results in inefficient inclusion of
exon 7 in SMN2 transcripts. Thus, the predominant form of SMN2 is a
truncated version, lacking exon 7, which is unstable and inactive
(Cartegni and Krainer, Nat. Genet., 2002, 30, 377-384). Expression
of the SMN2 gene results in approximately 10-20% of the SMN protein
and 80-90% of the unstable/non-functional SMNdelta7 protein. SMN
protein plays a well-established role in assembly of the
spliceosome and may also mediate mRNA trafficking in the axon and
nerve terminus of neurons.
[0007] Antisense technology is an effective means for modulating
the expression of one or more specific gene products, including
alternative splice products, and is uniquely useful in a number of
therapeutic, diagnostic, and research applications. The principle
behind antisense technology is that an antisense compound, which
hybridizes to a target nucleic acid, modulates gene expression
activities such as transcription, splicing or translation through
one of a number of antisense mechanisms. The sequence specificity
of antisense compounds makes them extremely attractive as tools for
target validation and gene functionalization, as well as
therapeutics to selectively modulate the expression of genes
involved in disease.
[0008] Certain antisense compounds complementary to SMN2 are known
in the art. See for example, WO 2007/002390; US 61/168,885; Hua et
al., American J. of Human Genetics (April 2008) 82, 1-15; Singh et
al., RNA Bio. 6:3, 1-10 (2009). Certain antisense compounds and
methods disclosed herein posses desirable characteristics compared
to such compounds and methods known in the art. Chimeric peptide
nucleic acid molecules designed to modulate splicing of SMN2 have
been described (WO 02/38738; Cartegni and Krainer, Nat. Struct.
Biol., 2003, 10, 120-125).
SUMMARY OF THE INVENTION
[0009] In certain embodiments, the present invention provides
methods comprising administering to a subject an antisense compound
comprising an antisense oligonucleotide complementary to intron 7
of a nucleic acid encoding human SMN2 pre-mRNA, wherein the
antisense compound is administered into the cerebrospinal fluid. In
certain embodiments, the administration is into the intrathecal
space. In certain embodiments, the administration is into the
cerebrospinal fluid in the brain. In certain embodiments, the
administration comprises a bolus injection. In certain embodiments,
the administration comprises infusion with a delivery pump.
[0010] In certain embodiments, the antisense compound is
administered at a dose from 0.01 to 10 milligrams of antisense
compound per kilogram of body weight of the subject. In certain
embodiments, the dose is from 0.01 to 10 milligrams of antisense
compound per kilogram of body weight of the subject. In certain
embodiments, the dose is from 0.01 to 5 milligrams of antisense
compound per kilogram of body weight of the subject. In certain
embodiments, the dose is from 0.05 to 1 milligrams of antisense
compound per kilogram of body weight of the subject. In certain
embodiments, the dose is from 0.01 to 0.5 milligrams of antisense
compound per kilogram of body weight of the subject. In certain
embodiments, the dose is from 0.05 to 0.5 milligrams of antisense
compound per kilogram of body weight of the subject.
[0011] In certain embodiments, the dose is administered daily. In
certain embodiments, the dose is administered weekly. In certain
embodiments, the antisense compound is administered continuously
and wherein the dose is the amount administered per day. In certain
embodiments, the method comprises administering at least one
induction dose during an induction phase and administering at least
one maintenance dose during a maintenance phase. In certain
embodiments, the induction dose is from 0.05 to 5.0 milligrams of
antisense compound per kilogram of body weight of the subject. In
certain embodiments, the maintenance dose is from 0.01 to 1.0
milligrams of antisense compound per kilogram of body weight of the
subject. In certain embodiments, the duration of the induction
phase is at least 1 week. In certain embodiments, the duration of
the maintenance phase is at least 1 week. In certain embodiments,
each induction dose and each maintenance dose comprises a single
injection. In certain embodiments, each induction dose and each
maintenance dose independently comprise two or more injections. In
certain embodiments, antisense compound is administered at least 2
times over a treatment period of at least 1 week. In certain
embodiments, the treatment period is at least one month. In certain
embodiments, the treatment period is at least 2 months. In certain
embodiments, the treatment period is at least 4 months. In certain
embodiments, the induction dose is administered by one or more
bolus injections and the maintenance dose is administered by an
infusion pump.
[0012] In certain embodiments, the method comprises assessing the
tolerability and/or effectiveness of the antisense compound. In
certain embodiments, dose amount or frequency of antisense compound
is reduced following an indication that administration of the
antisense compound is not tolerated. In certain embodiments, the
dose amount or frequency of antisense compound is maintained or
reduced following an indication that administration of the
antisense compound is effective. In certain embodiments, the dose
of antisense compound is increased following an indication that
administration of the antisense compound is not effective. In
certain embodiments, frequency of administration of antisense
compound is reduced following an indication that administration of
the antisense compound is effective. In certain embodiments,
frequency of administration of antisense compound is increased
following an indication that administration of the antisense
compound is not effective.
[0013] In certain embodiments, the methods comprise
co-administration of the antisense compound and at least one other
therapy. In certain embodiments, an antisense compound and at least
one other therapy are co-administered at the same time. In certain
embodiments, an antisense compound is administered prior to
administration of the at least one other therapy. In certain
embodiments, an antisense compound is administered after
administration of the at least one other therapy. In certain
embodiments, the at least one other therapy comprises
administration of one or more of valproic acid, riluzole,
hydroxyurea, and a butyrate. In certain embodiments, at least one
other therapy comprises administration of trichostatin-A. In
certain embodiments, the at least one other therapy comprises
administration of stem cells. In certain embodiments, at least one
other therapy is gene therapy. In certain embodiments, gene therapy
is administered to the CSF and an antisense compound is
administered systemically. In certain embodiments, gene therapy is
administered to the CSF and an antisense compound is administered
systemically and to the CSF. In certain embodiments, the invention
provides treatment regimens where initially, an antisense compound
is administered to the CSF and systemically, followed by gene
therapy administration to the CSF and systemic administration of
antisense compound. In certain such embodiments, the subject is an
infant at the time of initial treatment. In certain such
embodiments, the subject is less that 2 years old. In certain
embodiments, antisense compound is administered to the CNS of a
subject until the subject is old enough for gene therapy. In
certain such embodiments, antisense compound is administered
systemically throughout.
[0014] In certain embodiments, the antisense compound is
administered at a concentration of about 0.01 mg/ml, about 0.05
mg/ml, about 0.1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 5
mg/ml, about 10 mg/ml, about 50 mg/ml, or about 100 mg/ml.
[0015] In certain embodiments, inclusion of exon 7 of SMN2 mRNA in
a motoneuron in the subject is increased. In certain embodiments,
inclusion of exon 7 amino acids in SMN2 polypeptide in a motoneuron
in the subject is increased.
[0016] In certain embodiments, the invention provides methods of
increasing inclusion of exon 7 of SMN2 mRNA in a motoneuron in a
subject comprising administering to the subject an antisense
compound comprising an antisense oligonucleotide complementary to
intron 7 of a nucleic acid encoding human SMN2 and thereby
increasing inclusion of exon 7 of SMN2 mRNA in the motoneuron in
the subject.
[0017] In certain embodiments, the invention provides methods of
increasing inclusion of exon 7 amino acids in SMN2 polypeptide in a
motoneuron in a subject comprising administering to the subject an
antisense compound comprising an antisense oligonucleotide
complementary to intron 7 of a nucleic acid encoding human SMN2 and
thereby increasing inclusion of exon 7 amino acids in SMN2
polypeptide in the motoneuron in the subject.
[0018] In certain embodiments, the subject has SMA. In certain
embodiments, the subject has type I SMA. In certain embodiments,
the subject has type II SMA. In certain embodiments, the subject
has type III SMA.
[0019] In certain embodiments, a first dose is administered in
utero. In certain embodiments, the first dose is administered prior
to complete formation of the blood-brain-barrier. In certain
embodiments, a first dose is administered within 1 week of birth of
the subject. In certain embodiments, a first dose is administered
within 1 month of birth of the subject. In certain embodiments, a
first dose is administered within 3 months of birth of the subject.
In certain embodiments, a first dose is administered within 6
months of birth of the subject. In certain embodiments, a first
dose is administered when the subject is from 1 to 2 years of age.
In certain embodiments, a first dose is administered when the
subject is from 1 to 15 years of age. In certain embodiments, a
first dose is administered when the subject is older than 15 years
of age.
[0020] In certain embodiments, the subject is a mammal. In certain
embodiments, the subject is a human.
[0021] In certain embodiments, the methods comprise identifying a
subject having SMA. In certain embodiments, the subject is
identified by measuring electrical activity of one or more muscles
of the subject. In certain embodiments, the subject is identified
by a genetic test to determine whether the subject has a mutation
in the subject's SMN1 gene. In certain embodiments, the subject is
identified by muscle biopsy.
[0022] In certain embodiments, administering the antisense compound
results in an increase in the amount of SMN2 mRNA having exon 7 of
at least 10%. In certain embodiments, the increase in the amount of
SMN2 mRNA having exon 7 is at least 20%. In certain embodiments,
the increase in the amount of SMN2 mRNA having exon 7 is at least
50%. In certain embodiments, the amount of SMN2 mRNA having exon 7
is at least 70%.
[0023] In certain embodiments, administering of the antisense
compound results in an increase in the amount of SMN2 polypeptide
having exon 7 amino acids of at least 10%. In certain embodiments,
wherein the increase in the amount of SMN2 polypeptide having exon
7 amino acids is at least 20%. In certain embodiments, the increase
in the amount of SMN2 polypeptide having exon 7 amino acids is at
least 50%. In certain embodiments, the increase in the amount of
SMN2 polypeptide having exon 7 amino acids is at least 70%.
[0024] In certain embodiments, the administering of the antisense
compound ameliorates at least one symptom of SMA in the subject. In
certain embodiments, the administering of the antisense compound
results in improved motor function in the subject. In certain
embodiments, the administering of the antisense compound results in
delayed or reduced loss of motor function in the subject. In
certain embodiments, administering of the antisense compound
results in improved respiratory function. In certain embodiments,
the administering of the antisense compound results in improved
survival.
[0025] In certain embodiments, at least one nucleoside of the
antisense oligonucleotide comprises a modified sugar moiety. In
certain embodiments, at least one modified sugar moiety comprises a
2'-methoxyethyl sugar moiety. In certain embodiments, essentially
each nucleoside of the antisense oligonucleotide comprises a
modified sugar moiety. In certain embodiments, the nucleosides
comprising a modified sugar moiety all comprise the same sugar
modification. In certain embodiments, wherein each modified sugar
moiety comprises a 2'-methoxyethyl sugar moiety. In certain
embodiments, each nucleoside of the antisense oligonucleotide
comprises a modified sugar moiety. In certain embodiments, the
nucleosides all comprise the same sugar modification. In certain
embodiments, each modified sugar moiety comprises a 2'-methoxyethyl
sugar moiety. In certain embodiments, at least one internucleoside
linkage is a phosphorothioate internucleoside linkage. In certain
embodiments, each internucleoside linkage is a phosphorothioate
internucleoside linkage.
[0026] In certain embodiments, the antisense oligonucleotide
consists of 10 to 25 linked nucleosides. In certain embodiments,
the antisense oligonucleotide consists of 12 to 22 linked
nucleosides. In certain embodiments, the antisense oligonucleotide
consists of 15 to 20 linked nucleosides. In certain embodiments,
the antisense oligonucleotide consists of 18 linked
nucleosides.
[0027] In certain embodiments, the antisense oligonucleotide is at
least 90% complementary to the nucleic acid encoding human SMN2. In
certain embodiments, the antisense oligonucleotide is fully
complementary to the nucleic acid encoding human SMN2. In certain
embodiments, the oligonucleotide has a nucleobase sequence
comprising at least 10 contiguous nucleobases of the nucleobase
sequence SEQ ID NO: 1. In certain embodiments, the oligonucleotide
has a nucleobase sequence comprising at least 15 contiguous
nucleobases of the nucleobase sequence SEQ ID NO: 1. In certain
embodiments, the oligonucleotide has a nucleobase sequence
comprising the nucleobase sequence SEQ ID NO: 1. In certain
embodiments, the oligonucleotide has a nucleobase sequence
consisting of the nucleobase sequence SEQ ID NO: 1.
[0028] In certain embodiments, the antisense compound comprises a
conjugate group or terminal group.
[0029] In certain embodiments, the antisense compound consists of
the antisense oligonucleotide.
[0030] In certain embodiments, the antisense compound is also
administered systemically. In certain embodiments, the systemic
administration is by intravenous or intraperitoneal injection. In
certain embodiments, systemic administration and the administration
into the central nervous system are performed at the same time. In
certain embodiments, systemic administration and the administration
into the central nervous system are performed at different
times.
[0031] In certain embodiments, the invention provides systemic
administration of antisense compounds, either alone or in
combination with delivery into the CSF. In certain embodiments,
pharmaceutical compositions are administered systemically. In
certain embodiments, pharmaceutical compositions are administered
subcutaneously. In certain embodiments, pharmaceutical compositions
are administered intravenously. In certain embodiments,
pharmaceutical compositions are administered by intramuscular
injection.
[0032] In certain embodiments, pharmaceutical compositions are
administered both directly to the CSF (e.g., IT and/or ICV
injection and/or infusion) and systemically.
[0033] In certain embodiments, the invention provides methods of
administering to a subject having at least one symptom associated
with SMA, at least one dose of an antisense compound comprising an
oligonucleotide consisting of 15 to 20 linked nucleosides and
having a nucleobase sequence which is 100% complementary to SEQ ID
NO. 7 over its entire length, and wherein each nucleoside is a
2'-MOE modified nucleoside; and wherein at least one dose is
between 0.1 mg/kg and 5 mg/kg administered to the CSF. In certain
such embodiments, the dose is between 0.5 mg/kg and 2 mg/kg. In
certain embodiments, at least one dose is administered by bolus
injection. In certain such embodiments, the dose is administered by
bolus intrathecal injection. In certain embodiments, at least one
second dose is administered. In certain such embodiments, the
second dose is administered at least 2 weeks after the first dose.
In certain embodiments, the second dose is administered at least 4
weeks after the first dose. In certain embodiments, the second dose
is administered at least 8 weeks after the first dose. In certain
embodiments, the second dose is administered at least 12 weeks
after the first dose. In certain embodiments, the second dose is
administered at least 16 weeks after the first dose. In certain
embodiments, the second dose is administered at least 20 weeks
after the first dose. In certain embodiments, the subject is under
2 years old at the time of the first dose. In certain embodiments,
the subject is between 2 and 15 years old. In certain embodiments,
the subject is between 15 and 30 years old. In certain embodiments,
the subject is older than 30 years old. In certain embodiments, at
least one symptom associated with SMA is reduced its progression
has slowed. In certain embodiments, the oligonucleotide is
ISIS396443.
[0034] In certain embodiments, the invention provides methods of
administering to a subject having at least one symptom associated
with SMA, at least one dose of an antisense compound comprising an
oligonucleotide consisting of 15 to 20 linked nucleosides and
having a nucleobase sequence comprising which is 100% complementary
to SEQ ID NO. 7 over its entire length, and wherein each nucleoside
is a 2'-MOE modified nucleoside; and wherein at least one dose is
administered systemically. In certain such embodiments, at least
one dose is administered by bolus injection. In certain such
embodiments, the dose is administered by bolus subcutaneous
injection. In certain embodiments, the dose administered is between
0.5mg/kg and 50mg/kg. In certain embodiments, the dose is between 1
mg/kg and 10mg/kg. In certain embodiments, the dose is between 1
mg/kg and 5 mg/kg. In certain embodiments, the dose is between
0.5mg/kg and 1mg/kg. In certain embodiments, at least one second
dose is administered. In certain such embodiments, the second dose
is administered at least 2 weeks after the first dose. In certain
embodiments, the second dose is administered at least 4 weeks after
the first dose. In certain embodiments, the second dose is
administered at least 8 weeks after the first dose. In certain
embodiments, the second dose is administered at least 12 weeks
after the first dose. In certain embodiments, the second dose is
administered at least 16 weeks after the first dose. In certain
embodiments, the second dose is administered at least 20 weeks
after the first dose. In certain embodiments, the subject is under
2 years old at the time of the first dose. In certain embodiments,
the subject is between 2 and 15 years old. In certain embodiments,
the subject is between 15 and 30 years old. In certain embodiments,
the subject is older than 30 years old. In certain embodiments, at
least one symptom associated with SMA is reduced its progression
has slowed. In certain embodiments, the oligonucleotide is
ISIS396443.
[0035] In certain embodiments, the invention provides methods of
administering to a subject having at least one symptom associated
with SMA, at least one dose to the CSF and at least one systemic
dose of an antisense compound comprising an oligonucleotide
consisting of 15 to 20 linked nucleosides and having a nucleobase
sequence which is 100% complementary to SEQ ID NO. 7 over its
entire length, and wherein each nucleoside is a 2'-MOE modified
nucleoside. In certain such embodiments, the CSF dose is between
0.1 mg/kg and 5 mg/kg. In certain embodiments, the systemic dose is
between 0.5mg/kg and 50mg/kg. In certain embodiments, at least one
CSF dose is administered by bolus injection. In certain such
embodiments, at least one CSF dose is administered by bolus
intrathecal injection. In certain embodiments, at least one
systemic dose is administered by bolus injection. In certain such
embodiments, at least one systemic dose is administered by
subcutaneous injection. In certain embodiments, the CSF dose and
the systemic dose are administered at the same time. In certain
embodiments, the CSF dose and the systemic dose are administered at
different times. In certain embodiments, the subject is under 2
years old at the time of the first dose. In certain embodiments,
the subject is between 2 and 15 years old. In certain embodiments,
the subject is between 15 and 30 years old. In certain embodiments,
the subject is older than 30 years old. In certain embodiments, at
least one symptom associated with SMA is reduced its progression
has slowed. In certain embodiments, the oligonucleotide is
ISIS396443.
[0036] In certain embodiments, the invention provides methods of
administering to a subject having at least one symptom associated
with SMA, at least one systemic dose of an antisense compound
comprising an oligonucleotide consisting of 15 to 20 linked
nucleosides and having a nucleobase sequence which is 100%
complementary to SEQ ID NO. 7 over its entire length, and wherein
each nucleoside is a 2'-MOE modified nucleoside; and at least one
dose of a gene therapy agent. In certain embodiments, the systemic
dose is between 0.5mg/kg and 50mg/kg. In certain embodiments, at
least one systemic dose is administered by bolus injection. In
certain such embodiments, at least one systemic dose is
administered by subcutaneous injection. In certain embodiments, the
systemic dose and the gene therapy agent are administered at the
same time. In certain embodiments, the systemic dose and the gene
therapy agent are administered at different times. In certain
embodiments, the gene therapy agent is administered to the CSF. In
certain such embodiments, the gene therapy agent is administered by
intrathecal injection and/or infusion. In certain such embodiments,
the gene therapy agent is administered by intracerebroventricular
injection and/or infusion. In certain embodiments, the subject is
under 2 years old at the time of the first dose. In certain
embodiments, the subject is between 2 and 15 years old. In certain
embodiments, the subject is between 15 and 30 years old. In certain
embodiments, the subject is older than 30 years old. In certain
embodiments, at least one symptom associated with SMA is reduced or
its progression has slowed. In certain embodiments, the
oligonucleotide is ISIS396443.
[0037] In certain embodiments, the invention provides methods of
selecting a subject having at least one symptom associated with SMA
and administering an antisense compound according to any of the
methods above. In certain such embodiments, at least one symptom of
SMA is assessed after administration. In certain such embodiments,
at least one symptom of SMA is improved. In certain such
embodiments, at least one symptom of SMA does not progress or
progresses more slowly compared to a subject who has not received
administration of antisense compound.
[0038] In certain embodiments, the invention provides an antisense
compound comprising an antisense oligonucleotide complementary to
intron 7 of a nucleic acid encoding human SMN2, for use in any of
the above methods. In certain embodiments, the invention provides
such a compound for use in treating a disease or condition
associated with survival motor neuron 1 (SMN1).
[0039] In certain embodiments, the invention provides use of an
antisense compound comprising an antisense oligonucleotide
complementary to intron 7 of a nucleic acid encoding human SMN2 in
the manufacture of a medicament for use in any of the above
methods. In certain embodiments, the medicament is for treating a
disease or condition associated with survival motor neuron 1
(SMN1).
BRIEF DESCRIPTION OF THE FIGURES
[0040] FIG. 1 shows results from a duration of action study
discussed in Example 4, in which the percent of SMN2 that includes
exon 7 (y-axis) was assessed at 0, 2, 4, 6, and 8 weeks after
termination of 7 days of treatment (x-axis). The week "0" sample
was taken 1 day after termination of treatment. CON represents
saline treated mice. There was no difference in % inclusion among
control saline treated mice at different time points from 0 to 6
months.
[0041] FIG. 2 shows results from a duration of action study
discussed in Example 4, in which the percent of SMN2 that includes
exon 7 was assessed at 0, 0.5, 1, 2, 5, and 6 months after
termination of 7 days of treatment. The month "0" sample was taken
1 day after termination of treatment. CON represents saline treated
mice. There was no difference in % inclusion among control saline
treated mice at different time points from 0 to 6 months.
[0042] FIG. 3A shows the results from experiments discussed in
Example 6 measuring the effect of embryonic administration of
ISIS396443 on tail-length in Taiwan strain of SMA mice. This figure
shows the first such experiment. FIG. 3B shows a repeated
experiment testing a different concentration of antisense compound,
as noted and including data for normal mice for comparison.
[0043] FIGS. 4A-4C show results from western blots discussed in
Example 7. The Y axis is the percent of SMN in the various samples
that includes exon 7.
[0044] FIGS. 5A-5D and 6A-6B show results from experiments
discussed in Example 7. A number of assessments of SMA mice (Taiwan
strain) were performed following treatment with either an antisense
compound or with a control oligonucletotide.
[0045] FIG. 7 shows a survival curve from an experiment discussed
in Example 7.
[0046] FIGS. 8A-8C show results from an assessment of the number of
motor neurons in different portions of the spinal cord following
treatment with an antisense compound or with a control
oligonucleotide, as discussed in Example 7.
[0047] FIG. 9 shows results from an assessment of full SMN RNA
(including exon 7) in animals treated with antisense as discussed
in Example 7.
[0048] FIG. 10 shows a survival curve from an experiment discussed
in Example 7 in which animals were either (1) untreated; (2) given
a single dose of antisense compound at birth (Day P0); or (3) given
a first dose at P0 and a second dose at day 21 (P21).
[0049] FIG. 11 shows a survival curve from an experiment described
in Example 7 comparing animals that received the second dose with
animals that received only the first dose.
[0050] FIGS. 12A-12G show results from an experiment discussed in
Example 9 in which antisense compound was administered to monkeys
by intrathecal infusion and concentration of the compound was
assessed in different tissues 96 hours later.
[0051] FIG. 13 shows a survival curve for experiments discussed in
Example 12, in which different doses of antisense compound were
administered to severe SMA mice by subcutaneous injection.
DETAILED DESCRIPTION OF THE INVENTION
[0052] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
[0053] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
I. Definitions
[0054] Unless specific definitions are provided, the nomenclature
utilized in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Certain such techniques
and procedures may be found for example in "Carbohydrate
Modifications in Antisense Research" Edited by Sangvi and Cook,
American Chemical Society, Washington D.C., 1994; "Remington's
Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th
edition, 1990; and "Antisense Drug Technology, Principles,
Strategies, and Applications" Edited by Stanley T. Crooke, CRC
Press, Boca Raton, Fla.; and Sambrook et al., "Molecular Cloning, A
laboratory Manual," 2.sup.nd Edition, Cold Spring Harbor Laboratory
Press, 1989, which are hereby incorporated by reference for any
purpose. Where permitted, all patents, applications, published
applications and other publications and other data referred to
throughout in the disclosure herein are incorporated by reference
in their entirety.
[0055] Unless otherwise indicated, the following terms have the
following meanings:
[0056] "Nucleoside" means a compound comprising a heterocyclic base
moiety and a sugar moiety. Nucleosides include, but are not limited
to, naturally occurring nucleosides, modified nucleosides, and
nucleosides having mimetic bases and/or sugar groups. Nucleosides
may be modified with any of a variety of substituents.
[0057] "Sugar moiety" means a natural or modified sugar or sugar
surrogate.
[0058] "Natural sugar" means a ribofuranose moiety of DNA (2'-H) or
RNA (2'-OH).
[0059] "Modified sugar" means a ribofuranose moiety comprising at
least one substituent other than that of a natural sugar. "Sugar
surrogate" means a structure other than a ribofuranose ring which
is capable of substituting for the sugar of a nucleoside. Examples
of sugar surrogates include, but are not limited to, open ring
systems, 6-membered rings, sugars in which the oxygen is replace
with, for example, sulfur or nitrogen. For example, sugar
surrogates include, but are not limited to morpholinos and
4'-thio-containing sugars.
[0060] "Nucleobase" means the heterocyclic base portion of a
nucleoside. Nucleobases may be naturally occurring or may be
modified. In certain embodiments, a nucleobase may comprise any
atom or group of atoms capable of hydrogen bonding to a nucleobase
of another nucleic acid.
[0061] "Nucleotide" means a nucleoside comprising a phosphate
linking group. As used herein, nucleosides include nucleotides.
[0062] "Modified nucleoside" a nucleoside comprising at least one
modification compared to naturally occurring RNA or DNA
nucleosides. Such modification may be at the sugar moiety and/or at
the nucleobase.
[0063] "Bicyclic nucleoside" or "BNA" means a nucleoside wherein
the sugar moiety of the nucleoside comprises a bridge connecting
two carbon atoms of the sugar ring, thereby forming a bicyclic
sugar moiety.
[0064] "4'-2' bicyclic nucleoside" means 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.
[0065] "2'-modified" or "2'-substituted" means a nucleoside
comprising a sugar comprising a substituent at the 2' position
other than H or OH.
[0066] "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl" each means a
nucleoside comprising a sugar comprising an --OCH.sub.3 group at
the 2' position of the sugar ring.
[0067] "MOE" or "2'-MOE" or "2'-OCH.sub.2CH.sub.2OCH.sub.3" or
"2'-O-methoxyethyl" each means a nucleoside comprising a sugar
comprising a --OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position
of the sugar ring.
[0068] "Oligonucleotide" means a compound comprising a plurality of
linked nucleosides. In certain embodiments, one or more of the
plurality of nucleosides is modified. In certain embodiments, an
oligonucleotide comprises one or more ribonucleosides (RNA) and/or
deoxyribonucleosides (DNA).
[0069] "Oligonucleoside" means an oligonucleotide in which none of
the internucleoside linkages contains a phosphorus atom. As used
herein, oligonucleotides include oligonucleosides.
[0070] "Modified oligonucleotide" means an oligonucleotide
comprising at least one modified nucleoside and/or at least one
modified internucleoside linkage.
[0071] "Internucleoside linkage" means a covalent linkage between
adjacent nucleosides of an oligonucleotide.
[0072] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0073] "Modified internucleoside linkage" means any internucleoside
linkage other than a naturally occurring internucleoside
linkage.
[0074] "Oligomeric compound" means a compound comprising an
oligonucleotide. In certain embodiments, an oligomeric compound
consists of an oligonucleotide. In certain embodiments, an
oligomeric compound further comprises one or more conjugate and/or
terminal groups.
[0075] "Antisense compound" means an oligomeric compound, at least
a portion of which is at least partially complementary to a target
nucleic acid to which it hybridizes, wherein such hybridization
results at least one antisense activity.
[0076] "Antisense oligonucleotide" means an antisense compound
wherein the oligomeric compound consists of an oligonucleotide.
[0077] "Antisense activity" refers to any detectable and/or
measurable effect attributable to the hybridization of an antisense
compound to its target nucleic acid. In certain embodiments, such
antisense activity is an increase or decrease in an amount of a
nucleic acid or protein. In certain embodiments, such antisense
activity is a change in the ratio of splice variants of a nucleic
acid or protein. In certain embodiments, such antisense activity is
a phenotypic change in a cell and/or subject.
[0078] "Detecting" or "measuring" of antisense activity may be
direct or indirect. For example, in certain embodiments, antisense
activity is assessed by detecting and/or measuring the amount of
target nucleic acid or protein or the relative amounts of splice
variants of a target nucleic acid or protein. In certain
embodiments, antisense activity is detected by observing a
phenotypic change in a cell or animal. In connection with any
activity, response, or effect, the terms "detecting" and
"measuring," indicate that a test for detecting or measuring is
performed. Such detection and/or measuring may include values of
zero. Thus, if a test for detection or measuring results in a
finding of no activity (activity of zero), the step of detecting or
measuring the activity has nevertheless been performed. "Target
nucleic acid" refers to any nucleic acid molecule the expression,
amount, or activity of which is capable of being modulated by an
antisense compound.
[0079] "Target mRNA" means a pre-selected RNA molecule that encodes
a protein.
[0080] "Target pre-mRNA" means a pre-selected RNA transcript that
has not been fully processed into mRNA. Notably, pre-mRNA includes
one or more intron.
[0081] "Target protein" means a protein encoded by a target nucleic
acid.
[0082] "Modulation" means to a perturbation of function or
activity. In certain embodiments, modulation means an increase in
gene expression. In certain embodiments, modulation means a
decrease in gene expression.
[0083] "Expression" means any functions and steps by which a gene's
coded information is converted into structures present and
operating in a cell.
[0084] "Nucleobase sequence" means the order of contiguous
nucleobases, in a 5' to 3' orientation, independent of any sugar,
linkage, and/or nucleobase modification.
[0085] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other in a nucleic acid.
[0086] "Nucleobase complementarity" means the ability of two
nucleobases to pair non-covalently via hydrogen bonding.
[0087] "Complementary" means that a first nucleic acid is capable
of hybridizing to a second nucleic acid under stringent
hybridization conditions. For example, an antisense compound is
complementary to its target nucleic acid if it is capable of
hybridizing to the target nucleic acid under stringent
hybridization conditions.
[0088] "Fully complementary" means each nucleobase of a first
nucleic acid is capable of pairing with a nucleobase at each
corresponding contiguous position in a second nucleic acid.
[0089] "Percent complementarity" of an antisense compound means the
percentage of nucleobases of the antisense compound that are
complementary to an equal-length portion of a target nucleic acid.
Percent complementarity is calculated by dividing the number of
nucleobases of the antisense oligonucleotide that are complementary
to nucleobases at corresponding contiguous positions in the target
nucleic acid by the total length of the antisense compound.
[0090] "Percent identity" means the number of nucleobases in first
nucleic acid that are identical to nucleobases at corresponding
positions in a second nucleic acid, divided by the total number of
nucleobases in the first nucleic acid.
[0091] "Hybridize" means the annealing of complementary nucleic
acids that occurs through nucleobase complementarity.
[0092] "Mismatch" means a nucleobase of a first nucleic acid that
is not capable of pairing with a nucleobase at a corresponding
position of a second nucleic acid.
[0093] "Identical nucleobase sequence" means having the same
nucleobase sequence, independent of any chemical modifications to
the nucleosides.
[0094] "Different modifications" or "differently modified" refer to
nucleosides or internucleoside linkages that have different
nucleoside modifications or internucleoside linkages than one
another, including absence of modifications. Thus, for example, a
MOE nucleoside and an unmodified DNA nucleoside are "differently
modified," even though the DNA nucleoside is unmodified. Likewise,
DNA and RNA are "differently modified," even though both are
naturally-occurring unmodified nucleosides. Nucleosides that are
the same but for comprising different nucleobases are not
differently modified, unless otherwise indicated. For example, a
nucleoside comprising a 2'-OMe modified sugar and an adenine
nucleobase and a nucleoside comprising a 2'-OMe modified sugar and
a thymine nucleobase are not differently modified.
[0095] "The same modifications" refer to nucleosides and
internucleoside linkages (including unmodified nucleosides and
internucleoside linkages) that are the same as one another. Thus,
for example, two unmodified DNA nucleoside have "the same
modification," even though the DNA nucleoside is unmodified.
[0096] "Type of modification" or nucleoside of a "type" means the
modification of a nucleoside and includes modified and unmodified
nucleosides. Accordingly, unless otherwise indicated, a "nucleoside
having a modification of a first type" may be an unmodified
nucleoside.
[0097] "Separate regions" of an oligonucleotide means a portion of
an oligonucleotide wherein the nucleosides and internucleoside
linkages within the region all comprise the same modifications; and
the nucleosides and/or the internucleoside linkages of any
neighboring portions include at least one different
modification.
[0098] "Motif" means a pattern of modified and/or unmodified
nucleobases, sugars, and/or internucleoside linkages in an
oligonucleotide.
[0099] "Fully modified oligonucleotide" means each nucleobase, each
sugar, and/or each internucleoside linkage is modified.
[0100] "Uniformly modified oligonucleotide" means each nucleobase,
each sugar, and/or each internucleoside linkage has the same
modification throughout the modified oligonucleotide.
[0101] "Alternating motif" means an oligonucleotide or a portion
thereof, having at least four separate regions of modified
nucleosides in a pattern (AB)nAm where A represents a region of
nucleosides having a first type of modification; B represent a
region of nucleosides having a different type of modification; n is
2-15; and m is 0 or 1. Thus, in certain embodiments, alternating
motifs include 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 or more alternating regions. In certain embodiments,
each A region and each B region independently comprises 1-4
nucleosides.
[0102] "Subject" means a human or non-human animal selected for
treatment or therapy.
[0103] "Subject in need thereof" means a subject identified as in
need of a therapy or treatment. In such embodiments, a subject has
one or more indications of having or developing SMA.
[0104] "Administering" means providing a pharmaceutical agent or
composition to a subject, and includes, but is not limited to,
administering by a medical professional and self-administering.
[0105] "Parenteral administration," means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, or intramuscular administration.
[0106] "Systemic administration" means administration to an area
other than the intended locus of activity. Examples or systemic
administration are subcutaneous administration and intravenous
administration, and intraperitoneal administration.
[0107] "Subcutaneous administration" means administration just
below the skin.
[0108] "Intravenous administration" means administration into a
vein.
[0109] "Cerebrospinal fluid" or "CSF" means the fluid filling the
space around the brain and spinal cord.
[0110] "Administration into the cerebrospinal fluid" means any
administration that delivers a substance directly into the CSF.
[0111] "Intracerebroventricular" or "ICV" mean administration into
the ventricular system of the brain.
[0112] "Intrathecal" or "IT" means administration into the CSF
under the arachnoid membrane which covers the brain and spinal
cord. IT injection is performed through the theca of the spinal
cord into the subarachnoid space, where a pharmaceutical agent is
injected into the sheath surrounding the spinal cord.
[0113] "Induction phase" means a dosing phase during which
administration is initiated and steady state concentrations of
active pharmaceutical agent are achieved in a target tissue. For
example, an induction phase is a dosing phase during which steady
state concentrations of antisense oligonucleotide are achieved in
liver.
[0114] "Maintenance phase" means a dosing phase after target tissue
steady state concentrations of drug have been achieved.
[0115] "Duration" means the period of time during which an activity
or event continues. For example, the duration of an induction phase
is the period of time during which induction doses are
administered.
[0116] "Maintenance dose" means a dose administered at a single
administration during the maintenance phase. As used herein,
"induction dose" means a dose administered at a single
administration during the induction phase.
[0117] "Co-administration" means administration of two or more
pharmaceutical agents to a subject. The two or more pharmaceutical
agents may be in a single pharmaceutical composition, or may be in
separate pharmaceutical compositions. Each of the two or more
pharmaceutical agents may be administered through the same or
different routes of administration. Co-administration encompasses
administration in parallel or sequentially.
[0118] "Therapy" means a disease treatment method. In certain
embodiments, therapy includes, but is not limited to surgical
therapies, chemical therapies, and physical interventions, such as
assisted respiration, feeding tubes, and physical therapy for the
purpose of increasing strength.
[0119] "Treatment" means the application of one or more specific
procedures used for the cure or amelioration of a disease. In
certain embodiments, the specific procedure is the administration
of one or more pharmaceutical agents.
[0120] "Amelioration" means a lessening of severity of at least one
indicator of a condition or disease. In certain embodiments,
amelioration includes a delay or slowing in the progression of one
or more indicators of a condition or disease. The severity of
indicators may be determined by subjective or objective measures
which are known to those skilled in the art.
[0121] "Prevent the onset of" means to prevent the development a
condition or disease in a subject who is at risk for developing the
disease or condition. In certain embodiments, a subject at risk for
developing the disease or condition receives treatment similar to
the treatment received by a subject who already has the disease or
condition.
[0122] "Delay the onset of" means to delay the development of a
condition or disease in a subject who is at risk for developing the
disease or condition.
[0123] "Slow the progression of" means that the severity of at
least one symptom associated with a disease or condition worsens
less quickly.
[0124] "Exon 7 amino acids" means the portion of an SMN protein
that correspond to exon 7 of the SMN RNA. Exon 7 amino acids are
present in SMN protein expressed from SMN RNA where exon 7 was not
excluded during splicing.
[0125] "SMN protein" means normal full length survival motor neuron
protein. SMN may be expressed from either an SMN1 gene or from an
SMN2 gene, provided that exon 7 is present in the mature mRNA and
the exon 7 amino acids are present in the SMN protein.
[0126] "Dose" means a specified quantity of a pharmaceutical agent
provided in a single administration or over a specified amount of
time. In certain embodiments, a dose may be administered in two or
more boluses, tablets, or injections. For example, in certain
embodiments, where subcutaneous or inrathecal or ICV administration
is desired, the desired dose requires a volume not easily
accommodated by a single injection. In such embodiments, two or
more injections may be used to achieve the desired dose. In the
setting of continuous infusion, dose may be expressed as the
quantity of a pharmaceutical agent delivered per unit of time.
[0127] "Dosage unit" means a form in which a pharmaceutical agent
is provided. In certain embodiments, a dosage unit is a vial
containing lyophilized oligonucleotide. In certain embodiments, a
dosage unit is a vial containing reconstituted oligonucleotide.
[0128] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
animal.
[0129] "Pharmaceutical composition" means a mixture of substances
suitable for administering to an individual that includes a
pharmaceutical agent. For example, a pharmaceutical composition may
comprise a modified oligonucleotide and a sterile aqueous
solution.
[0130] "Acceptable safety profile" means a pattern of side effects
that is within clinically acceptable limits.
[0131] "Side effect" means a physiological response attributable to
a treatment other than desired effects.
1. Certain Modified Oligonucleotides
[0132] In certain embodiments, the present invention provides
methods and compositions involving antisense oligonucleotides
comprising one or more modification compared to oligonucleotides of
naturally occurring oligomers, such as DNA or RNA. Such modified
antisense oligonucleotides may possess one or more desirable
properties. Certain such modifications alter the antisense activity
of the antisense oligonucleotide, for example by increasing
affinity of the antisense oligonucleotide for its target nucleic
acid, increasing its resistance to one or more nucleases, and/or
altering the pharmacokinetics or tissue distribution of the
oligonucleotide. In certain embodiments, such modified antisense
oligonucleotides comprise one or more modified nucleosides and/or
one or more modified nucleoside linkages and/or one or more
conjugate groups.
[0133] a. Certain Modified Nucleosides
[0134] In certain embodiments, antisense oligonucleotides comprise
one or more modified nucleosides. Such modified nucleosides may
include a modified sugar and/or a modified 3 0 nucleobase. In
certain embodiments, incorporation of such modified nucleosides in
an oligonucleotide results in increased affinity for a target
nucleic acid and/or increased stability, including but not limited
to, increased resistance to nuclease degradation, and or improved
toxicity and/or uptake properties of the modified
oligonucleotide.
[0135] i. Certain Nucleobases
[0136] The naturally occurring base portion of nucleosides are
heterocyclic base, typically purines and pyrimidines. In addition
to "unmodified" or "natural" nucleobases such as the purine
nucleobases adenine (A) and guanine (G), and the pyrimidine
nucleobases thymine (T), cytosine (C) and uracil (U), many modified
nucleobases or nucleobase mimetics known to those skilled in the
art are amenable to incorporation into the compounds described
herein. In certain embodiments, a modified nucleobase is a
nucleobase that is fairly similar in structure to the parent
nucleobase, such as for example a 7-deaza purine, a 5-methyl
cytosine, or a G-clamp. In certain embodiments, nucleobase mimetic
include more complicated structures, such as for example a
tricyclic phenoxazine nucleobase mimetic. Methods for preparation
of the above noted modified nucleobases are well known to those
skilled in the art.
[0137] ii. Certain Modified Sugars and Sugar Surrogates
[0138] Antisense oligonucleotides of the present invention can
optionally contain one or more nucleosides wherein the sugar moiety
is modified, compared to a natural sugar. Oligonucleotides
comprising such sugar modified nucleosides may have enhanced
nuclease stability, increased binding affinity or some other
beneficial biological property. Such modifications include without
limitation, addition of substituent groups, bridging of non-geminal
ring atoms to form a bicyclic nucleic acid (BNA), replacement of
the ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R).sub.2
(R.dbd.H, C.sub.1-C.sub.12 alkyl or a protecting group) and
combinations of these such as for example a 2'-F-5'-methyl
substituted nucleoside (see PCT International Application WO
2008/101157 Published on Aug. 21, 2008 for other disclosed
5',2'-bis substituted nucleosides) or replacement of the ribosyl
ring oxygen atom with S with further substitution at the
2'-position (see published U.S. Patent Application US2005-0130923,
published on Jun. 16, 2005) or alternatively 5'-substitution of a
BNA (see PCT International Application WO 2007/134181 Published on
Nov. 22, 2007 wherein LNA is substituted with for example a
5'-methyl or a 5'-vinyl group).
[0139] Examples of nucleosides having modified sugar moieties
include without limitation nucleosides comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3 and
2'-O(CH.sub.2).sub.2OCH.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,
O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl.
[0140] Examples of bicyclic nucleic acids (BNAs) include without
limitation nucleosides comprising a bridge between the 4' and the
2' ribosyl ring atoms. In certain embodiments, antisense compounds
provided herein include one or more BNA nucleosides wherein the
bridge comprises one of the formulas: 4'-.beta.-D-(CH.sub.2)--O-2'
(.beta.-D-LNA); 4'-(CH.sub.2)--S-2'; 4'-.alpha.-L-(CH.sub.2)--O-2'
(.alpha.-L-LNA); 4'-(CH.sub.2).sub.2--O-2' (ENA);
4'-C(CH.sub.3).sub.2--O-2' (see PCT/US2008/068922);
4'-CH(CH.sub.3)--O-2' and 4'-C--H(CH.sub.2OCH.sub.3)--O-2' (see
U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008);
4'-CH.sub.2--N(OCH.sub.3)-2' (see PCT/US2008/064591);
4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent
Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2' (see U.S. Pat. No. 7,427,672, issued on
Sep. 23, 2008); 4'-CH.sub.2--C(CH.sub.3)-2'and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' (see PCT/US2008/066154); and
wherein R is, independently, H, C.sub.1-C.sub.12 alkyl, or a
protecting group.
[0141] In certain embodiments, the present invention provides
modified nucleosides comprising modified sugar moieties that are
not bicyclic sugar moieties. Certain such modified nucleosides are
known. In certain embodiments, the sugar ring of a nucleoside may
be modified at any position. Examples of sugar modifications useful
in this invention include, but are not limited to compounds
comprising a sugar substituent group selected from: OH, F, 0-alkyl,
S-alkyl, N-alkyl, or O-alkyl-O-alkyl, wherein the alkyl, alkenyl
and alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. In certain such
embodiments, such substituents are at the 2' position of the
sugar.
[0142] In certain embodiments, modified nucleosides comprise a
substituent at the 2' position of the sugar. In certain
embodiments, such substituents are selected from among: a halide
(including, but not limited to F), allyl, amino, azido, thio,
0-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3,
O--(CH.sub.2).sub.2--O--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--CH2-C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl.
[0143] In certain embodiments, modified nucleosides suitable for
use in the present invention are: 2-methoxyethoxy, 2'-O-methyl
(2'-O--CH.sub.3), 2'-fluoro (2'-F).
[0144] In certain embodiments, modified nucleosides having a sub
stituent group at the 2'-position selected from:
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, 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'-sugar substituent groups include:
C.sub.1 to C.sub.10 alkyl, substituted alkyl, alkenyl, alkynyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, 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 RNA cleaving group, a reporter group, an intercalator, a
group for improving pharmacokinetic properties, or a group for
improving the pharmacodynamic properties of an oligomeric compound,
and other substituents having similar properties.
[0145] In certain embodiments, modifed 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, P., Hely. 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).
[0146] In certain embodiments, 2'-sugar substituent groups are in
either the arabino (up) position or ribo (down) position. In
certain such embodiments, a 2'-arabino modification is 2'-F arabino
(FANA). Similar modifications can also be made at other positions
on the sugar, particularly the 3' position of the sugar on a 3'
terminal nucleoside or in 2'-5' linked oligonucleotides and the 5'
position of 5' terminal nucleotide.
[0147] In certain embodiments, nucleosides suitable for use in the
present invention have sugar surrogates such as cyclobutyl in place
of the ribofuranosyl sugar. Representative U.S. 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;
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; 5,658,873; 5,670,633; 5,792,747; and
5,700,920, each of which is herein incorporated by reference in its
entirety.
[0148] In certain embodiments, the present invention provides
nucleosides comprising a modification at the 2'-position of the
sugar. In certain embodiments, the invention provides nucleosides
comprising a modification at the 5'-position of the sugar. In
certain embodiments, the invention provides nucleosides comprising
modifications at the 2'-position and the 5'-position of the sugar.
In certain embodiments, modified nucleosides may be useful for
incorporation into oligonucleotides. In certain embodiment,
modified nucleosides are incorporated into oligonucleosides at the
5'-end of the oligonucleotide.
[0149] b. Certain Internucleoside Linkages
[0150] Antisense oligonucleotides of the present invention can
optionally contain one or more modified internucleoside linkages.
The two main classes of linking groups are defined by the presence
or absence of a phosphorus atom. Representative phosphorus
containing linkages include, but are not limited to,
phosphodiesters (P.dbd.O), phosphotriesters, methylphosphonates,
phosphoramidate, and phosphorothioates (P.dbd.S). Representative
non-phosphorus containing linking groups include, but are not
limited to, methylenemethylimino (--CH2-N(CH3)-O--CH2-),
thiodiester (--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--);
siloxane (--O--Si(H)2-O--); and N,N'-dimethylhydrazine
(--CH2-N(CH3)-N(CH3)-). Oligonucleotides having non-phosphorus
linking groups are referred to as oligonucleosides. Modified
linkages, compared to natural phosphodiester linkages, can be used
to alter, typically increase, nuclease resistance of the
oligonucleotides. In certain embodiments, linkages having a chiral
atom can be prepared as racemic mixtures, as separate enantomers.
Representative chiral linkages include, but are not limited to,
alkylphosphonates and phosphorothioates. Methods of preparation of
phosphorous-containing and non-phosphorous-containing linkages are
well known to those skilled in the art.
[0151] The antisense oligonucleotides described herein contain one
or more asymmetric centers and thus give rise to enantiomers,
diastereomers, and other stereoisomeric configurations that may be
defined, in terms of absolute stereochemistry, as (R) or (S), such
as for sugar anomers, or as (D) or (L) such as for amino acids et
al. Included in the antisense compounds provided herein are all
such possible isomers, as well as their racemic and optically pure
forms.
[0152] In certain embodiments, antisense oligonucleotides have at
least one modified internucleoside linkage. In certain embodiments,
antisense oligonucleotides have at least 2 modified internucleoside
linkages. In certain embodiments, antisense oligonucleotides have
at least 3 modified internucleoside linkages. In certain
embodiments, antisense oligonucleotides have at least 10 modified
internucleoside linkages. In certain embodiments, each
internucleoside linkage of an antisense oligonucleotide is a
modified internucleoside linkage. In certain embodiments, such
modified internucleoside linkages are phosphorothioate
linkages.
[0153] c. Lengths
[0154] In certain embodiments, the present invention provides
antisense oligonucleotides of any of a variety of ranges of
lengths. In certain embodiments, the invention provides antisense
compounds or antisense oligonucleotides comprising or consisting of
X-Y linked nucleosides, where X and Y are each independently
selected from 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, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that
X<Y. For example, in certain embodiments, the invention provides
antisense compounds or antisense oligonucleotides comprising or
consisting of: 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 8-16, 8-17,
8-18, 8-19, 8-20, 8-21, 8-22, 8-23, 8-24, 8-25, 8-26, 8-27, 8-28,
8-29, 8-30, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 9-16, 9-17, 9-18,
9-19, 9-20, 9-21, 9-22, 9-23, 9-24, 9-25, 9-26, 9-27, 9-28, 9-29,
9-30, 10-11, 10-12, 10-13, 10-14, 10-15, 10-16, 10-17, 10-18,
10-19, 10-20, 10-21, 10-22, 10-23, 10-24, 10-25, 10-26, 10-27,
10-28, 10-29, 10-30, 11-12, 11-13, 11-14, 11-15, 11-16, 11-17,
11-18, 11-19, 11-20, 11-21, 11-22, 11-23, 11-24, 11-25, 11-26,
11-27, 11-28, 11-29, 11-30, 12-13, 12-14, 12-15, 12-16, 12-17,
12-18, 12-19, 12-20, 12-21, 12-22, 12-23, 12-24, 12-25, 12-26,
12-27, 12-28, 12-29, 12-30, 13-14, 13-15, 13-16, 13-17, 13-18,
13-19, 13-20, 13-21, 13-22, 13-23, 13-24, 13-25, 13-26, 13-27,
13-28, 13-29, 13-30, 14-15, 14-16, 14-17, 14-18, 14-19, 14-20,
14-21, 14-22, 14-23, 14-24, 14-25, 14-26, 14-27, 14-28, 14-29,
14-30, 15-16, 15-17, 15-18, 15-19, 15-20, 15-21, 15-22, 15-23,
15-24, 15-25, 15-26, 15-27, 15-28, 15-29, 15-30, 16-17, 16-18,
16-19, 16-20, 16-21, 16-22, 16-23, 16-24, 16-25, 16-26, 16-27,
16-28, 16-29, 16-30, 17-18, 17-19, 17-20, 17-21, 17-22, 17-23,
17-24, 17-25, 17-26, 17-27, 17-28, 17-29, 17-30, 18-19, 18-20,
18-21, 18-22, 18-23, 18-24, 18-25, 18-26, 18-27, 18-28, 18-29,
18-30, 19-20, 19-21, 19-22, 19-23, 19-24, 19-25, 19-26, 19-29,
19-28, 19-29, 19-30, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26,
20-27, 20-28, 20-29, 20-30, 21-22, 21-23, 21-24, 21-25, 21-26,
21-27, 21-28, 21-29, 21-30, 22-23, 22-24, 22-25, 22-26, 22-27,
22-28, 22-29, 22-30, 23-24, 23-25, 23-26, 23-27, 23-28, 23-29,
23-30, 24-25, 24-26, 24-27, 24-28, 24-29, 24-30, 25-26, 25-27,
25-28, 25-29, 25-30, 26-27, 26-28, 26-29, 26-30, 27-28, 27-29,
27-30, 28-29, 28-30, or 29-30 linked nucleosides.
[0155] In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 15 nucleosides in
length. In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 16 nucleosides in
length. In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 17 nucleosides in
length. In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 18 nucleosides in
length. In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 19 nucleosides in
length. In certain embodiments, antisense compounds or antisense
oligonucleotides of the present invention are 20 nucleosides in
length.
[0156] d. Certain Oligonucleotide Motifs
[0157] In certain embodiments, antisense oligonucleotides have
chemically modified subunits arranged in specific orientations
along their length. In certain embodiments, antisense
oligonucleotides of the invention are fully modified. In certain
embodiments, antisense oligonucleotides of the invention are
uniformly modified. In certain embodiments, antisense
oligonucleotides of the invention are uniformly modified and each
nucleoside comprises a 2'-MOE sugar moiety. In certain embodiments,
antisense oligonucleotides of the invention are uniformly modified
and each nucleoside comprises a 2'-OMe sugar moiety. In certain
embodiments, antisense oligonucleotides of the invention are
uniformly modified and each nucleoside comprises a morpholino sugar
moiety.
[0158] In certain embodiments, oligonucleotides of the invention
comprise an alternating motif. In certain such embodiments, the
alternating modification types are selected from among 2'-MOE,
2'-F, a bicyclic sugar-modifed nucleoside, and DNA (unmodified
2'-deoxy). In certain such embodiments, each alternating region
comprises a single nucleoside.
[0159] In certain embodiments, oligonucleotides of the invention
comprise one or more block of nucleosides of a first type and one
or more block of nucleosides of a second type.
[0160] In certain embodiments, one or more alternating regions in
an alternating motif include more than a single nucleoside of a
type. For example, oligomeric compounds of the present invention
may include one or more regions of any of the following nucleoside
motifs:
[0161] Nu.sub.1 Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.1;
[0162] Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.2;
[0163] Nu.sub.1 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.1 Nu.sub.2;
[0164] Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1
Nu.sub.1 Nu.sub.2 Nu.sub.2;
[0165] Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.1;
[0166] Nu.sub.1 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1
Nu.sub.2;
[0167] Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.1;
[0168] Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.1 Nu.sub.2
Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.1;
[0169] Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.1
Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1
Nu.sub.1; or
[0170] Nu.sub.1 Nu.sub.2Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1
Nu.sub.1 Nu.sub.2 Nu.sub.2 Nu.sub.1 Nu.sub.2 Nu.sub.1 Nu.sub.2
Nu.sub.1 Nu.sub.1 ;
wherein Nu.sub.1 is a nucleoside of a first type and Nu.sub.2 is a
nucleoside of a second type. In certain embodiments, one of
Nu.sub.1 and Nu.sub.2 is a 2'-MOE nucleoside and the other of
Nu.sub.1 and Nu.sub.2 is a selected from: a 2'-OMe modified
nucleoside, BNA, and an unmodifed DNA or RNA nucleoside.
2. Oligomeric Compounds
[0171] In certain embodiments, the present invention provides
oligomeric compounds. In certain embodiments, oligomeric compounds
are comprised only of an oligonucleotide. In certain embodiments,
an oligomeric compound comprises an oligonucleotide and one or more
conjugate and/or terminal group. Such conjugate and/or terminal
groups may be added to oligonucleotides having any of the chemical
motifs discussed above. Thus, for example, an oligomeric compound
comprising an oligonucleotide having region of alternating
nucleosides may comprise a terminal group.
[0172] a. Certain Conjugate Groups
[0173] In certain embodiments, oligonucleotides of the present
invention are modified by attachment of one or more conjugate
groups. In general, conjugate groups modify one or more properties
of the attached oligomeric compound including but not limited to,
pharmacodynamics, pharmacokinetics, stability, binding, absorption,
cellular distribution, cellular uptake, charge and clearance.
Conjugate groups are routinely used in the chemical arts and are
linked directly or via an optional conjugate linking moiety or
conjugate linking group to a parent compound such as an oligomeric
compound, such as an oligonucleotide. Conjugate groups includes
without limitation, intercalators, reporter molecules, polyamines,
polyamides, polyethylene glycols, thioethers, polyethers,
cholesterols, thiocholesterols, cholic acid moieties, folate,
lipids, phospholipids, biotin, phenazine, phenanthridine,
anthraquinone, adamantane, acridine, fluoresceins, rhodamines,
coumarins and dyes. Certain conjugate groups have been described
previously, for example: cholesterol moiety (Letsinger et al.,
Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain,
e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al.,
EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990,
259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0174] In certain embodiments, a conjugate group comprises an
active drug substance, for example, aspirin, warfarin,
phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen,
(S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic. Oligonucleotide-drug conjugates and
their preparation are described in U.S. patent application Ser. No.
09/334,130.
[0175] Representative U.S. patents that teach the preparation of
oligonucleotide conjugates include, but are not limited to, U.S.
Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;
5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;
4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;
5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;
5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;
5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;
5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and
5,688,941.
[0176] Conjugate groups may be attached to either or both ends of
an oligonucleotide (terminal conjugate groups) and/or at any
internal position.
[0177] b. Terminal Groups
[0178] In certain embodiments, oligomeric compounds comprise
terminal groups at one or both ends. In certain embodiments, a
terminal group may comprise any of the conjugate groups discussed
above. In certain embodiments, terminal groups may comprise
additional nucleosides and/or inverted a basic nucleosides. In
certain embodiments, a terminal group is a stabilizing group.
[0179] In certain embodiments, oligomeric compounds comprise one or
more terminal stabilizing group that enhances properties such as
for example nuclease stability. Included in stabilizing groups are
cap structures. The terms "cap structure" or "terminal cap moiety,"
as used herein, refer to chemical modifications, which can be
attached to one or both of the termini of an oligomeric compound.
Certain such terminal modifications protect the oligomeric
compounds having terminal nucleic acid moieties from exonuclease
degradation, and can help in delivery and/or localization within a
cell. The cap can be present at the 5'-terminus (5'-cap) or at the
3'-terminus (3'-cap) or can be present on both termini. (for more
details see Wincott et al., International PCT publication No. WO
97/26270; Beaucage and Tyer, 1993, Tetrahedron 49, 1925; U.S.
Patent Application Publication No. US 2005/0020525; and WO
03/004602.
[0180] In certain embodiments, one or more additional nucleosides
is added to one or both terminal ends of an oligonucleotide of an
oligomeric compound. Such additional terminal nucleosides are
referred to herein as terminal-group nucleosides. In a
double-stranded compound, such terminal-group nucleosides are
terminal (3' and/or 5') overhangs. In the setting of
double-stranded antisense compounds, such terminal-group
nucleosides may or may not be complementary to a target nucleic
acid. In certain embodiments, the terminal group is a
non-nucleoside terminal group. Such non-terminal groups may be any
terminal group other than a nucleoside.
[0181] c. Oligomeric Compound Motifs
In certain embodiments, oligomeric compounds of the present
invention comprise a motif:
T.sub.1-(Nu.sub.1).sub.n1-(Nu.sub.2).sub.n2-(Nu.sub.1).sub.n3-(Nu.sub.2).-
sub.n4-(Nu.sub.1).sub.n5-T.sub.2, wherein: [0182] Nu.sub.1, is a
nucleoside of a first type; [0183] Nu.sub.2, is a nucleoside of a
second type; [0184] each of n1 and n5 is, independently from 0 to
3;
[0185] the sum of n2 plus n4 is between 10 and 25; [0186] n3 is
from 0 and 5; and [0187] each T.sub.1 and T.sub.2 is,
independently, H, a hydroxyl protecting group, an optionally linked
conjugate group or a capping group. In certain such embodiments,
the sum of n2 and n4 is 13 or 14; n1 is 2; n3 is 2 or 3; and n5 is
2. In certain such embodiments, oligomeric compounds of the present
invention comprise a motif selected from Table A.
TABLE-US-00001 [0187] TABLE A n1 n2 n3 n4 n5 2 16 0 0 2 2 2 3 11 2
2 5 3 8 2 2 8 3 5 2 2 11 3 2 2 2 9 3 4 2 2 10 3 3 2 2 3 3 10 2 2 4
3 9 2 2 6 3 7 2 2 7 3 6 2 2 8 6 2 2 2 2 2 12 2 2 3 2 11 2 2 4 2 10
2 2 5 2 9 2 2 6 2 8 2 2 7 2 7 2 2 8 2 6 2 2 9 2 5 2 2 10 2 4 2 2 11
2 3 2 2 12 2 2 2
[0188] Table A is intended to illustrate, but not to limit the
present invention. The oligomeric compounds depicted in Table A
each comprise 20 nucleosides. Oligomeric compounds comprising more
or fewer nucleosides can easily by designed by selecting different
numbers of nucleosides for one or more of n1-n5. In certain
embodiments, Nu.sub.1 and Nut are each selected from among: 2'-MOE,
2'-OMe, DNA, and a bicyclic nucleoside.
3. Antisense
[0189] In certain embodiments, oligomeric compounds of the present
invention are antisense compounds. Accordingly, in such
embodiments, oligomeric compounds hybridize with a target nucleic
acid, resulting in an antisense activity.
[0190] a. Hybridization
[0191] In certain embodiments, the invention provides antisense
compounds that specifically hybridize to a target nucleic acid when
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
nucleic acid sequences under conditions in which specific binding
is desired, i.e., under physiological conditions in the case of in
vivo assays or therapeutic treatment, and under conditions in which
assays are performed in the case of in vitro assays.
[0192] Thus, "stringent hybridization conditions" or "stringent
conditions" means conditions under which an antisense compounds
hybridize to a target sequence, but to a minimal number of other
sequences. Stringent conditions are sequence-dependent and will be
different in different circumstances, and "stringent conditions"
under which antisense oligonucleotides hybridize to a target
sequence are determined by the nature and composition of the
antisense oligonucleotides and the assays in which they are being
investigated.
[0193] It is understood in the art that incorporation of nucleotide
affinity modifications may allow for a greater number of mismatches
compared to an unmodified compound. Similarly, certain nucleobase
sequences may be more tolerant to mismatches than other nucleobase
sequences. One of ordinary skill in the art is capable of
determining an appropriate number of mismatches between
oligonucleotides, or between an antisense oligonucleotide and a
target nucleic acid, such as by determining melting temperature
(Tm). Tm or .DELTA.Tm can be calculated by techniques that are
familiar to one of ordinary skill in the art. For example,
techniques described in Freier et al. (Nucleic Acids Research,
1997, 25, 22: 4429-4443) allow one of ordinary skill in the art to
evaluate nucleotide modifications for their ability to increase the
melting temperature of an RNA:DNA duplex.
[0194] b. pre-mRNA Processing
[0195] In certain embodiments, antisense compounds provided herein
are complementary to a pre-mRNA. In certain embodiments, such
antisense compounds alter splicing of the pre-mRNA. In certain such
embodiments, the ratio of one variant of a mature mRNA
corresponding to a target pre-mRNA to another variant of that
mature mRNA is altered. In certain such embodiments, the ratio of
one variant of a protein expressed from the target pre-mRNA to
another variant of the protein is altered. Certain oligomeric
compounds and nucleobase sequences that may be used to alter
splicing of a pre-mRNA may be found for example in U.S. Pat. No.
6,210,892; U.S. Pat. No. 5,627,274; U.S. Pat. No. 5,665,593;
5,916,808; U.S. Pat. No. 5,976,879; US2006/0172962; US2007/002390;
US2005/0074801; US2007/0105807; US2005/0054836; WO 2007/090073;
WO2007/047913, Hua et al., PLoS Biol 5(4):e73; Vickers et al., J.
Immunol. 2006 Mar. 15; 176(6):3652-61; and Hua et al., American J.
of Human Genetics (April 2008) 82, 1-15, each of which is hereby
incorporated by reference in its entirety for any purpose. In
certain embodiments antisense sequences that alter splicing are
modified according to motifs of the present invention.
[0196] Antisense is an effective means for modulating the
expression of one or more specific gene products and is uniquely
useful in a number of therapeutic, diagnostic, and research
applications. Provided herein are antisense compounds useful for
modulating gene expression via antisense mechanisms of action,
including antisense mechanisms based on target occupancy. In one
aspect, the antisense compounds provided herein modulate splicing
of a target gene. Such modulation includes promoting or inhibiting
exon inclusion. Further provided herein are antisense compounds
targeted to cis splicing regulatory elements present in pre-mRNA
molecules, including exonic splicing enhancers, exonic splicing
silencers, intronic splicing enhancers and intronic splicing
silencers. Disruption of cis splicing regulatory elements is
thought to alter splice site selection, which may lead to an
alteration in the composition of splice products.
[0197] Processing of eukaryotic pre-mRNAs is a complex process that
requires a multitude of signals and protein factors to achieve
appropriate mRNA splicing. Exon definition by the spliceosome
requires more than the canonical splicing signals which define
intron-exon boundaries. One such additional signal is provided by
cis-acting regulatory enhancer and silencer sequences. Exonic
splicing enhancers (ESE), exonic splicing silencers (ESS), intronic
splicing enhancers (ISE) and intron splicing silencers (ISS) have
been identified which either repress or enhance usage of splice
donor sites or splice acceptor sites, depending on their site and
mode of action (Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A.
101(44):15700-15705). Binding of specific proteins (trans factors)
to these regulatory sequences directs the splicing process, either
promoting or inhibiting usage of particular splice sites and thus
modulating the ratio of splicing products (Scamborova et al. 2004,
Mol. Cell. Biol. 24(5):1855-1869; Hovhannisyan and Carstens, 2005,
Mol. Cell. Biol. 25(1):250-263; Minovitsky et al. 2005, Nucleic
Acids Res. 33(2):714-724).
4. Pharmaceutical Compositions
[0198] In certain embodiments, the present invention provides
pharmaceutical compositions comprising one or more antisense
compound. In certain embodiments, such pharmaceutical composition
comprises a sterile saline solution and one or more antisense
compound. In certain embodiments, such pharmaceutical composition
consists of a sterile saline solution and one or more antisense
compound.
[0199] In certain embodiments, antisense compounds may be admixed
with pharmaceutically acceptable active and/or inert substances for
the preparation of pharmaceutical compositions or formulations.
Compositions and methods for the formulation of pharmaceutical
compositions depend on a number of criteria, including, but not
limited to, route of administration, extent of disease, or dose to
be administered.
[0200] In certain embodiments antisense compounds, can be utilized
in pharmaceutical compositions by combining such oligomeric
compounds with a suitable pharmaceutically acceptable diluent or
carrier. A pharmaceutically acceptable diluent includes
phosphate-buffered saline (PBS). PBS is a diluent suitable for use
in compositions to be delivered parenterally. Accordingly, in
certain embodiments, employed in the methods described herein is a
pharmaceutical composition comprising an antisense compound and a
pharmaceutically acceptable diluent. In certain embodiments, the
pharmaceutically acceptable diluent is PBS.
[0201] Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters. In certain embodiments, pharmaceutical compositions
comprising antisense compounds comprise one or more oligonucleotide
which, upon administration to an animal, including a human, is
capable of providing (directly or indirectly) the biologically
active metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts.
[0202] A prodrug can include the incorporation of additional
nucleosides at one or both ends of an oligomeric compound which are
cleaved by endogenous nucleases within the body, to form the active
antisense oligomeric compound.
[0203] Lipid-based vectors have been used in nucleic acid therapies
in a variety of methods. For example, in one method, the nucleic
acid is introduced into preformed liposomes or lipoplexes made of
mixtures of cationic lipids and neutral lipids. In another method,
DNA complexes with mono- or poly-cationic lipids are formed without
the presence of a neutral lipid.
[0204] Certain preparations are described in Akinc et al., Nature
Biotechnology 26, 561-569 (1 May 2008), which is herein
incorporated by reference in its entirety.
5. Administration to a Subject
[0205] In certain embodiments, pharmaceutical compositions
comprising one or more antisense compound are administered to a
subject. In certain embodiments, such pharmaceutical compositions
are administered by injection. In certain embodiments, such
pharmaceutical compositions are administered by infusion.
[0206] In certain embodiments, pharmaceutical compositions are
administered by injection or infusion into the CSF. In certain such
embodiments, pharmaceutical compositions are administered by direct
injection or infusion into the spine. In certain embodiments,
pharmaceutical compositions are administered by injection or
infusion into the brain. In certain embodiments, pharmaceutical
compositions are administered by intrathecal injection or infusion
rather than into the spinal cord tissue itself. Without being
limited as to theory, in certain embodiments, the antisense
compound released into the surrounding CSF and may penetrate into
the spinal cord parenchyma. An additional advantage of intrathecal
delivery is that the intrathecal route mimics lumbar puncture
administration (i.e., spinal tap) already in routine use in
humans.
[0207] In certain embodiments, pharmaceutical compositions are
administered by intracerebroventricular (ICV) injection or
infusion. Intracerebroventricular, or intraventricular, delivery of
a pharmaceutical composition comprising one or more antisense
compounds may be performed in any one or more of the brain's
ventricles, which are filled with cerebrospinal fluid (CSF). CSF is
a clear fluid that fills the ventricles, is present in the
subarachnoid space, and surrounds the brain and spinal cord. CSF is
produced by the choroid plexuses and via the weeping or
transmission of tissue fluid by the brain into the ventricles. The
choroid plexus is a structure lining the floor of the lateral
ventricle and the roof of the third and fourth ventricles. Certain
studies have indicated that these structures are capable of
producing 400-600 ccs of fluid per day consistent with an amount to
fill the central nervous system spaces four times in a day. In
adult humans, the volume of this fluid has been calculated to be
from 125 to 150 ml (4-5 oz). The CSF is in continuous formation,
circulation and absorption. Certain studies have indicated that
approximately 430 to 450 ml (nearly 2 cups) of CSF may be produced
every day. Certain calculations estimate that production equals
approximately 0.35 ml per minute in adults and 0.15 per minute in
infant humans. The choroid plexuses of the lateral ventricles
produce the majority of CSF. It flows through the foramina of Monro
into the third ventricle where it is added to by production from
the third ventricle and continues down through the aqueduct of
Sylvius to the fourth ventricle. The fourth ventricle adds more
CSF; the fluid then travels into the subarachnoid space through the
foramina of Magendie and Luschka. It then circulates throughout the
base of the brain, down around the spinal cord and upward over the
cerebral hemispheres. The CSF empties into the blood via the
arachnoid villi and intracranial vascular sinuses.
[0208] In certain embodiments, such pharmaceutical compositions are
administered systemically. In certain embodiments, pharmaceutical
compositions are administered subcutaneously. In certain
embodiments, pharmaceutical compositions are administered
intravenously. In certain embodiments, pharmaceutical compositions
are administered by intramuscular injection.
[0209] In certain embodiments, pharmaceutical compositions are
administered both directly to the CSF (e.g., IT and/or ICV
injection and/or infusion) and systemically.
[0210] In certain embodiments, an antisense compound administered
systemically enters neurons. In certain embodiments, systemically
administered antisense compounds may penetrate the blood-brain
barrier, particularly in young subjects where the blood-brain
barrier is not fully formed (e.g., in subjects in eutero and/or in
newborn subjects). In certain embodiments, some amount of
systemically administered antisense compound may be taken up by
nerve cells, even in subjects in which the blood-brain barrier is
fully formed. For example, antisense compounds may enter a neuron
at or near the neuromuscular junction (retrograde uptake). In
certain embodiments, such retrograde uptake results in antisense
activity inside the neuron, including, but not limited to, a motor
neuron, and provides a therapeutic benefit by antisense activity
inside the neuron.
[0211] In certain embodiments, systemic administration provides
therapeutic benefit by antisense activity occurring in cells and/or
tissues other than neurons. While evidence suggests that functional
SMN inside neurons is required for normal neuron function, the
consequence of reduced functional SMN in other cells and tissues is
not well characterized. In certain embodiments, antisense activity
in non-neuronal cells results in restoration of SMN function in
those non-neuronal cells, which in turn results in therapeutic
benefit.
[0212] In certain embodiments, improved SMN function in
non-neuronal cells provides improved neuronal cell function,
whether or not SMN function inside neurons is improved. For
example, in certain embodiments, systemic administration of
pharmaceutical compositions of the present invention results in
antisense activity in muscle cells. Such antisense activity in
muscle cells may provide a benefit to the motor-neurons associated
with that muscle cell or to neurons generally. In such embodiments,
the muscle cell having restored SMN function may provide a factor
that improves neuronal viability and/or function. In certain
embodiments, such antisense activity is independent of benefit from
antisense activity occurring from antisense compounds inside
neurons. In certain embodiments, systemic administration of
pharmaceutical compositions of the present invention results in
antisense activity in other non-neuronal cells, including cells not
in immediate association with neurons. Such antisense activity in
non-neuronal cells may improve function of neurons. For example,
antisense activity in a non-neuronal cell (e.g., liver cell) may
result in that cell producing a factor that improves function of
neurons. Note: since the term "antisense activity" includes direct
and indirect activities, a benefit to neuronal function is an
"antisense activity" even if no antisense compound enters the
neuron.
[0213] In certain embodiments, systemic administration of a
pharmaceutical composition results in therapeutic benefit
independent of direct or indirect antisense activities in neurons.
Typically, in the setting of SMA, neuronal function is diminished,
resulting in significant symptoms. Additional symptoms may result
from diminished SMN activity in other cells. Certain such symptoms
may be masked by the relative severity of symptoms from diminished
neuronal function. In certain embodiments, systemic administration
results in restored or improved SMN function in non-neuronal cells.
In certain such embodiments, such restored or improved SMN function
in non-neuronal cells has therapeutic benefit. For example, in
certain instances, subjects having SMA have reduced growth. Such
reduced growth may not result from diminished function in neuronal
cells. Indeed, reduced growth may be related to impaired function
of cells in another organ, such as the pituitary gland, and/or may
be the result of SMN deficiencies throughout the cells of the body.
In such embodiments, systemic administration may result in improved
SMN activity in pituitary cells and/or other cells, resulting in
improved growth. In certain instances, administration to the CSF
restores sufficient neuronal function to allow a subject to live
longer, however one or more symptoms previously unknown because
subjects typically died before such symptoms appeared emerges,
because the subject lives longer. Certain such emergent symptoms
may be lethal. In certain embodiments, emergent symptoms are
treated by systemic administration. Regardless of mechanism, in
certain embodiments, a variety of symptoms of SMA, including, but
not limited to symptoms previously masked by more severe symptoms
associated with impaired neuronal function, may be treated by
systemic administration.
[0214] In certain embodiments, systemic administration of
pharmaceutical compositions of the present invention result in
increased SMN activity in muscle cells. In certain embodiments,
such improved SMN activity in muscle cells provides therapeutic
benefit. Improved SMN activity in muscle alone has been reported to
be insufficient to provide therapeutic benefit (e.g., Gravrilina,
et al., Hum Mol Genet 2008 17(8):1063-1075). In certain
embodiments, the present invention provides methods that result
improve SMN function in muscle and do provide therapeutic benefit.
In certain instances, therapeutic benefit may be attributable to
improved SMN function in other cells (alone or in combination with
muscle cells). In certain embodiments, improved SMN function in
muscle alone may provide benefit.
[0215] In certain embodiments, systemic administration results in
improved survival.
6. Spinal Muscular Atrophy (SMA)
[0216] SMA is a genetic disorder characterized by degeneration of
spinal motor neurons. SMA is caused by the homozygous loss of both
functional copies of the SMN1 gene. However, the SMN2 gene has the
potential to code for the same protein as SMN1 and thus overcome
the genetic defect of SMA patients. SMN2 contains a translationally
silent mutation (C.fwdarw.T) at position +6 of exon 7, which
results in inefficient inclusion of exon 7 in SMN2 transcripts.
Therefore, the predominant form of SMN2, one which lacks exon 7, is
unstable and inactive. Thus, therapeutic compounds capable of
modulating SMN2 splicing such that the percentage of SMN2
transcripts containing exon 7 is increased, would be useful for the
treatment of SMA.
[0217] In certain embodiments, the present invention provides
antisense compounds complementary to a pre-mRNA encoding SMN2. In
certain such embodiments, the antisense compound alters splicing of
SMN2. Certain sequences and regions useful for altering splicing of
SMN2 may be found in PCT/US06/024469, which is hereby incorporated
by reference in its entirety for any purpose. In certain
embodiments, oligomeric compounds having any motif described herein
have a nucleobase sequence complementary to intron 7 of SMN2.
Certain such nucleobase sequences are exemplified in the
non-limiting table below.
TABLE-US-00002 Sequence Length SEQ ID TGCTGGCAGACTTAC 15 3
CATAATGCTGGCAGA 15 4 TCATAATGCTGGCAG 15 5 TTCATAATGCTGGCA 15 6
TTTCATAATGCTGGC 15 2 ATTCACTTTCATAATGCTGG 20 7 TCACTTTCATAATGCTGG
18 1 CTTTCATAATGCTGG 15 8 TCATAATGCTGG 12 9 ACTTTCATAATGCTG 15 10
TTCATAATGCTG 12 11 CACTTTCATAATGCT 15 12 TTTCATAATGCT 12 13
TCACTTTCATAATGC 15 14 CTTTCATAATGC 12 15 TTCACTTTCATAATG 15 16
ACTTTCATAATG 12 17 ATTCACTTTCATAAT 15 18 CACTTTCATAAT 12 19
GATTCACTTTCATAA 15 20 TCACTTTCATAA 12 21 TTCACTTTCATA 12 22
ATTCACTTTCAT 12 23 AGTAAGATTCACTTT 15 24
[0218] Antisense compounds of the present invention can be used to
modulate the expression of SMN2 in a subject, such as a human. In
certain embodiments, the subject has spinal muscular atrophy. In
certain such subjects, the SMN1 gene is absent or otherwise fails
to produce sufficient amounts of functional SMN protein. In certain
embodiments, the antisense compounds of the present invention
effectively modulate splicing of SMN2, resulting in an increase in
exon 7 inclusion in SMN2 mRNA and ultimately in SMN2 protein that
includes the amino acids corresponding to exon 7. Such alternate
SMN2 protein resembles wild-type SMN protein. Antisense compounds
of the present invention that effectively modulate expression of
SMN2 mRNA or protein products of expression are considered active
antisense compounds.
[0219] Modulation of expression of SMN2 can be measured in a bodily
fluid, which may or may not contain cells; tissue; or organ of the
animal. Methods of obtaining samples for analysis, such as body
fluids (e.g., sputum, serum, CSF), tissues (e.g., biopsy), or
organs, and methods of preparation of the samples to allow for
analysis are well known to those skilled in the art. Methods for
analysis of RNA and protein levels are discussed above and are well
known to those skilled in the art. The effects of treatment can be
assessed by measuring biomarkers associated with the target gene
expression in the aforementioned fluids, tissues or organs,
collected from an animal contacted with one or more compounds of
the invention, by routine clinical methods known in the art.
[0220] Methods whereby bodily fluids, organs or tissues are
contacted with an effective amount of one or more of the antisense
compounds or compositions of the invention are also contemplated.
Bodily fluids, organs or tissues can be contacted with one or more
of the compounds of the invention resulting in modulation of SMN2
expression in the cells of bodily fluids, organs or tissues. An
effective amount can be determined by monitoring the modulatory
effect of the antisense compound or compounds or compositions on
target nucleic acids or their products by methods routine to the
skilled artisan.
[0221] The invention also provides an antisense compound as
described herein, for use in any of the methods as described
herein. For example, the invention provides an antisense compound
comprising an antisense oligonucleotide complementary to a nucleic
acid encoding human SMN2, for use in treating a disease or
condition associated with survival motor neuron protein (SMN), such
as spinal muscular atrophy (SMA). As a further example, the
invention provides an antisense compound comprising an antisense
oligonucleotide complementary to a nucleic acid encoding human
SMN2, for use in treating a disease or condition associated with
survival motor neuron protein (SMN) by administering the antisense
compound directly into the central nervous system (CNS) or CSF.
[0222] The invention also provides the use of an antisense compound
as described herein in the manufacture of a medicament for use in
any of the methods as described herein. For example, the invention
provides the use of an antisense compound comprising an antisense
oligonucleotide complementary to a nucleic acid encoding human SMN2
in the manufacture of a medicament for treating a disease or
condition associated with survival motor neuron protein (SMN), such
as spinal muscular atrophy (SMA). As a further example, the
invention provides the use of an antisense compound comprising an
antisense oligonucleotide complementary to a nucleic acid encoding
human SMN2 in the manufacture of a medicament for treating a
disease or condition associated with survival motor neuron protein
(SMN) by administration of the medicament directly into the central
nervous system (CNS) or CSF.
[0223] In certain embodiments, oligomeric compounds having any
motif described herein have a nucleobase sequence complementary to
exon 7 of SMN2.
[0224] In certain embodiments, oligomeric compounds having any
motif described herein have a nucleobase sequence complementary to
intron 6 of SMN2.
[0225] In certain embodiments, an antisense compound comprises an
antisense oligonucleotide having a nucleobase sequence comprising
at least 10 nucleobases of the sequence: TCACTTTCATAATGCTGG (SEQ ID
NO: 1). In certain embodiments, an antisense oligonucleotide has a
nucleobase sequence comprising at least 11 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 12 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 13 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 14 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 15 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 16 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising at least 17 nucleobases of such
sequence. In certain embodiments, an antisense oligonucleotide has
a nucleobase sequence comprising the nucleobases of such sequence.
In certain embodiments, an antisense oligonucleotide has a
nucleobase sequence consisting of the nucleobases of such sequence.
In certain embodiments, an antisense oligonucleotide consists of
10-18 linked nucleosides and has a nucleobase sequence 100%
identical to an equal-length portion of the sequence:
TABLE-US-00003 (SEQ ID NO: 1) TCACTTTCATAATGCTGG.
7. Certain Subjects
[0226] In certain embodiments, a subject has one or more indicator
of SMA. In certain embodiments, the subject has reduced electrical
activity of one or more muscles. In certain embodiments, the
subject has a mutant SMN1 gene. In certain embodiment, the
subject's SMN1 gene is absent or incapable of producing functional
SMN protein. In certain embodiments, the subject is diagnosed by a
genetic test. In certain embodiments, the subject is identified by
muscle biopsy. In certain embodiments, a subject is unable to sit
upright. In certain embodiments, a subject is unable to stand or
walk. In certain embodiments, a subject requires assistance to
breathe and/or eat. In certain embodiment, a subject is identified
by electrophysiological measurement of muscle and/or muscle
biopsy.
[0227] In certain embodiments, the subject has SMA type I. In
certain embodiments, the subject has SMA type II. In certain
embodiments, the subject has SMA type III. In certain embodiments,
the subject is diagnosed as having SMA in utero. In certain
embodiments, the subject is diagnosed as having SMA within one week
after birth. In certain embodiments, the subject is diagnosed as
having SMA within one month of birth. In certain embodiments, the
subject is diagnosed as having SMA by 3 months of age. In certain
embodiments, the subject is diagnosed as having SMA by 6 months of
age. In certain embodiments, the subject is diagnosed as having SMA
by 1 year of age. In certain embodiments, the subject is diagnosed
as having SMA between 1 and 2 years of age. In certain embodiments,
the subject is diagnosed as having SMA between 1 and 15 years of
age. In certain embodiments, the subject is diagnosed as having SMA
when the subject is older than 15 years of age.
[0228] In certain embodiments, the first dose of a pharmaceutical
composition according to the present invention is administered in
utero. In certain such embodiments, the first dose is administered
before complete development of the blood-brain-barrier. In certain
embodiments, the first dose is administered to the subject in utero
systemically. In certain embodiments, the first dose is
administered in utero after formation of the blood-brain-barrier.
In certain embodiments, the first dose is administered to the
CSF.
[0229] In certain embodiments, the first dose of a pharmaceutical
composition according to the present invention is administered when
the subject is less than one week old. In certain embodiments, the
first dose of a pharmaceutical composition according to the present
invention is administered when the subject is less than one month
old. In certain embodiments, the first dose of a pharmaceutical
composition according to the present invention is administered when
the subject is less than 3 months old. In certain embodiments, the
first dose of a pharmaceutical composition according to the present
invention is administered when the subject is less than 6 months
old. In certain embodiments, the first dose of a pharmaceutical
composition according to the present invention is administered when
the subject is less than one year old. In certain embodiments, the
first dose of a pharmaceutical composition according to the present
invention is administered when the subject is less than 2 years
old. In certain embodiments, the first dose of a pharmaceutical
composition according to the present invention is administered when
the subject is less than 15 years old. In certain embodiments, the
first dose of a pharmaceutical composition according to the present
invention is administered when the subject is older than 15 years
old.
[0230] 8. Certain Doses
[0231] In certain embodiments, the present invention provides dose
amounts and frequencies. In certain embodiments, pharmaceutical
compositions are administered as a bolus injection. In certain such
embodiments, the dose of the bolus injection is from 0.01 to 25
milligrams of antisense compound per kilogram body weight of the
subject. In certain such embodiments, the dose of the bolus
injection is from 0.01 to 10 milligrams of antisense compound per
kilogram body weight of the subject. In certain embodiments, the
dose is from 0.05 to 5 milligrams of antisense compound per
kilogram body weight of the subject. In certain embodiments, the
dose is from 0.1 to 2 milligrams of antisense compound per kilogram
body weight of the subject. In certain embodiments, the dose is
from 0.5 to 1 milligrams of antisense compound per kilogram body
weight of the subject. In certain embodiments, such doses are
administered twice monthly. In certain embodiments, such doses are
administered every month. In certain embodiments, such doses are
administered every 2 months. In certain embodiments, such doses are
administered every 6 months. In certain embodiments, such doses are
administered by bolus injection into the CSF. In certain
embodiments, such doses are administered by intrathecal bolus
injection. In certain embodiments, such doses are administered by
bolus systemic injection (e.g., subcutaneous, intramuscular, or
intravenous injection). In certain embodiments, subjects receive
bolus injections into the CSF and bolus systemic injections. In
such embodiments, the doses of the CSF bolus and the systemic bolus
may be the same or different from one another. In certain
embodiments, the CSF and systemic doses are administered at
different frequencies. In certain embodiments, the invention
provides a dosing regimen comprising at least one bolus intrathecal
injection and at least one bolus subcutaneous injection.
[0232] In certain embodiments, pharmaceutical compositions are
administered by continuous infusion. Such continuous infusion may
be accomplished by an infusion pump that delivers pharmaceutical
compositions to the CSF. In certain embodiments, such infusion pump
delivers pharmaceutical composition IT or ICV. In certain such
embodiments, the dose administered is between 0.05 and 25
milligrams of antisense compound per kilogram body weight of the
subject per day. In certain embodiments, the dose administered is
from 0.1 to 10 milligrams of antisense compound per kilogram body
weight of the subject per day. In certain embodiments, the dose
administered is from 0.5 to 10 milligrams of antisense compound per
kilogram body weight of the subject per day. In certain
embodiments, the dose administered is from 0.5 to 5 milligrams of
antisense compound per kilogram body weight of the subject per day.
In certain embodiments, the dose administered is from 1 to 5
milligrams of antisense compound per kilogram body weight of the
subject per day. In certain embodiments, the invention provides a
dosing regimen comprising infusion into the CNS and at least one
bolus systemic injection. In certain embodiments, the invention
provides a dosing regimen comprising infusion into the CNS and at
least one bolus subcutaneous injection. In certain embodiments, the
dose, whether by bolus or infusion, is adjusted to achieve or
maintain a concentration of antisense compound from 0.1 to 100
microgram per gram of CNS tissue. In certain embodiments, the dose,
whether by bolus or infusion, is adjusted to achieve or maintain a
concentration of antisense compound from 1 to 10 microgram per gram
of CNS tissue. In certain embodiments, the dose, whether by bolus
or infusion, is adjusted to achieve or maintain a concentration of
antisense compound from 0.1 to 1 microgram per gram of CNS
tissue.
[0233] In certain embodiments, dosing a subject is divided into an
induction phase and a maintenance phase. In certain such
embodiments, the dose administered during the induction phase is
greater than the dose administered during the maintenance phase. In
certain embodiments, the dose administered during the induction
phase is less than the dose administered during the maintenance
phase. In certain embodiments, the induction phase is achieved by
bolus injection and the maintenance phase is achieved by continuous
infusion.
[0234] In certain embodiments, the invention provides systemic
administration of antisense compounds, either alone or in
combination with delivery into the CSF. In certain embodiments, the
dose for systemic administration is from 0.1 mg/kg to 200 mg/kg. In
certain embodiments, the dose for systemic administration is from
0.1 mg/kg to 100 mg/kg. In certain embodiments, the dose for
systemic administration is from 0.5 mg/kg to 100 mg/kg. In certain
embodiments, the dose for systemic administration is from 1 mg/kg
to 100 mg/kg. In certain embodiments, the dose for systemic
administration is from 1 mg/kg to 50 mg/kg. In certain embodiments,
the dose for systemic administration is from 1 mg/kg to 25 mg/kg.
In certain embodiments, the dose for systemic administration is
from 0.1 mg/kg to 25 mg/kg. In certain embodiments, the dose for
systemic administration is from 0.1 mg/kg to 10 mg/kg. In certain
embodiments, the dose for systemic administration is from 1 mg/kg
to 10 mg/kg. In certain embodiments, the dose for systemic
administration is from 1 mg/kg to 5 mg/kg. In certain embodiments
comprising both systemic and CSF delivery, the doses for those two
routes are independently determined.
[0235] a. Calculation of Appropriate Human Doses
[0236] In certain embodiments, the subject is a human. In certain
embodiments, a human dose is calculated or estimated from data from
animal experiments, such as those described herein. In certain
embodiments, a human dose is calculated or estimated from data from
monkey and/or mouse experiments, such as those described herein. In
certain embodiments, a human dose is calculated or estimated from
data from mouse experiments, such as those described herein. In
certain embodiments, appropriate human doses can be calculated
using pharmacokinetic data from mouse along with knowledge of brain
weight and/or cerebrospinal fluid (CSF) turnover rates. For
example, the mouse brain weight is approximately 0.4 g, which is
approximately 2% of its body weight. In humans, the average brain
weight is 1.5 kg which is approximately 2.5% of body weight. In
certain embodiments, administration into the CSF results in
elimination of a portion of the compound through uptake in brain
tissue and subsequent metabolism. By using the ratio of human to
mouse brain weight as a scaling factor an estimate of the
elimination and clearance through the brain tissue can be
calculated. Additionally, the CSF turnover rate can be used to
estimate elimination of compound from the CSF to blood. Mouse CSF
turnover rate is approximately 10-12 times per day (0.04 mL
produced at 0.325 .mu.l/min). Human CSF turnover rate is
approximately 4 times per day (100-160 mL produced at 350-400
.mu.l/min). Clearance, and therefore dosing requirements, can be
based on brain weight elimination scaling, and/or the CSF turnover
scaling. The extrapolated human CSF clearance can be used to
estimate equivalent doses in humans that approximate doses in mice.
In this way, human doses can be estimated that account for
differences in tissue metabolism based on brain weight and CSF
turnover rates. Such methods of calculation and estimate are known
to those skilled in the art.
[0237] By way of non-limiting example, in certain embodiments, an
equivalent human dose can be estimated from a desired mouse dose by
multiplying the mg/kg mouse dose by a factor from about 0.25 to
about 1.25 depending on the determined clearance and elimination of
a particular compound. Thus, for example, in certain embodiments, a
human dose equivalent of a 0.01 mg dose for a 20 g mouse will range
from about 8.75 mg to about 43.75 mg total dose for a 70 kg human.
Likewise, in certain embodiments, a human dose equivalent of a 0.01
mg dose for a 4 g newborn mouse will range from about 1.9 mg to
about 9.4 mg total dose for a 3 kg newborn human. These example
doses are merely to illustrate how one of skill may determine an
appropriate human dose and are not intended to limit the present
invention.
[0238] In certain embodiments, a human dose for systemic delivery
(whether administered alone or in combination with CSF delivery) is
calculated or estimated from data from animal experiments, such as
those described herein. Typically, an appropriate human dose (in
mg/kg) for systemic dose is between 0.1 and 10 times an effective
dose in animals. Thus, solely for example, a subcutaneous dose of
50 .mu.g in a 2 g newborn mouse is a dose of 25 mg/kg. The
corresponding dose for a human is predicted to be between 2.5 mg/kg
and 250 mg/kg. For a 3 kilogram infant, the corresponding dose is
between 7.5 mg and 750 mg. For a 25 kg child, the corresponding
dose is from 62.5 mg to 6250 mg.
9. Treatment Regimens
[0239] In certain embodiments, the above dose amounts, dose
frequencies, routes of administration, induction and maintenance
phases, and timing of first dose are combined to provide dosing
regimens for subjects having SMA. Such dosing regimens may be
selected and adjusted to provide amelioration of one or more
symptom of SMA and/or to reduce or avoid toxicity or side effects
attributable to the administration of the pharmaceutical
composition. In certain embodiments, subjects are in utero or
newborn. In such embodiments, administration of pharmaceutical
compositions, particularly by continuous infusion, presents
particular challenges. Accordingly, in certain embodiments, the
present invention provides for administration of pharmaceutical
compositions by bolus administration while the subject is in utero
or very young, followed by continuous infusion via an implanted
infusion pump when the subject is older and placement of such pump
is more practical. Further, in certain embodiments, as a subject
grows, the absolute dose is increased to achieve the same or
similar dose:body-weight ratio. The following table is intended to
exemplify treatment regimens and is not intended to limit the
possible combinations of treatments which will be easily
accomplished by one of skill in the art.
TABLE-US-00004 Dosing period First Second Third Fourth Fifth
Regimen 1 Subject Age In utero, prior In utero, after >1 week 6
months 1.5 years to formation formation of blood- of blood-
brain-barrier brain-barrier Dose Amount 50 .mu.g 50 .mu.g 100 .mu.g
10 .mu.g/day 50 .mu.g/day Frequency Single admin Single admin
Monthly Continuous Continuous Route of Systemic IT IT IT IT
Administration injection injection injections infusion infusion
Duration N/A N/A 6 months 1 year Ongoing Regimen 2 Subject Age In
utero, after >1 week 6 months 1.5 years N/A formation of blood-
brain-barrier Dose Amount 50 .mu.g 100 .mu.g 5 mg/day 10 mg/day N/A
Frequency Single admin Monthly Continuous Continuous N/A Route of
ICV ICV ICV ICV N/A Administration injection injection infusion
infusion Duration N/A 6 months 1 year Ongoing N/A Regimen 3 Subject
Age >1 week 6 months 1.5 years 2.5 years* Dose Amount 100 .mu.g
500 .mu.g/day 20 mg/day 20 mg/day 100 mg Frequency 2xMonthly
Continuous Continuous Continuous 2xMonthly Route of ICV ICV ICV ICV
IP Administration injection infusion infusion infusion Duration 6
months 1 year 1 year Ongoing Ongoing *Note: the 4.sup.th dosing
period in regimen 3 exemplifies continuous CSF infusion combined
with periodic systemic administration. These treatment regimens are
intended to exemplify and not to limit the present invention.
[0240] In certain embodiments, the dosing regimen comprises a
systemic administration, either alone or in combination with
administration into the CSF (for example regimen 3, above). The
table, below further exemplifies such regimens.
TABLE-US-00005 Systemic administration CSF administration Dose
Route Frequency Dose Route Frequency 1-5 mg/kg subcutaneous weekly
5-10 mg/kg bolus IT monthly 1-5 mg/kg subcutaneous monthly 1-5
mg/kg bolus ICV 2 months 10-50 mg/kg subcutaneous monthly 0.5-1
mg/kg bolus IT 6 months 0.5-25 mg/kg subcutaneous monthly 10
mg/kg/day IT infusion continuous for 7 days every 6 months 0.1-10
mg/kg subcutaneous monthly none none 0.5-1 mg/kg bolus IT 6
months
These treatment regimens are intended to exemplify and not to limit
the present invention. One of skill in the art will be able to
select an appropriate combination of the doses and deliveries in
view of the present disclosure and based on a variety of factors,
such as the severity of the condition and the overall health and
age of the subject.
10. Co-Administration
[0241] In certain embodiments, pharmaceutical compositions of the
present invention are co-administered with at least one other
pharmaceutical composition for treating SMA and/or for treating one
or more symptom associated with SMA. In certain embodiments, such
other pharmaceutical composition is selected from trichostatin-A,
valproic acid, riluzole, hydroxyurea, and a butyrate or butyrate
derivative. In certain embodiments, pharmaceutical compositions of
the present invention are co-administered with trichostatin A. In
certain embodiments, pharmaceutical compositions of the present
invention are co-administered with a derivative of quinazoline, for
example as described in Thurmond, et al., J. Med Chem. 2008, 51,
449-469. In certain embodiments, a pharmaceutical composition of
the present invention and at least one other pharmaceutical
composition are co-administered at the same time. In certain
embodiments, a pharmaceutical composition of the present invention
and at least one other pharmaceutical composition are
co-administered at different times.
[0242] In certain embodiments, pharmaceutical compositions of the
present invention are co-administered with a gene therapy agent. In
certain such embodiments, the gene therapy agent is administered to
the CSF and the pharmaceutical composition of the present invention
is administered systemically. In certain such embodiments, the gene
therapy agent is administered to the CSF and the pharmaceutical
composition of the present invention is administered to the CSF and
systemically. In certain embodiments, a pharmaceutical composition
of the present invention and a gene therapy agent are
co-administered at the same time. In certain embodiments, a
pharmaceutical composition of the present invention and a gene
therapy agent are co-administered at different times. Certain gene
therapy approaches to SMA treatment have been reported (e.g., Coady
et al., PLoS ONE 2008 3(10): e3468; Passini et al., J Clin Invest
2010 Apr. 1, 120(4): 1253-64).
[0243] In certain embodiments, pharmaceutical compositions of the
present invention are co-administered with at least one other
therapy for SMA. In certain embodiments, such other therapy for SMA
is surgery. In certain embodiments, such other therapy is physical
therapy, including, but not limited to exercises designed to
strengthen muscles necessary for breathing, such as cough therapy.
In certain embodiments, other therapy is a physical intervention,
such as a feeding tube or device for assisted breathing.
[0244] In certain embodiments, pharmaceutical compositions of the
present invention are co-administered with one or more other
pharmaceutical compositions that reduce an undesired side-effect of
the pharmaceutical compositions of the present invention.
11. Phenotypic Effects
[0245] In certain embodiments, administration of at least one
pharmaceutical composition of the present invention results in a
phenotypic change in the subject. In certain embodiments, such
phenotypic changes include, but are not limited to: increased
absolute amount of SMN mRNA that includes exon 7; increase in the
ratio SMN mRNA that includes exon 7 to SMN mRNA lacking exon 7;
increased absolute amount of SMN protein that includes exon 7;
increase in the ratio SMN protein that includes exon 7 to SMN
protein lacking exon 7; improved muscle strength, improved
electrical activity in at least one muscle; improved respiration;
weight gain; and survival. In certain embodiments, at least one
phenotypic change is detected in a motoneuron of the subject. In
certain embodiments, administration of at least one pharmaceutical
composition of the present invention results in a subject being
able to sit-up, to stand, and/or to walk. In certain embodiments,
administration of at least one pharmaceutical composition of the
present invention results in a subject being able to eat, drink,
and/or breathe without assistance. In certain embodiments, efficacy
of treatment is assessed by electrophysiological assessment of
muscle. In certain embodiments, administration of a pharmaceutical
composition of the present invention improves at least one symptom
of SMA and has little or no inflammatory effect. In certain such
embodiment, absence of inflammatory effect is determined by the
absence of significant increase in Aif1 levels upon treatment.
[0246] In certain embodiments, administration of at least one
pharmaceutical composition of the present invention delays the
onset of at least one symptom of SMA. In certain embodiments,
administration of at least one pharmaceutical composition of the
present invention slows the progression of at least one symptom of
SMA. In certain embodiments, administration of at least one
pharmaceutical composition of the present invention reduces the
severity of at least one symptom of SMA.
[0247] In certain embodiments, administration of at least one
pharmaceutical composition of the present invention results in an
undesired side-effect. In certain embodiments, a treatment regimen
is identified that results in desired amelioration of symptoms
while avoiding undesired side-effects.
12. Dosage Units
[0248] In certain embodiments pharmaceutical compositions of the
present invention are prepared as dosage units for administration.
Certain such dosage units are at concentrations selected from 0.01
mg to 100 mg. In certain such embodiments, a pharmaceutical
composition of the present invention comprises a dose of antisense
compound selected from 0.01 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg,
20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, and 200 mg. In certain
embodiments, a pharmaceutical composition is comprises a dose of
oligonucleotide selected from 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 25
mg, and 50 mg.
13. Kits
[0249] In certain embodiments, the present invention provides kits
comprising at least one pharmaceutical composition. In certain
embodiments, such kits further comprise a means of delivery, for
example a syringe or infusion pump.
Nonlimiting Disclosure and Incorporation by Reference
[0250] While certain compounds, compositions and methods described
herein have been described with specificity in accordance with
certain embodiments, the following examples serve only to
illustrate the compounds described herein and are not intended to
limit the same. Each of the references, GenBank accession numbers,
and the like recited herein is hereby incorporated by reference in
its entirety.
[0251] Although the sequence listing accompanying this filing
identifies each sequence as either "RNA" or "DNA" as required, in
reality, those sequences may be modified with any combination of
chemical modifications. One of skill in the art will readily
appreciate that such designation as "RNA" or "DNA" to describe
modified oligonucleotides is, in certain instances, arbitrary. For
example, an oligonucleotide comprising a nucleoside comprising a
2'-OH sugar moiety and a thymine base could be described as a DNA
having a modified sugar (2'-OH for the natural 2'-H of DNA) or as
an RNA having a modified base (thymine (methylated uracil) for
natural uracil of RNA).
[0252] Accordingly, nucleic acid sequences provided herein,
including, but not limited to those in the sequence listing, are
intended to encompass nucleic acids containing any combination of
natural or modified RNA and/or DNA, including, but not limited to
such nucleic acids having modified nucleobases. By way of further
example and without limitation, an oligomeric compound having the
nucleobase sequence "ATCGATCG" encompasses any oligomeric compounds
having such nucleobase sequence, whether modified or unmodified,
including, but not limited to, such compounds comprising RNA bases,
such as those having sequence "AUCGAUCG" and those having some DNA
bases and some RNA bases such as "AUCGATCG" and oligomeric
compounds having other modified bases, such as "AT.sup.meCGAUCG,"
wherein .sup.meC indicates a cytosine base comprising a methyl
group at the 5-position.
EXAMPLE 1
Antisense Compounds Targeting SMN2
[0253] The following oligonucleotides were synthesized using
standard techniques previously reported.
TABLE-US-00006 Reference # Sequence Length Chemistry SEQ ID
ISIS396443 TCACTTTCATAATGCTGG 18 Full 2'-MOE; full PS 1 ISIS396449
TTTCATAATGCTGGC 15 Full 2'-MOE; full PS 2 PS = phosphorothioate
internucleoside linkages
EXAMPLE 2
Smn-/- SMN Transgenic Mice
[0254] Therapeutic effectiveness and safety using the antisense
compounds as described above can be tested in an appropriate animal
model. For example, animal models which appear most similar to
human disease include animal species which either spontaneously
develop a high incidence of the particular disease or those that
have been induced to do so.
[0255] In particular, animal models for SMA are known. As explained
above, the molecular basis of SMA, an autosomal recessive
neuromuscular disorder, is the homozygous loss of the survival
motor neuron gene 1 (SMN1). A nearly identical copy of the SMN1
gene, called SMN2 is found in humans and modulates the disease
severity. In contrast to humans, mice have a single gene (Smn) that
is equivalent to SMN1. Homozygous loss of this gene is lethal to
embryos and results in massive cell death, which indicates that the
Smn gene product is necessary for cellular survival and function.
The introduction of 2 copies of SMN2 into mice lacking SMN rescues
the embryonic lethality, resulting in mice with the SMA phenotype
(Monani et al., Hum. Mol. Genet. (2000) 9:333-339. A high copy
number of SMN2 rescues the mice because sufficient SMN protein is
produced in motor neurons. See, also, Hsieh-Li, et al., Nat. Genet.
(2000) 24:66-70, reporting the production of transgenic mouse lines
that expressed human SMN2. In particular, transgenic mice harboring
SMN2 in the Smn-/- background showed pathological changes in the
spinal cord and skeletal muscles similar to those of SMA patients.
The severity of the pathological changes in these mice correlated
with the amount of SMN protein that contained the region encoded by
exon 7. Heterozygous mice lacking one copy of Smn are designated
Smn-/+ and are a model for the less severe form of SMA, type
III.
[0256] The severity of the SMA phenotype is a function of the
number of copies of human SMN2 in the mice. The "Taiwan" strain has
4 copies of human SMN2, resulting in mice that have moderate to
severe SMA phenotype, similar to Type I or Type II.
[0257] Delta-7 mice (Smn.sup.-/-, hSMN2.sup.-/+,
SMN.DELTA.7.sup.+/+) also lack mouse Smn and express human SMN2.
Delta 7 mice have a more severe phenotype and die shortly after
birth, typically about 15-20 days after birth.
EXAMPLE 3
Systemic Administration of Antisense Compounds In Vivo in Smn-/-
SMN2 (Taiwan Strain)
[0258] Taiwan mice were treated by intraperitoneal injection with
saline or with 35 mg/kg of ISIS396443 or a mismatched antisense
oligonucleotide control once each day for 5 days and were
sacrificed 2 days later on day 7. Liver and kidney were collected
and RNA was isolated using standard techniques. SMN2 with and
without exon 7 was visualized by RT-PCR. Administration with
ISIS396443 resulted in substantial increase in exon 7 inclusion in
the SMN2 from kidney and liver compared to saline and mismatch
control treated animals.
EXAMPLE 4
Intracerebroventricular (ICV) Administration of Antisense Compounds
In Vivo in Smn-/- SMN2 (Taiwan Strain)
[0259] Taiwan mice were injected ICV either with saline or with 150
.mu.g of ISIS396443 each day for 7 days. The mice were sacrificed
on day 8 and RNA from brain and spinal cord was extracted. RT-PCR
analysis showed substantial increase in exon 7 inclusion in the
SMN2 in brain and spinal cord samples obtained from animals treated
with ISIS396443. These results indicate that ICV treatment with
antisense oligonucleotide targeting SMN can rescue the SMA
condition because exclusion of exon 7 is associated with the SMA
phenotype.
[0260] Dose-Response
[0261] Taiwan mice were injected ICV either with saline or with 10,
50, 100, or 150 .mu.g of ISIS396443 each day for 7 days (5 mice in
each treatment group) and were sacrificed on day 8. RNA was
isolated and analyzed by RT-PCR. The 10 .mu.g treatment group
showed moderate exon 7 inclusion. The 50 .mu.g, 100 .mu.g, and 150
.mu.g groups all showed substantial exon 7 inclusion.
[0262] Duration of Response
[0263] To determine the duration of effect, 24 mice were injected
ICV with 50 .mu.g of ISIS396443 each day for 7 days. Four mice were
sacrificed at the time of the final dose (time 0) and four mice
were sacrificed at each of: 1 week, 2 weeks, 4 weeks and 8 weeks
after the final dose. All treated mice showed substantial exon 7
inclusion by RT-PCR with the effect at week 8 showing no difference
with the other groups, as shown in FIG. 1:
[0264] These results indicate that ICV administration of ISIS396443
at 50 .mu.g per day for 7 days is effective for at least 8 weeks
following treatment.
[0265] The experiment was repeated to test longer time points. Type
III mice were treated by ICV infusion of ISIS396443 at 50 .mu.g/day
for 7 days. Mice were sacrificed 0, 0.5, 1, 2, 4, and 6 months
after the end of the 7 day infusion period. RNA was collected from
the spinal cords and analyzed by northern blot. As shown in the
graph below, the effect of ISIS396443 infusion persisted for 6
months after infusion, as shown in FIG. 2. This long duration of
effect has several possible explanations. It may reflect stability
of ISIS396443, stability of the corrected SMN protein and/or that
the dose was high enough that even after loss of compound to
metabolism, the remaining dose continued to provide benefit. Thus,
these data may support administration of lower doses as well as
infrequent doses.
EXAMPLE 5
Administration of Antisense Compounds by Continuous
Intracerebroventricular (ICV) Infusion
[0266] Using a micro-osmotic pump (Azlet Osmotic Pumps, Cupertino,
Calif., USA), ISIS396443 was delivered into cerebrospinal fluid
(CSF) through the right lateral ventricle in adult type-III Smn+/-
or Smn-/- SMA mice with a human SMN2 transgene (Taiwan strain).
Dose-response studies revealed that intracerebroventricular (ICV)
infusion of the ISIS396443 increased SMN2 exon 7 inclusion in
spinal cord to .about.90%, compared to .about.10% in saline-treated
mice. Western blotting and immunohistochemical analysis
demonstrated a robust increase of the human transgenic SMN protein
levels in spinal-cord motor neurons. These results indicate that
CNS infusion of antisense oligonucleotide ISIS396443 can rescue the
SMA condition because exclusion of exon 7 is associated with the
SMA phenotype.
EXAMPLE 6
Embryonic Administration
[0267] A single ICV injection of either 20 .mu.g or 10 .mu.g of
ISIS396443 was administered to embryonic Taiwan mice at day 15 of
gestation (E15). Animals were sacrificed at day 7 after birth (P7).
RNA was isolated from the lumbar spinal cord and analyzed by
RT-PCR. The single embryonic administration of ISIS 396443 resulted
in substantial exon 7 inclusion. These results indicate that
treatment with antisense oligonucleotide ISIS396443 in utero can
rescue the SMA condition because exclusion of exon 7 is associated
with the SMA phenotype.
[0268] The above experiment was repeated and the animals were
sacrificed at 11 weeks. Untreated Taiwan mice develop necrotic
tails, which shorten over time. The single embryonic injection of
20 of ISIS396443 significantly delayed the onset of tail
degradation, as shown in FIG. 3A. These results indicate that
embryonic treatment with antisense oligonucleotide targeting SMN
delays onset of SMA.
[0269] These results were confirmed in another study using the same
conditions, except the doses tested were 20 .mu.g and 10 .mu.g of
ISIS396443 and the study included normal mice for comparison.
[0270] Results from that experiment are shown in FIG. 3B.
EXAMPLE 7
In Vivo Administration in the Delta-7 Mouse Model
[0271] Heterozygote (SMN.sup.+/-, hSMN2.sup.-/+,
SMN.DELTA.7.sup.+/+) breeding pairs were mated and, on the day of
birth (P0), newborn pups were treated with ISIS396443 (18-mer, SEQ
ID NO. 1), ISIS396449 (15-mer, SEQ ID NO. 2), ISIS387954 (20-mer,
SEQ ID NO. 7) or a scrambled control ASO (ISIS439273; 18-mer). Mice
were injected bilaterally into the cerebral lateral ventricles for
a total dose of 8 .mu.g (4 .mu.g in each lateral ventricle). All
the injections were performed with a finely drawn glass
micropipette needle as described (Passini et al, J. Virol. (2001)
75:12382-12392). Following the injections, the pups were
toe-clipped and genotyped (Le et al., Hum. Mol. Genet. (2005)
14:845-857) to identify SMA (SMN.sup.-/-, hSMN2.sup.+/+,
SMN.DELTA.7.sup.+/+), heterozygote, and wild type (SMN.sup.+/-,
hSMN2.sup.+/-, SMN.DELTA.7.sup.+/-) mice. All the litters were
culled to 7 pups to control for litter size on survival. Some of
the litters were not injected in order to generate untreated
control groups.
[0272] Widespread distribution of the18-mer was detected in the
spinal cord at 14 days post-injection in SMA mice, including the
thoracic, lumbar, and cervical regions of the spinal cord.
Furthermore, co-localization studies with ChAT confirmed the vast
majority of the cells targeted by ISIS396443 in the spinal cord
were motor neurons. No signal was detected in control, untreated
mice.
[0273] Western blot analysis at 14 days showed the amount of SMN in
the brain and spinal cord were at 40-60% wild type levels, compared
to 10% in untreated SMA controls. No signal above background was
detected in control mice treated with a scrambled version of the
ASO. Results of the western blots are provided in FIG. 4.
[0274] SMA mice treated with SMA ASOs also exhibited a significant
increase in weight, ambulatory function (righting reflex and grip
strength), and coordination (hindlimb splay) regardless of the
length of the ASO as compared to untreated SMA mice or SMA mice
treated with a scrambled ASO. No significant increase in body
weight, ambulatory function (righting reflex and grip strength), or
coordination was observed in SMA mice treated with a scrambled ASO
as compared to untreated SMA mice. Results are provided in FIGS. 5
and 6.
[0275] Importantly, SMA mice treated with ASOs regardless of length
of ASO produced a significant increase in median survival as shown
in FIG. 7. Survival was from birth was 31.5 (15-mer), 27.0
(18-mer), and 28.0 (20-mer) days, compared to 16.0 days in
untreated SMA controls. In contrast, SMA mice treated with an
18-mer scrambled control did not improve survival. These results
demonstrate that treatment with antisense oligonucleotide targeting
SMN treatment increases lifespan in SMA affected subjects.
[0276] The SMA ASOs also increased motor neuron cell counts in the
spinal cord as shown in FIG. 8.
[0277] SMN RNA was measured by RT-PCR. Animals treated with SMA
ASOs had increased SMN RNA levels compared to untreated SMA mice.
Results from mice treated with the 20-mer ASO compared to untreated
SMA mice are shown in FIG. 9.
[0278] To determine whether survival could be further increased by
administration of a second dose, the above experiment was repeated
with an additional dose of 20 .mu.g at day 21. Results are shown in
FIG. 10. The graph above shows the effect of the first dose of 8
.mu.g at day 0. At day P21, half of the treated mice were given a
second treatment.
[0279] The effect of the second treatment compared to mice that
received only the first treatment is shown in FIG. 11. This result
indicates that a second ICV treatment with antisense
oligonucleotide further increases survival.
EXAMPLE 8
Activity in SMA type III Mice
[0280] Two antisense compounds and one control compound were tested
in a mouse model of SMA. The compounds are described in table
below.
TABLE-US-00007 Compounds Tested in Taiwan Strain SMA Mice ISIS#
Sequence Description SEQ ID 396443 TCACTTTCATAATGCTGG Uniform
2'-MOE, full PS; 18- 1 mer; complementary to intron 7 of human SMN2
449220 ATTCACTTTCATAATGCTGG Uniform 2-OMe; full PS; 20- 3 mer;
complementary to intron 7 of human SMN2 439272 TTAGTTTAATCACGCTCG
Uniform 2'-MOE full PS; 18- 4 mer; control sequence
[0281] Taiwan strain of SMA type III mice were obtained from The
Jackson Laboratory (Bar Harbor, Me.). These mice lack mouse SMN and
are homozygous for human SMN2 (mSMN-/-; hSMN2+/+). These mice have
been described in Hsieh-Li HM, et al., Nature Genet. 24, 66-70
2000.
[0282] Mice were treated with 3, 10, 30, or 100 .mu.g of ISIS396443
or ISIS449220 per day or with 30 or 100 .mu.g of control compound
ISIS439272 per day in phosphate buffered saline (PBS). Control mice
were treated with PBS alone (dose of 0). All treatments were
administered by intracerebroventricular (ICV) infusion using an
Azlet 1007D osmotic pump. There were five animals for each dose,
however, two of the mice from the highest dose of ISIS449220 died
prior to completion of the study. Animals were sacrificed on day 9
(two days after final dose) and brain and lumbar sections of the
spinal cords were collected from each animal. Real time PCR was
performed on each sample to determine the amount of human SMN2
message including exon 7 ((+)exon 7) and the amount of human SMN2
message lacking exon 7((-)exon 7). Real time PCR was also performed
to determine the expression levels of allograft inflammatory factor
(AIF1) and glyceraldehyde 3-phosphate dehyrogenase (GADPH).
[0283] Expression levels for (+)exon 7 and (-)exon 7were normalized
to GADPH levels. Those normalized expression levels were then
divided by the GADPH-normalized levels from the PBS treated control
mice. The resulting fold-control values are reported in Table 17,
below. Data represent mean fold of control for all five mice in
each group, except the highest dose of ISIS449220, which represent
the 3 surviving mice.
[0284] Administration of ISIS396443 resulted in a striking increase
in inclusion of exon 7. At 10 .mu.g/day, ISIS396443 resulted in
nearly twice as much (1.8 fold) exon 7 retained SMN2 message in
brain, and in lumbar spinal cord it was more than twice as much
compared to untreated control.
Ability of Antisense Compounds to Alter Splicing in SMA Mice
TABLE-US-00008 [0285] Brain Lumbar Cord Dose (+)exon (-)exon
(+)exon (-)exon Compound (.mu.g/day) 7 7 7 7 396443 0 1.0 1.0 1.0
1.0 (2'-MOE) 3 1.3 1.0 1.4 1.0 10 1.8 0.7 2.1 0.6 30 2.4 0.6 3.4
0.3 100 3.0 0.3 3.8 0.1 449220 0 1.0 1.0 1.0 1.0 (2'-OMe) 3 0.9 1.1
1.0 1.1 10 1.0 1.1 1.0 1.2 30 1.0 1.2 1.1 1.2 100* 1.0 1.0 1.2 1.1
439272 0 1.0 1.0 1.0 1.0 Control 30 1.0 1.1 0.9 1.1 100 1.0 1.0 1.0
1.0 *data from only 3 mice for this dose
[0286] Expression of allograft inflammatory factor (AIF1) was
tested as a measure of inflammation. After normalization of all
samples to (GADPH), the ratio of AIF1 for each treatment group was
divided by the value for the PBS control. ISIS396443 resulted in no
increase in AIF1, even at the highest dose. ISIS449220 resulted in
increased AIF1 in both brain and lumbar spinal cord. Data in Table
18 represent mean fold of control for all five mice in each group,
except the highest dose of ISIS449220, which represent the 3
surviving mice.
Toxicity of Antisense Compounds in SMA Mice
TABLE-US-00009 [0287] Dose AIF-1/GAPDH Compound (.mu.g/day) Brain
Lumbar 396443 0 1.0 1.0 (2'-MOE) 3 10 1.0 10 1.1 1.2 30 1.0 1.0 100
0.9 1.0 449220 0 1.0 1.0 (2'-OMe) 3 1.0 1.0 10 1.0 1.8 30 1.2 2.9
100* 1.8 3.3 439272 0 0.9 0.9 Control 30 0.9 1.0 100 0.9 1.2 *data
from only 3 mice for this dose
EXAMPLE 9
Administration to Monkeys
[0288] Cynomolgus monkeys were used to assess distribution of
ISIS395443 at different doses and routes of administration.
ISIS396443 was administered to 2 monkeys. One monkey received a
dose of 3 mg by ICV infusion and the other monkey received a dose
of 3 mg by IT infusion. Both infusions were delivered over a 24
hour period. The monkeys were sacrificed and tissues were harvested
96 hours after the end of the infusion period. The concentration of
ISIS396443 was measured in samples from Cervical, Thoracic, and
Lumbar sections of the spinal cord. Results are summarized in the
table below.
TABLE-US-00010 Concentration of ISIS396443 Animal # Dose Route
Tissue (.mu.g/g) 1 3 mg over ICV Cervical 21.5 24 hours infusion
Thoracic 9.4 Lumbar 23.9 2 3 mg over IT Cervical 12.5 24 hours
infusion Thoracic 22.6 Lumbar 42.6
Since cynololgus monkeys are approximately 3 kg, this dose is about
1 mg/kg.
[0289] To further assess distribution of ISIS39644, twenty-six
monkeys were divided into six groups as provided in the table
below.
TABLE-US-00011 Concentration Duration Day of compound of sacri-
Number of Group Dose Route (mg/ml) infusion ficed monkeys 1 0 ICV 0
14 days Day 19 2M/2F 2 3 mg ICV 0.09 14 days Day 19 2M/2F 3 3 mg IT
1.25 .sup. 1 day Day 6 3M/2F 4 3 mg IT 0.42 3 days Day 8 2M/2F 5 3
mg IT 0.18 7 days Day 12 3M/2F 6 3 mg IT 0.09 14 days Day 19
2M/2F
Infusion rate for all groups was 100 .mu.L/hour. All monkeys
received a total of 3 mg of ISIS39644 in saline, except for group
1, which received saline only. Monkeys were sacrificed and tissues
were harvested 5 days after the end of infusion.
[0290] Concentrations of ISIS39644 in tissue samples from the
monkeys were evaluated using standard techniques. Summaries of the
results are provided in the graphs in FIG. 12. Samples were also
evaluated by histology. The histology did not show any adverse
effect of treatment and confirmed presence of ISIS396443. There was
no evidence of Purkinje cell loss.
[0291] Rapid infusion appeared to have more ISIS396443 than slower
infusion. These results suggest that faster infusion rates or bolus
injection may be preferred in certain embodiments. Since bolus
administration has certain practical advantages over infusion, in
certain embodiments, it is the preferred method of administration
into the CSF. In certain embodiments, the preferred method of
administration into the CSF is by bolus IT injection.
EXAMPLE 10
Generation of a Mouse Model of Severe SMA and ICV Treatment
[0292] Mice having a severe SMA phenotype (sSMA mice) were
generated. Homozygote sSMA mice carry 2 copies of human SMN2 and no
mouse SMN. The average lifespan is about 10 days. In addition, the
SMA mice are smaller and have shorter tails. Heterozygotes carry
mouse SMN and develop normally.
[0293] To study the effect of antisense compounds in these sSMA
mice, 20 .mu.g of ISIS396443 was injected ICV at day P1. Treatment
resulted in an increase in average survival from 9.9 days (saline
treated control) to 16.7 days. RT-PCR analysis showed increased
full-length SMN RNA in tissues from the treated mice.
EXAMPLE 11
Systemic Administration of ISIS 396443
[0294] sSMA mice and healthy heterozygote control mice were divided
into groups to study the effect of ISIS396443 by bolus ICV
injection and/or bolus subcutaneous injection (SC) as follows:
[0295] Group 1--ICV+SC [0296] One ICV injection of 20 .mu.g at P1
or P2 (day 1 or 2 after birth); and two subcutaneous injections of
50 .mu.g/g delivered between P0 and P3. [0297] Group 2--SC+SC
[0298] Two SC injections of 50 .mu.g/g delivered between P0 and P3;
and one subcutaneous injection of 50 .mu.g/g delivered between P5
and P6; and subcutaneous injection of 50 .mu.g/g delivered between
P9 and P10. [0299] Group 3--SC [0300] Two SC injections of 50
.mu.g/g delivered between P0 and P3. [0301] Group 4--SMA saline
control [0302] One ICV injection of saline at P1 or P2; and two
subcutaneous injections of saline delivered between P0 and P3.
[0303] Group 5--Heterozygous control [0304] One ICV injection of 20
.mu.g at P1 or P2; and two subcutaneous injections of 50 .mu.g/g
delivered between P0 and P3 of heterozygous mice. Each group
included from 14 to 22 mice. Survival (in days) for individual mice
in each group is provided in the table, below. Many mice in this
study remain alive at the time that this patent application is
being prepared. Thus, a value proceeded by a ">" indicates that
the mouse has lived that number of days and is still alive.
TABLE-US-00012 [0304] Group 1 Group 2 Group 3 Group 4 Group 5 Mouse
ICV + SC SC + SC SC Saline Het 1 >141 >130 >103 8 >146
2 >141 127 94 8 >146 3 22 >114 61 8 >146 4 >140 73
>103 8 >146 5 117 27 >103 8 >145 6 >124 27 >103 8
>145 7 >111 18 34 8 >145 8 >111 >102 26 8 >145 9
>111 >98 31 8 >145 10 >111 >98 69 9 >144 11 29
>102 69 9 >144 12 >110 >102 67 9 >144 13 >110
>102 >91 9 >144 14 >110 >102 >90 9 >143 15
>110 ND >90 9 >143 16 >108 ND >90 9 >143 17
>108 ND >90 10 >129 18 >109 ND 86 10 >129 19 18 ND
>75 10 >129 20 ND ND 69 10 ND 21 ND ND 18 11 ND 22 ND ND
>71 12 ND 23 ND ND ND 12 ND 24 ND ND ND 13 ND 25 ND ND ND 13 ND
26 ND ND ND 14 ND
EXAMPLE 12
Dose-Response of SC Administration
[0305] Survival of sSMA mice receiving different doses of
subcutaneous ISIS396443 was assessed by the following dosing
groups. [0306] Group 1--SC400 (dose ranges from 80 mg/kg to 180
mg/kg) [0307] Two SC injections totaling 400 .mu.g per mouse
delivered between P0 to P3, first dose was 150 .mu.g at P0 or P1
(volume of 3 .mu.l) and second was 250 .mu.g delivered P2 or P3
(volume of 5 .mu.l). [0308] Group 2--SC200 (dose ranges from 40
mg/kg to 90 mg/kg) [0309] Two SC injections totaling 200 .mu.g per
mouse delivered between P0 and P3, first dose was 75 at P0-P1
(volume of 1.5 .mu.l) and second was 125 .mu.g delivered P2or P3
(volume of 2.5 .mu.l). [0310] Group 3--SC100 (dose ranges from 20
mg/kg to 45 mg/kg) [0311] Two SC injections totaling 100 .mu.g per
mouse delivered between P0 and P3, first dose was 40 at P0 or P1
(volume of 2 .mu.l) and second was 60 .mu.g delivered P2 or P3
(volume of 3 .mu.l). [0312] Group 4--SMA saline (negative controls)
[0313] Two SC injections of saline between P0 and P3, first was at
P0 or P1 (volume of 5 .mu.l) and second delivered P2 or P3 (volume
of 5 .mu.l). [0314] Group 5--Heterozygous control (positive
controls) [0315] Mice without any treatment. Each group included
from 14 to 26 mice. Survival (in days) for individual mice in each
group is provided in the table, below. Many mice in this study
remain alive at the time that this patent application is being
prepared. Thus, a value proceeded by a ">" indicates that the
mouse has lived that number of days and is still alive.
TABLE-US-00013 [0315] Group 1 Group 2 Group 3 Group 4 Group 5 Mouse
SC400 SC200 SC100 Saline Het 1 >82 >93 11 8 >87 2 >82
>91 11 8 >87 3 >82 >91 11 9 >87 4 >82 >91 11 9
>87 5 >82 14 11 9 >87 6 >82 25 12 9 >87 7 >82 92
18 9 >86 8 >82 >93 19 9 >86 9 >82 >93 22 9 >86
10 >82 >90 69 9 >86 11 >82 >90 >77 9 >86 12
>80 >91 >77 10 >86 13 >80 >91 >77 10 >86 14
25 >90 >77 10 >86 15 ND >90 >75 10 >85 16 ND
>90 >74 11 >85 17 ND 86 >74 11 >85 18 ND >90
>74 12 ND 19 ND >52 >74 12 ND 20 ND ND >74 13 ND 21 ND
ND >74 13 ND 22 ND ND >71 13 ND 23 ND ND >49 13 ND 24 ND
ND >49 14 ND 25 ND ND >49 15 ND 26 ND ND 23 ND ND
EXAMPLE 13
ICV Infusion vs. ICV Bolus
[0316] Administration by intracerebroventricular bolus injection
(ICV bolus) was compared to administration by continuous
intracerebroventricular infusion (ICV infusion). The SMA type III
transgenic mice were dosed with ISIS387954. ICV infusion mice were
given a total dose of 0 (PBS control), 87.5 .mu.g, 175 .mu.g, 350
.mu.g, or 700 .mu.g infused over 7 days and were sacrificed 2 days
later. ICV bolus mice were given the same total doses, 0 (PBS
control), 87.5 .mu.g, 175 .mu.g, 350 .mu.g, or 700 .mu.g, in a
single ICV injection and were sacrificed 9 days later. There were 5
mice in each group. RNA was collected from the lumbar spinal cord
and was analyzed by real time PCR. Intron 7 inclusion was
normalized to the saline-treated controls. Results are summarized
in the table below.
TABLE-US-00014 Fold increase in intron 7 Group Dose inclusion
relative to PBS 1 PBS (control) 1.0 2 87.5 .mu.g by ICV infusion
over 7 days 2.1 3 175 .mu.g by ICV infusion over 7 days 2.4 4 350
.mu.g by ICV infusion over 7 days 3.2 5 700 .mu.g by ICV infusion
over 7 days 3.6 6 PBS (control) 1.0 7 87.5 .mu.g by ICV bolus 3.1 8
175 .mu.g by ICV bolus 3.7 9 350 .mu.g by ICV bolus 3.8 10 700
.mu.g by ICV bolus 3.8
In this experiment, the same dose when delivered by ICV bolus
injection resulted in greater activity than when delivered by ICV
infusion over 7 days.
[0317] Real time PCR was also performed to determine the expression
levels of allograft inflammatory factor (AIF1) to assess
inflammation. None of the samples from treated mice showed a
significant difference from control mice.
EXAMPLE 14
Dose-Response by ICV Bolus
[0318] Administration by intracerebroventricular bolus was tested
at additional doses. The transgenic mice were administered 0, 10.9
.mu.g, 21.9 .mu.g, 43.4 .mu.g, 87.5 .mu.g, or 175 .mu.g of
ISIS387954 by single bolus ICV injection and were sacrificed 9 days
later as described in Example 13. Samples were collected from brain
and from lumbar spinal cord. RNA was prepared and analyzed by
RT-PCR for change in intron 7 inclusion and for change in AIF1.
None of the samples showed a change in AIF1 compared to control.
Results from intron 7 inclusion are summarized in the table below.
The ED50 is at around 22 .mu.g.
TABLE-US-00015 Fold increase in intron 7 inclusion relative to PBS
Lumbar Group Dose Brain spinal cord 1 PBS (control) 1.0 1.0 2 10.9
.mu.g by ICV bolus 2.4 2.2 3 21.9 .mu.g by ICV bolus 2.8 2.7 4 43.4
.mu.g by ICV bolus 3.2 3.4 5 87.5 .mu.g by ICV bolus 3.5 3.4 6 175
.mu.g by ICV bolus 4.4 3.7
Sequence CWU 1
1
24118DNAArtificial SequenceSynthetic Oligonucleotide 1tcactttcat
aatgctgg 18215DNAArtificial SequenceSynthetic Oligonucleotide
2tttcataatg ctggc 15315DNAArtificial SequenceSynthetic
Oligonucleotide 3tgctggcaga cttac 15415DNAArtificial
SequenceSynthetic Oligonucleotide 4cataatgctg gcaga
15515DNAArtificial SequenceSynthetic Oligonucleotide 5tcataatgct
ggcag 15615DNAArtificial SequenceSynthetic Oligonucleotide
6ttcataatgc tggca 15720DNAArtificial SequenceSynthetic
Oligonucleotide 7attcactttc ataatgctgg 20815DNAArtificial
SequenceSynthetic Oligonucleotide 8ctttcataat gctgg
15912DNAArtificial SequenceSynthetic Oligonucleotide 9tcataatgct gg
121015DNAArtificial SequenceSynthetic Oligonucleotide 10actttcataa
tgctg 151112DNAArtificial SequenceSynthetic Oligonucleotide
11ttcataatgc tg 121215DNAArtificial SequenceSynthetic
Oligonucleotide 12cactttcata atgct 151312DNAArtificial
SequenceSynthetic Oligonucleotide 13tttcataatg ct
121415DNAArtificial SequenceSynthetic Oligonucleotide 14tcactttcat
aatgc 151512DNAArtificial SequenceSynthetic Oligonucleotide
15ctttcataat gc 121615DNAArtificial SequenceSynthetic
Oligonucleotide 16ttcactttca taatg 151712DNAArtificial
SequenceSynthetic Oligonucleotide 17actttcataa tg
121815DNAArtificial SequenceSynthetic Oligonucleotide 18attcactttc
ataat 151912DNAArtificial SequenceSynthetic Oligonucleotide
19cactttcata at 122015DNAArtificial SequenceSynthetic
Oligonucleotide 20gattcacttt cataa 152112DNAArtificial
SequenceSynthetic Oligonucleotide 21tcactttcat aa
122212DNAArtificial SequenceSynthetic Oligonucleotide 22ttcactttca
ta 122312DNAArtificial SequenceSynthetic Oligonucleotide
23attcactttc at 122415DNAArtificial SequenceSynthetic
Oligonucleotide 24agtaagattc acttt 15
* * * * *