U.S. patent application number 16/893253 was filed with the patent office on 2021-03-25 for compositions and methods for inhibiting expression of the alas1 gene.
The applicant listed for this patent is ALNYLAM PHARMACEUTICALS, INC., ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI. Invention is credited to Brian Bettencourt, Robert J. Desnick, Kevin Fitzgerald, William Querbes, Makiko Yasuda.
Application Number | 20210087558 16/893253 |
Document ID | / |
Family ID | 1000005260731 |
Filed Date | 2021-03-25 |
View All Diagrams
United States Patent
Application |
20210087558 |
Kind Code |
A1 |
Bettencourt; Brian ; et
al. |
March 25, 2021 |
COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF THE ALAS1
GENE
Abstract
The invention relates to double-stranded ribonucleic acid
(dsRNA) compositions targeting the ALAS1 gene, and methods of using
such dsRNA compositions to alter (e.g., inhibit) expression of
ALAS1.
Inventors: |
Bettencourt; Brian; (Groton,
MA) ; Fitzgerald; Kevin; (Brookline, MA) ;
Querbes; William; (Cambridge, MA) ; Desnick; Robert
J.; (New York, NY) ; Yasuda; Makiko; (New
York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALNYLAM PHARMACEUTICALS, INC.
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI |
Cambridge
New York |
MA
NY |
US
US |
|
|
Family ID: |
1000005260731 |
Appl. No.: |
16/893253 |
Filed: |
June 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16142953 |
Sep 26, 2018 |
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16893253 |
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14814911 |
Jul 31, 2015 |
10125364 |
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16142953 |
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13835613 |
Mar 15, 2013 |
9133461 |
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14814911 |
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61622288 |
Apr 10, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1137 20130101;
C12N 2310/343 20130101; A61K 48/00 20130101; C12N 15/113 20130101;
C12N 2310/315 20130101; C12N 2310/321 20130101; C12N 2310/344
20130101; C12Y 203/01037 20130101; C12N 2310/322 20130101; C12N
2310/533 20130101; C12N 2310/3521 20130101; A61K 47/60 20170801;
C12N 2310/50 20130101; C12N 2310/351 20130101; C12N 2310/14
20130101; C12N 2310/3533 20130101; A61K 31/713 20130101; C12N
2320/30 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713; A61K 47/60 20060101
A61K047/60; A61K 48/00 20060101 A61K048/00 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting
expression of ALAS1, wherein said dsRNA comprises a sense strand
and an antisense strand 15-30 base pairs in length and the
antisense strand is complementary to at least 15 contiguous
nucleotides of SEQ ID NO: 1 or 382.
2. A double-stranded ribonucleic acid (dsRNA) for inhibiting
expression of ALAS1, wherein said dsRNA comprises a sense strand
and an antisense strand, the antisense strand comprising a region
of complementarity to an ALAS1 RNA transcript, which antisense
strand comprises at least 15 contiguous nucleotides differing by no
more than 3 nucleotides from the antisense sequence of AD-58882
(SEQ ID NO: 3434) or one of the antisense sequences listed in any
one of Tables 2, 3, 6, 7, 8, 9, 14, 15, 18, or 20.
3. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
4.-5. (canceled)
6. The dsRNA of claim 1, wherein the region of complementarity is
at least 17 nucleotides in length.
7. The dsRNA of claim 6, wherein the region of complementarity is
between 19 and 23 nucleotides in length.
8. (canceled)
9. The dsRNA of claim 1, wherein each strand is no more than 30
nucleotides in length.
10.-11. (canceled)
12. The dsRNA of claim 1, further comprising a ligand.
13. The dsRNA of claim 12, wherein said ligand is a GalNAc
ligand.
14. (canceled)
15. The dsRNA of claim 12, wherein the ligand is conjugated to the
3' end of the sense strand of the dsRNA.
16. (canceled)
17. The dsRNA of claim 1, wherein the dsRNA comprises a sense
strand consisting of a sense sequence selected from the sense
sequences disclosed in Tables 2, 3, 6, 7, 8, 9, 14, 15, 18 and 20,
and an antisense strand consisting of an antisense sequence
selected from the antisense sequences disclosed in Tables 2, 3, 6,
7, 8, 9, 14, 15, 18 and 20.
18. A cell containing the dsRNA of claim 1.
19. A pharmaceutical composition for inhibiting expression of an
ALAS1 gene, the composition comprising the dsRNA of claim 1.
20.-28. (canceled)
29. The pharmaceutical composition of claim 19 wherein said
composition is administered intravenously or subcutaneously.
30.-31. (canceled)
32. A method of inhibiting ALAS1 expression in a cell, the method
comprising: (a) introducing into the cell the dsRNA of claim 1, and
(b) maintaining the cell of step (a) for a time sufficient to
obtain degradation of the mRNA transcript of an ALAS1 gene, thereby
inhibiting expression of the ALAS1 gene in the cell.
33.-41. (canceled)
42. A method of treating a disorder related to ALAS1 expression
comprising administering to a subject in need of such treatment a
therapeutically effective amount of the dsRNA of claim.
43. (canceled)
44. The method of claim 42, wherein the subject is at risk for
developing, or is diagnosed with, a porphyria.
45.-58. (canceled)
59. A method for decreasing a level of a porphyrin or a porphyrin
precursor in a cell, comprising contacting the cell with the dsRNA
of claim 1, in an amount effective to decrease the level of the
porphyrin or the porphyrin precursor in the cell.
60.-61. (canceled)
62. A vector encoding at least one strand of a dsRNA of claim
1.
63.-65. (canceled)
66. A cell comprising the vector of claim 1.
67.-68. (canceled)
69. The dsRNA of claim 1, wherein the dsRNA comprises a sense
strand comprising a sequence selected from the group consisting of
SEQ ID NO: 202, SEQ ID NO: 451, SEQ ID NO:330, SEQ ID NO:334, SEQ
ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:356, SEQ ID
NO:358, SEQ ID NO:362, SEQ ID NO:366, SEQ ID NO:376, and SEQ ID
NO:380.
70. The dsRNA of claim 1, wherein the dsRNA comprises an antisense
strand comprising a sequence selected from the group consisting of
SEQ ID NO: 203, SEO ID NO: 452, SEQ ID NO:331, SEQ ID NO:335, SEQ
ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:357, SEQ ID
NO:359, SEQ ID NO:363, SEQ ID NO:367, SEQ ID NO:377, and SEQ ID
NO:381.
71. The dsRNA of claim 1, wherein the dsRNA comprises a sense
strand comprising a sequence selected from the group consisting of
SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:152, SEQ ID NO:154, SEQ ID
NO:156, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:172, SEQ ID NO:176,
SEQ ID NO:186, and SEQ ID NO:190.
72. The dsRNA of claim 1, wherein the dsRNA comprises an antisense
strand comprising a sequence selected from the group consisting of
SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:153, SEQ ID NO:155, SEQ ID
NO:157, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:173, SEQ ID NO:177,
SEQ ID NO:187, and SEQ ID NO:191.
73. The method of claim 42, wherein said method (i) ameliorates a
symptom associated with an ALAS1 related disorder (e.g., a
porphyria) (ii) inhibits ALAS1 expression in the subject, (iii)
decreases a level of a porphyrin precursor or a porphyrin in the
subject, (iv) decreases frequency of acute attacks of symptoms
associated with a porphyria in the subject, or (v) decreases
incidence of acute attacks of symptoms associated with a porphyria
in the subject when the subject is exposed to a precipitating
factor.
74.-77. (canceled)
78. A double stranded RNAi (dsRNA) comprising a sense strand
complementary to an antisense strand, wherein said antisense strand
comprises a region of complementarity to an ALAS1 RNA transcript,
wherein each strand has about 14 to about 30 nucleotides, wherein
said dsRNA is represented by formula (III): TABLE-US-00028 sense:
5'
n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.sub.a-n.-
sub.q 3' (III) antisense: 3'
n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub-
.l- N.sub.a'-n.sub.q' 5'
wherein: i, j, k, and l are each independently 0 or 1; p, p', q,
and q' are each independently 0-6; each N.sub.a and N.sub.a'
independently represents an oligonucleotide sequence comprising
0-25 nucleotides which are either modified or unmodified or
combinations thereof, each sequence comprising at least two
differently modified nucleotides; each N.sub.b and N.sub.b'
independently represents an oligonucleotide sequence comprising
0-10 nucleotides which are either modified or unmodified or
combinations thereof; each n.sub.p, n.sub.p', n.sub.q, and n.sub.q'
independently represents an overhang nucleotide; XXX, YYY, ZZZ,
X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif
of three identical modifications on three consecutive nucleotides;
modifications on N.sub.b differ from the modification on Y and
modifications on N.sub.b' differ from the modification on Y',
wherein said dsRNA comprises the sense and antisense sequences of a
duplex disclosed in Table 3, 8, 9, or 15.
79. The dsRNA of claim 78, wherein the sense strand is conjugated
to at least one ligand.
80.-88. (canceled)
89. The dsRNA of claim 78, wherein the modifications on the
nucleotides are selected from the group consisting of LNA, HNA,
CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl,
2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.
90. The dsRNA of claim 89, wherein the modifications on the
nucleotides are 2'-O-methyl, 2'-fluoro or both.
91. (canceled)
92. The dsRNA of claim 12, wherein the ligand is attached via a
linker.
93. (canceled)
94. The dsRNA of claim 12, wherein the ligand is ##STR00026##
95. The dsRNA of claim 12, wherein the ligand is attached via a
linker, and wherein the ligand and linker are as shown in Formula
XXIV: ##STR00027##
96.-111. (canceled)
112. The method of claim 42, wherein the porphyria is a hepatic
porphyria selected from acute intermittent porphyria (AIP)
hereditary coproporphyria (HCP), variegate porphyria (VP), ALA
deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic
porphyria.
113.-115. (canceled)
116. The method of claim 42, wherein the dsRNA or composition
comprising dsRNA is administered before, during, or after an acute
attack of porphyria.
117. (canceled)
118. The method of claim 42, wherein the dsRNA or composition
comprising dsRNA is administered during a prodrome.
119. The method of claim 118, wherein the prodrome is characterized
by pain, nausea, psychological symptoms, restlessness or
insomnia.
120.-123. (canceled)
124. The method of claim 42, wherein the subject has an elevated
level of ALA and/or PBG.
125.-150. (canceled)
151. A method of treating a subject with an elevated level of ALA
and/or PBG, the method comprising administering to the subject a
double-stranded ribonucleic acid (dsRNA), wherein said dsRNA
comprises a sense strand and an antisense strand 15-30 base pairs
in length and the antisense strand is complementary to at least 15
contiguous nucleotides of SEQ ID NO:1 or SEQ ID NO:382.
152.-154. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 16/142,953, filed Sep. 26, 2018, which is a divisional of U.S.
application Ser. No. 14/814,911, filed Jul. 31, 2015, now U.S. Pat.
No. 10,125,364, issued Nov. 13, 2018, which is a divisional of U.S.
application Ser. No. 13/835,613, filed Mar. 15, 2013, now U.S. Pat.
No. 9,133,461, issued Sep. 15, 2015, which claims priority to U.S.
Provisional Application No. 61/622,288, filed Apr. 10, 2012. The
entire contents of the aforesaid applications are hereby
incorporated in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 14, 2016, is named A2038-719640_SL.txt and is 596,779 bytes
in size.
FIELD OF THE INVENTION
[0003] The invention relates to the specific inhibition of the
expression of the ALAS1 gene.
BACKGROUND OF THE INVENTION
[0004] The inherited porphyrias are a family of disorders resulting
from the deficient activity of specific enzymes in the heme
biosynthetic pathway, also referred to herein as the porphyrin
pathway. Deficiency in the enzymes of the porphyrin pathway leads
to insufficient heme production and to an accumulation of
porphyrins, which are toxic to tissue in high concentrations.
[0005] Of the inherited porphyrias, acute intermittent porphyria
(AIP, e.g., autosomal dominant AIP), variegate porphyria (VP, e.g.,
autosomal dominant VP), hereditary coproporphyria (copropophyria or
HCP, e.g., autosomal dominant HCP), and 5' aminolevulinic acid
(also known as .delta.-aminolevulinic acid or ALA) dehydratase
deficiency porphyria (ADP, e.g., autosomal recessive ADP) are
classified as acute hepatic porphyrias and are manifested by acute
neurological attacks that can be life threatening. The acute
attacks are characterized by autonomic, peripheral, and central
nervous symptoms, including severe abdominal pain, hypertension,
tachycardias, constipation, motor weakness, paralysis, and
seizures. If not treated properly, quadriplegia, respiratory
impairment, and death may ensue. Various factors, including
cytrochrome P450-inducing drugs, dieting, and hormonoal changes can
precipitate acute attacks by increasing the activity of hepatic
5'-aminolevulinic acid synthase 1 (ALAS1), the first and
rate-limiting enzyme of the heme biosynthetic pathway. In the acute
porphyrias, e.g., AIP, VP, HCP and ADP, the respective enzyme
deficiencies result in hepatic production and accumulation of one
or more substances (e.g., porphyrins and/or porphyrin precursors,
e.g., ALA and/or PBG) that can be neurotoxic and can result in the
occurrence of acute attacks. See, e.g., Balwani, M and Desnick, R.
J., Blood, 120:4496-4504, 2012.
[0006] The current therapy for the actute neurological attacks in
the intravenous administration of hemin (Panhematin.RTM., Lundbeck
or Normosang.RTM., Orphan Europe), which provides exogenous heme
for the negative feedback inhibition of ALAS1, and thereby,
decreases production of ALA and PBG. Hemin is used for the
treatment during an acute attack and for prevention of attacks,
particularly in women with the acute porphyrias who experience
frequent attacks with the hormonal changes during their menstrual
cycles. While patients generally respond well, its effect is slow,
typically taking two to four days or longer to normalize urinary
ALA and PBG concentrations towards normal levels. As the
intravenous hemin is rapidly metabolized, three to four infusions
are usually necessary to effectively treat or prevent an acute
attack. In addition, repeated infusions may cause iron overload and
phlebitis, which may compromise peripheral venous access. Although
orthotrophic liver transplantation is curative, this procedure has
significant morbidity and mortality and the availability of liver
donors is limited. Therefore, an alternative therapeutic approach
that is more effective, fast-acting, and safe is needed. It would
be particularly advantageous if such treatment could be delivered
by subcutaneous administration, as this would preclude the need for
infusions and prolonged hospitalization.
[0007] AIP, also referred to as porphobilinogen deaminase
deficiency (PBGD), or hydroxymethylbilane synthase (HMBS)
deficiency, is the most common of the acute hepatic prophyrias. It
is an autosomal dominant disorder caused by mutations in the
HMB-synthase (HMBS) gene that result in reduced, e.g., half-normal
activity of the enzyme. Previously, a mouse model of AIP that has
.about.30% of wildtype HMBS activity was generated by homologous
recombination Like human patients, these mice increase hepatic
ALAS1 activity and accumulate large quantities of plasma and
urinary ALA and PBG when administered porphyrinogenic drugs, such
as phenobarbital. Thus, they serve as an excellent model to
evaluate the efficacy of novel therapeutics for the acute hepatic
porphyrias.
SUMMARY OF THE INVENTION
[0008] The present invention describes methods and iRNA
compositions for modulating the expression of an ALAS1 gene. In
certain embodiments, expression of an ALAS1 gene is reduced or
inhibited using an ALAS1-specific iRNA. Such inhibition can be
useful in treating disorders related to ALAS1 expression, such as
porphyrias.
[0009] Accordingly, described herein are compositions and methods
that effect the RNA-induced silencing complex (RISC)-mediated
cleavage of RNA transcripts of the ALAS1 gene, such as in a cell or
in a subject (e.g., in a mammal, such as a human subject). Also
described are compositions and methods for treating a disorder
related to expression of an ALAS1 gene, such as a porphyria, e.g.,
X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency
porphyria (Doss porphyria or ADP), acute intermittent porphyria
(AIP), congenital erythropoietic porphyria (CEP), prophyria cutanea
tarda (PCT), hereditary coproporphyria (coproporphyria, or HCP),
variegate porphyria (VP), erythropoietic protoporphyria (EPP), or
transient erythroporphyria of infancy. In some embodiments, the
disorder is an acute hepatic porphyria, e.g., ALA deyhdratase
deficiency porphyria (ADP), AIP, HCP, or VP. In certain
embodiments, the disorder is ALA deyhdratase deficiency porphyria
(ADP) or AIP.
[0010] In embodiments, the porphyria is a hepatic porphyria, e.g.,
a porphyria selected from acute intermittent porphyria (AIP)
hereditary coproporphyria (HCP), variegate porphyria (VP), ALA
deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic
porphyria. In embodiments, the porphyria is a homozygous dominant
hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or
hepatoerythropoietic porphyria, In embodiments, the porphyria is a
dual porphyria.
[0011] As used herein, the term "iRNA," "RNAi", "iRNA agent," or
"RNAi agent" refers to an agent that contains RNA as that term is
defined herein, and which mediates the targeted cleavage of an RNA
transcript, e.g., via an RNA-induced silencing complex (RISC)
pathway. In one embodiment, an iRNA as described herein effects
inhibition of ALAS1 expression in a cell or mammal.
[0012] The iRNAs included in the compositions featured herein
encompass a dsRNA having an RNA strand (the antisense strand)
having a region, e.g., a region that is 30 nucleotides or less,
generally 19-24 nucleotides in length, that is substantially
complementary to at least part of an mRNA transcript of an ALAS1
gene (e.g., a mouse or human ALAS1 gene) (also referred to herein
as an "ALAS1-specific iRNA"). Alternatively, or in combination,
iRNAs encompass a dsRNA having an RNA strand (the antisense strand)
having a region that is 30 nucleotides or less, generally 19-24
nucleotides in length, that is substantially complementary to at
least part of an mRNA transcript of an ALAS1 gene (e.g., a human
variant 1 or 2 of an ALAS1 gene) (also referred to herein as a
"ALAS1-specific iRNA").
[0013] In embodiments, the iRNA (e.g., dsRNA) described herein
comprises an antisense strand having a region that is substantially
complementary to a region of a human ALAS1. In embodiments, the
human ALAS1 has the sequence of NM_000688.4 (SEQ ID NO:1) or
NM_000688.5 (SEQ ID NO:382).
[0014] In other embodiments, an iRNA encompasses a dsRNA having an
RNA strand (the antisense strand) having a region that is
substantially complementary to a portion of an ALAS1 mRNA according
to any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In one
embodiment, the iRNA encompasses a dsRNA having an RNA strand (the
antisense strand) having a region that is substantially
complementary to a portion of an ALAS1 mRNA, e.g., a human ALAS1
mRNA (e.g., a human ALAS1 mRNA as provided in SEQ ID NO:1 or SEQ ID
NO:382).
[0015] In one embodiment, an iRNA for inhibiting expression of an
ALAS1 gene includes at least two sequences that are complementary
to each other. The iRNA includes a sense strand having a first
sequence and an antisense strand having a second sequence. The
antisense strand includes a nucleotide sequence that is
substantially complementary to at least part of an mRNA encoding an
ALAS1 transcript, and the region of complementarity is 30
nucleotides or less, and at least 15 nucleotides in length.
Generally, the iRNA is 19 to 24 nucleotides in length.
[0016] In some embodiments, the iRNA is 19-21 nucleotides in
length. In some embodiments, the iRNA is 19-21 nucleotides in
length and is in a lipid formulation, e.g. a lipid nanoparticle
(LNP) formulation (e.g., an LNP11 formulation).
[0017] In some embodiments, the iRNA is 21-23 nucleotides in
length. In some embodiments, the iRNA is 21-23 nucleotides in
length and is in the form of a conjugate, e.g., conjugated to one
or more GalNAc derivatives as described herein.
[0018] In some embodiments the iRNA is from about 15 to about 25
nucleotides in length, and in other embodiments the iRNA is from
about 25 to about 30 nucleotides in length. An iRNA targeting
ALAS1, upon contact with a cell expressing ALAS1, inhibits the
expression of an ALAS1 gene by at least 10%, at least 20%, at least
25%, at least 30%, at least 35% or at least 40% or more, such as
when assayed by a method as described herein. In one embodiment,
the iRNA targeting ALAS1 is formulated in a stable nucleic acid
lipid particle (SNALP).
[0019] In one embodiment, an iRNA (e.g., a dsRNA) featured herein
includes a first sequence of a dsRNA that is selected from the
group consisting of the sense sequences of Tables 2, 3, 6, 7, 8, 9,
14, and 15 and a second sequence that is selected from the group
consisting of the corresponding antisense sequences of Tables 2, 3,
6, 7, 8, 9, 14 and 15.
[0020] In one embodiment, an iRNA (e.g., a dsRNA) featured herein
has sense and/or antisense sequences selected from those of
AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856,
AD-59129, AD-59124, AD-58874, AD-59125, AD-59105, AD-59120,
AD-59122, AD-59106, AD-59126, and AD-59107 as disclosed herein in
the Examples. In embodiments, the iRNA (e.g., dsRNA) has sense
and/or antisense sequences selected from those of AD-58882,
AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129.
[0021] The iRNA molecules featured herein can include naturally
occurring nucleotides or can include at least one modified
nucleotide, including, but not limited to a 2'-O-methyl modified
nucleotide, a nucleotide having a 5'-phosphorothioate group, and a
terminal nucleotide linked to a cholesteryl derivative.
Alternatively, the modified nucleotide may be chosen from the group
of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified
nucleotide, a locked nucleotide, an abasic nucleotide,
2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
morpholino nucleotide, a phosphoramidate, and a non-natural base
comprising nucleotide. Such a modified sequence can be based, e.g.,
on a first sequence of said iRNA selected from the group consisting
of the sense sequences of Table 2, and a second sequence selected
from the group consisting of the corresponding antisense sequences
of Table 2.
[0022] In one embodiment, an iRNA (e.g., a dsRNA) featured herein
comprises a sense strand comprising a sequence selected from the
group consisting of SEQ ID NO:330, SEQ ID NO:334, SEQ ID NO:342,
SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:356, SEQ ID NO:358, SEQ ID
NO:362, SEQ ID NO:366, SEQ ID NO:376, and SEQ ID NO:380.
[0023] In one embodiment, an iRNA (e.g., a dsRNA) featured herein
comprises an antisense strand comprising a sequence selected from
the group consisting of SEQ ID NO:331, SEQ ID NO:335, SEQ ID
NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:357, SEQ ID NO:359,
SEQ ID NO:363, SEQ ID NO:367, SEQ ID NO:377, and SEQ ID NO:381.
[0024] In one embodiment, an iRNA (e.g., a dsRNA) featured herein
comprises a sense strand comprising a sequence selected from the
group consisting of SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:152,
SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:166, SEQ ID NO:168, SEQ ID
NO:172, SEQ ID NO:176, SEQ ID NO:186, and SEQ ID NO:190. In one
embodiment, an iRNA (e.g., a dsRNA) featured herein comprises an
antisense strand comprising a sequence selected from the group
consisting of SEQ ID NO:141, SEQ ID NO:145, SEQ ID NO:153, SEQ ID
NO:155, SEQ ID NO:157, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:173,
SEQ ID NO:177, SEQ ID NO:187, and SEQ ID NO:191.
[0025] In one embodiment, an iRNA as described herein targets a
wildtype ALAS1 RNA transcript variant, and in another embodiment,
the iRNA targets a mutant transcript (e.g., an ALAS1 RNA carrying
an allelic variant). For example, an iRNA featured in the invention
can target a polymorphic variant, such as a single nucleotide
polymorphism (SNP), of ALAS1. In another embodiment, the iRNA
targets both a wildtype and a mutant ALAS1 transcript. In yet
another embodiment, the iRNA targets a particular transcript
variant of ALAS1 (e.g., human ALAS1 variant 1). In yet another
embodiment, the iRNA agent targets multiple transcript variants
(e.g., both variant 1 and variant 2 of human ALAS1).
[0026] In one embodiment, an iRNA featured in the invention targets
a non-coding region of an ALAS1 RNA transcript, such as the 5' or
3' untranslated region of a transcript.
[0027] In some embodiments, an iRNA as described herein is in the
form of a conjugate, e.g., a carbohydrate conjugate, which may
serve as a targeting moiety and/or ligand, as described herein. In
one embodiment, the conjugate is attached to the 3' end of the
sense strand of the dsRNA. In some embodiments, the conjugate is
attached via a linker, e.g., via a bivalent or trivalent branched
linker.
[0028] In some embodiments, the conjugate comprises one or more
N-acetylgalactosamine (GalNAc) derivatives. Such a conjugate is
also referred to herein as a GalNAc conjugate. In some embodiments,
the conjugate targets the RNAi agent to a particular cell, e.g., a
liver cell, e.g., a hepatocyte. The GalNAc derivatives can be
attached via a linker, e.g., a bivalent or trivalent branched
linker. In particular embodiments, the conjugate is
##STR00001##
[0029] In some embodiments, the RNAi agent is attached to the
carbohydrate conjugate via a linker, e.g., a linker as shown in the
following schematic, wherein X is O or S
##STR00002##
[0030] In some embodiments, X is O. In some embodiments, X is
S.
[0031] In some embodiments, the RNAi agent is conjugated to L96 as
defined in Table 1 and shown below
##STR00003##
[0032] In an aspect provided herein is a pharmaceutical composition
for inhibiting the expression of an ALAS1 gene in an organism,
generally a human subject. The composition typically includes one
or more of the iRNAs described herein and a pharmaceutically
acceptable carrier or delivery vehicle. In one embodiment, the
composition is used for treating a porphyria, e.g., AIP.
[0033] In one aspect, an iRNA provided herein is a double-stranded
ribonucleic acid (dsRNA) for inhibiting expression of ALAS1,
wherein said dsRNA comprises a sense strand and an antisense strand
15-30 base pairs in length and the antisense strand is
complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1
or 382.
[0034] In a further aspect, an iRNA provided herein is a double
stranded RNAi (dsRNA) comprising a sense strand complementary to an
antisense strand, wherein said antisense strand comprises a region
of complementarity to an ALAS1 RNA transcript, wherein each strand
has about 14 to about 30 nucleotides, wherein said double stranded
RNAi agent is represented by formula (III):
TABLE-US-00001 (III) sense: 5'
n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-Na-n.sub.q
3' antisense: 3'
n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub-
.l- N.sub.a'-n.sub.q' 5'
[0035] wherein: [0036] i, j, k, and 1 are each independently 0 or
1; [0037] p, p', q, and q' are each independently 0-6; [0038] each
N.sub.a and N.sub.a' independently represents an oligonucleotide
sequence comprising 0-25 nucleotides which are either modified or
unmodified or combinations thereof, each sequence comprising at
least two differently modified nucleotides; [0039] each N.sub.b and
N.sub.b' independently represents an oligonucleotide sequence
comprising 0-10 nucleotides which are either modified or unmodified
or combinations thereof, [0040] each n.sub.p, n.sub.p', n.sub.q,
and n.sub.q' independently represents an overhang nucleotide;
[0041] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently
represent one motif of three identical modifications on three
consecutive nucleotides; [0042] modifications on N.sub.b differ
from the modification on Y and modifications on N.sub.b' differ
from the modification on Y'.
[0043] In embodiments, the sense strand is conjugated to at least
one ligand.
[0044] In embodiments, i is 1; j is 1; or both i and j are 1.
[0045] In embodiments, k is 1; l is 1; or both k and 1 are 1.
[0046] In embodiments, XXX is complementary to X'X'X', YYY is
complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.
[0047] In embodiments, the Y'Y'Y' motif occurs at the 11, 12 and 13
positions of the antisense strand from the 5'-end.
[0048] In embodiments, the Y' is 2'-O-methyl.
[0049] In embodiments, the duplex region is 15-30 nucleotide pairs
in length.
[0050] In embodiments, the duplex region is 17-23 nucleotide pairs
in length.
[0051] In embodiments, the duplex region is 19-21 nucleotide pairs
in length.
[0052] In embodiments, the duplex region is 21-23 nucleotide pairs
in length.
[0053] In embodiments, the modifications on the nucleotides are
selected from the group consisting of LNA, HNA, CeNA,
2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro,
2'-deoxy, 2'-hydroxyl, and combinations thereof.
[0054] In embodiments, the modifications on the nucleotides are
2'-O-methyl, 2'-fluoro or both.
[0055] In embodiments, the ligand comprises a carbohydrate.
[0056] In embodiments, the ligand is attached via a linker.
[0057] In embodiments, the linker is a bivalent or trivalent
branched linker.
[0058] In embodiments, the ligand is
##STR00004##
[0059] In embodiments, the ligand and linker are as shown in
Formula XXIV:
##STR00005##
[0060] In embodiments, the ligand is attached to the 3' end of the
sense strand.
[0061] In embodiments, the dsRNA has a nucleotide sequence selected
from the group of sequences provided in Tables 2 and 3. In
embodiments, the dsRNA has a nucleotide sequence selected from the
group of sequences provided in Tables 2, 3, 6, 7, 8 and 9. In
embodiments, the dsRNA has a nucleotide sequence selected from the
group of sequences provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15.
In embodiments, the dsRNA has a nucleotide sequence selected from
the group of sequences provided in Tables 14 and 15.
[0062] In embodiments, dsRNA has a nucleotide sequence selected
from the group of sequences provided in Tables 3 and 8.
[0063] In a further aspect, an iRNA provided herein is a
double-stranded ribonucleic acid (dsRNA) for inhibiting expression
of ALAS1, wherein said dsRNA comprises a sense strand and an
antisense strand, the antisense strand comprising a region of
complementarity to an ALAS1 RNA transcript, which antisense strand
comprises at least 15 contiguous nucleotides differing by no more
than 3 nucleotides from one of the antisense sequences listed in
any one of Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some such
embodiments, the sense and antisense sequences are selected from
those of the duplexes AD-58882, AD-58878, AD-58886, AD-58877,
AD-59115, AD-58856, AD-59129, AD-59124, AD-58874, AD-59125,
AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, and AD-59107 as
disclosed herein in the Examples. In embodiments, the sense and
antisense sequences are selected from those of the duplexes
AD-58882, AD-58878, AD-58886, AD-58877, AD-59115, AD-58856, and
AD-59129.
[0064] In some embodiments, the dsRNA comprises at least one
modified nucleotide.
[0065] In some embodiments, at least one of the modified
nucleotides is chosen from the group consisting of: a 2'-O-methyl
modified nucleotide, a nucleotide comprising a 5'-phosphorothioate
group, and a terminal nucleotide linked to a cholesteryl derivative
or dodecanoic acid bisdecylamide group.
[0066] In some embodiments, the modified nucleotide is chosen from
the group consisting of: a 2'-deoxy-2'-fluoro modified nucleotide,
a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
[0067] In some embodiments, the region of complementarity is at
least 17 nucleotides in length.
[0068] In some embodiments, the region of complementarity is
between 19 and 21 nucleotides in length.
[0069] In some embodiments, the region of complementarity is 19
nucleotides in length.
[0070] In some embodiments, each strand is no more than 30
nucleotides in length.
[0071] In some embodiments, at least one strand comprises a 3'
overhang of at least 1 nucleotide.
[0072] In some embodiments, at least one strand comprises a 3'
overhang of at least 2 nucleotides.
[0073] In some embodiments, a dsRNA described herein further
comprises a ligand.
[0074] In some embodiments, the ligand is a GalNAc ligand.
[0075] In some embodiments, the ligand targets the dsRNA to
hepatocytes.
[0076] In some embodiments, the ligand is conjugated to the 3' end
of the sense strand of the dsRNA.
[0077] In some embodiments, the region of complementarity consists
of an antisense sequence selected from Table 2 or Table 3. In
embodiments, the region of complementarity consists of an antisense
sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or 15. In some
embodiments, the region of complementarity consists of an antisense
sequence selected from that of AD-58882, AD-58878, AD-58886,
AD-58877, AD-59115, AD-58856, AD-59129, AD-59124, AD-58874,
AD-59125, AD-59105, AD-59120, AD-59122, AD-59106, AD-59126, or
AD-59107 as disclosed herein in the Examples.
[0078] In some embodiments, the dsRNA comprises a sense strand
consisting of a sense strand sequence selected from Table 2 or
Table 3, and an antisense strand consisting of an antisense
sequence selected from Table 2 or Table 3.
[0079] In some embodiments, the dsRNA comprises a sense strand
consisting of a sense strand sequence selected from Tables 2, 3, 6,
7, 8, 9, 14, or 15, and an antisense strand consisting of an
antisense sequence selected from Tables 2, 3, 6, 7, 8, 9, 14, or
15. In embodiments, the dsRNA comprises a pair of corresponding
sense and antisense sequences selected from those of the duplexes
disclosed in Tables 2, 3, 6, 7, 8, 9, 14, and 15.
[0080] In one aspect, the invention provides a cell containing at
least one of the iRNAs (e.g., dsRNAs) featured herein. The cell is
generally a mammalian cell, such as a human cell. In some
embodiments, the cell is an erythroid cell. In other embodiments,
the cell is a liver cell (e.g., a hepatocyte).
[0081] In an aspect provided herein is a pharmaceutical composition
for inhibiting expression of an ALAS1 gene, the composition
comprising an iRNA (e.g., a dsRNA) described herein.
[0082] In embodiments of the pharmaceutical compositions described
herein, the iRNA (e.g., dsRNA) is administered in an unbuffered
solution. In embodiments, the unbuffered solution is saline or
water.
[0083] In embodiments of the pharmaceutical compositions described
herein, the iRNA (e.g., dsRNA is administered with a buffer
solution. In embodiments, the buffer solution comprises acetate,
citrate, prolamine, carbonate, or phosphate or any combination
thereof. In embodiments, the buffer solution is phosphate buffered
saline (PBS).
[0084] In embodiments of the pharmaceutical compositions described
herein, the iRNA (e.g., dsRNA) is targeted to hepatocytes.
[0085] In embodiments of the pharmaceutical compositions described
herein, the composition is administered intravenously.
[0086] In embodiments of the pharmaceutical compositions described
herein, the composition is administered subcutaneously.
[0087] In embodiments, a pharmaceutical composition comprises an
iRNA (e.g., a dsRNA) described herein that comprises a ligand
(e.g., a GalNAc ligand) that targets the iRNA (e.g., dsRNA) to
hepatocytes.
[0088] In embodiments, a pharmaceutical composition comprises an
iRNA (e.g., a dsRNA) described herein that comprises a ligand
(e.g., a GalNAc ligand), and the pharmaceutical composition is
administered subcutaneously. In embodiments, the ligand targets the
iRNA (e.g., dsRNA) to hepatocytes.
[0089] In certain embodiments, a pharmaceutical composition, e.g.,
a composition described herein, includes a lipid formulation. In
some embodiments, the RNAi agent is in a LNP formulation, e.g., a
MC3 formulation. In some embodiments, the LNP formulation targets
the RNAi agent to a particular cell, e.g., a liver cell, e.g., a
hepatocyte. In embodiments, the lipid formulation is a LNP11
formulation. In embodiments, the composition is administered
intravenously.
[0090] In another embodiment, the pharmaceutical composition is
formulated for administration according to a dosage regimen
described herein, e.g., not more than once every four weeks, not
more than once every three weeks, not more than once every two
weeks, or not more than once every week. In another embodiment, the
administration of the pharmaceutical composition can be maintained
for a month or longer, e.g., one, two, three, or six months, or one
year or longer.
[0091] In another embodiment, a composition containing an iRNA
featured in the invention, e.g., a dsRNA targeting ALAS1, is
administered with a non-iRNA therapeutic agent, such as an agent
known to treat a porphyria (e.g., AIP), or a symptom of a porphyria
(e.g., pain). In another embodiment, a composition containing an
iRNA featured in the invention, e.g., a dsRNA targeting AIP, is
administered along with a non-iRNA therapeutic regimen, such as
hemin or glucose (e.g., glucose infusion (e.g., IV glucose)). For
example, an iRNA featured in the invention can be administered
before, after, or concurrent with glucose, dextrose, or a similar
treatment that serves to restore energy balance (e.g., total
parenteral nutrition). An iRNA featured in the invention can also
be administered before, after, or concurrent with the
administration of a heme product (e.g., hemin, heme arginate, or
heme albumin), and optionally also in combination with a glucose
(e.g. IV glucose) or the like.
[0092] Typically, glucose administered for the treatment of a
porphyria is administered intravenously (IV). Administration of
glucose intravenously is referred to herein as "IV glucose."
However, alternative embodiments in which glucose is administered
by other means are also encompassed.
[0093] In one embodiment, an ALAS1 iRNA is administered to a
patient, and then the non-iRNA agent or therapeutic regimen (e.g.,
glucose and/or a heme product) is administered to the patient (or
vice versa). In another embodiment, an ALAS1 iRNA and the non-iRNA
therapeutic agent or therapeutic regimen are administered at the
same time.
[0094] In an aspect provided herein is a method of inhibiting ALAS1
expression in a cell, the method comprising: (a) introducing into
the cell an iRNA (e.g. a dsRNA) described herein and (b)
maintaining the cell of step (a) for a time sufficient to obtain
degradation of the mRNA transcript of an ALAS1 gene, thereby
inhibiting expression of the ALAS1 gene in the cell.
[0095] In an aspect provided herein is a method for reducing or
inhibiting the expression of an ALAS1 gene in a cell (e.g., an
erythroid cell or a liver cell, such as, e.g., a hepatocyte). The
method includes: [0096] (a) introducing into the cell a
double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
includes at least two sequences that are complementary to each
other. The dsRNA has a sense strand having a first sequence and an
antisense strand having a second sequence; the antisense strand has
a region of complementarity that is substantially complementary to
at least a part of an mRNA encoding ALAS1, and where the region of
complementarity is 30 nucleotides or less, i.e., 15-30 nucleotides
in length, and generally 19-24 nucleotides in length, and where the
dsRNA upon contact with a cell expressing ALAS1, inhibits
expression of an ALAS1 gene by at least 10%, e.g., at least 20%, at
least 30%, at least 40% or more; and [0097] (b) maintaining the
cell of step (a) for a time sufficient to obtain degradation of the
mRNA transcript of the ALAS1 gene, thereby reducing or inhibiting
expression of an ALAS1 gene in the cell.
[0098] In embodiments of the foregoing methods of inhibiting ALAS1
expression in a cell, the cell is treated ex vivo, in vitro, or in
vivo. In embodiments, the cell is a hepatocyte.
[0099] In embodiments, the cell is present in a subject in need of
treatment, prevention and/or management of a disorder related to
ALAS1 expression.
[0100] In embodiments, the disorder is a porphyria. In embodiments,
the porphyria is acute intermittent porphyria or ALA-dehydratase
deficiency porphyria.
[0101] In embodiments, the porphyria is a hepatic porphyria, e.g.,
a porphyria selected from acute intermittent porphyria (AIP)
hereditary coproporphyria (HCP), variegate porphyria (VP), ALA
deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic
porphyria. In embodiments, the porphyria is a homozygous dominant
hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or
hepatoerythropoietic porphyria, In embodiments, the porphyria is a
dual porphyria.
[0102] In embodiments, the expression of ALAS1 is inhibited by at
least 30%.
[0103] In embodiments, the iRNA (e.g., dsRNA) has an IC.sub.50 in
the range of 0.01-1 nM.
[0104] In certain embodiments, the cell (e.g., the hepatocyte) is a
mammalian cell (e.g., a human, non-human primate, or rodent
cell).
[0105] In one embodiment, the cell is treated ex vivo, in vitro, or
in vivo (e.g., the cell is present in a subject (e.g., a patient in
need of treatment, prevention and/or management of a disorder
related to ALAS1 expression).
[0106] In one embodiment, the subject is a mammal (e.g., a human)
at risk, or diagnosed with a porphyria, e.g., X-linked
sideroblastic anemia (XLSA), ALA deyhdratase deficiency porphyria
(ADP or Doss porphyria), acute intermittent porphyria (AIP),
congenital erythropoietic porphyria (CEP), prophyria cutanea tarda
(PCT), hereditary coproporphyria (coproporphyria, or HCP),
variegate porphyria (VP), erythropoietic protoporphyria (EPP), or
transient erythroporphyria of infancy. In some embodiments, the
disorder is an acute hepatic porphyria, e.g., ALA deyhdratase
deficiency porphyria (ADP), AIP, HCP, or VP. In specific
embodiments, the disorder is ALA deyhdratase deficiency porphyria
(ADP) or AIP.
[0107] In embodiments, the porphyria is a hepatic porphyria, e.g.,
a porphyria selected from acute intermittent porphyria (AIP)
hereditary coproporphyria (HCP), variegate porphyria (VP), ALA
deyhdratase deficiency porphyria (ADP), and hepatoerythropoietic
porphyria. In embodiments, the porphyria is a homozygous dominant
hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or
hepatoerythropoietic porphyria, In embodiments, the porphyria is a
dual porphyria.
[0108] In one embodiment, the dsRNA introduced reduces or inhibits
expression of an ALAS1 gene in the cell.
[0109] In one embodiment, the dsRNA introduced reduces or inhibits
expression of an ALAS1 gene, or the level of one or more porphyrins
or porphyrin precursors (e.g., .delta.-aminolevulinic acid (ALA),
porphopilinogen (PBG), hydroxymethylbilane (HMB), uroporphyrinogen
I or III, coproporphyrinogen I or III, protoporphrinogen IX, and
protoporphyrin IX) or porphyrin products or metabolites, by at
least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more
compared to a reference, (e.g., an untreated cell or a cell treated
with a non-targeting control dsRNA). Without being bound by theory,
ALAS1 is the first enzyme of the porphyrin pathway. Thus, reducing
expression of the ALAS1 gene is likely to reduce the level of one
or more porphyrin precursors, porphyrins or porphyrin products or
metabolites.
[0110] In other aspects, the invention provides methods for
treating, preventing or managing pathological processes related to
ALAS1 expression (e.g., pathological processes involving
porphyrins, porphyrin precuorsors, or defects in the porphyrin
pathway, such as, for example, porphyrias). In one embodiment, the
method includes administering to a subject, e.g., a patient in need
of such treatment, prevention or management, an effective (e.g., a
therapeutically or prophylactically effective) amount of one or
more of the iRNAs featured herein.
[0111] In an aspect provided herein is a method of treating and/or
preventing a disorder related to ALAS1 expression comprising
administering to a subject in need of such treatment a
therapeutically effective amount of an iRNA (e.g., a dsRNA)
described herein, or a composition comprising an iRNA (e.g., a
dsRNA) described herein.
[0112] In an aspect provided herein is a method of treating and/or
preventing a porphyria comprising administering to a subject in
need of such treatment a double-stranded ribonucleic acid (dsRNA),
wherein said dsRNA comprises a sense strand and an antisense strand
15-30 base pairs in length and the antisense strand is
complementary to at least 15 contiguous nucleotides of SEQ ID NO:1
or SEQ ID NO:382.
[0113] In one embodiment, subject (e.g., the patient) has a
porphyria. In another embodiment, the subject (e.g., patient) is at
risk for developing a porphyria. In some embodiments,
administration of the iRNA targeting ALAS1 alleviates or relieves
the severity of at least one symptom of a disorder related to ALAS1
in the patient.
[0114] In one embodiment, the subject is a mammal (e.g., a human)
at risk, or that has been diagnosed with, a disorder related to
ALAS1 expression, e.g., a porphyria, e.g., X-linked sideroblastic
anemia (XLSA), ALA deyhdratase deficiency porphyria (Doss
porphyria), acute intermittent porphyria (AIP), congenital
erythropoietic porphyria (CEP), prophyria cutanea tarda (PCT),
hereditary coproporphyria (coproporphyria, or HCP), variegate
porphyria (VP), erythropoietic protoporphyria (EPP), or transient
erythroporphyria of infancy. In a further embodiment, the porphyria
is an acute hepatic porphyria, e.g., ALA deyhdratase deficiency
porphyria (ADP), AIP, HCP, or VP. In some such embodiments, the
disorder is ALA deyhdratase deficiency porphyria (ADP) or AIP.
[0115] In embodiments the subject has, or is at risk for
developing, a porphyria. In embodiments, the porphyria is a hepatic
porphyria, e.g., a porphyria selected from acute intermittent
porphyria (AIP) hereditary coproporphyria (HCP), variegate
porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and
hepatoerythropoietic porphyria. In embodiments, the porphyria is a
homozygous dominant hepatic porphyria (e.g., homozygous dominant
AP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments,
the porphyria is a dual porphyria.
[0116] In embodiments, a porphyria, a symptom of porphyria, a
prodrome, or an attack of porphyria is induced by exposure to a
precipitating factor, as described herein. In some embodiments, the
precipitating factor is a chemical exposure. In some embodiments,
the precipitating factor is a drug, e.g., a prescription drug or an
over the counter drug. In some embodiments, the precipitating
factor is the menstrual cycle, e.g., a particular phase of the
menstrual cycle, e.g., the luteal phase.
[0117] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered after an acute attack of
porphyria.
[0118] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered during an acute attack of
porphyria.
[0119] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered prophylactically to prevent an
acute attack of porphyria.
[0120] In embodiments, the iRNA (e.g., dsRNA) is formulated as an
LNP formulation.
[0121] In embodiments, the iRNA (e.g., dsRNA) is in the form of a
GalNAc conjugate.
[0122] In embodiments, iRNA (e.g., dsRNA) is administered at a dose
of 0.05-50 mg/kg.
[0123] In embodiments, the iRNA (e.g., dsRNA) is administered at a
concentration of 0.01 mg/kg-5 mg/kg bodyweight of the subject.
[0124] In embodiments, the iRNA (e.g., dsRNA) is formulated as an
LNP formulation and is administered at a dose of 0.05-5 mg/kg.
[0125] In embodiments, the iRNA (e.g., dsRNA) is in the form of a
GalNAc conjugate and is administered at a dose of 0.5-50 mg/kg.
[0126] In embodiments, the method decreases a level of a porphyrin
or a porphyrin precursor in the subject.
[0127] In embodiments, the level is decreased by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, or 90%. In an embodiment, the level
is decreased by at least 30%.
[0128] In embodiments, the porphyrin precursor is
.delta.-aminolevulinic acid (ALA) or porphopilinogen (PBG).
[0129] In embodiments, the iRNA (e.g., dsRNA) has an IC.sub.50 in
the range of 0.01-1 nM.
[0130] In embodiments, a method described herein [0131] (i)
ameliorates a symptom associated with an ALAS1 related disorder
(e.g., a porphyria) [0132] (ii) inhibits ALAS1 expression in the
subject, [0133] (iii) decreases a level of a porphyrin precursor
(e.g., ALA or PBG) or a porphyrin in the subject, [0134] (iv)
decreases frequency of acute attacks of symptoms associated with a
porphyria in the subject, or [0135] (v) decreases incidence of
acute attacks of symptoms associated with a porphyria in the
subject when the subject is exposed to a precipitating factor
(e.g., the premenstrual phase or the luteal phase).
[0136] In embodiments, the method ameliorates pain and/or
progressive neuropathy.
[0137] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered according to a dosing
regimen.
[0138] In some embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered before or during an acute
attack of porphyria. In some embodiments, the iRNA is administered
before an acute attack of porphyria.
[0139] In some embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered during a prodrome. In
embodiments, the prodrome is characterized by abdominal pain,
nausea, psychological symptoms (e.g., anxiety), restlessness and/or
insomnia.
[0140] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered during a particular phase of
the menstrual cycle, e.g., during the luteal phase.
[0141] In embodiments, the method ameliorates or prevents cyclical
attacks of porphyria, e.g., by reducing the severity, duration, or
frequency of attacks. In embodiments, the cyclical attacks are
associated with a precipitating factor. In embodiments, the
precipitating factor is the menstrual cycle, e.g., a particular
phase of the menstrual cycle, e.g., the luteal phase.
[0142] In embodiments, the subject has an elevated level of ALA
and/or PBG. In embodiments, the subject has or is at risk for
developing a porphyria, e.g., a hepatic porphyria. In embodiments,
the subject is asymptomatic. In embodiments, the subject carries a
genetic alteration (e.g., a gene mutation) associated with a
porphyria, as described herein.
[0143] In embodiments, the subject has or is at risk for developing
a porphyria and suffers from pain (e.g., chronic pain, e.g.,
chronic neuropathic pain) and/or neuropathy (e.g., progressive
neuropathy). In embodiments, the subject does not suffer from acute
attacks but suffers from pain (e.g., chronic pain, e.g., chronic
neuropathic pain) and/or neuropathy (e.g., progressive neuropathy).
In embodiments, the pain is abdominal pain.
[0144] In embodiments, the subject (a) has an elevated level of ALA
and/or PBG and (b) suffers from pain (e.g., chronic pain, e.g.,
chronic neuropathic pain) and/or neuropathy (e.g., progressive
neuropathy). In embodiments, the pain is abdominal pain.
[0145] In embodiments, the subject has a plasma level and/or a
urine level of ALA and/or PBG that is elevated. In embodiments, the
elevated level of ALA and/or PBG is accompanied by other symptoms,
e.g., pain (e.g., chronic pain, e.g., chronic neuropathic pain) or
neuropathy (e.g., progressive neuropathy). In embodiments, the pain
is abdominal pain. In embodiments, the subject is asymptomatic. In
embodiments, the subject has a genetic mutation associated with a
porphyria, e.g., a mutation as described herein.
[0146] In embodiments, the subject has a level (e.g., a plasma
level or a urine level) of a porphyrin precursor, e.g., ALA and/or
PBG, that is elevated, e.g., the level is greater than, or greater
than or equal to, a reference value. In embodiments, the level is
greater than the reference value. In embodiments, the reference
value is two standard deviations above the mean level in a sample
of healthy individuals. In embodiments, the reference value is an
upper reference limit.
[0147] In embodiments, the subject has a plasma level and/or a
urine level of ALA and/or PBG that is greater than, or greater than
or or equal to, 2 times, 3 times, 4 times, or 5 times that of an
upper reference limit. As used herein, an "upper reference limit"
refers to a level that is the upper limit of the 95% confidence
interval for a reference sample, e.g., a sample of normal (e.g.,
wild type) or healthy individuals, e.g., individuals who do not
carry a genetic mutation associated with a porphyria and/or
individuals who do not suffer from a porphyria. In embodiments, the
subject has a urine level of ALA and/or PBG that is greater than 2
to 4 times that of an upper reference limit. In embodiments, the
subject has a urine level of ALA and/or PBG that is greater than 4
times that of an upper reference limit.
[0148] In embodiments, the reference value for plasma PBG is 0.12
.mu.mol/L. In embodiments, the subject is a human and has a plasma
PBG level that is greater than, or greater than or equal to, 0.12
.mu.mol/L, 0.24 .mu.mol/L, 0.36 .mu.mol/L, 0.48 .mu.mol/L, or 0.60
.mu.mol/L. In embodiments, the subject is a human and has a plasma
level of PBG that is greater than, or greater than or equal to,
0.48 .mu.mol/L.
[0149] In embodiments, the reference value for urine PBG is 1.2
mmol/mol creatinine. In embodiments, the subject is a human and has
a urine PBG level that is greater than, or greater than or equal
to, 1.2 mmol/mol creatinine, 2.4 mmol/mol creatinine, 3.6 mmol/mol
creatinine, 4.8 mmol/mol creatinine, or 6.0 mmol/mol creatinine. In
embodiments, the subject is a human and has a urine level of PBG
that is greater than, or greater than or equal to, 4.8 mmol/mol
creatinine.
[0150] In embodiments, the reference value for plasma ALA is 0.12
.mu.mol/L. In embodiments, the subject is a human and has a plasma
ALA level that is greater than, or greater than or equal to, 0.12
.mu.mol/L, 0.24 .mu.mol/L, 0.36 .mu.mol/L, 0.48 .mu.mol/L, or 0.60
.mu.mol/L. In embodiments, the subject is a human and has a plasma
ALA level that is greater than, or greater than or equal to 0.48
.mu.mol/L.
[0151] In embodiments, the reference value for urine ALA is 3.1
mmol/mol creatinine. In embodiments, the subject is a human and has
a urine ALA level that is greater than, or greater than or equal
to, 3.1 mmol/mol creatinine, 6.2 mmol/mol creatinine, 9.3 mmol/mol
creatinine, 12.4 mmol/mol creatinine, or 15.5 mmol/mol
creatinine.
[0152] In embodiments, the method decreases an elevated level of
ALA and/or PBG. In embodiments, the method decreases pain (e.g.,
chronic pain, e.g. chronic neuropathic pain) and/or neuropathy
(e.g., progressive neuropathy). In embodiments, the pain is
abdominal pain.
[0153] In embodiments, the pain is neuropathic pain (e.g., pain
associated with the progressive neuropathy of acute porphyrias).
The decrease in pain can include, e.g., prevention of pain, delay
in the onset of pain, reduction in the frequency of pain, and/or
reduction in severity of pain.
[0154] In embodiments, the method ameliorates or prevents acute
attacks of porphyria, e.g., by reducing the severity, duration, or
frequency of attacks.
[0155] In embodiments, the method decreases or prevents nerve
damage.
[0156] In embodiments, the method prevents deterioration (e.g.,
prevents development of abnormalities) of or results in an
improvement of clinical measures, e.g., clinical measures of muscle
and/or nerve function, e.g., EMG and/or nerve conduction
velocities.
[0157] In embodiments, the method is effective to reduce a level of
ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG).
In embodiments, the method is effective to produce a predetermined
reduction in the elevated level of ALA and/or PBG.
[0158] In embodiments, the predetermined reduction is a reduction
to a value that is less than or equal to a reference value. In some
embodiments, the reference value is an upper reference limit.
[0159] In some embodiments, the reference value is the value that
is two standard deviations above the mean level in a reference
sample.
[0160] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered repeatedly, e.g., according to
a dosing regimen.
[0161] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered prophylactically to a subject
who is at risk for developing a porphyria. In embodiments, the iRNA
(e.g., dsRNA) or composition comprising the iRNA is administered
prophylactically beginning at puberty. In embodiments, the subject
carries a genetic mutation associated with a porphyria and/or has
an elevated level of ALA and/or PBG (e.g., an elevated plasma or
urine level of ALA and/or PBG). In embodiments, the mutation makes
an individual susceptible to an acute attack (e.g., upon exposure
to a precipitating factor, e.g., a drug, dieting or other
precipitating factor, e.g., a precipitating factor as disclosed
herein). In embodiments, the mutation is associated with elevated
levels of a porphyrin or a porphyrin precursor (e.g., ALA and/or
PBG). In embodiments, the mutation is associated with chronic pain
(e.g., chronic neuropathic pain) and/or neuropathy (e.g.,
progressive neuropathy).
[0162] In embodiments, the mutation is a mutation in the ALAS1
gene. In embodiments, the mutation is a mutation in the ALAS1 gene
promoter, or in regions upstream or downstream from the ALAS1 gene.
In embodiments, the mutation is a mutation in transcription factors
or other genes that interact with ALAS1. In embodiments, the
mutation is a mutation in a gene that encodes an enzyme in the heme
biosynthetic pathway.
[0163] In embodiments, the iRNA (e.g., dsRNA) or composition
comprising the iRNA is administered subcutaneously. In embodiments,
the iRNA is in the form of a GalNAc conjugate. In embodiments, the
iRNA (e.g., the dsRNA) is administered at a dose of 0.5-50
mg/kg.
[0164] In one aspect provided herein is a method of treating a
subject with an elevated level of ALA and/or PBG, the method
comprising administering to the subject a double-stranded
ribonucleic acid (dsRNA), wherein said dsRNA comprises a sense
strand and an antisense strand 15-30 base pairs in length and the
antisense strand is complementary to at least 15 contiguous
nucleotides of SEQ ID NO:1 or SEQ ID NO:382.
[0165] In one aspect provided herein is a method of treating a
subject with an elevated level of ALA and/or PBG, the method
comprising administering to the subject a therapeutically effective
amount of an dsRNA or a composition comprising a dsRNA, as
described herein.
[0166] In some embodiments, the methods described herein are
effective to decrease the level of ALA and/or PBG. In some
embodiments, the level of ALA and/or PBG is decreased such that it
is less than, or less than or equal to, a reference value, e.g., an
upper reference limit. In another aspect, the invention provides
methods for decreasing a level of a porphyrin or a porphyrin
precursor in a cell (e.g., an erythroid cell or a liver cell, such
as, e.g., a hepatocyte). In one embodiment, the cell is treated ex
vivo, in vitro, or in vivo (e.g., the cell is present in a subject
(e.g., a patient in need of treatment, prevention and/or management
of a disorder related to ALAS1 expression). The method includes
contacting the cell with an effective amount of one or more of the
iRNAs targeting ALAS1, e.g., one or more of the iRNAs disclosed
herein, thereby decreasing the level of a porphyrin or a porphyrin
precursor in the cell; or decreasing the level of a porphyrin or a
porphyrin precursor in other cells, tissues, or fluids within a
subject in which the cell is located; relative to the level prior
to contacting. Such methods can be used to treat (e.g., ameliorate
the severity) of disorders related to ALAS1 expression, such as
porphyrias, e.g., AIP or ALA dehydratase deficiency porphyria.
[0167] In one embodiment, the contacting step is effected ex vivo,
in vitro, or in vivo. For example, the cell can be present in a
subject, e.g., a mammal (e.g., a human) at risk, or that has been
diagnosed with, a porphyria. In an embodiment, the porphyria is an
acute hepatic porphyria. In embodiments, the porphyria is a hepatic
porphyria, e.g., a porphyria selected from acute intermittent
porphyria (AIP), hereditary coproporphyria (HCP), variegate
porphyria (VP), ALA deyhdratase deficiency porphyria (ADP), and
hepatoerythropoietic porphyria. In embodiments, the porphyria is a
homozygous dominant hepatic porphyria (e.g., homozygous dominant
AIP, HCP, or VP) or hepatoerythropoietic porphyria, In embodiments,
the porphyria is a dual porphyria.
[0168] In an aspect provided herein is a method for decreasing a
level of a porphyrin or a porphyrin precursor (e.g., ALA or PBG) in
a cell, comprising contacting the cell with an iRNA (e.g. a dsRNA),
as described herein, in an amount effective to decrease the level
of the porphyrin or the porphyrin precursor in the cell. In
embodiments, the cell is a hepatocyte. In embodiments, the
porphyrin or porphyrin precursor is .delta.-aminolevulinic acid
(ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB),
uroporphyrinogen I or III, coproporphyrinogen I or III,
protoporphrinogen IX, or protoporphyrin IX. In embodiments, the
porphyrin precursor is ALA or PBG.
[0169] In one embodiment, the cell is an erythroid cell. In a
further embodiment, the cell is a liver cell (e.g., a
hepatocyte).
[0170] In an aspect provided herein is a vector encoding at least
one strand of an iRNA (e.g., a dsRNA) as described herein.
[0171] In an aspect provided herein is a vector encoding at least
one strand of a dsRNA, wherein said dsRNA comprises a region of
complementarity to at least a part of an mRNA encoding ALAS1,
wherein said dsRNA is 30 base pairs or less in length, and wherein
said dsRNA targets said mRNA for cleavage.
[0172] In embodiments, the region of complementarity is at least 15
nucleotides in length.
[0173] In embodiments, the region of complementarity is 19 to 21
nucleotides in length. In one aspect, the invention provides a
vector for inhibiting the expression of an ALAS1 gene in a
cell.
[0174] In one embodiment, the vector comprises an iRNA as described
herein. In one embodiment, the vector includes at least one
regulatory sequence operably linked to a nucleotide sequence that
encodes at least one strand of an iRNA as described herein. In one
embodiment the vector comprises at least one strand of an ALAS1
iRNA.
[0175] In an aspect provided herein is a cell comprising a vector
as described herein. In an aspect provided herein is a cell
containing a vector for inhibiting the expression of an ALAS1 gene
in a cell. The vector includes a regulatory sequence operably
linked to a nucleotide sequence that encodes at least one strand of
one of the iRNAs as described herein. In one embodiment, the cell
is a liver cell (e.g., a hepatocyte). In another embodiment, the
cell is an erythroid cell.
[0176] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety.
[0177] The details of various embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS
[0178] FIG. 1 depicts the heme biosynthetic pathway.
[0179] FIG. 2A is a table that summarizes certain porphyrias
associated with genetic errors in heme metabolism.
[0180] FIG. 2B is a continuation of the table in FIG. 2A.
[0181] FIG. 3A depicts nucleotides 1-2280 of the sequence of a
human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq.
NM_000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO:
1).
[0182] FIG. 3B depicts nucleotides 2281-2407 of the sequence of a
human ALAS1 mRNA sequence transcript variant 1 (Ref. Seq.
NM_000688.4 (GI:40316942, record dated Nov. 19, 2011), SEQ ID NO:
1).
[0183] FIG. 4A depicts nucleotides 1-2280 of the sequence of a
human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq.
NM_000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO:
382).
[0184] FIG. 4B depicts nucleotides 2281-2458 of the sequence of a
human ALAS1 mRNA sequence transcript variant 2 (Ref. Seq.
NM_000688.5 (GI: 362999011, record dated Apr. 1, 2012), SEQ ID NO:
382).
[0185] FIG. 5 shows the dose-response of the siRNA AD-53558 in
suppressing mouse ALAS1 (mALAS1) mRNA relative to a PBS control.
Results for a luciferase (LUC) AD-1955 control are also shown.
[0186] FIG. 6 shows the dose-response of the siRNA AD-53558 in
suppressing ALAS1 mRNA in rats relative to a PBS control. Results
for a luciferase (LUC) AD-1955 control are also shown.
[0187] FIG. 7 shows the durability of suppression of mouse ALAS1
(mALAS1) mRNA by the siRNA AD-53558 relative to a PBS control.
[0188] FIG. 8 shows means standard deviations of plasma ALA levels
(in .mu.M) at baseline, and after phenobarbitol treatment in the
experimental (ALAS1 siRNA) and control (LUC siRNA) groups.
[0189] FIG. 9 shows shows the plasma ALA levels (in .mu.M) of
individual animals at baseline, and after phenobarbitol treatment
in animals that received ALAS1 siRNA and control (LUC siRNA)
treatment.
[0190] FIG. 10 shows means standard deviations of plasma PBG levels
(in .mu.M) at baseline, and after phenobarbitol treatment in
animals that received ALAS1 siRNA and control (LUC siRNA)
treatment.
[0191] FIG. 11 shows shows the plasma PBG levels (in .mu.M) of
individual animals at baseline, and after phenobarbitol treatment
in animals that received ALAS1 siRNA and control (LUC siRNA)
treatment.
[0192] FIG. 12 shows the relative mALAS1 mRNA level in liver at
baseline, and after phenobarbitol treatment in select
representative experimental (ALAS1 siRNA) and control (PBS)
animals.
[0193] FIG. 13 shows the effects of three GalNAc conjugated mALAS1
siRNAs on mALAS1 expression (relative to a PBS control) in mouse
liver tissue.
[0194] FIG. 14 shows plasma ALA and PBG levels over time after
phenobarbitol administration and treatment with ALAS1 siRNA or
control LUC siRNA.
DETAILED DESCRIPTION OF THE INVENTION
[0195] iRNA directs the sequence-specific degradation of mRNA
through a process known as RNA interference (RNAi). Described
herein are iRNAs and methods of using them for inhibiting the
expression of an ALAS1 gene in a cell or a mammal where the iRNA
targets an ALAS1 gene. Also provided are compositions and methods
for disorders related to ALAS1 expression, such as porphyrias
(e.g., ALA deyhdratase deficiency porphyria (ADP or Doss
porphyria), acute intermittent porphyria, congenital erythropoietic
porphyria, prophyria cutanea tarda, hereditary coproporphyria
(coproporphyria), variegate porphyria, erythropoietic
protoporphyria (EPP), X-linked sideroblastic anemia (XLSA), and and
transient erythroporphyria of infancy).
[0196] Porphyrias are inherited or acquired disorders that can be
caused by decreased or enhanced activity of specific enzymes in the
heme biosynthetic pathway, also referred to herein as the porphyrin
pathway (See FIG. 1). Porphyrins are the main precursors of heme.
Porphyrins and porphyrin precursors include .delta.-aminolevulinic
acid (ALA), porphopilinogen (PBG), hydroxymethylbilane (HMB),
uroporphyrinogen I or III, coproporphyrinogen I or III,
protoporphrinogen IX, and protoporphyrin IX. Heme is an essential
part of hemoglobin, myoglobin, catalases, peroxidases, and
cytochromes, the latter including the respiratory and P450 liver
cytochromes. Heme is synthesized in most or all human cells. About
85% of heme is made in erythroid cells, primarily for hemoglobin.
Most of the remaining heme is made in the liver, 80% of which is
used for the synthesis of cytochromes. Deficiency of specific
enzymes in the porphyrin pathway leads to insufficient heme
production and also to an accumulation of porphyrin precursors
and/or porphyrins, which can be toxic to cell or organ function in
high concentrations.
[0197] Porphyrias may manifest with neurological complications
("acute"), skin problems ("cutaneous") or both. Porphyrias may be
classified by the primary site of the overproduction and
accumulation of porphyrins or their precursors. In hepatic
porphyrias, porphyrins and porphyrin precursors are overproduced
predominantly in the liver, whereas in erythropoietic porphyrias,
porphyrins are overproduced in the erythroid cells in the bone. The
acute or hepatic porphyrias lead to dysfunction of the the nervous
system and neurologic manifestations that can affect both the
central and peripheral nervous system, resulting in symptoms such
as, for example, pain (e.g., abdominal pain and/or chronic
neuropathic pain), vomiting, neuropathy (e.g., acute neuropathy
progressive neuropathy), muscle weakness, seizures, mental
disturbances (e.g., hallucinations, depression anxiety, paranoia),
cardiac arrhythmias, tachycardia, constipation, and diarrhea. The
cutaneous or erythropoietic porphyrias primarily affect the skin,
causing symptoms such as photosensitivity that can be painful,
blisters, necrosis, itching, swelling, and increased hair growth on
areas such as the forehead. Subsequent infection of skin lesions
can lead to bone and tissue loss, as well as scarring,
disfigurement, and loss of digits (e.g., fingers, toes). Most
porphyrias are caused by mutations that encode enzymes in the heme
biosynthetic pathway. A summary of porphyrias associated with
genetic errors in heme metabolism is provided in FIG. 2.
[0198] Not all porphyrias are genetic. For example, patients with
liver disease may develop porphyria as a result of liver
dysfunction, and a transient form of erythroporphria (transient
erythroporphyria of infancy) has been described in infancy (see
Crawford, R. I. et al, J Am Acad Dermatol. 1995 August; 33(2 Pt
2):333-6.) Patients with PCT can acquire the deficient activity of
uroporphyrinogen decarboxylase (URO-D), due to the formation of a
ORO-D enzyme with lower than normal enzymatic activity (see
Phillips et al. Blood, 98:3179-3185, 2001.)
[0199] Acute intermittent porphyria (AIP) (also be referred to as
porphobilinogen (PBG) deaminase deficiency, or hydroxymethylbilane
synthase (HMBS) deficiency), is the most common type of acute
hepatic porphyria. Other types of acute hepatic porphyrias include
hereditary coproporphyria (HCP), variegate porphyria (VP), and ALA
deyhdratase deficiency porphyria (ADP). Acute hepatic porphyrias
are described, e.g., in Balwani, M and Desnick, R. J., Blood,
120:4496-4504, 2012.
[0200] AIP is typically an autosomal dominant disease that is
characterized by a deficiency of the enzyme porphobilinogen
deaminase (PBG deaminase); this enzyme is also known as
hydroxymethylbilane synthase (HMB synthase or HMBS). PBG deaminase
is the third enzyme of the heme biosynthetic pathway (see FIG. 1)
and catalyzes the head to tail condensation of four porphobilinogen
molecules into the linear tetrapyrrole, hydroxymethylbilane (HMB).
Alternatively spliced transcript variants encoding different
isoforms of PBG deaminase have been described. Mutations in the PBG
deaminase gene are associated with AP. Such mutations may lead to
decreased amounts of PBG deaminase and/or decreased activity of PBG
deaminase (affected individuals typically have a 50% reduction in
PBG deaminase activity).
[0201] There are at least two different models of the
pathophysiology of AP and other acute hepatic porphyrias (see,
e.g., Lin CS-Y et al., Clinical Neurophysiology, 2011;
122:2336-44). According to one model, the decreased heme production
resulting from PBG deaminase deficiency causes energy failure and
axonal degeneration. According to the other, currently more favored
model, the buildup of porphyrin precursors (e.g., ALA and PBG)
results in neurotoxicity.
[0202] AIP has been found to have a prevalence as high as 1 in
10,000 in certain populations (e.g., in Northern Sweden; see
Floderus Y, et al. Clin Genet. 2002; 62:288-97). The prevalence in
the general population in United States and Europe, excluding the
U.K., is estimated to be about 1 in 10,000 to 1 in 20,000. Clinical
disease manifests itself in only approximately 10-15% of
individuals who carry mutations that are known to be associated
with AP. However, the penetrance is as high as 40% in individuals
with certain mutations (e.g., the W198X mutation). AIP is typically
latent prior to puberty. Symptoms are more common in females than
in males. The prevalence of the disease is probably underestimated
due to its incomplete penetrance and long periods of latency. In
the United States, it is estimated that there are about 2000
patients who have suffered at least one attack. It is estimated
that there are about 150 active recurrent cases in France, Sweden,
the U.K., and Poland; these patients are predominantly young women,
with a median age of 30. See, e.g., Elder et al, J Inherit Metab
Dis., published online Nov. 1, 2012.
[0203] AIP affects, for example, the visceral, peripheral,
autonomic, and central nervous systems. Symptoms of AIP are
variable and include gastrointestinal symptoms (e.g., severe and
poorly localized abdominal pain, nausea/vomiting, constipation,
diarrhea, ileus), urinary symptoms (dysuria, urinary
retention/incontinence, or dark urine), neurologic symptoms (e.g.,
sensory neuropathy, motor neuropathy (e.g., affecting the cranial
nerves and/or leading to weakness in the arms or legs), seizures,
neuropathic pain (e.g., pain associated with progressive
neuropathy, e.g., chronic neuropathic pain), neuropsychiatric
symptoms (e.g., mental confusion, anxiety, agitation,
hallucination, hysteria, delirium, apathy, depression, phobias,
psychosis, insomnia, somnolence, coma), autonomic nervous system
involvement (resulting e.g., in cardiovascular symptoms such as
tachycardia, hypertension, and/or arrhythmias, as well as other
symptoms, such as, e.g., increased circulating catecholamine
levels, sweating, restlessness, and/or tremor), dehydration, and
electrolyte abnormalities. The most common symptoms are abdominal
pain and tachycardia. In addition, patients frequently have chronic
neuropathic pain and develop a progressive neuropathy. Patients
with recurring attacks often have a prodrome. Permanent paralysis
may occur after a severe attack. Recovery from severe attacks that
are not promptly treated may take weeks or months. An acute attack
may be fatal, for example, due to paralysis of respiratory muscles
or cardiovascular failure from electrolyte imbalance. (See, e.g.,
Thunell S. Hydroxymethylbilane Synthase Deficiency. 2005 Sep. 27
[Updated 2011 Sep. 1]. In: Pagon R A, Bird T D, Dolan C R, et al.,
editors. GeneReviews.TM. [Internet]. Seattle (Wash.): University of
Washington, Seattle; 1993--(hereinafter Thunell (1993)), which is
hereby incorporated by reference in its entirety.) Prior to the
availability of Hemin treatments, up to 20% of patients with AIP
died from the disease.
[0204] In individuals who carry genes for AIP, the risk of
hepatocellular cancer is increased. In those with recurrent
attacks, the risk of hepatocellular cancer is particularly grave:
after the age of 50, the risk is nearly 100-fold greater than in
the general population.
[0205] Attacks of acute porphyria may be precipitated by endogenous
or exogenous factors. The mechanisms by which such factors induce
attacks may include, for example, increased demand for hepatic P450
enzymes and/or induction of ALAS1 activity in the liver. Increased
demand for hepatic P450 enzymes results in decreased hepatic free
heme, thereby inducing the synthesis of hepatic ALAS1.
[0206] Precipitating factors include fasting (or other forms of
reduced or inadequate caloric intake, due to crash diets,
long-distance athletics, etc.), metabolic stresses (e.g.,
infections, surgery, international air travel, and psychological
stress), endogenous hormones (e.g., progesterone), cigarette
smoking, lipid-soluble foreign chemicals (including, e.g.,
chemicals present in tobacco smoke, certain prescription drugs,
organic solvents, biocides, components in alcoholic beverages),
endocrine factors (e.g., reproductive hormones (women may
experience exacerbations during the premenstrual period), synthetic
estrogens, progesterones, ovulation stimulants, and hormone
replacement therapy). See, for example, Thunell (1993).
[0207] Over 1000 drugs are contraindicated in the acute hepatic
porphyrias (e.g., AIP, HCP, ADP, and VP) including, for example,
alcohol, barbiturates, Carbamazepine, Carisoprodol, Clonazepam
(high doses), Danazol, Diclofenac and possibly other NSAIDS,
Ergots, estrogens, Ethyclorvynol, Glutethimide, Griseofulvin,
Mephenytoin, Meprobamate (also mebutamate and tybutamate),
Methyprylon, Metodopramide, Phenytoin, Primidone, progesterone and
synthetic progestins, Pyrazinamide, Pyrazolones (aminopyrine and
antipyrine), Rifampin, Succinimides (ethosuximide and
methsuximide), sulfonamide antibiotics, and Valproic acid.
[0208] Objective signs of AIP include discoloration of the urine
during an acute attack (the urine may appear red or red-brown), and
increased concentrations of PBG and ALA in urine during an acute
attack. Molecular genetic testing identifies mutations in the PBG
deaminase (also known as HMBS) gene in more than 98% of affected
individuals. Thunell (1993).
[0209] The differential diagnosis of porphyrias may involve
determining the type of porphyria by measuring individual levels of
porphyrins or porphyrin precursors (e.g., ALA, PBG) in the urine,
feces, and/or plasma (e.g., by chromatography and fluorometry)
during an attack. The diagnosis of AIP can be confirmed by
establishing that erythrocyte PBG deaminase activity is at 50% or
less of the normal level. DNA testing for mutations may be carried
out in patients and at-risk family members. The diagnosis of AIP is
typically confirmed by DNA testing to identify a specific
caustative gene mutation (e.g., an HMBS mutation).
[0210] Treatment of acute attacks typically requires
hospitalization to control and treat acute symptoms, including,
e.g., abdominal pain, seizures, dehydration/hyponatremia,
nausea/vomiting, tachycardia/hypertension, urinary retention/ileus.
For example, abdominal pain may be treated, e.g., with narcotic
analgesics, seizures may be treated with seizure precautions and
possibly medications (although many anti-seizure medications are
contraindicated), nausea/vomiting may be treated, e.g., with
phenothiazines, and tachycardia/hypertension may be treated, e.g.,
with beta blockers. Treatment may include withdrawal of unsafe
medications, monitoring of respiratory function, as well as muscle
strength and neurological status. Mild attacks (e.g., those with no
paresis or hyponatremia) may be treated with at least 300 g
intravenous 10% glucose per day, although increasingly hemin is
provided immediately. Severe attacks should be treated as soon as
possible with intravenous hemin (3-4 mg/kg daily for 4-14 days) and
with IV glucose while waiting for the IV hemin to take effect.
Typically, attacks are treated with IV hemin for 4 days and with IV
glucose while waiting for administration of the IV hemin.
[0211] Hemin (Panhematin.RTM. or hemin for injection, previously
known as hematin) is the only heme product approved for use in the
United States and was the first drug approved under the Orphan Drug
Act. Panhematin.RTM. is hemin derived from processed red blood
cells (PRBCs), and is Protoporphyrin IX containing a ferric iron
ion (Heme B) with a chloride ligand. Heme acts to limit the hepatic
and/or marrow synthesis of porphyrin. The exact mechanism by which
hemin produces symptomatic improvement in patients with acute
episodes of the hepatic porphyrias has not been elucidated;
however, its action is likely due to the (feedback) inhibition of
.delta.-aminolevulinic acid (ALA) synthase, the enzyme which limits
the rate of the porphyrin/heme biosynthetic pathway. See
Panhematin.RTM. product label, Lundbeck, Inc., October 2010.
Inhibition of ALA synthase should result in reduced production of
ALA and PBG as well as porphyrins and porphyrin intermediates.
[0212] Drawbacks of hemin include its delayed impact on clinical
symptoms and its failure to prevent the recurrence of attacks.
Adverse reactions associated with hemin administration may include
thrombophlebitis, anticoagulation, thrombocytopenia, renal shut
down, or iron overload, which is particularly likely in patients
requiring multiple courses of hemin treatment for recurrent
attacks. To prevent phlebitis, an indwelling venous catheter is
needed for access in patients with recurrent attacks. Uncommonly
reported side effects include fever, aching, malaise, hemolysis,
anaphalaxis, and circulatory collapse. See Anderson, K. E.,
Approaches to Treatment and Prevention of Human Porphyrias, in The
Porphyrin Handbook: Medical Aspects of Porphyrins, Edited by Karl
M. Kadish, Kevin M. Smith, Roger Guilard (2003) (hereinafter
Anderson).
[0213] Heme is difficult to prepare in a stable form for
intravenous administration. It is insoluble at neutral pH but can
be prepared as heme hydroxide at pH 8 or higher. Anderson.
Panhematin is a lyophilized hemin preparation. When lyophilized
hemin is solubilized for intravenous administration, degradation
products form rapidly; these degradation products are responsible
for a transient anticoagulant effect and for phlebitis at the site
of infusion. Anderson. Heme albumin and heme arginate (Normosang,
the European version of hemin) are more stable and may potentially
cause less thrombophlebitis. However, heme arginate is not approved
for use in the United States. Panhemin may be stabilized by
solubilizing it for infusion in 30% human albumin rather than in
sterile water; however, albumin adds intravascular volume-expanding
effects and increases the cost of treatment as well as risk of
pathogens since it is isolated from human blood. See, e.g.,
Anderson.
[0214] The successful treatment of an acute attack does not prevent
or delay recurrence. There is a question of whether hemin itself
can trigger recurring attacks due to induction of heme oxygenase.
Nonetheless, in some areas (especially France), young women with
multiply recurrent attacks are being treated with weekly hemin with
the goal of achieving prophylaxis.
[0215] Limited experience with liver transplantation suggests that
if successful, it is an effective treatment for AIP. There have
been approximately 12 transplants in Europe in human patients, with
curative or varying effects. Liver transplantation can restore
normal excretion of ALA and PBG and prevent acute attacks. See,
e.g., Dar, F. S. et al. Hepatobiliary Pancreat. Dis. Int.,
9(1):93-96(2010). Furthermore, if the liver of a patient with AP is
transplanted into another patient ("domino transplant"), the
patient receiving the transplant may develop AIP.
[0216] Among the long-term clinical effects of acute porphyrias is
chronic neuropathic pain that may result from a progressive
neuropathy due to neurotoxic effects, e.g., of elevated porphyrin
precursors (e.g., ALA and/or PBG). Patients may suffer from
neuropathic pain prior to or during an acute attack. Older patients
may experience increased neuropathic pain with age for which
various narcotic drugs are typically prescribed. Electromyogram
abnormalities and decreased conduction times have been documented
in patients with acute hepatic porphyrias. Of note, untreated,
uninduced mice with AIP (PBG deaminase deficiency) develop a
progressive motor neuropathy that has been shown to cause
progressive quadriceps nerve axon degeneration and loss presumably
due to constitutively elevated porphyrin precursor (ALA & PBG)
levels, porphyrins and/or heme deficiency (Lindberg et al., J.
Clin. Invest., 103(8): 1127-1134, 1999). In patients with acute
porphyria (e.g., ADP, AIP, HCP, or VP), levels of porphyrin
precursors (ALA & PBG) are often elevated in asymptomatic
patients and in symptomatic patients between attacks. Thus,
reduction of the porphyrin precursors and resumption of normal heme
biosynthesis by reducing the level of ALAS1 expression and/or
activity is expected to prevent and/or minimize development of
chronic and progressive neuropathy. Treatment, e.g., chronic
treatment (e.g., periodic treatment with iRNA as described herein,
e.g., treatment according to a dosing regimen as described herein,
e.g., weekly or biweekly treatment) can continuously reduce the
ALAS1 expression in acute porphyria patients who have elevated
levels of porphyrin precursors, porphyrins, porphyrin products or
their metabolites. Such treatment may be provided as needed to
prevent or reduce the frequency or severity of an individual
patient's symptoms (e.g., pain and/or neuropathy) and/or to reduce
a level of a porphyrin precursor, porphyrin, porphyrin product or
metabolite.
[0217] The need exists for identifying novel therapeutics that can
be used for the treatment of porphyrias. As discussed above,
existing treatments such as hemin have numerous drawbacks. For
example, the impact of hemin on clinical symptoms is delayed, it is
expensive, and it may have side effects (e.g., thrombophlebitis,
anticoagulation, thrombocytopenia, iron overload, renal shutdown).
Novel therapeutics such as those described herein can address these
drawbacks and the unmet needs of patients by, for example, acting
faster, not inducing phlebitis, providing the convenience of
subcutaneous administration, successfully preventing recurrent
attacks, preventing or ameliorating pain (e.g., chronic neuropathic
pain) and/or progressive neuropathy, and/or not causing certain
adverse effects associated with hemin (e.g., iron overload,
increased risk of hepatocellular cancer).
[0218] The present disclosure provides methods and iRNA
compositions for modulating the expression of an ALAS1 gene. In
certain embodiments, expression of ALAS1 is reduced or inhibited
using an ALAS1-specific iRNA, thereby leading to a decreased
expression of an ALAS1 gene. Reduced expression of an ALAS1 gene
may reduce the level of one or more porphyrin precursors,
porphyrins, or porphyrin products or metabolites. Decreased
expression of an ALAS1 gene, as well as related decreases in the
level of one or more porphyrin precursors and/or porphyrins, can be
useful in treating disorders related to ALAS1 expression, e.g.,
porphyrias.
[0219] The iRNAs of the compositions featured herein include an RNA
strand (the antisense strand) having a region which is 30
nucleotides or less in length, i.e., 15-30 nucleotides in length,
generally 19-24 nucleotides in length, which region is
substantially complementary to at least part of an mRNA transcript
of an ALAS1 gene (also referred to herein as an "ALAS1-specific
iRNA"). The use of such an iRNA enables the targeted degradation of
mRNAs of genes that are implicated in pathologies associated with
ALAS1 expression in mammals, e.g., porphyrias such as ALA
dehydratase deficiency porphyria (Doss porphyria) or acute
intermittent porphyria. Very low dosages of ALAS1-specific iRNAs
can specifically and efficiently mediate RNAi, resulting in
significant inhibition of expression of an ALAS1 gene. iRNAs
targeting ALAS1 can specifically and efficiently mediate RNAi,
resulting in significant inhibition of expression of an ALAS1 gene,
e.g., in cell based assays. Thus, methods and compositions
including these iRNAs are useful for treating pathological
processes related to ALAS1 expression, such as porphyrias (e.g.,
X-linked sideroblastic anemia (XLSA), ALA deyhdratase deficiency
porphyria (Doss porphyria), acute intermittent porphyria (AIP),
congenital erythropoietic porphyria, prophyria cutanea tarda,
hereditary coproporphyria (coproporphyria), variegate porphyria,
erythropoietic protoporphyria (EPP), and transient erythroporphyria
of infancy).
[0220] The following description discloses how to make and use
compositions containing iRNAs to inhibit the expression of an ALAS1
gene, as well as compositions and methods for treating diseases and
disorders caused by or modulated by the expression of this gene.
Embodiments of the pharmaceutical compositions featured in the
invention include an iRNA having an antisense strand comprising a
region which is 30 nucleotides or less in length, generally 19-24
nucleotides in length, which region is substantially complementary
to at least part of an RNA transcript of an ALAS1 gene, together
with a pharmaceutically acceptable carrier. Embodiments of
compositions featured in the invention also include an iRNA having
an antisense strand having a region of complementarity which is 30
nucleotides or less in length, generally 19-24 nucleotides in
length, and is substantially complementary to at least part of an
RNA transcript of an ALAS1 gene.
[0221] Accordingly, in some aspects, pharmaceutical compositions
containing an ALAS1 iRNA and a pharmaceutically acceptable carrier,
methods of using the compositions to inhibit expression of an ALAS1
gene, and methods of using the pharmaceutical compositions to treat
disorders related to ALAS1 expression are featured in the
invention.
I. Definitions
[0222] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0223] "G," "C," "A," "T" and "U" each generally stand for a
nucleotide that contains guanine, cytosine, adenine, thymidine and
uracil as a base, respectively. However, it will be understood that
the term "ribonucleotide" or "nucleotide" can also refer to a
modified nucleotide, as further detailed below, or a surrogate
replacement moiety. The skilled person is well aware that guanine,
cytosine, adenine, and uracil may be replaced by other moieties
without substantially altering the base pairing properties of an
oligonucleotide comprising a nucleotide bearing such replacement
moiety. For example, without limitation, a nucleotide comprising
inosine as its base may base pair with nucleotides containing
adenine, cytosine, or uracil. Hence, nucleotides containing uracil,
guanine, or adenine may be replaced in the nucleotide sequences of
dsRNA featured in the invention by a nucleotide containing, for
example, inosine. In another example, adenine and cytosine anywhere
in the oligonucleotide can be replaced with guanine and uracil,
respectively to form G-U Wobble base pairing with the target mRNA.
Sequences containing such replacement moieties are suitable for the
compositions and methods featured in the invention.
[0224] As used herein, "ALAS1" (also known as ALAS-1;
.delta.-aminolevulinate synthase 1; .delta.-ALA synthase 1;
5'-aminolevulinic acid synthase 1; ALAS-H; ALASH; ALAS-N; ALAS3;
EC2.3.1.37; 5-aminolevulinate synthase, nonspecific, mitochondrial;
ALAS; MIG4; OTTHUMP00000212619; OTTHUMP00000212620;
OTTHUMP00000212621; OTTHUMP00000212622; migration-inducing protein
4; EC 2.3.1) refers to a nuclear-encoded mitochondrial enzyme that
is the first and typically rate-limiting enzyme in the mammalian
heme biosynthetic pathway. ALAS1 catalyzes the condensation of
glycine with succinyl-CoA to form S-aminolevulinic acid (ALA). The
human ALAS1 gene is expressed ubiquitously, is found on chromosome
3p21.1 and typically encodes a sequence of 640 amino acids. In
contrast, the ALAS-2 gene, which encodes an isozyme, is expressed
only in erythrocytes, is found on chromoxome Xp11.21, and typically
encodes a sequence of 550 amino acids. As used herein an "ALAS1
protein" means any protein variant of ALAS1 from any species (e.g.,
human, mouse, non-human primate), as well as any mutants and
fragments thereof that retain an ALAS1 activity. Similarly, an
"ALAS1 transcript" refers to any transcript variant of ALAS1, from
any species (e.g., human, mouse, non-human primate). A sequence of
a human ALAS1 variant 1 mRNA transcript can be found at NM_000688.4
(FIG. 3; SEQ ID NO:1). Another version, a human ALAS1 variant 2
mRNA transcript, can be found at NM_000688.5 (FIG. 4; SEQ ID
NO:382). The level of the mature encoded ALAS1 protein is regulated
by heme: high levels of heme down-regulate the mature enzyme in
mitochondria while low heme levels up-regulate. Multiple
alternatively spliced variants, encoding the same protein, have
been identified.
[0225] As used herein, the term "iRNA," "RNAi", "iRNA agent," or
"RNAi agent" refers to an agent that contains RNA as that term is
defined herein, and which mediates the targeted cleavage of an RNA
transcript, e.g., via an RNA-induced silencing complex (RISC)
pathway. In one embodiment, an iRNA as described herein effects
inhibition of ALAS1 expression. Inhibition of ALAS1 expression may
be assessed based on a reduction in the level of ALAS1 mRNA or a
reduction in the level of the ALAS1 protein. As used herein,
"target sequence" refers to a contiguous portion of the nucleotide
sequence of an mRNA molecule formed during the transcription of an
ALAS1 gene, including mRNA that is a product of RNA processing of a
primary transcription product. The target portion of the sequence
will be at least long enough to serve as a substrate for
iRNA-directed cleavage at or near that portion. For example, the
target sequence will generally be from 9-36 nucleotides in length,
e.g., 15-30 nucleotides in length, including all sub-ranges
therebetween. As non-limiting examples, the target sequence can be
from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22
nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19
nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30
nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22
nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30
nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22
nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30
nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24
nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21
nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25
nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22
nucleotides.
[0226] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0227] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0228] Complementary sequences within an iRNA, e.g., within a dsRNA
as described herein, include base-pairing of the oligonucleotide or
polynucleotide comprising a first nucleotide sequence to an
oligonucleotide or polynucleotide comprising a second nucleotide
sequence over the entire length of one or both nucleotide
sequences. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but generally not more
than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a
duplex up to 30 base pairs, while retaining the ability to
hybridize under the conditions most relevant to their ultimate
application, e.g., inhibition of gene expression via a RISC
pathway. However, where two oligonucleotides are designed to form,
upon hybridization, one or more single stranded overhangs, such
overhangs shall not be regarded as mismatches with regard to the
determination of complementarity. For example, a dsRNA comprising
one oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes described
herein.
[0229] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled. Such non-Watson-Crick base pairs includes,
but are not limited to, G:U Wobble or Hoogstein base pairing.
[0230] The terms "complementary," "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of an iRNA agent and a
target sequence, as will be understood from the context of their
use.
[0231] As used herein, a polynucleotide that is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide that is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., an mRNA encoding
an ALAS1 protein). For example, a polynucleotide is complementary
to at least a part of an ALAS1 mRNA if the sequence is
substantially complementary to a non-interrupted portion of an mRNA
encoding ALAS1. As another example, a polynucleotide is
complementary to at least a part of an ALAS1 mRNA if the sequence
is substantially complementary to a non-interrupted portion of an
mRNA encoding ALAS1.
[0232] The term "double-stranded RNA" or "dsRNA," as used herein,
refers to an iRNA that includes an RNA molecule or complex of
molecules having a hybridized duplex region that comprises two
anti-parallel and substantially complementary nucleic acid strands,
which will be referred to as having "sense" and "antisense"
orientations with respect to a target RNA. The duplex region can be
of any length that permits specific degradation of a desired target
RNA, e.g., through a RISC pathway, but will typically range from 9
to 36 base pairs in length, e.g., 15-30 base pairs in length.
Considering a duplex between 9 and 36 base pairs, the duplex can be
any length in this range, for example, 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, or 36 and any sub-range therein between, including, but
not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base
pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19
base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs,
18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base
pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23
base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs,
20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base
pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30
base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs,
21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell
by processing with Dicer and similar enzymes are generally in the
range of 19-22 base pairs in length. One strand of the duplex
region of a dsDNA comprises a sequence that is substantially
complementary to a region of a target RNA. The two strands forming
the duplex structure can be from a single RNA molecule having at
least one self-complementary region, or can be formed from two or
more separate RNA molecules. Where the duplex region is formed from
two strands of a single molecule, the molecule can have a duplex
region separated by a single stranded chain of nucleotides (herein
referred to as a "hairpin loop") between the 3'-end of one strand
and the 5'-end of the respective other strand forming the duplex
structure. The hairpin loop can comprise at least one unpaired
nucleotide; in some embodiments the hairpin loop can comprise at
least 3, at least 4, at least 5, at least 6, at least 7, at least
8, at least 9, at least 10, at least 20, at least 23 or more
unpaired nucleotides. Where the two substantially complementary
strands of a dsRNA are comprised by separate RNA molecules, those
molecules need not, but can be covalently connected. Where the two
strands are connected covalently by means other than a hairpin
loop, the connecting structure is referred to as a "linker." The
term "siRNA" is also used herein to refer to a dsRNA as described
above.
[0233] In another embodiment, the iRNA agent may be a
"single-stranded siRNA" that is introduced into a cell or organism
to inhibit a target mRNA. Single-stranded RNAi agents bind to the
RISC endonuclease Argonaute 2, which then cleaves the target mRNA.
The single-stranded siRNAs are generally 15-30 nucleotides and are
chemically modified. The design and testing of single-stranded
siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al.,
(2012) Cell 150: 883-894, the entire contents of each of which are
hereby incorporated herein by reference. Any of the antisense
nucleotide sequences described herein (e.g., sequences provided in
Tables 2, 3, 6, 7, 8, 9, 14, and 15) may be used as a
single-stranded siRNA as described herein or as chemically modified
by the methods described in Lima et al., (2012) Cell
150:883-894.
[0234] In another aspect, the RNA agent is a "single-stranded
antisense RNA molecule". An single-stranded antisense RNA molecule
is complementary to a sequence within the target mRNA.
Single-stranded antisense RNA molecules can inhibit translation in
a stoichiometric manner by base pairing to the mRNA and physically
obstructing the translation machinery, see Dias, N. et al., (2002)
Mol Cancer Ther 1:347-355. Alternatively, the single-stranded
antisense molecules inhibit a target mRNA by hybridizing to the
target and cleaving the target through an RNaseH cleavage event.
The single-stranded antisense RNA molecule may be about 10 to about
30 nucleotides in length and have a sequence that is complementary
to a target sequence. For example, the single-stranded antisense
RNA molecule may comprise a sequence that is at least about 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides
from any one of the antisense nucleotide sequences described
herein, e.g., sequences provided in any one of Tables 2, 3, 6, 7,
8, 9, 14, and 15.
[0235] The skilled artisan will recognize that the term "RNA
molecule" or "ribonucleic acid molecule" encompasses not only RNA
molecules as expressed or found in nature, but also analogs and
derivatives of RNA comprising one or more
ribonucleotide/ribonucleoside analogs or derivatives as described
herein or as known in the art. Strictly speaking, a
"ribonucleoside" includes a nucleoside base and a ribose sugar, and
a "ribonucleotide" is a ribonucleoside with one, two or three
phosphate moieties. However, the terms "ribonucleoside" and
"ribonucleotide" can be considered to be equivalent as used herein.
The RNA can be modified in the nucleobase structure or in the
ribose-phosphate backbone structure, e.g., as described herein
below. However, the molecules comprising ribonucleoside analogs or
derivatives must retain the ability to form a duplex. As
non-limiting examples, an RNA molecule can also include at least
one modified ribonucleoside including but not limited to a
2'-O-methyl modified nucleostide, a nucleoside comprising a 5'
phosphorothioate group, a terminal nucleoside linked to a
cholesteryl derivative or dodecanoic acid bisdecylamide group, a
locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro
modified nucleoside, a 2'-amino-modified nucleoside,
2'-alkyl-modified nucleoside, morpholino nucleoside, a
phosphoramidate or a non-natural base comprising nucleoside, or any
combination thereof. Alternatively, an RNA molecule can comprise at
least two modified ribonucleosides, at least 3, at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 15, at least 20 or more, up to the entire length of
the dsRNA molecule. The modifications need not be the same for each
of such a plurality of modified ribonucleosides in an RNA molecule.
In one embodiment, modified RNAs contemplated for use in methods
and compositions described herein are peptide nucleic acids (PNAs)
that have the ability to form the required duplex structure and
that permit or mediate the specific degradation of a target RNA,
e.g., via a RISC pathway.
[0236] In one aspect, a modified ribonucleoside includes a
deoxyribonucleoside. In such an instance, an iRNA agent can
comprise one or more deoxynucleosides, including, for example, a
deoxynucleoside overhang(s), or one or more deoxynucleosides within
the double stranded portion of a dsRNA. However, it is self evident
that under no circumstances is a double stranded DNA molecule
encompassed by the term "iRNA."
[0237] In one aspect, an RNA interference agent includes a single
stranded RNA that interacts with a target RNA sequence to direct
the cleavage of the target RNA. Without wishing to be bound by
theory, long double stranded RNA introduced into cells is broken
down into siRNA by a Type III endonuclease known as Dicer (Sharp et
al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like
enzyme, processes the dsRNA into 19-23 base pair short interfering
RNAs with characteristic two base 3' overhangs (Bernstein, et al.,
(2001) Nature 409:363). The siRNAs are then incorporated into an
RNA-induced silencing complex (RISC) where one or more helicases
unwind the siRNA duplex, enabling the complementary antisense
strand to guide target recognition (Nykanen, et al., (2001) Cell
107:309). Upon binding to the appropriate target mRNA, one or more
endonucleases within the RISC cleaves the target to induce
silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in
one aspect the invention relates to a single stranded RNA that
promotes the formation of a RISC complex to effect silencing of the
target gene.
[0238] As used herein, the term "nucleotide overhang" refers to at
least one unpaired nucleotide that protrudes from the duplex
structure of an iRNA, e.g., a dsRNA. For example, when a 3'-end of
one strand of a dsRNA extends beyond the 5'-end of the other
strand, or vice versa, there is a nucleotide overhang. A dsRNA can
comprise an overhang of at least one nucleotide; alternatively the
overhang can comprise at least two nucleotides, at least three
nucleotides, at least four nucleotides, at least five nucleotides
or more. A nucleotide overhang can comprise or consist of a
nucleotide/nucleoside analog, including a
deoxynucleotide/nucleoside. The overhang(s) may be on the sense
strand, the antisense strand or any combination thereof.
Furthermore, the nucleotide(s) of an overhang can be present on the
5' end, 3' end or both ends of either an antisense or sense strand
of a dsRNA.
[0239] In one embodiment, the antisense strand of a dsRNA has a
1-10 nucleotide overhang at the 3' end and/or the 5' end. In one
embodiment, the sense strand of a dsRNA has a 1-10 nucleotide
overhang at the 3' end and/or the 5' end. In another embodiment,
one or more of the nucleotides in the overhang is replaced with a
nucleoside thiophosphate.
[0240] The terms "blunt" or "blunt ended" as used herein in
reference to a dsRNA mean that there are no unpaired nucleotides or
nucleotide analogs at a given terminal end of a dsRNA, i.e., no
nucleotide overhang. One or both ends of a dsRNA can be blunt.
Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt
ended. To be clear, a "blunt ended" dsRNA is a dsRNA that is blunt
at both ends, i.e., no nucleotide overhang at either end of the
molecule. Most often such a molecule will be double-stranded over
its entire length.
[0241] The term "antisense strand" or "guide strand" refers to the
strand of an iRNA, e.g., a dsRNA, which includes a region that is
substantially complementary to a target sequence. As used herein,
the term "region of complementarity" refers to the region on the
antisense strand that is substantially complementary to a sequence,
for example a target sequence, as defined herein. Where the region
of complementarity is not fully complementary to the target
sequence, the mismatches may be in the internal or terminal regions
of the molecule. Generally, the most tolerated mismatches are in
the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the
5' and/or 3' terminus.
[0242] The term "sense strand," or "passenger strand" as used
herein, refers to the strand of an iRNA that includes a region that
is substantially complementary to a region of the antisense strand
as that term is defined herein.
[0243] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP represents a vesicle of lipids coating
a reduced aqueous interior comprising a nucleic acid such as an
iRNA or a plasmid from which an iRNA is transcribed. SNALPs are
described, e.g., in U.S. Patent Application Publication Nos.
20060240093, 20070135372, and in International Application No. WO
2009082817. These applications are incorporated herein by reference
in their entirety.
[0244] "Introducing into a cell," when referring to an iRNA, means
facilitating or effecting uptake or absorption into the cell, as is
understood by those skilled in the art. Absorption or uptake of an
iRNA can occur through unaided diffusive or active cellular
processes, or by auxiliary agents or devices. The meaning of this
term is not limited to cells in vitro; an iRNA may also be
"introduced into a cell," wherein the cell is part of a living
organism. In such an instance, introduction into the cell will
include the delivery to the organism. For example, for in vivo
delivery, iRNA can be injected into a tissue site or administered
systemically. In vivo delivery can also be by a .beta.-glucan
delivery system, such as those described in U.S. Pat. Nos.
5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781,
which are hereby incorporated by reference in their entirety. In
vitro introduction into a cell includes methods known in the art
such as electroporation and lipofection. Further approaches are
described herein below or known in the art.
[0245] As used herein, the term "modulate the expression of,"
refers to at an least partial "inhibition" or partial "activation"
of an ALAS1 gene expression in a cell treated with an iRNA
composition as described herein compared to the expression of ALAS1
in a control cell. A control cell includes an untreated cell, or a
cell treated with a non-targeting control iRNA.
[0246] The terms "activate," "enhance," "up-regulate the expression
of," "increase the expression of," and the like, in so far as they
refer to an ALAS1 gene, herein refer to the at least partial
activation of the expression of an ALAS1 gene, as manifested by an
increase in the amount of ALAS1 mRNA, which may be isolated from or
detected in a first cell or group of cells in which an ALAS1 gene
is transcribed and which has or have been treated such that the
expression of an ALAS1 gene is increased, as compared to a second
cell or group of cells substantially identical to the first cell or
group of cells but which has or have not been so treated (control
cells).
[0247] In one embodiment, expression of an ALAS1 gene is activated
by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by
administration of an iRNA as described herein. In some embodiments,
an ALAS1 gene is activated by at least about 60%, 70%, or 80% by
administration of an iRNA featured in the invention. In some
embodiments, expression of an ALAS1 gene is activated by at least
about 85%, 90%, or 95% or more by administration of an iRNA as
described herein. In some embodiments, the ALAS1 gene expression is
increased by at least 1-fold, at least 2-fold, at least 5-fold, at
least 10-fold, at least 50-fold, at least 100-fold, at least
500-fold, at least 1000 fold or more in cells treated with an iRNA
as described herein compared to the expression in an untreated
cell. Activation of expression by small dsRNAs is described, for
example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A.
103:17337-42, and in US20070111963 and US2005226848, each of which
is incorporated herein by reference.
[0248] The terms "silence," "inhibit expression of," "down-regulate
expression of," "suppress expression of," and the like, in so far
as they refer to an ALAS1 gene, herein refer to the at least
partial suppression of the expression of an ALAS1 gene, as
assessed, e.g., based on on ALAS1 mRNA expression, ALAS1 protein
expression, or another parameter functionally linked to ALAS1 gene
expression (e.g., ALA or PBG concentrations in plasma or urine).
For example, inhibition of ALAS1 expression may be manifested by a
reduction of the amount of ALAS1 mRNA which may be isolated from or
detected in a first cell or group of cells in which an ALAS1 gene
is transcribed and which has or have been treated such that the
expression of an ALAS1 gene is inhibited, as compared to a control.
The control may be a second cell or group of cells substantially
identical to the first cell or group of cells, except that the
second cell or group of cells have not been so treated (control
cells). The degree of inhibition is usually expressed as a
percentage of a control level, e.g.,
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0249] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
ALAS1 gene expression, e.g., the amount of protein encoded by an
ALAS1 gene, or the level of one or more porphyrins. The reduction
of a parameter functionally linked to ALAS1 gene expression may
similarly be expressed as a percentage of a control level. In
principle, ALAS1 gene silencing may be determined in any cell
expressing ALAS1, either constitutively or by genomic engineering,
and by any appropriate assay. However, when a reference is needed
in order to determine whether a given iRNA inhibits the expression
of the ALAS1 gene by a certain degree and therefore is encompassed
by the instant invention, the assays provided in the Examples below
shall serve as such reference.
[0250] For example, in certain instances, expression of an ALAS1
gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, or 50% by administration of an iRNA featured in the
invention. In some embodiments, an ALAS1 gene is suppressed by at
least about 60%, 65%, 70%, 75%, or 80% by administration of an iRNA
featured in the invention. In some embodiments, an ALAS1 gene is
suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by
administration of an iRNA as described herein.
[0251] As used herein in the context of ALAS1 expression, the terms
"treat," "treating," "treatment," and the like, refer to relief
from or alleviation of pathological processes related to ALAS1
expression (e.g., pathological processes involving porphyrins or
defects in the porphyrin pathway, such as, for example,
porphyrias). In the context of the present invention insofar as it
relates to any of the other conditions recited herein below (other
than pathological processes related to ALAS1 expression), the terms
"treat," "treatment," and the like mean to prevent, relieve or
alleviate at least one symptom associated with such condition, or
to slow or reverse the progression or anticipated progression of
such condition. For example, the methods featured herein, when
employed to treat porphyria, may serve to reduce or prevent one or
more symptoms associated with porphyria (e.g., pain), to reduce the
severity or frequency of attacks associated with porphyria, to
reduce the likelihood that an attack of one or more symptoms
associated with porphyria will occur upon exposure to a
precipitating condition, to shorten an attack associated with
porphyria, and/or to reduce the risk of developing conditions
associated with porphyria (e.g., hepatocellular cancer or
neuropathy (e.g., progressive neuropathy),). Thus, unless the
context clearly indicates otherwise, the terms "treat,"
"treatment," and the like are intended to encompass prophylaxis,
e.g., prevention of disorders and/or symptoms of disorders related
to ALAS1 expression.
[0252] By "lower" in the context of a disease marker or symptom is
meant a statistically or clinically significant decrease in such
level. The decrease can be, for example, at least 10%, at least
20%, at least 30%, at least 40% or more, and is typically down to a
level accepted as within the range of normal for an individual
without such disorder.
[0253] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pathological processes related to ALAS1
expression. The specific amount that is therapeutically effective
can be readily determined by an ordinary medical practitioner, and
may vary depending on factors known in the art, such as, for
example, the type of pathological process, the patient's history
and age, the stage of pathological process, and the administration
of other agents.
[0254] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of an iRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
iRNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, in a method of treating a
disorder related to ALAS1 expression (e.g., in a method of treating
a porphyria), an effective amount includes an amount effective to
reduce one or more symptoms associated with a porphyria, an amount
effective to reduce the frequency of attacks, an amount effective
to reduce the likelihood that an attack of one or more symptoms
associated with porphyria will occur upon exposure to a
precipitating factor, or an amount effective to reduce the risk of
developing conditions associated with porphyria (e.g., neuropathy
(e.g., progressive neuropathy), hepatocellular cancer). For
example, if a given clinical treatment is considered effective when
there is at least a 10% reduction in a measurable parameter
associated with a disease or disorder, a therapeutically effective
amount of a drug for the treatment of that disease or disorder is
the amount necessary to effect at least a 10% reduction in that
parameter. For example, a therapeutically effective amount of an
iRNA targeting ALAS1 can reduce ALAS1 protein levels by any
measurable amount, e.g., by at least 10%, 20%, 30%, 40% or 50%.
[0255] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract. Agents included in drug formulations
are described further herein below.
[0256] The term "about" when referring to a number or a numerical
range means that the number or numerical range referred to is an
approximation within experimental variability (or within
statistical experimental error), and thus the number or numerical
range may vary from, for example, between 1% and 15% of the stated
number or numerical range.
II. Double-Stranded Ribonucleic Acid (dsRNA)
[0257] Described herein are iRNA agents that inhibit the expression
of an ALAS1 gene. In one embodiment, the iRNA agent includes
double-stranded ribonucleic acid (dsRNA) molecules for inhibiting
the expression of an ALAS1 gene in a cell or in a subject (e.g., in
a mammal, e.g., in a human having a porphyria), where the dsRNA
includes an antisense strand having a region of complementarity
which is complementary to at least a part of an mRNA formed in the
expression of an ALAS1 gene, and where the region of
complementarity is 30 nucleotides or less in length, generally
19-24 nucleotides in length, and where the dsRNA, upon contact with
a cell expressing the ALAS1 gene, inhibits the expression of the
ALAS1 gene by at least 10% as assayed by, for example, a PCR or
branched DNA (bDNA)-based method, or by a protein-based method,
such as by Western blot. In one embodiment, the iRNA agent
activates the expression of an ALAS1 gene in a cell or mammal.
Expression of an ALAS1 gene in cell culture, such as in COS cells,
HeLa cells, primary hepatocytes, HepG2 cells, primary cultured
cells or in a biological sample from a subject can be assayed by
measuring ALAS1 mRNA levels, such as by bDNA or TaqMan assay, or by
measuring protein levels, such as by immunofluorescence analysis,
using, for example, Western Blotting or flow cytometric
techniques.
[0258] A dsRNA includes two RNA strands that are sufficiently
complementary to hybridize to form a duplex structure under
conditions in which the dsRNA will be used. One strand of a dsRNA
(the antisense strand) includes a region of complementarity that is
substantially complementary, and generally fully complementary, to
a target sequence, derived from the sequence of an mRNA formed
during the expression of an ALAS1 gene. The other strand (the sense
strand) includes a region that is complementary to the antisense
strand, such that the two strands hybridize and form a duplex
structure when combined under suitable conditions. Generally, the
duplex structure is between 15 and 30 inclusive, more generally
between 18 and 25 inclusive, yet more generally between 19 and 24
inclusive, and most generally between 19 and 21 base pairs in
length, inclusive. Similarly, the region of complementarity to the
target sequence is between 15 and 30 inclusive, more generally
between 18 and 25 inclusive, yet more generally between 19 and 24
inclusive, and most generally between 19 and 21 nucleotides in
length, inclusive. In some embodiments, the dsRNA is between 15 and
20 nucleotides in length, inclusive, and in other embodiments, the
dsRNA is between 25 and 30 nucleotides in length, inclusive. As the
ordinarily skilled person will recognize, the targeted region of an
RNA targeted for cleavage will most often be part of a larger RNA
molecule, often an mRNA molecule. Where relevant, a "part" of an
mRNA target is a contiguous sequence of an mRNA target of
sufficient length to be a substrate for RNAi-directed cleavage
(i.e., cleavage through a RISC pathway). dsRNAs having duplexes as
short as 9 base pairs can, under some circumstances, mediate
RNAi-directed RNA cleavage. Most often a target will be at least 15
nucleotides in length, e.g., 15-30 nucleotides in length.
[0259] One of skill in the art will also recognize that the duplex
region is a primary functional portion of a dsRNA, e.g., a duplex
region of 9 to 36, e.g., 15-30 base pairs. Thus, in one embodiment,
to the extent that it becomes processed to a functional duplex of
e.g., 15-30 base pairs that targets a desired RNA for cleavage, an
RNA molecule or complex of RNA molecules having a duplex region
greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled
artisan will recognize that in one embodiment, then, an miRNA is a
dsRNA. In another embodiment, a dsRNA is not a naturally occurring
miRNA. In another embodiment, an iRNA agent useful to target ALAS1
expression is not generated in the target cell by cleavage of a
larger dsRNA.
[0260] A dsRNA as described herein may further include one or more
single-stranded nucleotide overhangs. The dsRNA can be synthesized
by standard methods known in the art as further discussed below,
e.g., by use of an automated DNA synthesizer, such as are
commercially available from, for example, Biosearch, Applied
Biosystems, Inc. In one embodiment, an ALAS1 gene is a human ALAS1
gene. In another embodiment the ALAS1 gene is a mouse or a rat
ALAS1 gene. In specific embodiments, the first sequence is a sense
strand of a dsRNA that includes a sense sequence from Table 2 or
Table 3, and the second sequence is an antisense strand of a dsRNA
that includes an antisense sequence from Table 2 or Table 3. In
embodiments, the first sequence is a sense strand of a dsRNA that
includes a sense sequence from Table 2, 3, 6, 7, 8, 9, 14, or 15,
and the second sequence is an antisense strand of a dsRNA that
includes an antisense sequence from Table 2, 3, 6, 7, 8, 9, 14, or
15. Alternative dsRNA agents that target sequences other than those
of the dsRNAs of Table 2 or Table 3 can readily be determined using
the target sequence and the flanking ALAS1 sequence.
[0261] In one aspect, a dsRNA will include at least sense and
antisense nucleotide sequences, whereby the sense strand is
selected from the groups of sequences provided in Tables 2 and 3,
and the corresponding antisense strand of the sense strand is
selected from Tables 2 and 3. In a further aspect, a dsRNA will
include at least sense and antisense nucleotide sequences, whereby
the sense strand is selected from the groups of sequences provided
in Tables 2, 3, 6, 7, 8, 9, 14, and 15, and the corresponding
antisense strand of the sense strand is selected from Tables 2, 3,
6, 7, 8, 9, 14, and 15. In these aspects, one of the two sequences
is complementary to the other of the two sequences, with one of the
sequences being substantially complementary to a sequence of an
mRNA generated by the expression of an ALAS1 gene gene. As such, a
dsRNA will include two oligonucleotides, where one oligonucleotide
is described as the sense strand in Table 2, 3, 6, 7, 8, 9, 14, or
15, and the second oligonucleotide is described as the
corresponding antisense strand of the sense strand from 2, 3, 6, 7,
8, 9, 14, or 15. As described elsewhere herein and as known in the
art, the complementary sequences of a dsRNA can also be contained
as self-complementary regions of a single nucleic acid molecule, as
opposed to being on separate oligonucleotides.
[0262] The skilled person is well aware that dsRNAs having a duplex
structure of between 20 and 23, but specifically 21, base pairs
have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer RNA duplex structures can
be effective as well. In the embodiments described above, by virtue
of the nature of the oligonucleotide sequences provided in Tables
2, 3, 6, 7, 8, 9, 14, and 15, dsRNAs described herein can include
at least one strand of a length of minimally 21 nucleotides. It can
be reasonably expected that shorter duplexes having one of the
sequences of Table 2, 3, 6, 7, 8, 9, 14, or 15 minus only a few
nucleotides on one or both ends may be similarly effective as
compared to the dsRNAs described above. Hence, dsRNAs having a
partial sequence of at least 15, 16, 17, 18, 19, 20, or more
contiguous nucleotides from one of the sequences of Table 2, 3, 6,
7, 8, 9, 14, or 15, and differing in their ability to inhibit the
expression of an ALAS1 gene by not more than 5, 10, 15, 20, 25, or
30% inhibition from a dsRNA comprising the full sequence, are
contemplated according to the invention.
[0263] In addition, the RNAs provided in Tables 2 and 3, as well as
the RNAs provided in Tables 2, 3, 6, 7, 8, 9, 14, and 15, identify
a site in an ALAS1 transcript that is susceptible to RISC-mediated
cleavage. As such, the present invention further features iRNAs
that target within one of such sequences. As used herein, an iRNA
is said to target within a particular site of an RNA transcript if
the iRNA promotes cleavage of the transcript anywhere within that
particular site. Such an iRNA will generally include at least 15
contiguous nucleotides from one of the sequences provided in Tables
2, 3, 6, 7, 8, 9, 14, and 15 coupled to additional nucleotide
sequences taken from the region contiguous to the selected sequence
in an ALAS1 gene.
[0264] While a target sequence is generally 15-30 nucleotides in
length, there is wide variation in the suitability of particular
sequences in this range for directing cleavage of any given target
RNA. Various software packages and the guidelines set out herein
provide guidance for the identification of optimal target sequences
for any given gene target, but an empirical approach can also be
taken in which a "window" or "mask" of a given size (as a
non-limiting example, 21 nucleotides) is literally or figuratively
(including, e.g., in silico) placed on the target RNA sequence to
identify sequences in the size range that may serve as target
sequences. By moving the sequence "window" progressively one
nucleotide upstream or downstream of an initial target sequence
location, the next potential target sequence can be identified,
until the complete set of possible sequences is identified for any
given target size selected. This process, coupled with systematic
synthesis and testing of the identified sequences (using assays as
described herein or as known in the art) to identify those
sequences that perform optimally can identify those RNA sequences
that, when targeted with an iRNA agent, mediate the best inhibition
of target gene expression. Thus, while the sequences identified,
for example, in Tables 2, 3, 6, 7, 8, 9, 14, and 15, represent
effective target sequences, it is contemplated that further
optimization of inhibition efficiency can be achieved by
progressively "walking the window" one nucleotide upstream or
downstream of the given sequences to identify sequences with equal
or better inhibition characteristics.
[0265] Further, it is contemplated that for any sequence
identified, e.g., in Tables 2, 3, 6, 7, 8, 9, 14, and 15, further
optimization can be achieved by systematically either adding or
removing nucleotides to generate longer or shorter sequences and
testing those and sequences generated by walking a window of the
longer or shorter size up or down the target RNA from that point.
Again, coupling this approach to generating new candidate targets
with testing for effectiveness of iRNAs based on those target
sequences in an inhibition assay as known in the art or as
described herein can lead to further improvements in the efficiency
of inhibition. Further still, such optimized sequences can be
adjusted by, e.g., the introduction of modified nucleotides as
described herein or as known in the art, addition or changes in
overhang, or other modifications as known in the art and/or
discussed herein to further optimize the molecule (e.g., increasing
serum stability or circulating half-life, increasing thermal
stability, enhancing transmembrane delivery, targeting to a
particular location or cell type, increasing interaction with
silencing pathway enzymes, increasing release from endosomes, etc.)
as an expression inhibitor.
[0266] An iRNA as described herein can contain one or more
mismatches to the target sequence. In one embodiment, an iRNA as
described herein contains no more than 3 mismatches. If the
antisense strand of the iRNA contains mismatches to a target
sequence, it is preferable that the area of mismatch not be located
in the center of the region of complementarity. If the antisense
strand of the iRNA contains mismatches to the target sequence, it
is preferable that the mismatch be restricted to be within the last
5 nucleotides from either the 5' or 3' end of the region of
complementarity. For example, for a 23 nucleotide iRNA agent RNA
strand which is complementary to a region of an ALAS1 gene, the RNA
strand generally does not contain any mismatch within the central
13 nucleotides. The methods described herein or methods known in
the art can be used to determine whether an iRNA containing a
mismatch to a target sequence is effective in inhibiting the
expression of an ALAS1 gene. Consideration of the efficacy of iRNAs
with mismatches in inhibiting expression of an ALAS1 gene is
important, especially if the particular region of complementarity
in an ALAS1 gene is known to have polymorphic sequence variation
within the population.
[0267] In one embodiment, at least one end of a dsRNA has a
single-stranded nucleotide overhang of 1 to 4, generally 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties relative to their
blunt-ended counterparts. In yet another embodiment, the RNA of an
iRNA, e.g., a dsRNA, is chemically modified to enhance stability or
other beneficial characteristics. The nucleic acids featured in the
invention may be synthesized and/or modified by methods well
established in the art, such as those described in "Current
protocols in nucleic acid chemistry," Beaucage, S. L. et al.
(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is
hereby incorporated herein by reference. Modifications include, for
example, (a) end modifications, e.g., 5' end modifications
(phosphorylation, conjugation, inverted linkages, etc.) 3' end
modifications (conjugation, DNA nucleotides, inverted linkages,
etc.), (b) base modifications, e.g., replacement with stabilizing
bases, destabilizing bases, or bases that base pair with an
expanded repertoire of partners, removal of bases (abasic
nucleotides), or conjugated bases, (c) sugar modifications (e.g.,
at the 2' position or 4' position) or replacement of the sugar, as
well as (d) backbone modifications, including modification or
replacement of the phosphodiester linkages. Specific examples of
RNA compounds useful in this invention include, but are not limited
to RNAs containing modified backbones or no natural internucleoside
linkages. RNAs having modified backbones include, among others,
those that do not have a phosphorus atom in the backbone. For the
purposes of this specification, and as sometimes referenced in the
art, modified RNAs that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides. In particular embodiments, the modified RNA will
have a phosphorus atom in its internucleoside backbone.
[0268] Modified RNA backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates,
phosphotriesters, aminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal
3'-5' linkages, 2'-5' linked analogs of these, and those) having
inverted polarity wherein the adjacent pairs of nucleoside units
are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed
salts and free acid forms are also included.
[0269] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445;
6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199;
6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167;
6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933;
7,321,029; and U.S. Pat. RE39464, each of which is herein
incorporated by reference.
[0270] Modified RNA backbones that do not include a phosphorus atom
therein have backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or
cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages. These
include those having morpholino linkages (formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide,
sulfoxide and sulfone backbones; formacetyl and thioformacetyl
backbones; methylene formacetyl and thioformacetyl backbones;
alkene containing backbones; sulfamate backbones; methyleneimino
and methylenehydrazino backbones; sulfonate and sulfonamide
backbones; amide backbones; and others having mixed N, O, S and
CH.sub.2 component parts.
[0271] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; and, 5,677,439, each of which is herein incorporated by
reference.
[0272] In other RNA mimetics suitable or contemplated for use in
iRNAs, both the sugar and the internucleoside linkage, i.e., the
backbone, of the nucleotide units are replaced with novel groups.
The base units are maintained for hybridization with an appropriate
nucleic acid target compound. One such oligomeric compound, an RNA
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar backbone of an RNA is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative U.S. patents that teach the preparation of PNA
compounds include, but are not limited to, U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be
found, for example, in Nielsen et al., Science, 1991, 254,
1497-1500.
[0273] Some embodiments featured in the invention include RNAs with
phosphorothioate backbones and oligonucleosides with heteroatom
backbones, and in particular --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--N(CH.sub.3)--CH.sub.2--CH.sub.2--[wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above-referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above-referenced U.S. Pat. No. 5,602,240. In some
embodiments, the RNAs featured herein have morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0274] Modified RNAs may also contain one or more substituted sugar
moieties. The iRNAs, e.g., dsRNAs, featured herein can include one
of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-,
S-, or N-alkenyl; O-, S- or N-alkynyl; 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. Exemplary suitable modifications include
O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2)..sub.nOCH.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, 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. In other embodiments, dsRNAs include one of
the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, 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 the
pharmacokinetic properties of an iRNA, or a group for improving the
pharmacodynamic properties of an iRNA, and other substituents
having similar properties. In some embodiments, the modification
includes a 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another
exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples herein below, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples herein below.
[0275] Other modifications include 2'-methoxy (2'-OCH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and
2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the RNA of an iRNA, particularly the 3' position of
the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs
and the 5' position of 5' terminal nucleotide. iRNAs may also have
sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl 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; and 5,700,920, certain
of which are commonly owned with the instant application, and each
of which is herein incorporated by reference.
[0276] An iRNA may also include nucleobase (often referred to in
the art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl
anal other 8-substituted adenines and guanines, 5-halo,
particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylguanine and 7-methyladenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine
and 3-deazaguanine and 3-deazaadenine. Further nucleobases include
those disclosed in U.S. Pat. No. 3,687,808, those disclosed in
Modified Nucleosides in Biochemistry, Biotechnology and Medicine,
Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed
by Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA
Research and Applications, pages 289-302, Crooke, S. T. and Lebleu,
B., Ed., CRC Press, 1993. Certain of these nucleobases are
particularly useful for increasing the binding affinity of the
oligomeric compounds featured in the invention. These include
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6
substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca
Raton, 1993, pp. 276-278) and are exemplary base substitutions,
even more particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0277] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;
5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200;
6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;
6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is
herein incorporated by reference, and U.S. Pat. No. 5,750,692, also
herein incorporated by reference.
[0278] The RNA of an iRNA can also be modified to include one or
more locked nucleic acids (LNA). A locked nucleic acid is a
nucleotide having a modified ribose moiety in which the ribose
moiety comprises an extra bridge connecting the 2' and 4' carbons.
This structure effectively "locks" the ribose in the 3'-endo
structural conformation. The addition of locked nucleic acids to
siRNAs has been shown to increase siRNA stability in serum, and to
reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids
Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther
6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research
31(12):3185-3193).
[0279] Representative U.S. Patents that teach the preparation of
locked nucleic acid nucleotides include, but are not limited to,
the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499;
6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is
herein incorporated by reference in its entirety.
[0280] Potentially stabilizing modifications to the ends of RNA
molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol
(Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6),
N-(acetyl-4-hydroxyprolinol (Hyp-NHAc),
thymidine-2'-0-deoxythymidine (ether),
N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),
2-docosanoyl-uridine-3''-phosphate, inverted base dT(idT) and
others. Disclosure of this modification can be found in PCT
Publication No. WO 2011/005861.
iRNA Motifs
[0281] In one embodiment, the sense strand sequence may be
represented by formula (I):
TABLE-US-00002 (I) 5'
n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.j-N.sub.a-n.-
sub.q 3'
[0282] wherein:
[0283] i and j are each independently 0 or 1;
[0284] p and q are each independently 0-6;
[0285] each N.sub.a independently represents an oligonucleotide
sequence comprising 0-25 modified nucleotides, each sequence
comprising at least two differently modified nucleotides;
[0286] each N.sub.b independently represents an oligonucleotide
sequence comprising 0-10 modified nucleotides;
[0287] each n.sub.p and n.sub.q independently represent an overhang
nucleotide;
[0288] wherein Nb and Y do not have the same modification; and
[0289] XXX, YYY and ZZZ each independently represent one motif of
three identical modifications on three consecutive nucleotides.
Preferably YYY is all 2'-F modified nucleotides.
[0290] In one embodiment, the N.sub.a and/or N.sub.b comprise
modifications of alternating pattern.
[0291] In one embodiment, the YYY motif occurs at or near the
cleavage site of the sense strand.
[0292] For example, when the RNAi agent has a duplex region of
17-23 nucleotides in length, the YYY motif can occur at or the
vicinity of the cleavage site (e.g.: can occur at positions 6, 7,
8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12 or 11, 12, 13) of - the
sense strand, the count starting from the 1.sup.st nucleotide, from
the 5'-end; or optionally, the count starting at the 1.sup.st
paired nucleotide within the duplex region, from the 5'-end.
[0293] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1,
or both i and j are 1. The sense strand can therefore be
represented by the following formulas:
TABLE-US-00003 (Ib) 5'
n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q 3'; (Ic) 5'
n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q 3'; or (Id) 5'
n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q 3'.
[0294] When the sense strand is represented by formula (Ib),
N.sub.b represents an oligonucleotide sequence comprising 0-10,
0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N.sub.a
independently can represent an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0295] When the sense strand is represented as formula (Ic),
N.sub.b represents an oligonucleotide sequence comprising 0-10,
0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N.sub.a can
independently represent an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0296] When the sense strand is represented as formula (Id), each
N.sub.b independently represents an oligonucleotide sequence
comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.
Preferably, N.sub.b is 0, 1, 2, 3, 4, 5 or 6. Each N.sub.a can
independently represent an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0297] Each of X, Y and Z may be the same or different from each
other.
[0298] In other embodiments, i is 0 and j is 0, and the sense
strand may be represented by the formula:
TABLE-US-00004 (Ia) 5' n.sub.p-N.sub.a-YYY-N.sub.a-n.sub.q 3'.
[0299] When the sense strand is represented by formula (Ia), each
N.sub.a independently can represent an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0300] In one embodiment, the antisense strand sequence of the RNAi
may be represented by formula (II):
TABLE-US-00005 (II) 5'
n.sub.q'-N.sub.a'-(Z'Z'Z').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(X'X'X').sub.-
l-N'.sub.a- n.sub.p' 3'
[0301] wherein:
[0302] k and l are each independently 0 or 1;
[0303] p' and q' are each independently 0-6;
[0304] each N.sub.a' independently represents an oligonucleotide
sequence comprising 0-25 modified nucleotides, each sequence
comprising at least two differently modified nucleotides;
[0305] each N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10 modified nucleotides;
[0306] each n.sub.p' and n.sub.q' independently represent an
overhang nucleotide;
[0307] wherein N.sub.b` and Y` do not have the same
modification;
[0308] and
[0309] X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one
motif of three identical modifications on three consecutive
nucleotides.
[0310] In one embodiment, the N.sub.a' and/or N.sub.b' comprise
modifications of alternating pattern.
[0311] The Y'Y'Y' motif occurs at or near the cleavage site of the
antisense strand. For example, when the RNAi agent has a duplex
region of 17-23 nucleotide in length, the Y'Y'Y' motif can occur at
positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14,
15 of the antisense strand, with the count starting from the
1.sup.st nucleotide, from the 5'-end; or optionally, the count
starting at the 1.sup.st paired nucleotide within the duplex
region, from the 5'-end.
[0312] Preferably, the Y'Y'Y' motif occurs at positions 11, 12,
13.
[0313] In one embodiment, Y'Y'Y' motif is all 2'-OMe modified
nucleotides.
[0314] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1,
or both k and l are 1.
[0315] The antisense strand can therefore be represented by the
following formulas:
TABLE-US-00006 (IIb) 5'
n.sub.q'-N.sub.a'-Z'Z'Z'-N.sub.b'-Y'Y'Y'-N.sub.a'-n.sub.p' 3';
(IIc) 5' n.sub.q'-N.sub.a'-Y'Y'Y'-N.sub.b'-X'X'X'-n.sub.p' 3'; or
(IId) 5'
n.sub.q'-N.sub.a'-Z'Z'Z'-N.sub.b'-Y'Y'Y'-N.sub.b'-X'X'X'-N.sub.a'-
n.sub.p' 3'.
[0316] When the antisense strand is represented by formula (IIb),
N.sub.b' represents an oligonucleotide sequence comprising 0-10,
0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N.sub.a'
independently represents an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0317] When the antisense strand is represented as formula (IIc),
N.sub.b' represents an oligonucleotide sequence comprising 0-10,
0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N.sub.a'
independently represents an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0318] When the antisense strand is represented as formula (IId),
each N.sub.b' independently represents an oligonucleotide sequence
comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each
N.sub.a' independently represents an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably,
N.sub.b is 0, 1, 2, 3, 4, 5 or 6.
[0319] In other embodiments, k is 0 and l is 0 and the antisense
strand may be represented by the formula:
TABLE-US-00007 (Ia) 5' n.sub.p'-N.sub.a'-Y'Y'Y'-N.sub.a'-n.sub.q'
3'.
[0320] When the antisense strand is represented as formula (IIa),
each N.sub.a' independently represents an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0321] Each of X', Y' and Z' may be the same or different from each
other.
[0322] Each nucleotide of the sense strand and antisense strand may
be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl,
2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For
example, each nucleotide of the sense strand and antisense strand
is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y,
Z, X', Y' and Z', in particular, may represent a 2'-O-methyl
modification or a 2'-fluoro modification.
[0323] In one embodiment, the sense strand of the RNAi agent may
contain YYY motif occurring at 9, 10 and 11 positions of the strand
when the duplex region is 21 nt, the count starting from the
1.sup.st nucleotide from the 5'-end, or optionally, the count
starting at the 1.sup.st paired nucleotide within the duplex
region, from the 5'-end; and Y represents 2'-F modification. The
sense strand may additionally contain XXX motif or ZZZ motifs as
wing modifications at the opposite end of the duplex region; and
XXX and ZZZ each independently represents a 2'-OMe modification or
2'-F modification.
[0324] In one embodiment the antisense strand may contain Y'Y'Y'
motif occurring at positions 11, 12, 13 of the strand, the count
starting from the 1.sup.st nucleotide from the 5'-end, or
optionally, the count starting at the 1.sup.st paired nucleotide
within the duplex region, from the 5'-end; and Y' represents
2'-O-methyl modification. The antisense strand may additionally
contain X'X'X' motif or Z'Z'Z' motifs as wing modifications at the
opposite end of the duplex region; and X'X'X' and Z'Z'Z' each
independently represents a 2'-OMe modification or 2'-F
modification.
[0325] The sense strand represented by any one of the above
formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense
strand being represented by any one of formulas (IIa), (IIb),
(IIc), and (IId), respectively.
[0326] Accordingly, the RNAi agents for use in the methods of the
invention may comprise a sense strand and an antisense strand, each
strand having 14 to 30 nucleotides, the RNAi duplex represented by
formula (III):
TABLE-US-00008 (III) sense: 5'
n.sub.p-N.sub.a-(XXX).sub.i-N.sub.b-YYY-N.sub.b-(ZZZ).sub.i-N.sub.a-n.s-
ub.q 3' antisense: 3'
n.sub.p'-N.sub.a'-(X'X'X').sub.k-N.sub.b'-Y'Y'Y'-N.sub.b'-(Z'Z'Z').sub.-
i-N.sub.a'- n.sub.q' 5'
[0327] wherein:
[0328] i, j, k, and l are each independently 0 or 1;
[0329] p, p', q, and q' are each independently 0-6;
[0330] each N.sub.a and N.sub.a' independently represents an
oligonucleotide sequence comprising 0-25 modified nucleotides, each
sequence comprising at least two differently modified
nucleotides;
[0331] each N.sub.b and N.sub.b' independently represents an
oligonucleotide sequence comprising 0-10 modified nucleotides;
[0332] wherein
[0333] each n.sub.p', n.sub.p, n.sub.q', and n.sub.q, each of which
may or may not be present, independently represents an overhang
nucleotide; and
[0334] XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently
represent one motif of three identical modifications on three
consecutive nucleotides.
[0335] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0;
or i is 0 and j is 1; or both i and j are 0; or both i and j are 1.
In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k
is 0 and l is 1; or both k and 1 are 0; or both k and 1 are 1.
[0336] Exemplary combinations of the sense strand and antisense
strand forming a RNAi duplex include the formulas below:
TABLE-US-00009 (IIIa) 5' n.sub.p-N.sub.a-YYY-N.sub.a-n.sub.q 3' 3'
n.sub.p'-N.sub.a'-Y'Y'Y'-N.sub.a'n.sub.q' 5' (IIIb) 5'
n.sub.p-N.sub.a-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q 3' 3'
n.sub.p'-N.sub.a'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.an.sub.q 5' (IIIc)
5' n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.a-n.sub.q 3' 3'
n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.a'-n.sub.q' 5'
(IIId) 5'
n.sub.p-N.sub.a-XXX-N.sub.b-YYY-N.sub.b-ZZZ-N.sub.a-n.sub.q 3' 3'
n.sub.p'-N.sub.a'-X'X'X'-N.sub.b'-Y'Y'Y'-N.sub.b'-Z'Z'Z'-N.sub.a-n.sub.-
q' 5'
[0337] When the RNAi agent is represented by formula (IIIa), each
N.sub.a independently represents an oligonucleotide sequence
comprising 2-20, 2-15, or 2-10 modified nucleotides.
[0338] When the RNAi agent is represented by formula (IIIb), each
N.sub.b independently represents an oligonucleotide sequence
comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N.sub.a
independently represents an oligonucleotide sequence comprising
2-20, 2-15, or 2-10 modified nucleotides.
[0339] When the RNAi agent is represented as formula (IIIc), each
N.sub.b, N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified
nucleotides. Each N.sub.a independently represents an
oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified
nucleotides.
[0340] When the RNAi agent is represented as formula (IIId), each
N.sub.b, N.sub.b' independently represents an oligonucleotide
sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified
nucleotides. Each N.sub.a, N.sub.a' independently represents an
oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified
nucleotides. Each of N.sub.a, N.sub.a', N.sub.b and N.sub.b'
independently comprises modifications of alternating pattern.
[0341] Each of X, Y and Z in formulas (III), (IIIa), (IIIb),
(IIIc), and (IIId) may be the same or different from each
other.
[0342] When the RNAi agent is represented by formula (III), (IIIa),
(IIIb), (IIc), and (IId), at least one of the Y nucleotides may
form a base pair with one of the Y' nucleotides. Alternatively, at
least two of the Y nucleotides form base pairs with the
corresponding Y' nucleotides; or all three of the Y nucleotides all
form base pairs with the corresponding Y' nucleotides.
[0343] When the RNAi agent is represented by formula (IIIb) or
(IIId), at least one of the Z nucleotides may form a base pair with
one of the Z' nucleotides. Alternatively, at least two of the Z
nucleotides form base pairs with the corresponding Z' nucleotides;
or all three of the Z nucleotides all form base pairs with the
corresponding Z' nucleotides.
[0344] When the RNAi agent is represented as formula (IIIc) or
(IIId), at least one of the X nucleotides may form a base pair with
one of the X' nucleotides. Alternatively, at least two of the X
nucleotides form base pairs with the corresponding X' nucleotides;
or all three of the X nucleotides all form base pairs with the
corresponding X' nucleotides.
[0345] In one embodiment, the modification on the Y nucleotide is
different than the modification on the Y' nucleotide, the
modification on the Z nucleotide is different than the modification
on the Z' nucleotide, and/or the modification on the X nucleotide
is different than the modification on the X' nucleotide.
[0346] In one embodiment, when the RNAi agent is represented by
formula (IId), the N.sub.a modifications are 2'-O-methyl or
2'-fluoro modifications. In another embodiment, when the RNAi agent
is represented by formula (IIId), the N.sub.a modifications are
2'-O-methyl or 2'-fluoro modifications and n.sub.p'>0 and at
least one n.sub.p' is linked to a neighboring nucleotide a via
phosphorothioate linkage. In yet another embodiment, when the RNAi
agent is represented by formula (IIId), the N.sub.a modifications
are 2'-O-methyl or 2'-fluoro modifications, n.sub.p'>0 and at
least one n.sub.p' is linked to a neighboring nucleotide via
phosphorothioate linkage, and the sense strand is conjugated to one
or more GalNAc derivatives attached through a bivalent or trivalent
branched linker. In another embodiment, when the RNAi agent is
represented by formula (IIId), the N.sub.a modifications are
2'-O-methyl or 2'-fluoro modifications, n.sub.p'>0 and at least
one n.sub.p' is linked to a neighboring nucleotide via
phosphorothioate linkage, the sense strand comprises at least one
phosphorothioate linkage, and the sense strand is conjugated to one
or more GalNAc derivatives attached through a bivalent or trivalent
branched linker.
[0347] In one embodiment, when the RNAi agent is represented by
formula (IIIa), the N.sub.a modifications are 2'-O-methyl or
2'-fluoro modifications, n.sub.p'>0 and at least one n.sub.p' is
linked to a neighboring nucleotide via phosphorothioate linkage,
the sense strand comprises at least one phosphorothioate linkage,
and the sense strand is conjugated to one or more GalNAc
derivatives attached through a bivalent or trivalent branched
linker.
[0348] In one embodiment, the RNAi agent is a multimer containing
at least two duplexes represented by formula (III), (IIIa), (IIIb),
(IIIc), and (IIId), wherein the duplexes are connected by a linker.
The linker can be cleavable or non-cleavable. Optionally, the
multimer further comprises a ligand. Each of the duplexes can
target the same gene or two different genes; or each of the
duplexes can target same gene at two different target sites.
[0349] In one embodiment, the RNAi agent is a multimer containing
three, four, five, six or more duplexes represented by formula
(III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are
connected by a linker. The linker can be cleavable or
non-cleavable. Optionally, the multimer further comprises a ligand.
Each of the duplexes can target the same gene or two different
genes; or each of the duplexes can target same gene at two
different target sites.
[0350] In one embodiment, two RNAi agents represented by formula
(III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other
at the 5' end, and one or both of the 3' ends and are optionally
conjugated to to a ligand. Each of the agents can target the same
gene or two different genes; or each of the agents can target same
gene at two different target sites.
iRNA Conjugates
[0351] The iRNA agents disclosed herein can be in the form of
conjugates. The conjugate may be attached at any suitable location
in the iRNA molecule, e.g., at the 3' end or the 5' end of the
sense or the antisense strand. The conjugates are optionally
attached via a linker.
[0352] In some embodiments, an iRNA agent described herein is
chemically linked to one or more ligands, moieties or conjugates,
which may confer functionality, e.g., by affecting (e.g.,
enhancing) the activity, cellular distribution or cellular uptake
of the iRNA. Such moieties include but are not limited to lipid
moieties such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan
et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether,
e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,
3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids
Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol 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-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-carbonyloxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
[0353] In one embodiment, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In some embodiments, a ligand provides an enhanced
affinity for a selected target, e.g., molecule, cell or cell type,
compartment, e.g., a cellular or organ compartment, tissue, organ
or region of the body, as, e.g., compared to a species absent such
a ligand. Typical ligands will not take part in duplex pairing in a
duplexed nucleic acid.
[0354] Ligands can include a naturally occurring substance, such as
a protein (e.g., human serum albumin (HSA), low-density lipoprotein
(LDL), or globulin); carbohydrate (e.g., a dextran, pullulan,
chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a
lipid. The ligand may also be a recombinant or synthetic molecule,
such as a synthetic polymer, e.g., a synthetic polyamino acid.
Examples of polyamino acids include polyamino acid is a polylysine
(PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic
acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer,
divinyl ether-maleic anhydride copolymer,
N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene
glycol (PEG), polyvinyl alcohol (PVA), polyurethane,
poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or
polyphosphazine. Example of polyamines include: polyethylenimine,
polylysine (PLL), spermine, spermidine, polyamine,
pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer
polyamine, arginine, amidine, protamine, cationic lipid, cationic
porphyrin, quaternary salt of a polyamine, or an a helical
peptide.
[0355] Ligands can also include targeting groups, e.g., a cell or
tissue targeting agent, e.g., a lectin, glycoprotein, lipid or
protein, e.g., an antibody, that binds to a specified cell type
such as a kidney cell. A targeting group can be a thyrotropin,
melanotropin, lectin, glycoprotein, surfactant protein A, Mucin
carbohydrate, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
multivalent fucose, glycosylated polyaminoacids, multivalent
galactose, transferrin, bisphosphonate, polyglutamate,
polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate,
vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
[0356] In some embodiments, the ligand is a GalNAc ligand that
comprises one or more N-acetylgalactosamine (GalNAc) derivatives.
Additional description of GalNAc ligands is provided in the section
titled Carbohydrate Conjugates.
[0357] Other examples of ligands include dyes, intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol,
cholic acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, borneol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2,
polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes,
haptens (e.g. biotin), transport/absorption facilitators (e.g.,
aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,
imidazole, bisimidazole, histamine, imidazole clusters,
acridine-imidazole conjugates, Eu3+ complexes of
tetraazamacrocycles), dinitrophenyl, HRP, or AP.
[0358] Ligands can be proteins, e.g., glycoproteins, or peptides,
e.g., molecules having a specific affinity for a co-ligand, or
antibodies e.g., an antibody, that binds to a specified cell type
such as a cancer cell, endothelial cell, or bone cell. Ligands may
also include hormones and hormone receptors. They can also include
non-peptidic species, such as lipids, lectins, carbohydrates,
vitamins, cofactors, multivalent lactose, multivalent galactose,
N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose,
or multivalent fucose. The ligand can be, for example, a
lipopolysaccharide, an activator of p38 MAP kinase, or an activator
of NF-.kappa.B.
[0359] The ligand can be a substance, e.g., a drug, which can
increase the uptake of the iRNA agent into the cell, for example,
by disrupting the cell's cytoskeleton, e.g., by disrupting the
cell's microtubules, microfilaments, and/or intermediate filaments.
The drug can be, for example, taxon, vincristine, vinblastine,
cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin,
swinholide A, indanocine, or myoservin.
[0360] In some embodiments, a ligand attached to an iRNA as
described herein acts as a pharmacokinetic modulator (PK
modulator). PK modulators include lipophiles, bile acids, steroids,
phospholipid analogues, peptides, protein binding agents, PEG,
vitamins etc. Exemplary PK modulators include, but are not limited
to, cholesterol, fatty acids, cholic acid, lithocholic acid,
dialkylglycerides, diacylglyceride, phospholipids, sphingolipids,
naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that
comprise a number of phosphorothioate linkages are also known to
bind to serum protein, thus short oligonucleotides, e.g.,
oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,
comprising multiple of phosphorothioate linkages in the backbone
are also amenable to the present invention as ligands (e.g. as PK
modulating ligands). In addition, aptamers that bind serum
components (e.g. serum proteins) are also suitable for use as PK
modulating ligands in the embodiments described herein.
[0361] Ligand-conjugated oligonucleotides of the invention may be
synthesized by the use of an oligonucleotide that bears a pendant
reactive functionality, such as that derived from the attachment of
a linking molecule onto the oligonucleotide (described below). This
reactive oligonucleotide may be reacted directly with
commercially-available ligands, ligands that are synthesized
bearing any of a variety of protecting groups, or ligands that have
a linking moiety attached thereto.
[0362] The oligonucleotides used in the conjugates of the present
invention may be conveniently and routinely made through the
well-known technique of solid-phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is also known to use similar techniques to prepare
other oligonucleotides, such as the phosphorothioates and alkylated
derivatives.
[0363] In the ligand-conjugated oligonucleotides and
ligand-molecule bearing sequence-specific linked nucleosides of the
present invention, the oligonucleotides and oligonucleosides may be
assembled on a suitable DNA synthesizer utilizing standard
nucleotide or nucleoside precursors, or nucleotide or nucleoside
conjugate precursors that already bear the linking moiety,
ligand-nucleotide or nucleoside-conjugate precursors that already
bear the ligand molecule, or non-nucleoside ligand-bearing building
blocks.
[0364] When using nucleotide-conjugate precursors that already bear
a linking moiety, the synthesis of the sequence-specific linked
nucleosides is typically completed, and the ligand molecule is then
reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. In some embodiments, the oligonucleotides or
linked nucleosides of the present invention are synthesized by an
automated synthesizer using phosphoramidites derived from
ligand-nucleoside conjugates in addition to the standard
phosphoramidites and non-standard phosphoramidites that are
commercially available and routinely used in oligonucleotide
synthesis.
[0365] Lipid Conjugates
[0366] In one embodiment, the ligand is a lipid or lipid-based
molecule. Such a lipid or lipid-based molecule can typically bind a
serum protein, such as human serum albumin (HSA). An HSA binding
ligand allows for distribution of the conjugate to a target tissue,
e.g., a non-kidney target tissue of the body. For example, the
target tissue can be the liver, including parenchymal cells of the
liver. Other molecules that can bind HSA can also be used as
ligands. For example, neproxin or aspirin can be used. A lipid or
lipid-based ligand can (a) increase resistance to degradation of
the conjugate, (b) increase targeting or transport into a target
cell or cell membrane, and/or (c) can be used to adjust binding to
a serum protein, e.g., HSA.
[0367] A lipid based ligand can be used to modulate, e.g., control
(e.g., inhibit) the binding of the conjugate to a target tissue.
For example, a lipid or lipid-based ligand that binds to HSA more
strongly will be less likely to be targeted to the kidney and
therefore less likely to be cleared from the body. A lipid or
lipid-based ligand that binds to HSA less strongly can be used to
target the conjugate to the kidney.
[0368] In one embodiment, the lipid based ligand binds HSA. For
example, the ligand can bind HSA with a sufficient affinity such
that distribution of the conjugate to a non-kidney tissue is
enhanced. However, the affinity is typically not so strong that the
HSA-ligand binding cannot be reversed.
[0369] In another embodiment, the lipid based ligand binds HSA
weakly or not at all, such that distribution of the conjugate to
the kidney is enhanced. Other moieties that target to kidney cells
can also be used in place of or in addition to the lipid based
ligand.
[0370] In another aspect, the ligand is a moiety, e.g., a vitamin,
which is taken up by a target cell, e.g., a proliferating cell.
These are particularly useful for treating disorders characterized
by unwanted cell proliferation, e.g., of the malignant or
non-malignant type, e.g., cancer cells.
[0371] Exemplary vitamins include vitamin A, E, and K. Other
exemplary vitamins include are B vitamin, e.g., folic acid, B12,
riboflavin, biotin, pyridoxal or other vitamins or nutrients taken
up by cancer cells. Also included are HSA and low density
lipoprotein (LDL).
[0372] Cell Permeation Agents
[0373] In another aspect, the ligand is a cell-permeation agent,
such as a helical cell-permeation agent. In one embodiment, the
agent is amphipathic. An exemplary agent is a peptide such as tat
or antennopedia. If the agent is a peptide, it can be modified,
including a peptidylmimetic, invertomers, non-peptide or
pseudo-peptide linkages, and use of D-amino acids. The helical
agent is typically an .alpha.-helical agent, and can have a
lipophilic and a lipophobic phase. The ligand can be a peptide or
peptidomimetic. A peptidomimetic (also referred to herein as an
oligopeptidomimetic) is a molecule capable of folding into a
defined three-dimensional structure similar to a natural peptide.
The attachment of peptide and peptidomimetics to iRNA agents can
affect pharmacokinetic distribution of the iRNA, such as by
enhancing cellular recognition and absorption. The peptide or
peptidomimetic moiety can be about 5-50 amino acids long, e.g.,
about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids
long.
[0374] A peptide or peptidomimetic can be, for example, a cell
permeation peptide, cationic peptide, amphipathic peptide, or
hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or
Phe). The peptide moiety can be a dendrimer peptide, constrained
peptide or crosslinked peptide. In another alternative, the peptide
moiety can include a hydrophobic membrane translocation sequence
(MTS). An exemplary hydrophobic MTS-containing peptide is RFGF
having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:3367).
An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID
NO:3368)) containing a hydrophobic MTS can also be a targeting
moiety. The peptide moiety can be a "delivery" peptide, which can
carry large polar molecules including peptides, oligonucleotides,
and protein across cell membranes. For example, sequences from the
HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3369)) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 3370)) have been
found to be capable of functioning as delivery peptides. A peptide
or peptidomimetic can be encoded by a random sequence of DNA, such
as a peptide identified from a phage-display library, or
one-bead-one-compound (OBOC) combinatorial library (Lam et al.,
Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic
tethered to a dsRNA agent via an incorporated monomer unit is a
cell targeting peptide such as an arginine-glycine-aspartic acid
(RGD)-peptide, or RGD mimic. A peptide moiety can range in length
from about 5 amino acids to about 40 amino acids. The peptide
moieties can have a structural modification, such as to increase
stability or direct conformational properties. Any of the
structural modifications described below can be utilized.
[0375] An RGD peptide for use in the compositions and methods of
the invention may be linear or cyclic, and may be modified, e.g.,
glycosylated or methylated, to facilitate targeting to a specific
tissue(s). RGD-containing peptides and peptidiomimemtics may
include D-amino acids, as well as synthetic RGD mimics. In addition
to RGD, one can use other moieties that target the integrin ligand.
Preferred conjugates of this ligand target PECAM-1 or VEGF.
[0376] An RGD peptide moiety can be used to target a particular
cell type, e.g., a tumor cell, such as an endothelial tumor cell or
a breast cancer tumor cell (Zitzmann et al., Cancer Res.,
62:5139-43, 2002). An RGD peptide can facilitate targeting of an
dsRNA agent to tumors of a variety of other tissues, including the
lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy
8:783-787, 2001). Typically, the RGD peptide will facilitate
targeting of an iRNA agent to the kidney. The RGD peptide can be
linear or cyclic, and can be modified, e.g., glycosylated or
methylated to facilitate targeting to specific tissues. For
example, a glycosylated RGD peptide can deliver a iRNA agent to a
tumor cell expressing .alpha..sub.v.beta..sub.3 (Haubner et al.,
Jour. Nucl. Med., 42:326-336, 2001).
[0377] A "cell permeation peptide" is capable of permeating a cell,
e.g., a microbial cell, such as a bacterial or fungal cell, or a
mammalian cell, such as a human cell. A microbial cell-permeating
peptide can be, for example, an .alpha.-helical linear peptide
(e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide
(e.g., .alpha.-defensin, .beta.-defensin or bactenecin), or a
peptide containing only one or two dominating amino acids (e.g.,
PR-39 or indolicidin). A cell permeation peptide can also include a
nuclear localization signal (NLS). For example, a cell permeation
peptide can be a bipartite amphipathic peptide, such as MPG, which
is derived from the fusion peptide domain of HIV-1 gp41 and the NLS
of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.
31:2717-2724, 2003).
[0378] Carbohydrate Conjugates
[0379] In some embodiments of the compositions and methods of the
invention, an iRNA oligonucleotide further comprises a
carbohydrate. The carbohydrate conjugated iRNA are advantageous for
the in vivo delivery of nucleic acids, as well as compositions
suitable for in vivo therapeutic use, as described herein. As used
herein, "carbohydrate" refers to a compound which is either a
carbohydrate per se made up of one or more monosaccharide units
having at least 6 carbon atoms (which can be linear, branched or
cyclic) with an oxygen, nitrogen or sulfur atom bonded to each
carbon atom; or a compound having as a part thereof a carbohydrate
moiety made up of one or more monosaccharide units each having at
least six carbon atoms (which can be linear, branched or cyclic),
with an oxygen, nitrogen or sulfur atom bonded to each carbon atom.
Representative carbohydrates include the sugars (mono-, di-, tri-
and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9
monosaccharide units), and polysaccharides such as starches,
glycogen, cellulose and polysaccharide gums. Specific
monosaccharides include C5 and above (e.g., C5, C6, C7, or C8)
sugars; di- and trisaccharides include sugars having two or three
monosaccharide units (e.g., C5, C6, C7, or C8).
[0380] In one embodiment, a carbohydrate conjugate comprises a
monosaccharide. In one embodiment, the monosaccharide is an
N-acetylgalactosamine (GalNAc). GalNAc conjugates are described,
for example, in U.S. Pat. No. 8,106,022, the entire content of
which is hereby incorporated herein by reference. In some
embodiments, the GalNAc conjugate serves as a ligand that targets
the iRNA to particular cells. In some embodiments, the GalNAc
conjugate targets the iRNA to liver cells, e.g., by serving as a
ligand for the asialoglycoprotein receptor of liver cells (e.g.,
hepatocytes).
[0381] In some embodiments, the carbohydrate conjugate comprises
one or more GalNAc derivatives. The GalNAc derivatives may be
attached via a linker, e.g., a bivalent or trivalent branched
linker. In some embodiments the GalNAc conjugate is conjugated to
the 3' end of the sense strand. In some embodiments, the GalNAc
conjugate is conjugated to the iRNA agent (e.g., to the 3' end of
the sense strand) via a linker, e.g., a linker as described
herein.
[0382] In some embodiments, the GalNAc conjugate is
##STR00006##
[0383] In some embodiments, the RNAi agent is attached to the
carbohydrate conjugate via a linker as shown in the following
schematic, wherein X is O or S
##STR00007##
[0384] In some embodiments, the RNAi agent is conjugated to L96 as
defined in Table 1 and shown below
##STR00008##
[0385] In some embodiments, a carbohydrate conjugate for use in the
compositions and methods of the invention is selected from the
group consisting of:
##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013##
[0386] Another representative carbohydrate conjugate for use in the
embodiments described herein includes, but is not limited to,
##STR00014##
[0387] (Formula XXIII), when one of X or Y is an oligonucleotide,
the other is a hydrogen.
[0388] In some embodiments, the carbohydrate conjugate further
comprises one or more additional ligands as described above, such
as, but not limited to, a PK modulator and/or a cell permeation
peptide.
[0389] In one embodiment, an iRNA of the invention is conjugated to
a carbohydrate through a linker. Non-limiting examples of iRNA
carbohydrate conjugates with linkers of the compositions and
methods of the invention include, but are not limited to,
##STR00015## ##STR00016##
when one of X or Y is an oligonucleotide, the other is a
hydrogen.
[0390] Linkers
[0391] In some embodiments, the conjugate or ligand described
herein can be attached to an iRNA oligonucleotide with various
linkers that can be cleavable or non-cleavable.
[0392] The term "linker" or "linking group" means an organic moiety
that connects two parts of a compound, e.g., covalently attaches
two parts of a compound. Linkers typically comprise a direct bond
or an atom such as oxygen or sulfur, a unit such as NR8, C(O),
C(O)NH, SO, SO.sub.2, SO.sub.2NH or a chain of atoms, such as, but
not limited to, substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl,
heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl,
heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,
alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,
alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,
alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,
alkylheteroarylalkenyl, alkylheteroarylalkynyl,
alkenylheteroarylalkyl, alkenylheteroarylalkenyl,
alkenylheteroarylalkynyl, alkynylheteroarylalkyl,
alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,
alkylheterocyclylalkyl, alkylheterocyclylalkenyl,
alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,
alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,
alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,
alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,
alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or
more methylenes can be interrupted or terminated by O, S, S(O),
SO.sub.2, N(R8), C(O), substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic
or substituted aliphatic. In one embodiment, the linker is between
about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18
atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.
[0393] In one embodiment, a dsRNA of the invention is conjugated to
a bivalent or trivalent branched linker selected from the group of
structures shown in any of formula (XXXI)-(XXXIV):
##STR00017##
wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent
independently for each occurrence 0-20 and wherein the repeating
unit can be the same or different; P.sup.2A, P.sup.2B, P.sup.3A,
P.sup.3B, P.sup.4A, P.sup.4B, P.sup.5A, P.sup.5B, P.sup.5C,
T.sup.2A, T.sup.2B, T.sup.3A, T.sup.3B, T.sup.4A, T.sup.4B,
T.sup.4A, T.sup.5B, T.sup.5C are each independently for each
occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH.sub.2,
CH.sub.2NH or CH.sub.2O; Q.sup.2A, Q.sup.2B, Q.sup.3A, Q.sup.3B,
Q.sup.4A, Q.sup.4B, Q.sup.5A, Q.sup.5B, Q.sup.5C, are independently
for each occurrence absent, alkylene, substituted alkylene wherein
one or more methylenes can be interrupted or terminated by one or
more of O, S, S(O), SO.sub.2, N(R.sup.N), C(R').dbd.C(R''),
C.ident.C or C(O); R.sup.2A, R.sup.2B, R.sup.3A, R.sup.3B,
R.sup.4A, R.sup.4B, R.sup.5A, R.sup.5B, R.sup.5C are each
independently for each occurrence absent, NH, O, S, CH.sub.2,
C(O)O, C(O)NH, NHCH(R.sup.a)C(O), --C(O)--CH(R.sup.a)--NH--, CO,
CH.dbd.N--O,
##STR00018##
heterocyclyl;
[0394] L.sup.2A, L.sup.2B, L.sup.3A, L.sup.3B, L.sup.4A, L.sup.4B,
L.sup.5A, L.sup.5B and L.sup.5C represent the ligand; i.e. each
independently for each occurrence a monosaccharide (such as
GalNAc), disaccharide, trisaccharide, tetrasaccharide,
oligosaccharide, or polysaccharide; and R.sup.a is H or amino acid
side chain. Trivalent conjugating GalNAc derivatives are
particularly useful for use with RNAi agents for inhibiting the
expression of a target gene, such as those of formula (XXXV):
##STR00019##
[0395] wherein L.sup.5A, L.sup.5B and L.sup.5C represent a
monosaccharide, such as GalNAc derivative.
[0396] Examples of suitable bivalent and trivalent branched linker
groups conjugating GalNAc derivatives include, but are not limited
to, the structures recited above as formulas II, VII, XI, X, and
XIII.
[0397] A cleavable linking group is one which is sufficiently
stable outside the cell, but which upon entry into a target cell is
cleaved to release the two parts the linker is holding together. In
a preferred embodiment, the cleavable linking group is cleaved at
least about 10 times, 20, times, 30 times, 40 times, 50 times, 60
times, 70 times, 80 times, 90 times or more, or at least about 100
times faster in a target cell or under a first reference condition
(which can, e.g., be selected to mimic or represent intracellular
conditions) than in the blood of a subject, or under a second
reference condition (which can, e.g., be selected to mimic or
represent conditions found in the blood or serum).
[0398] Cleavable linking groups are susceptible to cleavage agents,
e.g., pH, redox potential or the presence of degradative molecules.
Generally, cleavage agents are more prevalent or found at higher
levels or activities inside cells than in serum or blood. Examples
of such degradative agents include: redox agents which are selected
for particular substrates or which have no substrate specificity,
including, e.g., oxidative or reductive enzymes or reductive agents
such as mercaptans, present in cells, that can degrade a redox
cleavable linking group by reduction; esterases; endosomes or
agents that can create an acidic environment, e.g., those that
result in a pH of five or lower; enzymes that can hydrolyze or
degrade an acid cleavable linking group by acting as a general
acid, peptidases (which can be substrate specific), and
phosphatases.
[0399] A cleavable linkage group, such as a disulfide bond can be
susceptible to pH. The pH of human serum is 7.4, while the average
intracellular pH is slightly lower, ranging from about 7.1-7.3.
Endosomes have a more acidic pH, in the range of 5.5-6.0, and
lysosomes have an even more acidic pH at around 5.0. Some linkers
will have a cleavable linking group that is cleaved at a preferred
pH, thereby releasing a cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0400] A linker can include a cleavable linking group that is
cleavable by a particular enzyme. The type of cleavable linking
group incorporated into a linker can depend on the cell to be
targeted. For example, a liver-targeting ligand can be linked to a
cationic lipid through a linker that includes an ester group. Liver
cells are rich in esterases, and therefore the linker will be
cleaved more efficiently in liver cells than in cell types that are
not esterase-rich. Other cell-types rich in esterases include cells
of the lung, renal cortex, and testis.
[0401] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0402] In general, the suitability of a candidate cleavable linking
group can be evaluated by testing the ability of a degradative
agent (or condition) to cleave the candidate linking group. It will
also be desirable to also test the candidate cleavable linking
group for the ability to resist cleavage in the blood or when in
contact with other non-target tissue. Thus, one can determine the
relative susceptibility to cleavage between a first and a second
condition, where the first is selected to be indicative of cleavage
in a target cell and the second is selected to be indicative of
cleavage in other tissues or biological fluids, e.g., blood or
serum. The evaluations can be carried out in cell free systems, in
cells, in cell culture, in organ or tissue culture, or in whole
animals. It can be useful to make initial evaluations in cell-free
or culture conditions and to confirm by further evaluations in
whole animals. In preferred embodiments, useful candidate compounds
are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80,
90, or about 100 times faster in the cell (or under in vitro
conditions selected to mimic intracellular conditions) as compared
to blood or serum (or under in vitro conditions selected to mimic
extracellular conditions).
[0403] Redox Cleavable Linking Groups
[0404] In one embodiment, a cleavable linking group is a redox
cleavable linking group that is cleaved upon reduction or
oxidation. An example of reductively cleavable linking group is a
disulphide linking group (--S--S--). To determine if a candidate
cleavable linking group is a suitable "reductively cleavable
linking group," or for example is suitable for use with a
particular iRNA moiety and particular targeting agent one can look
to methods described herein. For example, a candidate can be
evaluated by incubation with dithiothreitol (DTT), or other
reducing agent using reagents know in the art, which mimic the rate
of cleavage which would be observed in a cell, e.g., a target cell.
The candidates can also be evaluated under conditions which are
selected to mimic blood or serum conditions. In one, candidate
compounds are cleaved by at most about 10% in the blood. In other
embodiments, useful candidate compounds are degraded at least about
2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster
in the cell (or under in vitro conditions selected to mimic
intracellular conditions) as compared to blood (or under in vitro
conditions selected to mimic extracellular conditions). The rate of
cleavage of candidate compounds can be determined using standard
enzyme kinetics assays under conditions chosen to mimic
intracellular media and compared to conditions chosen to mimic
extracellular media.
[0405] Phosphate-Based Cleavable Linking Groups
[0406] In another embodiment, a cleavable linker comprises a
phosphate-based cleavable linking group. A phosphate-based
cleavable linking group is cleaved by agents that degrade or
hydrolyze the phosphate group. An example of an agent that cleaves
phosphate groups in cells are enzymes such as phosphatases in
cells. Examples of phosphate-based linking groups are
--O--P(O)(ORk)-O--, --O--P(S)(ORk)-O--, --O--P(S)(SRk)-O--,
--S--P(O)(ORk)-O--, --O--P(O)(ORk)-S--, --S--P(O)(ORk)-S--,
--O--P(S)(ORk)-S--, --S--P(S)(ORk)-O--, --O--P(O)(Rk)-O--,
--O--P(S)(Rk)-O--, --S--P(O)(Rk)-O--, --S--P(S)(Rk)-O--,
--S--P(O)(Rk)-S--, --O--P(S)(Rk)-S--. Preferred embodiments are
--O--P(O)(OH)--O--, --O--P(S)(OH)--O--, --O--P(S)(SH)--O--,
--S--P(O)(OH)--O--, --O--P(O)(OH)--S--, --S--P(O)(OH)--S--,
--O--P(S)(OH)--S--, --S--P(S)(OH)--O--, --O--P(O)(H)--O--,
--O--P(S)(H)--O--, --S--P(O)(H)--O, --S--P(S)(H)--O--,
--S--P(O)(H)--S--, --O--P(S)(H)--S--. A preferred embodiment is
--O--P(O)(OH)--O--. These candidates can be evaluated using methods
analogous to those described above.
[0407] Acid Cleavable Linking Groups
[0408] In another embodiment, a cleavable linker comprises an acid
cleavable linking group. An acid cleavable linking group is a
linking group that is cleaved under acidic conditions. In preferred
embodiments acid cleavable linking groups are cleaved in an acidic
environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75,
5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can
act as a general acid. In a cell, specific low pH organelles, such
as endosomes and lysosomes can provide a cleaving environment for
acid cleavable linking groups. Examples of acid cleavable linking
groups include but are not limited to hydrazones, esters, and
esters of amino acids. Acid cleavable groups can have the general
formula --C.dbd.NN--, C(O)O, or --OC(O). A preferred embodiment is
when the carbon attached to the oxygen of the ester (the alkoxy
group) is an aryl group, substituted alkyl group, or tertiary alkyl
group such as dimethyl pentyl or t-butyl. These candidates can be
evaluated using methods analogous to those described above.
[0409] Ester-Based Cleavable Linking Groups
[0410] In another embodiment, a cleavable linker comprises an
ester-based cleavable linking group. An ester-based cleavable
linking group is cleaved by enzymes such as esterases and amidases
in cells. Examples of ester-based cleavable linking groups include
but are not limited to esters of alkylene, alkenylene and
alkynylene groups. Ester cleavable linking groups have the general
formula --C(O)O--, or --OC(O)--. These candidates can be evaluated
using methods analogous to those described above.
[0411] Peptide-Based Cleavable Linking Groups
[0412] In yet another embodiment, a cleavable linker comprises a
peptide-based cleavable linking group. A peptide-based cleavable
linking group is cleaved by enzymes such as peptidases and
proteases in cells. Peptide-based cleavable linking groups are
peptide bonds formed between amino acids to yield oligopeptides
(e.g., dipeptides, tripeptides etc.) and polypeptides.
Peptide-based cleavable groups do not include the amide group
(--C(O)NH--). The amide group can be formed between any alkylene,
alkenylene or alkynelene. A peptide bond is a special type of amide
bond formed between amino acids to yield peptides and proteins. The
peptide based cleavage group is generally limited to the peptide
bond (i.e., the amide bond) formed between amino acids yielding
peptides and proteins and does not include the entire amide
functional group. Peptide-based cleavable linking groups have the
general formula --NHCHRAC(O)NHCHRBC(O)-- (SEQ ID NO: 13), where RA
and RB are the R groups of the two adjacent amino acids. These
candidates can be evaluated using methods analogous to those
described above.
Representative U.S. patents that teach the preparation of RNA
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; 5,591,584; 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; 6,294,664;
6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022,
the entire contents of each of which is herein incorporated by
reference.
[0413] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an iRNA. The present
invention also includes iRNA compounds that are chimeric
compounds.
[0414] "Chimeric" iRNA compounds, or "chimeras," in the context of
the present invention, are iRNA compounds, e.g., dsRNAs, that
contain two or more chemically distinct regions, each made up of at
least one monomer unit, i.e., a nucleotide in the case of a dsRNA
compound. These iRNAs typically contain at least one region wherein
the RNA is modified so as to confer upon the iRNA increased
resistance to nuclease degradation, increased cellular uptake,
and/or increased binding affinity for the target nucleic acid. An
additional region of the iRNA may serve as a substrate for enzymes
capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example,
RNase H is a cellular endonuclease which cleaves the RNA strand of
an RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of iRNA inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter iRNAs when
chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs
hybridizing to the same target region. Cleavage of the RNA target
can be routinely detected by gel electrophoresis and, if necessary,
associated nucleic acid hybridization techniques known in the
art.
[0415] In certain instances, the RNA of an iRNA can be modified by
a non-ligand group. A number of non-ligand molecules have been
conjugated to iRNAs in order to enhance the activity, cellular
distribution or cellular uptake of the iRNA, and procedures for
performing such conjugations are available in the scientific
literature. Such non-ligand moieties have included lipid moieties,
such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm.,
2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan
et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic
chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,
259:327; Svinarchuk et a., Biochimie, 1993, 75:49), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res.,
1990, 18:3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,
1995, 1264:229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such RNA conjugates have been
listed above. Typical conjugation protocols involve the synthesis
of an RNAs bearing an aminolinker at one or more positions of the
sequence. The amino group is then reacted with the molecule being
conjugated using appropriate coupling or activating reagents. The
conjugation reaction may be performed either with the RNA still
bound to the solid support or following cleavage of the RNA, in
solution phase. Purification of the RNA conjugate by HPLC typically
affords the pure conjugate.
[0416] Delivery of iRNA
[0417] The delivery of an iRNA to a subject in need thereof can be
achieved in a number of different ways. In vivo delivery can be
performed directly by administering a composition comprising an
iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be
performed indirectly by administering one or more vectors that
encode and direct the expression of the iRNA. These alternatives
are discussed further below.
[0418] Direct Delivery
[0419] In general, any method of delivering a nucleic acid molecule
can be adapted for use with an iRNA (see e.g., Akhtar S. and Julian
R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which
are incorporated herein by reference in their entireties). However,
there are three factors that are important to consider in order to
successfully deliver an iRNA molecule in vivo: (a) biological
stability of the delivered molecule, (2) preventing non-specific
effects, and (3) accumulation of the delivered molecule in the
target tissue. The non-specific effects of an iRNA can be minimized
by local administration, for example by direct injection or
implantation into a tissue (as a non-limiting example, a tumor) or
topically administering the preparation. Local administration to a
treatment site maximizes local concentration of the agent, limits
the exposure of the agent to systemic tissues that may otherwise be
harmed by the agent or that may degrade the agent, and permits a
lower total dose of the iRNA molecule to be administered. Several
studies have shown successful knockdown of gene products when an
iRNA is administered locally. For example, intraocular delivery of
a VEGF dsRNA by intravitreal injection in cynomolgus monkeys
(Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal
injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216)
were both shown to prevent neovascularization in an experimental
model of age-related macular degeneration. In addition, direct
intratumoral injection of a dsRNA in mice reduces tumor volume
(Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong
survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther.
14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA
interference has also shown success with local delivery to the CNS
by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49;
Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al
(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004)
Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl.
Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J.
Neurophysiol. 93:594-602) and to the lungs by intranasal
administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484;
Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V.,
et al (2005) Nat. Med. 11:50-55). For administering an iRNA
systemically for the treatment of a disease, the RNA can be
modified or alternatively delivered using a drug delivery system;
both methods act to prevent the rapid degradation of the dsRNA by
endo- and exo-nucleases in vivo.
[0420] Modification of the RNA or the pharmaceutical carrier can
also permit targeting of the iRNA composition to the target tissue
and avoid undesirable off-target effects. iRNA molecules can be
modified by chemical conjugation to other groups, e.g., a lipid or
carbohydrate group as described herein. Such conjugates can be used
to target iRNA to particular cells, e.g., liver cells, e.g.,
hepatocytes. For example, GalNAc conjugates or lipid (e.g., LNP)
formulations can be used to target iRNA to particular cells, e.g.,
liver cells, e.g., hepatocytes.
[0421] Lipophilic groups such as cholesterol to enhance cellular
uptake and prevent degradation. For example, an iRNA directed
against ApoB conjugated to a lipophilic cholesterol moiety was
injected systemically into mice and resulted in knockdown of apoB
mRNA in both the liver and jejunum (Soutschek, J., et al (2004)
Nature 432:173-178). Conjugation of an iRNA to an aptamer has been
shown to inhibit tumor growth and mediate tumor regression in a
mouse model of prostate cancer (McNamara, J O., et al (2006) Nat.
Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA
can be delivered using drug delivery systems such as a
nanoparticle, a dendrimer, a polymer, liposomes, or a cationic
delivery system. Positively charged cationic delivery systems
facilitate binding of an iRNA molecule (negatively charged) and
also enhance interactions at the negatively charged cell membrane
to permit efficient uptake of an iRNA by the cell. Cationic lipids,
dendrimers, or polymers can either be bound to an iRNA, or induced
to form a vesicle or micelle (see e.g., Kim S H., et al (2008)
Journal of Controlled Release 129(2):107-116) that encases an iRNA.
The formation of vesicles or micelles further prevents degradation
of the iRNA when administered systemically. Methods for making and
administering cationic-iRNA complexes are well within the abilities
of one skilled in the art (see e.g., Sorensen, D R., et al (2003)
J. Mol. Biol 327:761-766; Verma, U N., et al (2003) Clin. Cancer
Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens.
25:197-205, which are incorporated herein by reference in their
entirety). Some non-limiting examples of drug delivery systems
useful for systemic delivery of iRNAs include DOTAP (Sorensen, D
R., et al (2003), supra; Verma, U N., et al (2003), supra),
Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T
S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et
al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int
J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al
(2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006)
J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S.
(2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A.,
et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999)
Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a
complex with cyclodextrin for systemic administration. Methods for
administration and pharmaceutical compositions of iRNAs and
cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is
herein incorporated by reference in its entirety.
[0422] Vector Encoded iRNAs
[0423] In another aspect, iRNA targeting the ALAS1 gene can be
expressed from transcription units inserted into DNA or RNA vectors
(see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A.,
et al., International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299). Expression can be transient (on the order of
hours to weeks) or sustained (weeks to months or longer), depending
upon the specific construct used and the target tissue or cell
type. These transgenes can be introduced as a linear construct, a
circular plasmid, or a viral vector, which can be an integrating or
non-integrating vector. The transgene can also be constructed to
permit it to be inherited as an extrachromosomal plasmid (Gassmann,
et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
[0424] The individual strand or strands of an iRNA can be
transcribed from a promoter on an expression vector. Where two
separate strands are to be expressed to generate, for example, a
dsRNA, two separate expression vectors can be co-introduced (e.g.,
by transfection or infection) into a target cell. Alternatively
each individual strand of a dsRNA can be transcribed by promoters
both of which are located on the same expression plasmid. In one
embodiment, a dsRNA is expressed as an inverted repeat joined by a
linker polynucleotide sequence such that the dsRNA has a stem and
loop structure.
[0425] An iRNA expression vector is typically a DNA plasmid or
viral vector. An expression vector compatible with eukaryotic
cells, e.g., with vertebrate cells, can be used to produce
recombinant constructs for the expression of an iRNA as described
herein. Eukaryotic cell expression vectors are well known in the
art and are available from a number of commercial sources.
Typically, such vectors contain convenient restriction sites for
insertion of the desired nucleic acid segment. Delivery of iRNA
expressing vectors can be systemic, such as by intravenous or
intramuscular administration, by administration to target cells
ex-planted from the patient followed by reintroduction into the
patient, or by any other means that allows for introduction into a
desired target cell.
[0426] An iRNA expression plasmid can be transfected into a target
cell as a complex with a cationic lipid carrier (e.g.,
Oligofectamine) or a non-cationic lipid-based carrier (e.g.,
Transit-TKO.TM.). Multiple lipid transfections for iRNA-mediated
knockdowns targeting different regions of a target RNA over a
period of a week or more are also contemplated by the invention.
Successful introduction of vectors into host cells can be monitored
using various known methods. For example, transient transfection
can be signaled with a reporter, such as a fluorescent marker, such
as Green Fluorescent Protein (GFP). Stable transfection of cells ex
vivo can be ensured using markers that provide the transfected cell
with resistance to specific environmental factors (e.g.,
antibiotics and drugs), such as hygromycin B resistance. Viral
vector systems which can be utilized with the methods and
compositions described herein include, but are not limited to, (a)
adenovirus vectors; (b) retrovirus vectors, including but not
limited to lentiviral vectors, moloney murine leukemia virus, etc.;
(c) adeno-associated virus vectors; (d) herpes simplex virus
vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma
virus vectors; (h) picornavirus vectors; (i) pox virus vectors such
as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary
pox or fowl pox; and (j) a helper-dependent or gutless adenovirus.
Replication-defective viruses can also be advantageous. Different
vectors will or will not become incorporated into the cells'
genome. The constructs can include viral sequences for
transfection, if desired. Alternatively, the construct may be
incorporated into vectors capable of episomal replication, e.g. EPV
and EBV vectors. Constructs for the recombinant expression of an
iRNA will generally require regulatory elements, e.g., promoters,
enhancers, etc., to ensure the expression of the iRNA in target
cells. Other aspects to consider for vectors and constructs are
further described below.
[0427] Vectors useful for the delivery of an iRNA will include
regulatory elements (promoter, enhancer, etc.) sufficient for
expression of the iRNA in the desired target cell or tissue. The
regulatory elements can be chosen to provide either constitutive or
regulated/inducible expression.
[0428] Expression of the iRNA can be precisely regulated, for
example, by using an inducible regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of dsRNA expression in cells or in mammals include, for
example, regulation by ecdysone, by estrogen, progesterone,
tetracycline, chemical inducers of dimerization, and
isopropyl-.beta.-D1-thiogalactopyranoside (IPTG). A person skilled
in the art would be able to choose the appropriate
regulatory/promoter sequence based on the intended use of the iRNA
transgene.
[0429] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding an iRNA can be used. For example, a
retroviral vector can be used (see Miller et al., Meth. Enzymol.
217:581-599 (1993)). These retroviral vectors contain the
components necessary for the correct packaging of the viral genome
and integration into the host cell DNA. The nucleic acid sequences
encoding an iRNA are cloned into one or more vectors, which
facilitates delivery of the nucleic acid into a patient. More
detail about retroviral vectors can be found, for example, in
Boesen et al., Biotherapy 6:291-302 (1994), which describes the use
of a retroviral vector to deliver the mdr1 gene to hematopoietic
stem cells in order to make the stem cells more resistant to
chemotherapy. Other references illustrating the use of retroviral
vectors in gene therapy are: Clowes et al., J. Clin. Invest.
93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons
and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and
Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993).
Lentiviral vectors contemplated for use include, for example, the
HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557;
and 5,981,276, which are herein incorporated by reference.
[0430] Adenoviruses are also contemplated for use in delivery of
iRNAs. Adenoviruses are especially attractive vehicles, e.g., for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, Current Opinion in Genetics and
Development 3:499-503 (1993) present a review of adenovirus-based
gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994)
demonstrated the use of adenovirus vectors to transfer genes to the
respiratory epithelia of rhesus monkeys. Other instances of the use
of adenoviruses in gene therapy can be found in Rosenfeld et al.,
Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155
(1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT
Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783
(1995). A suitable AV vector for expressing an iRNA featured in the
invention, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0431] Use of Adeno-associated virus (AAV) vectors is also
contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300
(1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can
be expressed as two separate, complementary single-stranded RNA
molecules from a recombinant AAV vector having, for example, either
the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter.
Suitable AAV vectors for expressing the dsRNA featured in the
invention, methods for constructing the recombinant AV vector, and
methods for delivering the vectors into target cells are described
in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et
al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J.
Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941;
International Patent Application No. WO 94/13788; and International
Patent Application No. WO 93/24641, the entire disclosures of which
are herein incorporated by reference.
[0432] Another typical viral vector is a pox virus such as a
vaccinia virus, for example an attenuated vaccinia such as Modified
Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary
pox.
[0433] The tropism of viral vectors can be modified by pseudotyping
the vectors with envelope proteins or other surface antigens from
other viruses, or by substituting different viral capsid proteins,
as appropriate. For example, lentiviral vectors can be pseudotyped
with surface proteins from vesicular stomatitis virus (VSV),
rabies, Ebola, Mokola, and the like. AAV vectors can be made to
target different cells by engineering the vectors to express
different capsid protein serotypes; see, e.g., Rabinowitz J E et
al. (2002), J Virol 76:791-801, the entire disclosure of which is
herein incorporated by reference.
[0434] The pharmaceutical preparation of a vector can include the
vector in an acceptable diluent, or can include a slow release
matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
III. Pharmaceutical Compositions Containing iRNA
[0435] In one embodiment, the invention provides pharmaceutical
compositions containing an iRNA, as described herein, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
containing the iRNA is useful for treating a disease or disorder
related to the expression or activity of an ALAS1 gene (e.g., a
disorder involving the porphyrin pathway). Such pharmaceutical
compositions are formulated based on the mode of delivery. For
example, compositions can be formulated for systemic administration
via parenteral delivery, e.g., by intravenous (IV) delivery. In
some embodiments, a composition provided herein (e.g., an LNP
formulation) is formulated for intravenous delivery. In some
embodiments, a composition provided herein (e.g., a composition
comprising a GalNAc conjugate) is formulated for subcutaneous
delivery.
[0436] The pharmaceutical compositions featured herein are
administered in a dosage sufficient to inhibit expression of an
ALAS1 gene. In general, a suitable dose of iRNA will be in the
range of 0.01 to 200.0 milligrams per kilogram body weight of the
recipient per day, generally in the range of 1 to 50 mg per
kilogram body weight per day. For example, the dsRNA can be
administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg,
3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per
single dose. The pharmaceutical composition may be administered
once daily, or the iRNA may be administered as two, three, or more
sub-doses at appropriate intervals throughout the day or even using
continuous infusion or delivery through a controlled release
formulation. In that case, the iRNA contained in each sub-dose must
be correspondingly smaller in order to achieve the total daily
dosage. The dosage unit can also be compounded for delivery over
several days, e.g., using a conventional sustained release
formulation which provides sustained release of the iRNA over a
several day period. Sustained release formulations are well known
in the art and are particularly useful for delivery of agents at a
particular site, such as can be used with the agents of the present
invention. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0437] The effect of a single dose on ALAS1 levels can be long
lasting, such that subsequent doses are administered at not more
than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4
week intervals.
[0438] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
iRNAs encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0439] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
pathological processes related to ALAS1 expression (e.g.,
pathological processes involving porphyrins or defects in the
porphyrin pathway, such as, for example, porphyrias). Such models
can be used for in vivo testing of iRNA, as well as for determining
a therapeutically effective dose and/or an effective dosing
regimen.
[0440] A suitable mouse model is, for example, a mouse containing a
transgene expressing human ALAS1. Mice that have knock-in mutations
(e.g., mutations that are associated with acute hepatic porphyrias
in humans) can be used to determine the therapeutically effective
dosage and/or duration of administration of ALAS1 siRNA. The
present invention also includes pharmaceutical compositions and
formulations that include the iRNA compounds featured in the
invention. The pharmaceutical compositions of the present invention
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. Administration may be topical (e.g., by a transdermal
patch), pulmonary, e.g., by inhalation or insufflation of powders
or aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal, oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; subdermal,
e.g., via an implanted device; or intracranial, e.g., by
intraparenchymal, intrathecal or intraventricular,
administration.
[0441] The iRNA can be delivered in a manner to target a particular
tissue, such as a tissue that produces erythrocytes. For example,
the iRNA can be delivered to bone marrow, liver (e.g., hepatocyes
of liver), lymph glands, spleen, lungs (e.g., pleura of lungs) or
spine. In one embodiment, the iRNA is delivered to bone marrow.
[0442] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Suitable
topical formulations include those in which the iRNAs featured in
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Suitable lipids and liposomes
include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, iRNAs may be complexed to lipids, in particular to
cationic lipids. Suitable fatty acids and esters include but are
not limited to arachidonic acid, oleic acid, eicosanoic acid,
lauric acid, caprylic acid, capric acid, myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-20 alkyl ester (e.g., isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S. Pat.
No. 6,747,014, which is incorporated herein by reference.
[0443] Liposomal Formulations
[0444] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0445] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0446] In order to traverse intact mammalian skin, lipid vesicles
must pass through a series of fine pores, each with a diameter less
than 50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0447] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0448] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes and as the merging of the liposome and cell progresses,
the liposomal contents are emptied into the cell where the active
agent may act.
[0449] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0450] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex.
[0451] The positively charged DNA/liposome complex binds to the
negatively charged cell surface and is internalized in an endosome.
Due to the acidic pH within the endosome, the liposomes are
ruptured, releasing their contents into the cell cytoplasm (Wang et
al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
[0452] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0453] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0454] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g., as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0455] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
[0456] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0457] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al).
[0458] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0459] A number of liposomes comprising nucleic acids are known in
the art. WO 96/40062 to Thierry et al. discloses methods for
encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising dsRNAs targeted to the raf gene.
[0460] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g., they are self-optimizing (adaptive to the shape of
pores in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0461] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0462] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0463] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0464] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0465] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0466] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0467] Nucleic Acid Lipid Particles
[0468] In one embodiment, an ALAS1 dsRNA featured in the invention
is fully encapsulated in the lipid formulation, e.g., to form a
SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used
herein, the term "SNALP" refers to a stable nucleic acid-lipid
particle, including SPLP. As used herein, the term "SPLP" refers to
a nucleic acid-lipid particle comprising plasmid DNA encapsulated
within a lipid vesicle. SNALPs and SPLPs typically contain a
cationic lipid, a non-cationic lipid, and a lipid that prevents
aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs
and SPLPs are extremely useful for systemic applications, as they
exhibit extended circulation lifetimes following intravenous (i.v.)
injection and accumulate at distal sites (e.g., sites physically
separated from the administration site). SPLPs include "pSPLP,"
which include an encapsulated condensing agent-nucleic acid complex
as set forth in PCT Publication No. WO 00/03683. The particles of
the present invention typically have a mean diameter of about 50 nm
to about 150 nm, more typically about 60 nm to about 130 nm, more
typically about 70 nm to about 110 nm, most typically about 70 nm
to about 90 nm, and are substantially nontoxic. In addition, the
nucleic acids when present in the nucleic acid-lipid particles of
the present invention are resistant in aqueous solution to
degradation with a nuclease. Nucleic acid-lipid particles and their
method of preparation are disclosed in, e.g., U.S. Pat. Nos.
5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT
Publication No. WO 96/40964.
[0469] In one embodiment, the lipid to drug ratio (mass/mass ratio)
(e.g., lipid to dsRNA ratio) will be in the range of from about 1:1
to about 50:1, from about 1:1 to about 25:1, from about 3:1 to
about 15:1, from about 4:1 to about 10:1, from about 5:1 to about
9:1, or about 6:1 to about 9:1.
[0470] The cationic lipid may be, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA) or analogs thereof,
(3aR,5s,6aS)--N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydr-
o-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100),
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (MC3),
1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami-
no)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G), or
a mixture thereof. The cationic lipid may comprise from about 20
mol % to about 50 mol % or about 40 mol % of the total lipid
present in the particle.
[0471] In another embodiment, the compound
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to
prepare lipid-siRNA nanoparticles. Synthesis of
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in
U.S. provisional patent application No. 61/107,998 filed on Oct.
23, 2008, which is herein incorporated by reference.
[0472] In one embodiment, the lipid-siRNA particle includes 40% 2,
2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%
Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of
63.0.+-.20 nm and a 0.027 siRNA/Lipid Ratio.
[0473] The non-cationic lipid may be an anionic lipid or a neutral
lipid including, but not limited to, distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof. The non-cationic lipid may be from about 5 mol %
to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol
is included, of the total lipid present in the particle.
[0474] The conjugated lipid that inhibits aggregation of particles
may be, for example, a polyethyleneglycol (PEG)-lipid including,
without limitation, a PEG-diacylglycerol (DAG), a
PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide
(Cer), or a mixture thereof. The PEG-DAA conjugate may be, for
example, a PEG-dilauryloxypropyl (Ci.sub.2), a
PEG-dimyristyloxypropyl (Ci.sub.4), a PEG-dipalmityloxypropyl
(Ci.sub.6), or a PEG-distearyloxypropyl (C].sub.8). The conjugated
lipid that prevents aggregation of particles may be from 0 mol % to
about 20 mol % or about 2 mol % of the total lipid present in the
particle.
[0475] In some embodiments, the nucleic acid-lipid particle further
includes cholesterol at, e.g., about 10 mol % to about 60 mol % or
about 48 mol % of the total lipid present in the particle.
[0476] In some embodiments, the iRNA is formulated in a lipid
nanoparticle (LNP).
[0477] LNP01
[0478] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see
U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008,
which is herein incorporated by reference), Cholesterol
(Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be
used to prepare lipid-dsRNA nanoparticles (e.g., LNP01 particles).
Stock solutions of each in ethanol can be prepared as follows:
ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100
mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions
can then be combined in a, e.g., 42:48:10 molar ratio. The combined
lipid solution can be mixed with aqueous dsRNA (e.g., in sodium
acetate pH 5) such that the final ethanol concentration is about
35-45% and the final sodium acetate concentration is about 100-300
mM. Lipid-dsRNA nanoparticles typically form spontaneously upon
mixing. Depending on the desired particle size distribution, the
resultant nanoparticle mixture can be extruded through a
polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a
thermobarrel extruder, such as Lipex Extruder (Northern Lipids,
Inc). In some cases, the extrusion step can be omitted. Ethanol
removal and simultaneous buffer exchange can be accomplished by,
for example, dialysis or tangential flow filtration. Buffer can be
exchanged with, for example, phosphate buffered saline (PBS) at
about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about
pH 7.2, about pH 7.3, or about pH 7.4.
##STR00020##
[0479] LNP01 formulations are described, e.g., in International
Application Publication No. WO 2008/042973, which is hereby
incorporated by reference.
[0480] Additional exemplary lipid-dsRNA formulations are provided
in the following table.
TABLE-US-00010 TABLE 10 Examplary lipid formulations cationic
lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Cationic
Lipid Lipid:siRNA ratio SNALP 1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA dimethylaminopropane (DLinDMA)
(57.1/7.1/34.4/1.4) lipid: siRNA ~ 7:1 S-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA
[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid: siRNA ~ 7:1 LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxo1ane (XTC) 57.5/7.5/31.5/3.5 lipid: siRNA ~ 6:1 LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1 LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid: siRNA ~ 6:1 LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~ 11:1 LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMG
di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-
Lipid: siRNA 10:1 cyclopenta[d][1,3]dioxo1-5-amine (ALN100) LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG
6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate
(MC3) Lipid:siRNA 10:1 LNP12 1,1'-(2-(4-(2-((2-(bis(2-
C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA
10:1 1-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTC
XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3
MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3
MC3/DSPC/Chol/PEG-DSG/Ga1NAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA:
11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA:
7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA:
10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA:
12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1
LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1
LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA:
7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA:
10:1 DSPC: distearoylphosphatidylcholine DPPC:
dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol
(C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG:
PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of
2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG
with avg mol wt of 2000) SNALP
(1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising
formulations are described in International Publication No.
W02009/127060, filed Apr. 15, 2009, which is hereby incorporated by
reference. XTC comprising formulations are described, e.g., in U.S.
Provisional Serial No. 61/148,366, filed Jan. 29, 2009; U.S.
Provisional Serial No. 61/156,851, filed Mar. 2, 2009; U.S.
Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Serial
No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Serial No.
61/239,686, filed Sep. 3, 2009, and International Application No.
PCT/U52010/022614, filed Jan. 29, 2010, which are hereby
incorporated by reference. MC3 comprising formulations are
described, e.g., in U.S. Provisional Serial No. 61/244,834, filed
Sep. 22, 2009, U.S. Provisional Serial No. 61/185,800, filed Jun.
10, 2009, and International Application No. PCT/US10/28224, filed
Jun. 10, 2010, which are hereby incorporated by reference. ALNY-100
comprising formulations are described, e.g., International patent
application number PCT/U509/63933, filed on Nov. 10, 2009, which is
hereby incorporated by reference. C12-200 comprising formulations
are described in U.S. Provisional Serial No. 61/175,770, filed May
5, 2009 and International Application No. PCT/US10/33777, filed May
5, 2010, which are hereby incorporated by reference.
[0481] Synthesis of Cationic Lipids
[0482] Any of the compounds, e.g., cationic lipids and the like,
used in the nucleic acid-lipid particles featured in the invention
may be prepared by known organic synthesis techniques, including
the methods described in more detail in the Examples. All
substituents are as defined below unless indicated otherwise.
[0483] "Alkyl" means a straight chain or branched, noncyclic or
cyclic, saturated aliphatic hydrocarbon containing from 1 to 24
carbon atoms. Representative saturated straight chain alkyls
include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and
the like; while saturated branched alkyls include isopropyl,
sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.
[0484] Representative saturated cyclic alkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like.
[0485] "Alkenyl" means an alkyl, as defined above, containing at
least one double bond between adjacent carbon atoms. Alkenyls
include both cis and trans isomers. Representative straight chain
and branched alkenyls include ethylenyl, propylenyl, 1-butenyl,
2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,
3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and
the like.
[0486] "Alkynyl" means any alkyl or alkenyl, as defined above,
which additionally contains at least one triple bond between
adjacent carbons. Representative straight chain and branched
alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl,
1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
[0487] "Acyl" means any alkyl, alkenyl, or alkynyl wherein the
carbon at the point of attachment is substituted with an oxo group,
as defined below. For example, --C(.dbd.O)alkyl,
--C(.dbd.O)alkenyl, and --C(.dbd.O)alkynyl are acyl groups.
[0488] "Heterocycle" means a 5- to 7-membered monocyclic, or 7- to
10-membered bicyclic, heterocyclic ring which is either saturated,
unsaturated, or aromatic, and which contains from 1 or 2
heteroatoms independently selected from nitrogen, oxygen and
sulfur, and wherein the nitrogen and sulfur heteroatoms may be
optionally oxidized, and the nitrogen heteroatom may be optionally
quaternized, including bicyclic rings in which any of the above
heterocycles are fused to a benzene ring. The heterocycle may be
attached via any heteroatom or carbon atom. Heterocycles include
heteroaryls as defined below. Heterocycles include morpholinyl,
pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,
hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,
tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
[0489] The terms "optionally substituted alkyl", "optionally
substituted alkenyl", "optionally substituted alkynyl", "optionally
substituted acyl", and "optionally substituted heterocycle" means
that, when substituted, at least one hydrogen atom is replaced with
a substituent. In the case of an oxo substituent (.dbd.O) two
hydrogen atoms are replaced. In this regard, substituents include
oxo, halogen, heterocycle, --CN, --OR.sup.x, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R.sup.x, --C(.dbd.O)OR.sup.x,
--C(.dbd.O)NR.sup.xR.sup.y, --SO.sub.nR.sup.x and
--SO.sub.nNR.sup.xR.sup.y, wherein n is 0, 1 or 2, R.sup.x and
R.sup.y are the same or different and independently hydrogen, alkyl
or heterocycle, and each of said alkyl and heterocycle substituents
may be further substituted with one or more of oxo, halogen, --OH,
--CN, alkyl, --OR.sup.x, heterocycle, --NR.sup.xR.sup.y,
--NR.sup.xC(.dbd.O)R.sup.y, --NR.sup.xSO.sub.2R.sup.y,
--C(.dbd.O)R, --C(.dbd.O)OR, --C(.dbd.O)NR.sup.xR.sup.y,
--SO.sub.nR.sup.x and --SO.sub.nNR.sup.xR.sup.y.
[0490] "Halogen" means fluoro, chloro, bromo and iodo.
[0491] In some embodiments, the methods featured in the invention
may require the use of protecting groups. Protecting group
methodology is well known to those skilled in the art (see, for
example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et
al., Wiley-Interscience, New York City, 1999). Briefly, protecting
groups within the context of this invention are any group that
reduces or eliminates unwanted reactivity of a functional group. A
protecting group can be added to a functional group to mask its
reactivity during certain reactions and then removed to reveal the
original functional group. In some embodiments an "alcohol
protecting group" is used. An "alcohol protecting group" is any
group which decreases or eliminates unwanted reactivity of an
alcohol functional group. Protecting groups can be added and
removed using techniques well known in the art.
[0492] Synthesis of Formula A
[0493] In one embodiments, nucleic acid-lipid particles featured in
the invention are formulated using a cationic lipid of formula
A:
##STR00021##
where R1 and R2 are independently alkyl, alkenyl or alkynyl, each
can be optionally substituted, and R3 and R4 are independently
lower alkyl or R3 and R4 can be taken together to form an
optionally substituted heterocyclic ring. In some embodiments, the
cationic lipid is XTC
(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general,
the lipid of formula A above may be made by the following Reaction
Schemes 1 or 2, wherein all substituents are as defined above
unless indicated otherwise.
##STR00022##
[0494] Lipid A, where R.sub.1 and R.sub.2 are independently alkyl,
alkenyl or alkynyl, each can be optionally substituted, and R.sub.3
and R.sub.4 are independently lower alkyl or R.sub.3 and R.sub.4
can be taken together to form an optionally substituted
heterocyclic ring, can be prepared according to Scheme 1. Ketone 1
and bromide 2 can be purchased or prepared according to methods
known to those of ordinary skill in the art. Reaction of 1 and 2
yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of
formula A. The lipids of formula A can be converted to the
corresponding ammonium salt with an organic salt of formula 5,
where X is anion counter ion selected from halogen, hydroxide,
phosphate, sulfate, or the like.
##STR00023##
[0495] Alternatively, the ketone 1 starting material can be
prepared according to Scheme 2. Grignard reagent 6 and cyanide 7
can be purchased or prepared according to methods known to those of
ordinary skill in the art. Reaction of 6 and 7 yields ketone 1.
Conversion of ketone 1 to the corresponding lipids of formula A is
as described in Scheme 1.
[0496] Synthesis of MC3
[0497] Preparation of DLin-M-C3-DMA (i.e.,
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate) was as follows. A solution of
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),
4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),
4-N,N-dimethylaminopyridine (0.61 g) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53
g) in dichloromethane (5 mL) was stirred at room temperature
overnight. The solution was washed with dilute hydrochloric acid
followed by dilute aqueous sodium bicarbonate. The organic
fractions were dried over anhydrous magnesium sulphate, filtered
and the solvent removed on a rotovap. The residue was passed down a
silica gel column (20 g) using a 1-5% methanol/dichloromethane
elution gradient. Fractions containing the purified product were
combined and the solvent removed, yielding a colorless oil (0.54
g).
[0498] Synthesis of ALNY-100
[0499] Synthesis of ketal 519 [ALNY-100] was performed using the
following scheme 3:
##STR00024##
[0500] Synthesis of 515:
[0501] To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in
200 ml anhydrous THE in a two neck RBF (1 L), was added a solution
of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0.degree. C.
under nitrogen atmosphere. After complete addition, reaction
mixture was warmed to room temperature and then heated to reflux
for 4 h. Progress of the reaction was monitored by TLC. After
completion of reaction (by TLC) the mixture was cooled to 0.degree.
C. and quenched with careful addition of saturated Na2SO4 solution.
Reaction mixture was stirred for 4 h at room temperature and
filtered off. Residue was washed well with THF. The filtrate and
washings were mixed and diluted with 400 mL dioxane and 26 mL conc.
HCl and stirred for 20 minutes at room temperature. The
volatilities were stripped off under vacuum to furnish the
hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR
(DMSO, 400 MHz): .delta.=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m,
1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
[0502] Synthesis of 516:
[0503] To a stirred solution of compound 515 in 100 mL dry DCM in a
250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and
cooled to 0.degree. C. under nitrogen atmosphere. After a slow
addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007
mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room
temperature. After completion of the reaction (2-3 h by TLC)
mixture was washed successively with 1N HCl solution (1.times.100
mL) and saturated NaHCO.sub.3 solution (1.times.50 mL). The organic
layer was then dried over anhyd. Na2SO4 and the solvent was
evaporated to give crude material which was purified by silica gel
column chromatography to get 516 as sticky mass. Yield: 11 g (89%).
1H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.36-7.27 (m, 5H), 5.69 (s,
2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H),
2.30-2.25 (m, 2H). LC-MS [M+H]-232.3 (96.94%).
[0504] Synthesis of 517A and 517B:
[0505] The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a
solution of 220 mL acetone and water (10:1) in a single neck 500 mL
RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492
mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108
mol) in tert-butanol at room temperature. After completion of the
reaction (.about.3 h), the mixture was quenched with addition of
solid Na2SO3 and resulting mixture was stirred for 1.5 h at room
temperature. Reaction mixture was diluted with DCM (300 mL) and
washed with water (2.times.100 mL) followed by saturated
NaHCO.sub.3(1.times.50 mL) solution, water (1.times.30 mL) and
finally with brine (1.times.50 mL). Organic phase was dried over
an.Na2SO4 and solvent was removed in vacuum. Silica gel column
chromatographic purification of the crude material was afforded a
mixture of diastereomers, which were separated by prep HPLC. Yield:
-6 g crude
[0506] 517A--Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400
MHz): .delta.=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H),
4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m,
4H). LC-MS--[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%.
Stereochemistry confirmed by X-ray.
[0507] Synthesis of 518:
[0508] Using a procedure analogous to that described for the
synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained
as a colorless oil. 1H-NMR (CDCl.sub.3, 400 MHz): .delta.=7.35-7.33
(m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75
(m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H),
1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m,
36H), 0.87 (m, 6H). HPLC-98.65%.
[0509] General Procedure for the Synthesis of Compound 519:
[0510] A solution of compound 518 (1 eq) in hexane (15 mL) was
added in a drop-wise fashion to an ice-cold solution of LAH in THF
(1 M, 2 eq). After complete addition, the mixture was heated at
40.degree. C. over 0.5 h then cooled again on an ice bath. The
mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then
filtered through celite and reduced to an oil. Column
chromatography provided the pure 519 (1.3 g, 68%) which was
obtained as a colorless oil. 13C NMR=130.2, 130.1 (.times.2), 127.9
(.times.3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9
(.times.2), 29.7, 29.6 (.times.2), 29.5 (.times.3), 29.3
(.times.2), 27.2 (.times.3), 25.6, 24.5, 23.3, 226, 14.1;
Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.
654.6, Found 654.6.
[0511] Formulations prepared by either the standard or
extrusion-free method can be characterized in similar manners. For
example, formulations are typically characterized by visual
inspection. They should be whitish translucent solutions free from
aggregates or sediment. Particle size and particle size
distribution of lipid-nanoparticles can be measured by light
scattering using, for example, a Malvern Zetasizer Nano ZS
(Malvern, USA). Particles should be about 20-300 nm, such as 40-100
nm in size. The particle size distribution should be unimodal. The
total dsRNA concentration in the formulation, as well as the
entrapped fraction, is estimated using a dye exclusion assay. A
sample of the formulated dsRNA can be incubated with an RNA-binding
dye, such as Ribogreen (Molecular Probes) in the presence or
absence of a formulation disrupting surfactant, e.g., 0.5%
Triton-X100. The total dsRNA in the formulation can be determined
by the signal from the sample containing the surfactant, relative
to a standard curve. The entrapped fraction is determined by
subtracting the "free" dsRNA content (as measured by the signal in
the absence of surfactant) from the total dsRNA content. Percent
entrapped dsRNA is typically >85%. For SNALP formulation, the
particle size is at least 30 nm, at least 40 nm, at least 50 nm, at
least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at
least 100 nm, at least 110 nm, and at least 120 nm. The suitable
range is typically about at least 50 nm to about at least 110 nm,
about at least 60 nm to about at least 100 nm, or about at least 80
nm to about at least 90 nm.
[0512] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. In some embodiments, oral formulations are those
in which dsRNAs featured in the invention are administered in
conjunction with one or more penetration enhancers surfactants and
chelators. Suitable surfactants include fatty acids and/or esters
or salts thereof, bile acids and/or salts thereof. Suitable bile
acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusidate and sodium
glycodihydrofusidate. Suitable fatty acids include arachidonic
acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or
a pharmaceutically acceptable salt thereof (e.g., sodium). In some
embodiments, combinations of penetration enhancers are used, for
example, fatty acids/salts in combination with bile acids/salts.
One exemplary combination is the sodium salt of lauric acid, capric
acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
DsRNAs featured in the invention may be delivered orally, in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. DsRNA complexing agents include
poly-amino acids; polyimines; polyacrylates; polyalkylacrylates,
polyoxethanes, polyalkylcyanoacrylates; cationized gelatins,
albumins, starches, acrylates, polyethyleneglycols (PEG) and
starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,
pollulans, celluloses and starches. Suitable complexing agents
include chitosan, N-trimethylchitosan, poly-L-lysine,
polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE),
polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate),
poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),
DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide,
DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for dsRNAs and their
preparation are described in detail in U.S. Pat. No. 6,887,906, US
Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which
is incorporated herein by reference.
[0513] Compositions and formulations for parenteral,
intraparenchymal (into the brain), intrathecal, intraventricular or
intrahepatic administration may include sterile aqueous solutions
which may also contain buffers, diluents and other suitable
additives such as, but not limited to, penetration enhancers,
carrier compounds and other pharmaceutically acceptable carriers or
excipients.
[0514] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0515] The pharmaceutical formulations featured in the present
invention, which may conveniently be presented in unit dosage form,
may be prepared according to conventional techniques well known in
the pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0516] The compositions featured in the present invention may be
formulated into any of many possible dosage forms such as, but not
limited to, tablets, capsules, gel capsules, liquid syrups, soft
gels, suppositories, and enemas. The compositions may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0517] Additional Formulations
[0518] Emulsions
[0519] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogeneous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,
Popovich N G., and Ansel H C., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;
Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising two immiscible liquid phases intimately
mixed and dispersed with each other. In general, emulsions may be
of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
When an aqueous phase is finely divided into and dispersed as
minute droplets into a bulk oily phase, the resulting composition
is called a water-in-oil (w/o) emulsion. Alternatively, when an
oily phase is finely divided into and dispersed as minute droplets
into a bulk aqueous phase, the resulting composition is called an
oil-in-water (o/w) emulsion. Emulsions may contain additional
components in addition to the dispersed phases, and the active drug
which may be present as a solution in either the aqueous phase,
oily phase or itself as a separate phase. Pharmaceutical excipients
such as emulsifiers, stabilizers, dyes, and anti-oxidants may also
be present in emulsions as needed. Pharmaceutical emulsions may
also be multiple emulsions that are comprised of more than two
phases such as, for example, in the case of oil-in-water-in-oil
(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex
formulations often provide certain advantages that simple binary
emulsions do not. Multiple emulsions in which individual oil
droplets of an o/w emulsion enclose small water droplets constitute
a w/o/w emulsion. Likewise a system of oil droplets enclosed in
globules of water stabilized in an oily continuous phase provides
an o/w/o emulsion.
[0520] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,
Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0521] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,
Popovich N G., and Ansel H C., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker,
Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are
typically amphiphilic and comprise a hydrophilic and a hydrophobic
portion. The ratio of the hydrophilic to the hydrophobic nature of
the surfactant has been termed the hydrophile/lipophile balance
(HLB) and is a valuable tool in categorizing and selecting
surfactants in the preparation of formulations. Surfactants may be
classified into different classes based on the nature of the
hydrophilic group: nonionic, anionic, cationic and amphoteric (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,
Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
285).
[0522] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0523] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0524] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0525] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0526] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (see e.g., Ansel's Pharmaceutical
Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G.,
and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.),
New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 199). Emulsion formulations for oral delivery
have been very widely used because of ease of formulation, as well
as efficacy from an absorption and bioavailability standpoint (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,
Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;
Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins
and high fat nutritive preparations are among the materials that
have commonly been administered orally as o/w emulsions.
[0527] In one embodiment of the present invention, the compositions
of iRNAs and nucleic acids are formulated as microemulsions. A
microemulsion may be defined as a system of water, oil and
amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (see e.g., Ansel's
Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV.,
Popovich N G., and Ansel H C., 2004, Lippincott Williams &
Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions
are systems that are prepared by first dispersing an oil in an
aqueous surfactant solution and then adding a sufficient amount of
a fourth component, generally an intermediate chain-length alcohol
to form a transparent system. Therefore, microemulsions have also
been described as thermodynamically stable, isotropically clear
dispersions of two immiscible liquids that are stabilized by
interfacial films of surface-active molecules (Leung and Shah, in:
Controlled Release of Drugs: Polymers and Aggregate Systems,
Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).
Microemulsions commonly are prepared via a combination of three to
five components that include oil, water, surfactant, cosurfactant
and electrolyte. Whether the microemulsion is of the water-in-oil
(w/o) or an oil-in-water (o/w) type is dependent on the properties
of the oil and surfactant used and on the structure and geometric
packing of the polar heads and hydrocarbon tails of the surfactant
molecules (Schott, in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 271).
[0528] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions (see
e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery
Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,
Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;
Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0529] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0530] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802;
7,157,099; Constantinides et al., Pharmaceutical Research, 1994,
11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993,
13, 205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (see e.g., U.S.
Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099;
Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho
et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions
may form spontaneously when their components are brought together
at ambient temperature. This may be particularly advantageous when
formulating thermolabile drugs, peptides or iRNAs. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of iRNAs and nucleic acids from the gastrointestinal
tract, as well as improve the local cellular uptake of iRNAs and
nucleic acids.
[0531] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
iRNAs and nucleic acids of the present invention. Penetration
enhancers used in the microemulsions of the present invention may
be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0532] Penetration Enhancers
[0533] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly iRNAs, to the skin of animals. Most drugs are
present in solution in both ionized and nonionized forms. However,
usually only lipid soluble or lipophilic drugs readily cross cell
membranes. It has been discovered that even non-lipophilic drugs
may cross cell membranes if the membrane to be crossed is treated
with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers
also enhance the permeability of lipophilic drugs.
[0534] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (see
e.g., Malmsten, M. Surfactants and polymers in drug delivery,
Informa Health Care, New York, N.Y., 2002; Lee et al., Critical
Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of
the above mentioned classes of penetration enhancers are described
below in greater detail.
[0535] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of iRNAs through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.
Surfactants and polymers in drug delivery, Informa Health Care, New
York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, p. 92); and perfluorochemical emulsions,
such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40,
252).
[0536] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-20 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC
Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El
Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
[0537] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug
delivery, Informa Health Care, New York, N.Y., 2002; Brunton,
Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of
Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York,
1996, pp. 934-935). Various natural bile salts, and their synthetic
derivatives, act as penetration enhancers. Thus the term "bile
salts" includes any of the naturally occurring components of bile
as well as any of their synthetic derivatives. Suitable bile salts
include, for example, cholic acid (or its pharmaceutically
acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium
dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic
acid (sodium glucholate), glycholic acid (sodium glycocholate),
glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid
(sodium taurocholate), taurodeoxycholic acid (sodium
taurodeoxycholate), chenodeoxycholic acid (sodium
chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium
tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate
and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M.
Surfactants and polymers in drug delivery, Informa Health Care, New
York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug
Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:
Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack
Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,
1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25;
Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
[0538] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of iRNAs through the mucosa is enhanced.
With regards to their use as penetration enhancers in the present
invention, chelating agents have the added advantage of also
serving as DNase inhibitors, as most characterized DNA nucleases
require a divalent metal ion for catalysis and are thus inhibited
by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
Suitable chelating agents include but are not limited to disodium
ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g.,
sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl
derivatives of collagen, laureth-9 and N-amino acyl derivatives of
.beta.-diketones (enamines)(see e.g., Katdare, A. et al., Excipient
development for pharmaceutical, biotechnology, and drug delivery,
CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,
Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7,
1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
[0539] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This
class of penetration enhancers include, for example, unsaturated
cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0540] Agents that enhance uptake of iRNAs at the cellular level
may also be added to the pharmaceutical and other compositions of
the present invention. For example, cationic lipids, such as
lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (Lollo et al., PCT Application WO 97/30731), are also
known to enhance the cellular uptake of dsRNAs. Examples of
commercially available transfection reagents include, for example
Lipofectamine.TM. (Invitrogen; Carlsbad, Calif.), Lipofectamine
2000.TM. (Invitrogen; Carlsbad, Calif.), 293fectin.TM. (Invitrogen;
Carlsbad, Calif.), Cellfectin.TM. (Invitrogen; Carlsbad, Calif.),
DMRIE-C.TM. (Invitrogen; Carlsbad, Calif.), FreeStyle.TM. MAX
(Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. 2000 CD
(Invitrogen; Carlsbad, Calif.), Lipofectamine.TM. (Invitrogen;
Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.),
Oligofectamine.TM. (Invitrogen; Carlsbad, Calif.), Optifect.TM.
(Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent
(Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal
Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER
Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or
Fugene (Grenzacherstrasse, Switzerland), Transfectam.RTM. Reagent
(Promega; Madison, Wis.), TransFast.TM. Transfection Reagent
(Promega; Madison, Wis.), Tfx.TM.-20 Reagent (Promega; Madison,
Wis.), Tfx.TM.-50 Reagent (Promega; Madison, Wis.), DreamFect.TM.
(OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences;
Marseille, France), TransPass.sup.a D1 Transfection Reagent (New
England Biolabs; Ipswich, Mass., USA), LyoVec.TM./LipoGen.TM.
(Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent
(Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection
Reagent (Genlantis; San Diego, Calif., USA), GenePORTER
Transfection reagent (Genlantis; San Diego, Calif., USA),
GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif.,
USA), Cytofectin Transfection Reagent (Genlantis; San Diego,
Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San
Diego, Calif., USA), TroganPORTER.TM. transfection Reagent
(Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton,
Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR
(B-Bridge International; Mountain View, Calif., USA), SureFECTOR
(B-Bridge International; Mountain View, Calif., USA), or HiFect.TM.
(B-Bridge International, Mountain View, Calif., USA), among
others.
[0541] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0542] Carriers
[0543] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extracirculatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate dsRNA in hepatic tissue can be reduced
when it is coadministered with polyinosinic acid, dextran sulfate,
polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA
& Nucl. Acid Drug Dev., 1996, 6, 177-183.
[0544] Excipients
[0545] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc.).
[0546] Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0547] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0548] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0549] Other Components
[0550] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0551] Aqueous suspensions may contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0552] In some embodiments, pharmaceutical compositions featured in
the invention include (a) one or more iRNA compounds and (b) one or
more biologic agents which function by a non-RNAi mechanism.
Examples of such biologic agents include agents that interfere with
an interaction of ALAS1 and at least one ALAS1 binding partner.
[0553] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds that exhibit
high therapeutic indices are typical.
[0554] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of compositions featured in the invention lies
generally within a range of circulating concentrations that include
the ED50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods
featured in the invention, the therapeutically effective dose can
be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range of the compound or, when appropriate, of the
polypeptide product of a target sequence (e.g., achieving a
decreased concentration of the polypeptide) that includes the IC50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0555] In addition to their administration, as discussed above, the
iRNAs featured in the invention can be administered in combination
with other known agents effective in treatment of diseases or
disorders related to ALAS1 expression. In any event, the
administering physician can adjust the amount and timing of iRNA
administration on the basis of results observed using standard
measures of efficacy known in the art or described herein.
[0556] Methods for Treating Diseases Related to Expression of an
ALAS1 Gene
[0557] The invention relates in particular to the use of an iRNA
targeting ALAS1 to inhibit ALAS1 expression and/or to treat a
disease, disorder, or pathological process that is related to ALAS1
expression.
[0558] As used herein, "a disorder related to ALAS1 expression," a
"disease related to ALAS1 expression, a "pathological process
related to ALAS1 expression," or the like includes any condition,
disorder, or disease in which ALAS1 expression is altered (e.g.,
elevated), the level of one or more porphyrins is altered (e.g.,
elevated), the level or activity of one or more enzymes in the heme
biosynthetic pathway (porphyrin pathway) is altered, or other
mechanisms that lead to pathological changes in the heme
biosynthetic pathway. For example, an iRNA targeting an ALAS1 gene,
or a combination thereof, may be used for treatment of conditions
in which levels of a porphyrin or a porphyrin precursor (e.g., ALA
or PBG) are elevated (e.g., certain porphyrias), or conditions in
which there are defects in the enzymes of the heme biosynthetic
pathway (e.g., certain porphyrias). Disorders related to ALAS1
expression include, for example, X-linked sideroblastic anemia
(XLSA), ALA deyhdratase deficiency porphyria (Doss porphyria),
acute intermittent porphyria (AIP), congenital erythropoietic
porphyria, prophyria cutanea tarda, hereditary coproporphyria
(coproporphyria), variegate porphyria, erythropoietic
protoporphyria (EPP), and transient erythroporphyria of
infancy.
[0559] As used herein, a "subject" to be treated according to the
methods described herein, includes a human or non-human animal,
e.g., a mammal. The mammal may be, for example, a rodent (e.g., a
rat or mouse) or a primate (e.g., a monkey). In some embodiments,
the subject is a human.
[0560] In some embodiments, the subject is suffering from a
disorder related to ALAS1 expression (e.g., has been diagnosed with
a porphyria or has suffered from one or more symptoms of porphyria
and is a carrier of a mutation associated with porphyria) or is at
risk of developing a disorder related to ALAS1 expression (e.g., a
subject with a family history of porphyria, or a subject who is a
carrier of a genetic mutation associated with porphyria).
[0561] Classifications of porphyrias, including acute hepatic
porphyrias, are described, e.g., in Balwani, M. & Desnick, R.
J., Blood, 120(23), published online as Blood First Edition paper,
July 12, 102; DOI 10.1182/blood-2012-05-423186. As described in
Balwain & Desnick, acute intermittent porphyria (AIP)
hereditary coproporphyria (HCP), variegate porphyria (VP) are
autosomal dominant porphyrias and ALA deyhdratase deficiency
porphyria (ADP) is autosomal recessive. In rare cases, AIP, HCP,
and VP occur as homozygous dominant forms. In addition, there is a
rare homozygous recessive form of porphyria cutanea tarda (PCT),
which is the single hepatic cutaneous porphyria, and is also known
as hepatoerythropoietic porphyria. The clinical and laboratory
features of these porphyrias are described in Table 11 below.
TABLE-US-00011 TABLE 11 Human hepatic porphyrias: clinical and
laboratory features Enzyme Principal activity, Deficient symptoms,
% of Increased porphyrin precursors and/or porphyrins* Porphyria
enzyme Inheritance NV or CP normal Erythrocytes Urine Stool Acute
hepatic porphyrias ADP ALA- AR NV ~5 Zn-protoporphyrin ALA, --
dehydratase coproporphyrin III AIP HMB- AD NV ~50 -- ALA, PBG, --
synthase uroporphyrin HCP COPRO- AD NV and CP ~50 -- ALA, PBG,
coproporphyrin oxidase coproporphyrin III III VP PROTO- AD NV and
CP ~50 -- ALA, PBG coproporphyrin oxidase coproporphyrin III, III
protoporphyrin Hepatic cutaneous porphyrias PCT URO- Sporadic or CP
<20 -- uroporphyrin, uroporphyrin, 7- decarboxylase AD
7-carboxylate carboxylate porphyrin porphyrin AR indicates
autosomal recessive; AD, autosomal dominant; NV, neurovisceral; CP,
cutaneous photosensitivity; and --, not applicable. *Increases that
may be important for diagnosis.
[0562] In some embodiments, the subject has or is at risk for
developing a porphyria, e.g., a hepatic porphyria, e.g., AIP, HCP,
VP, ADP, or hepatoerythropoietic porphyria.
[0563] In some embodiments, the porphyria is an acute hepatic
porphyria, e.g., an acute hepatic porphyria is selected from acute
intermittent porphyria (AIP), hereditary coproporphyria (HCP),
variegate porphyria (VP), and ALA deyhdratase deficiency porphyria
(ADP).
[0564] In some embodiments, the porphyria is a dual porphyria,
e.g., at least two porphyrias. In some embodiments, the dual
porphyria comprises two or more porphyrias selected from acute
intermittent porphyria (AIP) hereditary coproporphyria (HCP),
variegate porphyria (VP), and ALA deyhdratase deficiency porphyria
(ADP).
[0565] In some embodiments, the porphyria is a homozygous dominant
hepatic porphyria (e.g., homozygous dominant AIP, HCP, or VP) or
hepatoerythropoietic porphyria, In some embodiments, the porphyria
is AIP, HCP, VP, or hepatoerythropoietic porphyria, or a
combination thereof (e.g., a dual porphyria). In embodiments, the
AIP, HCP, or VP is either heterozygous dominant or homozygous
dominant.
[0566] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., ADP, and shows an elevated level (e.g., an
elevated urine level) of ALA and/or coproporphyrin III. In
embodiments, the subject has or is at risk for developing a
porphyria, e.g., ADP, and shows an elevated level of erythrocyte
Zn-protoporphyrin.
[0567] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., AP, and shows an elevated level (e.g., an
elevated urine level) of ALA, PBG, and/or uroporphyrin.
[0568] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., HCP, and shows an elevated level (e.g., an
elevated urine level) of ALA, PBG, and/or coproporphyrin III. In
embodiments, the subject has or is at risk for developing a
porphyria, e.g., HCP, and shows an elevated level (e.g., an
elevated stool level) of coproporphyrin III.
[0569] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., VP, and shows an elevated level (e.g., an
elevated urine level) of ALA, PBG, and/or coproporphyrin III.
[0570] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., HCP, and shows an elevated level (e.g., an
elevated stool level) of coproporphyrin III and/or
protoporphyrin.
[0571] In embodiments, the subject has or is at risk for developing
a porphyria, e.g., PCT, (e.g., hepatoerythropoietic porphyria) and
shows an elevated level (e.g., an elevated urine level) of
uroporphyrin and/or 7-carboxylate porphyrin. In embodiments, the
subject has or is at risk for developing a porphyria, e.g., PCT,
(e.g., hepatoerythropoietic porphyria) and shows an elevated level
(e.g., an elevated stool level) of uroporphyrin and/or
7-carboxylate porphyrin.
[0572] A mutation associated with porphyria includes any mutation
in a gene encoding an enzyme in the heme biosynthetic pathway
(porphyrin pathway) or a gene which alters the expression of a gene
in the heme biosynthetic pathway. In many embodiments, the subject
carries one or more mutations in an enzyme of the porphyrin pathway
(e.g., a mutation in ALA dehydratase or PBG deaminase). In some
embodiments, the subject is suffering from an acute porphyria
(e.g., AIP, ALA deydratase deficiency porphyria).
[0573] In some cases, patients with an acute hepatic porphyria
(e.g., AIP), or patients who carry mutations associated with an
acute hepatic porphyria (e.g., AIP) but who are asymptomatic, have
elevated ALA and/or PBG levels compared with healthy individuals.
See, e.g., Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707,
2006; Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349,
2007. In such cases, the level of ALA and/or PBG can be elevated
even when the patient is not having, or has never had, an attack.
In some such cases, the patient is otherwise completely
asymptomatic. In some such cases, the patient suffers from pain,
e.g., neuropathic pain, which can be chronic pain (e.g., chronic
neuropathic pain). In some cases, the patient has a neuropathy. In
some cases, the patient has a progressive neuropathy.
[0574] In some embodiments, the subject to be treated according to
the methods described herein has an elevated level of a porphyrin
or a porphyrin precursor, e.g., ALA and/or PBG. Levels of a
porphyrin or a porphyrin precursor can be assessed using methods
known in the art or methods described herein. For example, methods
of assessing using and plasma ALA and PBG levels, as well as urine
and plasma porphyrin levels, are disclosed in Floderus, Y. et al,
Clinical Chemistry, 52(4): 701-707, 2006; and Sardh et al.,
Clinical Pharmacokinetics, 46(4): 335-349, 2007, the entire
contents of which are hereby incorporated in their entirety.
[0575] In some embodiments, the subject is an animal model of a
porphyria, e.g., a mouse model of a porphyria (e.g., a mutant mouse
as described in Lindberg et al. Nature Genetics, 12: 195-199,
1996). In some embodiments, the subject is a human, e.g., a human
who has or is at risk for developing a porphyria, as described
herein. In some embodiments, the subject is not having an acute
attack of porphyria. In some embodiments, the subject has never had
an attack. In some embodiments, the patient suffers from chronic
pain. In some embodiments, the patient has nerve damage. In
embodiments, the subject has EMG changes and/or changes in nerve
conduction velocity. In some embodiments, the subject is
asymptomatic. In some embodiments, the subject is at risk for
developing a porphyria (e.g., carries a gene mutation associated
with a porphyria) and is asymptomatic. In some embodiments, the
subject has previously had an acute attack but is asymptomatic at
the time of treatment.
[0576] In some embodiments, the subject is at risk for developing a
porphyria and is treated prophylactically to prevent the
development of a porphyria. In some embodiments the subject has an
elevated level of a porphyrin or a porphyrin precursor, e.g., ALA
and/or PBG. In some embodiments, the prophylactic treatment begins
at puberty. In some embodiments the treatment lowers the level
(e.g., the plasma level or the urine level) of a porphyrin or a
porphyrin precursor, e.g., ALA and/or PBG. In some embodiments, the
treatment prevents the development of an elevated level of a
porphyrin or a porphyrin precursor, e.g., ALA and/or PBG. In some
embodiments, the treatment prevents the development of, or
decreases the frequency or severity of, a symptom associated with a
porphyria, e.g., pain or nerve damage.
[0577] In some embodiments, the level of a porphyrin or a porphyrin
precursor, e.g., ALA or PBG, is elevated, e.g., in a sample of
plasma or urine from the subject. In some embodiments, the level of
a porphyrin or a porphyrin precursor, e.g., ALA or PBG, in the
subject is assessed based on the absolute level of the porphyrin or
the porphyrin precursor, e.g., ALA or PBG in a sample from the
subject. In some embodiments, the level of a porphyrin or a
porphyrin precursor, e.g., ALA or PBG, in the subject is assessed
based on the relative level of the porphyrin or porphyrin
precursor, e.g., ALA or PBG, in a sample from the subject. In some
embodiments, the relative level is relative to the level of another
protein or compound, e.g., the level of creatinine, in a sample
from the subject. In some embodiments, the sample is a urine
sample. In some embodiments, the sample is a plasma sample. In some
embodiments, the sample is a stool sample.
[0578] An elevated level of a porphyrin or a porphyrin precursor,
e.g., ALA and/or PBG, can be established, e.g., by showing that the
subject has a level of a porphyrin or a porphyrin precursor, e.g.,
ALA and/or PBG (e.g., a plasma or urine level of ALA and/or PBG)
that is greater than, or greater than or equal to, a reference
value. A physician with expertise in the treatment of porphyrias
would be able to determine whether the level of a porphyrin or a
porphyrin precursor, (e.g., ALA and/or PBG) is elevated, e.g., for
the purpose of diagnosing a porphyria or for determining whether a
subject is at risk for developing a porphyria, e.g., a subject may
be predisposed to an acute attack or to pathology associated with a
porphyria, such as, e.g., chronic pain (e.g., neuropathic pain) and
neuropathy (e.g., progressive neuropathy).
[0579] As used herein, a "reference value" refers to a value from
the subject when the subject is not in a disease state, or a value
from a normal or healthy subject, or a value from a reference
sample or population, e.g., a group of normal or healthy subjects
(e.g., a group of subjects that does not carry a mutation
associated with a porphyria and/or a group of subjects that does
not suffer from symptoms associated with a porphyria).
[0580] In some embodiments, the reference value is a pre-disease
level in the same individual. In some embodiments, the reference
value is a level in a reference sample or population. In some
embodiments, the reference value is the mean or median value in a
reference sample or population. In some embodiments, the reference
value the value that is is two standard deviations above the mean
in a reference sample or population. In some embodiments, the
reference value is the value that is 2.5, 3, 3.5, 4, 4.5, or 5
standard deviations above the mean in a reference sample or
population.
[0581] In some embodiments, wherein the subject has an elevated
level of a porphyrin or a porphyrin precursor, e.g., ALA and/or
PBG, the subject has a level of ALA and/or PBG that is at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher than a
reference value. In some embodiments, the subject has a level of a
porphyrin or a porphyrin precursor, e.g., ALA and/or PBG, that is
at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold higher than a reference
value.
[0582] In some embodiments, the reference value is an upper
reference limit. As used herein, an "upper reference limit" refers
to a level that is the upper limit of the 95% confidence interval
for a reference sample or population, e.g., a group of normal
(e.g., wild type) or healthy individuals, e.g., individuals who do
not carry a genetic mutation associated with a porphyria and/or
individuals who do not suffer from a porphyria. Accordingly, a
lower reference limit refers to a level that is the lower limit of
the same 95% confidence interval.
[0583] In some embodiments wherein the subject has an elevated
level, e.g., a plasma level or a urine level, of a porphyrin or a
porphyrin precursor, e.g., ALA or PBG, the level is greater than or
equal to 2 times, 3 times, 4 times, or 5 times that of a reference
value, e.g., an upper reference limit. In some embodiments, the
subject has a urine level of a porphyrin or a porphyrin precursor,
e.g., ALA or PBG, that is greater than 4 times that of an upper
reference limit.
[0584] In some embodiments, the reference value is a value provided
in Floderus, Y. et al, Clinical Chemistry, 52(4): 701-707, 2006 or
Sardh et al., Clinical Pharmacokinetics, 46(4): 335-349, 2007. In
some embodiments, the reference value is a value provided in Table
1 of Sardh et al.
[0585] In some embodiments, the subject is a human and has a urine
level of PBG that is greater than or equal to 4.8 mmol/mol
creatinine. In certain embodiments, the subject is a human and has
a urine level of PBG that is greater than, or greater than or equal
to, about 3, 4, 5, 6, 7, or 8 mmol/mol creatinine.
[0586] In embodiments, the reference value for plasma PBG is 0.12
.mu.mol/L. In embodiments, the subject is a human and has a plasma
PBG level that is greater than, or greater than or equal to, 0.10
.mu.mol/L, 0.12 .mu.mol/L, 0.24 .mu.mol/L, 0.36 .mu.mol/L, 0.48
.mu.mol/L, or 0.60 .mu.mol/L. In embodiments, the subject is a
human and has a plasma level of PBG that is greater than, or
greater than or equal to, 0.48 .mu.mol/L.
[0587] In embodiments, the reference value for urine PBG is 1.2
mmol/mol creatinine. In embodiments, the subject is a human and has
a urine PBG level that is greater than, or greater than or equal
to, 1.0 mmol/mol creatinine, 1.2 mmol/mol creatinine, 2.4 mmol/mol
creatinine, 3.6 mmol/mol creatinine, 4.8 mmol/mol creatinine, or
6.0 mmol/mol creatinine. In embodiments, the subject is a human and
has a urine level of PBG that is greater than, or greater than or
equal to, 4.8 mmol/mol creatinine.
[0588] In embodiments, the reference value for plasma ALA is 0.12
.mu.mol/L. In embodiments, the subject is a human and has a plasma
ALA level that is greater than, or greater than or equal to, 0.10
.mu.mol/L, 0.12 .mu.mol/L, 0.24 .mu.mol/L, 0.36 .mu.mol/L, 0.48
.mu.mol/L, or 0.60 .mu.mol/L. In embodiments, the subject is a
human and has a plasma ALA level that is greater than, or greater
than or equal to 0.48 .mu.mol/L.
[0589] In embodiments, the reference value for urine ALA is 3.1
mmol/mol creatinine. In embodiments, the subject is a human and has
a urine ALA level that is greater than, or greater than or equal
to, 2.5 mmol/mol creatinine, 3.1 mmol/mol creatinine, 6.2 mmol/mol
creatinine, 9.3 mmol/mol creatinine, 12.4 mmol/mol creatinine, or
15.5 mmol/mol creatinine.
[0590] In embodiments, the reference value for plasma porphyrin is
10 nmol/L. In embodiments, the subject is a human and has a plasma
porphyrin level that is greater than, or greater than or equal to,
10 nmol/L. In embodiments, the subject is a human and has a plasma
porphyrin level that is greater than, or greater than or equal to,
8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nmol/L. the subject is a
human and has a plasma porphyrin level that is greater than, or
greater than or equal to 40 nmol/L. In embodiments, the reference
value for urine porphyrin is 25 .mu.mol/mol creatinine. In
embodiments, the subject is a human and has a urine porphyrin level
that is greater than, or greater than or equal to, 25 .mu.mol/mol
creatinine. In embodiments, the subject is a human and has a urine
porphyrin level that is greater than, or equal to, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, or 80 .mu.mol/mol creatinine.
[0591] In some embodiments, the subject has a level, e.g., a plasma
level or a urine level, of a porphyrin or a porphyrin precursor,
e.g., ALA or PBG, that is greater than that of 99% of individuals
in a sample of healthy individuals.
[0592] In some embodiments, the subject has a level, e.g., a plasma
level or a urine level, of ALA or PBG that is greater than two
standard deviations above the mean level in a sample of healthy
individuals.
[0593] In some embodiments, the subject has a urine level of ALA
that is 1.6 or more times that of the mean level in a normal
subject (e.g., a subject that does not carry a mutation associated
with a porphyria). In some embodiments, the subject has a plasma
level of ALA that is 2 or 3 times that of the mean level in a
normal subject. In some embodiments, the subject has a urine level
of PBG that is four or more times that of the mean level in a
normal subject. In some embodiments, the subject has a plasma level
of PBG that is four or more times that of the mean level in a
normal subject.
[0594] In some embodiments, the method is effective to decrease the
level of a porphyrin or a porphyrin precursor, e.g., ALA and/or
PBG. In embodiments, the method is effective to produce a
predetermined reduction in the elevated level of the porphyrin or
porphyrin precursor, e.g., ALA or PBG. In some embodiments, the
predetermined reduction is a decrease of at least 10%, 20%, 30%,
40%, or 50%. In some embodiments, the predetermined reduction is a
reduction that is effective to prevent or ameliorate symptoms,
e.g., pain or recurring attacks.
[0595] In some embodiments, the predetermined reduction is a
reduction that is at least 1, 2, 3, or more standard deviations,
wherein the standard deviation is determined based on the values
from a reference sample, e.g., a reference sample as described
herein.
[0596] In some embodiments, the predetermined reduction is a
reduction that brings the level of the porphyrin or porphyrin
precursor to a level that is less than, or to a level that is less
than or equal to, a reference value (e.g., a reference value as
described herein).
[0597] In some embodiments, the subject to be treated according to
the methods described suffers from pain, e.g., chronic pain. In
some embodiments, the subject has or is at risk for developing a
porphyria, e.g. an acute hepatic porphyria, e.g., AIP. In
embodiments, the method is effective to treat the pain, e.g., by
reducing the severity of the pain or curing the pain. In
embodiments, the method is effective to decrease or prevent nerve
damage.
[0598] In some embodiments, the subject to be treated according to
the methods described herein (a) has an elevated level of ALA
and/or PBG and (b) suffers from pain, e.g., chronic pain. In
embodiments, the method is effective to decrease an elevated level
of ALA and/or PBG and/or to treat the pain, e.g., by reducing the
severity of the pain or curing the pain.
[0599] In some embodiments, the subject is an animal that serves as
a model for a disorder related to ALAS1 expression.
[0600] In some embodiments the subject is an animal that serves as
a model for porphyria (e.g., a genetically modified animal with one
or more mutations. In some embodiments, the porphyria is AIP and
the subject is an animal model of AIP. In one such embodiment, the
subject is a genetically modified mouse that is deficient in
porphobilinogen deaminase, such as, for example, the mouse
described in Lindberg et al., Nature Genetics, 12:195-199, 1996, or
the homozygous R167Q mouse described in Yasuda, M., Yu, C. Zhang,
J., Clavero, S., Edelmann, W., Gan, L., Phillips, J. D., &
Desnick, R. J. Acute intermittent porphyria: A severely affected
knock-in mouse that mimics the human homozygous dominant phenotype.
(Abstract of Presentation on Oct. 14, 2011 at the American Society
of Human Genetics; Program No. 1308F; accessed online on Apr. 4,
2012 at ichg2011.org/cgi-bin/showdetail.pl?absno=21167); both of
these references are hereby incorporated herein in their entirety.
Several knock-in models for mutations causing homozygous dominant
AIP in humans have been generated. The mutations employed include,
e.g., R167Q, R173Q, and R173W in PBG deaminase. Viable homozygotes
included the R167Q/R176Q and R167Q/R173Q, both of which exhibit
constitutively elevated ALA and PBG levels analogous to the
phenotype in human homozygous dominant AIP; in some embodiments,
such a viable homozygous ATP mouse model is the subject.
[0601] In one embodiment, a subject to be treated according to the
methods described herein, (e.g., a human subject or patient), is at
risk of developing, or has been diagnosed, with a disorder related
to ALAS1 expression, e.g. a porphyria. In some embodiments, the
subject is a subject who has suffered one or more acute attacks of
one or more porphyric symptoms. In other embodiments, the subject
is a subject who has suffered chronically from one or more symptoms
of porphyria (e.g., pain, e.g., neuropathic pain and or neuropathy,
e.g., progressive neuropathy). In some embodiments, the subject
carries a genetic alteration (e.g., a mutation) as described herein
but is otherwise asymptomatic. In some embodiments, the subject has
previously been treated with a heme product (e.g., hemin, heme
arginate, or heme albumin), as described herein.
[0602] In some embodiments, a subject (e.g., a subject with a
porphyria, such as, e.g., AP) to be treated according to the
methods described herein has recently experienced or is currently
experiencing a prodrome. In some such embodiments, the subject is
administered a combination treatment, e.g., an iRNA as described
herein, and one or more additional treatments known to be effective
against porphyria (e.g., glucose and/or a heme product such as
hemin, as described herein) or its associated symptoms.
[0603] In one embodiment, an iRNA as described herein is
administered in combination with glucose or dextrose. For example,
10-20% dextrose in normal saline may be provided intravenously.
Typically, when glucose is administered, at least 300 g of 10%
glucose is administered intravenously daily. The iRNA (e.g., an
iRNA in an LNP formulation) may also be administered intravenously,
as part of the same infusion that is used to administer the glucose
or dextrose, or as a separate infusion that is administered before,
concurrently, or after the administration of the glucose or
dextrose. In some embodiments, the iRNA is administered via a
different route of administration (e.g., subcutaneously). In yet
another embodiment, the iRNA is administered in combination with
total parenteral nutrition. The iRNA may be administered before,
concurrent with, or after the administration of total parenteral
nutrition.
[0604] In one embodiment, the iRNA is administered in combination
with a heme product (e.g., hemin, heme arginate, or heme albumin).
In a further embodiment, the iRNA is administered in combination
with a heme product and glucose, a heme product and dextrose, or a
heme product and total parenteral nutrition.
[0605] A "prodrome," as used herein, includes any symptom that the
individual subject has previously experienced immediately prior to
developing an acute attack. Typical symptoms of a prodrome include,
e.g., abdominal pain, nausea, headaches, psychological symptoms
(e.g., anxiety), restlessness and/or insomnia. In some embodiments,
the subject experiences pain (e.g., abdominal pain and/or a
headache) during the prodrome. In some embodiments, the subject
experiences nausea during the prodrome. In some embodiments, the
subject experiences psychological symptoms (e.g., anxiety) during
the prodrome. In some embodiments, the subject becomes restless
and/or suffers from insomnia during the prodrome.
[0606] An acute "attack" of porphyria involves the onset of one or
more symptoms of porphyria, typically in a patient who carries a
mutation associated with porphyria (e.g., a mutation in a gene that
encodes an enzyme in the porphyrin pathway).
[0607] In certain embodiments, administration of an ALAS1 iRNA
results in a decrease in the level of one or more porphyrins or
porphyrin precursors, as described herein (e.g., ALA and/or PBG).
The decrease may be measured relative to any appropriate control or
reference value. For example, the decrease in the level of one or
more porphyrins or porphyrin precursors may be established in an
individual subject, e.g., as a decrease of at least 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50% or more compared with the level
prior to treatment (e.g., immediately prior to treatment). A
decrease in the level of a porphyrin precursor, a porphyrin, or or
a porphyrin metabolite may be measured using any method known in
the art. For example, the level of PBG and/or ALA in urine or
plasma may be assessed, using the Watson-Schwartz test, ion
exchange chromatography, or high-performance liquid
chromatography-mass spectrometry. See, e.g., Thunell (1993).
[0608] In some embodiments, administration of an ALAS1 siRNA is
effective to reduce the level of ALA and/or PBG in the subject. The
level of ALA or PBG in the subject can be assessed, e.g., based on
the absolute level of ALA or PBG, or based on the relative level of
ALA or PBG (e.g., relative to the level of another protein or
compound, e.g., the level of creatinine) in a sample from the
subject. In some embodiments, the sample is a urine sample. In some
embodiments, the sample is a plasma sample.
[0609] In certain embodiments, an iRNA that targets ALAS1 is
administered in combination one or more additional treatments,
e.g., another treatment known to be effective in treating porphyria
or symptoms of porphyria. For example, the other treatment may be
glucose (e.g., IV glucose) or a heme product (e.g., hemin, heme
arginate, or heme albumin). The additional treatment(s) may be
administered before, after, or concurrent with the administration
of iRNA.
[0610] The iRNA and an additional therapeutic agent can be
administered in combination in the same composition, e.g.,
intravenously, or the additional therapeutic agent can be
administered as part of a separate composition or by another method
described herein.
[0611] In some embodiments, administration of iRNA, or
administration of iRNA in combination one or more additional
treatments (e.g., glucose, dextrose or the like), decreases the
frequency of acute attacks (e.g., by preventing acute attacks so
that they no longer occur, or by reducing the number of attacks
that occur in a certain time period, e.g., fewer attacks occur per
year). In some such embodiments, the iRNA is administered according
to a regular dosing regimen, e.g., daily, weekly, biweekly, or
monthly.
[0612] In some embodiments, the iRNA is administered after an acute
attack of porphyria. In some such embodiments, the iRNA is in a
composition, e.g. a composition comprising a lipid formulation,
e.g. an LNP formulation.
[0613] In some embodiments, the iRNA is administered during an
acute attack of porphyria. In some such embodiments, the iRNA is in
a composition, e.g. a composition comprising a lipid formulation
(e.g., an LNP formulation) or a composition comprising a GalNAc
conjugate.
[0614] In some embodiments, administration of an ALAS1 siRNA is
effective to lessen the severity of the attack (e.g., by
ameliorating one or more signs or symptoms associated with the
attack). In some embodiments, administration of an ALAS1 siRNA is
effective to shorten the duration of an attack. In some
embodiments, administration of an ALAS1 siRNA is effective to stop
an attack. In some embodiments, the iRNA is administered
prophylactically to prevent an acute attack of porphyria. In some
such embodiments, the iRNA is in the form of a GalNAc conjugate,
e.g., in a composition comprising a GalNAc conjugate. In some
embodiments, the prophylactic administration is before, during, or
after exposure to or occurrence of a precipitating factor. In some
embodiments, the subject is at risk of developing porphyria.
[0615] In some embodiments, the siRNA is administered during a
prodrome. In some embodiments, the prodrome is characterized by
pain (e.g., headache and/or abdominal pain), nausea, psychological
symptoms (e.g., anxiety), restlessness and/or insomnia.
[0616] In some embodiments, the siRNA is administered during a
particular phase of the menstrual cycle, e.g., during the luteal
phase.
[0617] In some embodiments, administration of an ALAS1 siRNA is
effective to prevent attacks (e.g., recurrent attacks that are
associated with a prodrome and/or with a precipitating factor,
e.g., with a particular phase of the menstrual cycle, e.g., the
luteal phase). In some embodiments, administration of an ALAS1
siRNA is effective to reduce the frequency of attacks. In
embodiments, administration of an ALAS1 siRNA is effective to
lessen the severity of the attack (e.g., by ameliorating one or
more signs or symptoms associated with the attack). In some
embodiments, administration of an ALAS1 siRNA is effective to
shorten the duration of an attack. In some embodiments,
administration of an ALAS1 siRNA is effective to stop an
attack.
[0618] In some embodiments administration of an ALAS1 siRNA is
effective to prevent or decrease the frequency or severity of pain,
e.g., neuropathic pain.
[0619] In some embodiments administration of an ALAS1 siRNA is
effective to prevent or decrease the frequency or severity of
neuropathy
[0620] Effects of administration of an ALAS1 siRNA can be
established, for example, by comparison with an appropriate
control. For example, a decrease in the frequency of acute attacks,
as well as a decrease in the level of one or more porphyrins or
porphyrin precursors, may be established, for example, in a group
of patients with AIP, as a decreased frequency compared with an
appropriate control group. A control group (e.g., a group of
similar individuals or the same group of individuals in a crossover
design) may include, for example, an untreated population, a
population that has been treated with a conventional treatment for
porphyria (e.g., a conventional treatment for AIP may include
glucose, hemin, or both); a population that has been treated with
placebo, or a non-targeting iRNA, optionally in combination with
one or more conventional treatments for porphyria (e.g., glucose,
e.g., IV glucose), and the like.
[0621] A subject "at risk" of developing porphyria, as used herein,
includes a subject with a family history of porphyria and/or a
history of one or more recurring or chronic porphyric symptoms,
and/or a subject who carries a genetic alteration (e.g., a
mutation) in a gene encoding an enzyme of the heme biosynthetic
pathway, and a subject who carries a genetic alteration, e.g., a
mutation. known to be associated with porphyria.
[0622] In embodiments, the alteration, e.g., the mutation, makes an
individual susceptible to an acute attack (e.g., upon exposure to a
precipitating factor, e.g., a drug, dieting or other precipitating
factor, e.g., a precipitating factor as disclosed herein). In
embodiments, the alteration, e.g., the mutation, is associated with
elevated levels of a porphyrin or a porphyrin precursor (e.g., ALA
and/or PBG). In embodiments, the alteration, e.g., the mutation, is
associated with chronic pain (e.g., chronic neuropathic pain)
and/or neuropathy (e.g., progressive neuropathy). In embodiments,
the, the alteration, e.g., the mutation, is associated with changes
in EMG and/or nerve conduction velocities.
[0623] In embodiments, the alteration is a mutation in the ALAS1
gene. In embodiments, the alteration is a mutation in the ALAS1
gene promoter, or in regions upstream or downstream from the ALAS1
gene. In embodiments, the alteration is a mutation in transcription
factors or other genes that interact with ALAS1. In embodiments,
the alteration is an alteration, e.g., a mutation, in a gene that
encodes an enzyme in the heme biosynthetic pathway.
[0624] In some embodiments, the subject has an genetic alteration
as described herein (e.g., a genetic mutation known to be
associated with a porphyria). In some such embodiments, the subject
has an elevated level (e.g., urine or plasma level) of ALA and/or
PBG. In some such embodiments, the subject does not have an
elevated level of ALA and/or PBG. In embodiments, the subject has a
genetic alteration as described herein and has other symptoms,
e.g., chronic pain, EMG changes, changes in nerve conduction
velocity, and/or other symptoms associated with a porphyria. In
embodiments, the subject has a genetic alteration but does not
suffer from acute attacks.
[0625] In embodiments, the subject has a mutation associated with
AIP, HCP, VP, or ADP.
[0626] In some embodiments, the porphyria is AIP. In some such
embodiments, the subject has an alteration, e.g., at least one
mutation, in the PBG deaminase gene. Many PBG deaminase mutations
are known in the art, for example, as reported in Hrdinka, M. et
al. Physiological Research, 55 (Suppl2):S119-136 (2006). In some
embodiments, the subject is heterozygous for a PBG deaminase
mutation. In other embodiments, the subject is homozygous for a PBG
deaminase mutation. A homozygous subject may carry two identical
mutations or two different mutations in the PBG deaminase gene.
[0627] In some embodiments, the porphyria is HCP. In some such
embodiments, the subject has an alteration, e.g., at least one
mutation, in the gene that encodes the enzyme coproporphyrinogen
III oxidase.
[0628] In some embodiments, the porphyria is VP. In some such
embodiments, the subject has an alteration, e.g., at least one
mutation, in the gene that encodes protoporphrinogen oxidase.
[0629] In embodiments, the porphyria is ADP, e.g., autosomal
recessive ADP. In some such embodiments, the subject has an
alteration, e.g., at least one mutation, in the gene that encodes
ALA deydratase.
[0630] Methods of treatment provided herein may serve to ameliorate
one or more symptoms associated with porphyria, to reduce the
frequency of attacks associated with porphyria, to reduce the
likelihood that an attack of one or more symptoms associated with
porphyria will occur upon exposure to a precipitating factor, or to
reduce the risk of developing conditions associated with porphyria
(e.g., neuropathy (e.g., progressive neuropathy), hepatocellular
cancer). Additionally, the methods provided herein may serve to
decrease the level of one or more porphyrin precursors, porphyrins
and/or related porphyrin products or metabolites. The level of a
porphyrin precursor or a porphyrin may be measured in any
biological sample, such as, e.g., urine, blood, feces,
cerebrospinal fluid, or a tissue sample. The sample may be present
within a subject or may be obtained or extracted from the subject.
In some embodiments, the porphyria is AIP, and the level of PBG
and/or ALA is decreased. In some embodiments, the porphyrin product
or metabolite is porphobilin, porphobilinogen, or uroporphyrin. A
decrease in the level of a porphyrin product or metabolite may be
measured using any method known in the art. For example, the level
of PBG and/or ALA in urine or plasma may be assessed, using the
Watson-Schwartz test, ion exchange chromatography, or
high-performance liquid chromatography--mass spectrometry. See,
e.g., Thunell (1993).
[0631] Methods described herein may also serve to reduce
chronically elevated levels of porphyrin precursors (e.g., ALA
and/or PBG) in subjects suffering from a porphyria (e.g., an acute
hepatic porphyria, e.g., AIP) or at risk for developing a
porphyria. Methods for assessing plasma and urine levels (e.g.,
chronically elevated levels) of porphyrin precursors include, e.g.,
HPLC-mass spectrometry and ion-exchange chromatography. The levels
of porphyrin precursors may be expressed as the level relative to
another protein or compound, e.g., creatinine. See, e.g., Floderus,
Y. et al, Clinical Chemistry, 52(4): 701-707, 2006; Sardh et al.,
Clinical Pharmacokinetics, 46(4): 335-349, 2007
[0632] A "precipitating factor" as used herein, refers to an
endogenous or exogenous factor that may induce an acute attack of
one or more symptoms associated with porphyria. Precipitating
factors include fasting (or other forms of reduced or inadequate
caloric intake, due to crash diets, long-distance athletics, etc.),
metabolic stresses (e.g., infections, surgery, international air
travel, and psychological stress), endogenous hormones (e.g.,
progesterone), cigarette smoking, lipid-soluble foreign chemicals
(including, e.g., chemicals present in tobacco smoke, certain
prescription drugs, organic solvents, biocides, components in
alcoholic beverages), endocrine factors (e.g., reproductive
hormones (women may experience exacerbations during the
premenstrual period), synthetic estrogens, progesterones, ovulation
stimulants, and hormone replacement therapy). See, for example,
Thunell (1993). Common precipitating factors include cytochrome
P450 inducing drugs and phenobarbitol.
[0633] Symptoms associated with porphyria may include abdominal
pain or cramping, headaches, effects caused by nervous system
abnormalities, and light sensitivity, causing rashes, blistering,
and scarring of the skin (photodermatitis). In certain embodiments,
the porphyria is AIP. Symptoms of AIP include gastrointestinal
symptoms (e.g., severe and poorly localized abdominal pain,
nausea/vomiting, constipation, diarrhea, ileus), urinary symptoms
(dysuria, urinary retention/incontinence, or dark urine),
neurologic symptoms (e.g., sensory neuropathy, motor neuropathy
(e.g., affecting the cranial nerves and/or leading to weakness in
the arms or legs), seizures, neuropathic pain, progressive
neuropathy, headaches, neuropsychiatric symptoms (e.g., mental
confusion, anxiety, agitation, hallucination, hysteria, delirium,
apathy, depression, phobias, psychosis, insomnia, somnolence,
coma), autonomic nervous system involvement (resulting e.g., in
cardiovascular sysmptoms such as tachycardia, hypertension, and/or
arrhythmias, as well as other symptoms, such as, e.g., increased
circulating catecholamine levels, sweating, restlessness, and/or
tremor), dehydration, and electrolyte abnormalities.
[0634] In some embodiments, an iRNA targeting ALAS1 is administered
together with (e.g., before, after, or concurrent with) another
treatment that may serve to alleviate one or more of the above
symptoms. For example, abdominal pain may be treated, e.g., with
narcotic analgesics, seizures may be treated, e.g., with
anti-seizure medications, nausea/vomiting may be treated, e.g.,
with phenothiazines, and tachycardia/hypertension may be treated,
e.g., with beta blockers.
[0635] The term "decrease" (or "increase") is intended to refer to
a measurable change, e.g., a statistically significant change. The
change may be, for example, at least 5%, 10%, 20%, 30%, 40%, 50% or
more change (e.g., decrease (or increase) relative to a reference
value, e.g., a reference where no iRNA is provided).
[0636] The invention further relates to the use of an iRNA or a
pharmaceutical composition thereof, e.g., for treating a disorder
related to ALAS1 expression, in combination with other
pharmaceuticals and/or other therapeutic methods, e.g., with known
pharmaceuticals and/or known therapeutic methods, such as, for
example, those which are currently employed for treating the
disorder. In one embodiment, the iRNA or pharmaceutical composition
thereof can be administered in conjunction with a heme product
(e.g., hemin, heme arginate, or heme albumin, as described herein)
and/or in conjunction with intravenous glucose infusions. In some
embodiments, the iRNA or pharmaceutical composition thereof is used
prophylactically, e.g., to prevent or ameliorate symptoms of an
anticipated attack of acute porphyria. The prophylactic use may be
timed according to the exposure or anticipated exposure of the
subject to a precipitating factor. As described herein, a
precipitating factor may be any endogenous or exogenous factor
known to precipitate an acute attack. For example, the premenstrual
phase is an endogenous precipitating factor, and a cytochrome P450
inducing drug is an exogenous precipitating factor.
[0637] The effective amount for the treatment of a disorder related
to ALAS1 expression (e.g., a porphyria such as AIP) depends on the
type of disorder to be treated, the severity of the symptoms, the
subject being treated, the sex, age and general condition of the
subject, the mode of administration and so forth. For any given
case, an appropriate "effective amount" can be determined by one of
ordinary skill in the art using routine experimentation. It is well
within the ability of one skilled in the art to monitor efficacy of
treatment or prevention by measuring any one of such parameters, or
any combination of parameters. In connection with the
administration of an iRNA targeting ALAS1 or pharmaceutical
composition thereof, "effective against" a disorder related to
ALAS1 expression indicates that administration in a clinically
appropriate manner results in a beneficial effect, e.g., for an
individual patient or for at least a fraction of patients, e.g., a
statistically significant fraction of patients. Beneficial effects
include, e.g., prevention of or reduction of symptoms or other
effects. For example, beneficial effects include, e.g., an
improvement (e.g., decrease in the severity or frequency) of
symptoms, a reduction in the severity or frequency of attacks, a
reduced risk of developing associated disease (e.g., neuropathy
(e.g., progressive neuropathy), hepatocellular cancer), an improved
ability to tolerate a precipitating factor, an improvement in
quality of life, a reduction in the expression of ALAS1, a
reduction in a level (e.g., a plasma or urine level) of a porphyrin
or a porphyrin precursor (e.g., ALA and/or PBG) or other effect
generally recognized as positive by medical doctors familiar with
treating the particular type of disorder.
[0638] A treatment or preventive effect is evident when there is an
improvement, e.g., a statistically significant improvement in one
or more parameters of disease status, or by a failure to worsen or
to develop symptoms where they would otherwise be anticipated. As
an example, a favorable change of at least 10% in a measurable
parameter of disease, e.g., at least 20%, 30%, 40%, 50% or more can
be indicative of effective treatment. Efficacy for a given iRNA
drug or formulation of that drug can also be judged using an
experimental animal model for the given disease as known in the
art. When using an experimental animal model, efficacy of treatment
is evidenced when a statistically significant reduction in a marker
(e.g., plasma or urinary ALA or PBG) or symptom is observed.
[0639] Patients can be administered a therapeutic amount of iRNA.
The therapeutic amount can be, e.g., 0.05-50 mg/kg. For example,
the therapeutic amount can be 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.5, 2.0, or 2.5, 3.0, 3.5, 4.0, 4.5, 5, 10,
15, 20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA.
[0640] In some embodiments, the iRNA is formulated as a lipid
formulation, e.g., an LNP formulation as described herein. In some
such embodiments, the therapeutic amount is 0.05-5 mg/kg, e.g.,
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0,
2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/kg dsRNA. In some embodiments,
the lipid formulation, e.g., LNP formulation, is administered
intravenously.
[0641] In some embodiments, the iRNA is administered by intravenous
infusion over a period of time, such as over a 5 minute, 10 minute,
15 minute, 20 minute, or 25 minute period.
[0642] In some embodiments, the iRNA is in the form of a GalNAc
conjugate as described herein. In some such embodiments, the
therapeutic amount is 0.5-50 mg, e.g., 0.5, 0.6, 0.7, 0.8, 0.9,
1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9, 10, 15,
20, 25, 30, 35, 40, 45, or 50 mg/kg dsRNA. In some embodiments, the
GalNAc conjugate is administered subcutaneously.
[0643] In some embodiments, the administration is repeated, for
example, on a regular basis, such as, daily, biweekly (i.e., every
two weeks) for one month, two months, three months, four months or
longer. After an initial treatment regimen, the treatments can be
administered on a less frequent basis. For example, after
administration biweekly for three months, administration can be
repeated once per month, for six months or a year or longer.
[0644] In some embodiments, the iRNA agent is administered in two
or more doses. In some embodiments, the number or amount of
subsequent doses is dependent on the achievement of a desired
effect, e.g., suppression of a ALAS gene, reduction of a level of a
porphyrin or porphyrin precursor (e.g., ALA and/or PBG), or the
achievement of a therapeutic or prophylactic effect, e.g.,
reduction or prevention of one or more symptoms associated with
porphyria (e.g., pain, e.g., neuropathic pain), and/or prevention
of attacks or reduction in the frequency and/or severity of attacks
associated with porphyria.
[0645] In some embodiments, the iRNA agent is administered
according to a schedule. For example, the iRNA agent may be
administered once per week, twice per week, three times per week,
four times per week, or five times per week. In some embodiments,
the schedule involves regularly spaced administrations, e.g.,
hourly, every four hours, every six hours, every eight hours, every
twelve hours, daily, every 2 days, every 3 days, every 4 days,
every 5 days, weekly, biweekly, or monthly. In embodiments, the
iRNA agent is administered weekly or biweekly to achieve a desired
effect, e.g., to decrease the level of ALA and/or PBG, to decrease
pain, and/or to prevent acute attacks.
[0646] In embodiments, the schedule involves closely spaced
administrations followed by a longer period of time during which
the agent is not administered. For example, the schedule may
involve an initial set of doses that are administered in a
relatively short period of time (e.g., about every 6 hours, about
every 12 hours, about every 24 hours, about every 48 hours, or
about every 72 hours) followed by a longer time period (e.g., about
1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks,
about 6 weeks, about 7 weeks, or about 8 weeks) during which the
iRNA agent is not administered. In one embodiment, the iRNA agent
is initially administered hourly and is later administered at a
longer interval (e.g., daily, weekly, biweekly, or monthly). In
another embodiment, the iRNA agent is initially administered daily
and is later administered at a longer interval (e.g., weekly,
biweekly, or monthly). In certain embodiments, the longer interval
increases over time or is determined based on the achievement of a
desired effect. In a specific embodiment, the iRNA agent is
administered once daily during an acute attack, followed by weekly
dosing starting on the eighth day of administration. In another
specific embodiment, the iRNA agent is administered every other day
during a first week followed by weekly dosing starting on the
eighth day of administration.
[0647] In one embodiment, the iRNA agent is administered to prevent
or reduce the severity or frequency of recurring attacks, e.g.,
cyclical attacks associated with a precipitating factor. In some
embodiments, the precipitating factor is the menstrual cycle. In
some embodiments, the iRNA is administered repeatedly, e.g., at
regular intervals to prevent or reduce the severity or frequency of
recurring attacks, e.g., cyclical attacks associated with a
precipitating factor, e.g., the menstrual cycle, e.g., a particular
phase of the menstrual cycle, e.g., the luteal phase. In some
embodiments, the iRNA is administered during a particular phase of
the menstrual cycle or based on hormone levels of the patient being
treated (e.g., based on hormone levels that are associated with a
particular phase of the menstrual cycle). In some embodiments, the
iRNA is administered on one or more particular days of the
menstrual cycle, e.g., on day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or
on day 28 (or later day for subjects who have a longer menstrual
cycle). In some embodiments, the iRNA is administered during the
luteal phase, e.g., on one or more days between days 14-28 of the
menstrual cycle (or later, in subjects who have a menstrual cycle
longer than 28 days). In some embodiments, ovulation of the subject
is assessed (e.g., using a blood or urine test that detects a
hormone associated with ovulation, e.g., LH) and the iRNA is
administered at a predetermined interval after ovulation. In some
embodiments, the iRNA is administered immediately after ovulation.
In some embodiments, the iRNA is administered 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 days after ovulation.
Any of these schedules may optionally be repeated for one or more
iterations. The number of iterations may depend on the achievement
of a desired effect, e.g., the suppression of a ALAS1 gene and/or
the achievement of a therapeutic or prophylactic effect, e.g.,
reduce or prevent one or more symptoms associated with porphyria,
to reduce the frequency of attacks associated with porphyria.
[0648] In some embodiments, an initial dose of the iRNA agent is
administered and the level of ALA or PBG is tested, e.g., 1-48
hours, e.g., 2, 4, 8, 12, or 24 hours following administration of
the initial dose. In some embodiments, if the level of ALA and/or
PBG has decreased (e.g., to achieve a predetermined reduction,
e.g., a normalization), and/or if the symptoms associated with
porphyria (e.g., pain) have improved (e.g., such that the patient
is asymptomatic), no further dose is administered, whereas if the
level of ALA and/or PBG has not decreased (e.g., has not achieved a
predetermined reduction, e.g., has not normalized), a further dose
of ALA or PBG is administered. In some embodiments, the further
dose is administered 12, 24, 36, 48, 60, or 72 hours after the
initial dose. In some embodiments, if the initial dose is not
effective to decrease the level of ALA and/or PBG, the further dose
is modified, e.g., increased to achieve a desired decrease (e.g., a
predetermined reduction, e.g., a normalization) in ALA or PBG
levels.
[0649] In some embodiments, the predetermined reduction is a
decrease of at least 10%, 20%, 30%, 40%, or 50%. In some
embodiments, the predetermined reduction is a reduction that is
effective to prevent or ameliorate symptoms, e.g., pain, prodromal
symptoms, or recurring attacks.
[0650] In some embodiments, the predetermined reduction is a
reduction of at least 1, 2, 3, or more standard deviations, wherein
the standard deviation is determined based on the values from a
reference sample, e.g., a reference sample as described herein.
[0651] In some embodiments, the predetermined reduction is a
reduction that brings the level of the porphyrin or porphyrin
precursor to a level that is less than, or to a level that is less
than or equal to, a reference value (e.g., a reference value as
described herein).
[0652] As used herein, a "normalization" in ALA or PBG levels (or a
"normal" or "normalized" level) refers to a level (e.g., a urine
and/or plasma level) of either ALA, or PBG, or both, that is within
the expected range for a healthy individual, an individual who is
asymptomatic (e.g., an individual who does not experience pain
and/or suffer from neuropathy), or an individual who does not have
a mutation associated with a porphyria. For example, in some
embodiments, a normalized level is within two standard deviations
of the normal mean. In some embodiments, a normalized level is
within normal reference limits, e.g., within the 95% confidence
interval for an appropriate control sample, e.g., a sample of
healthy individuals or individuals who do not carry a gene mutation
associated with a porphyria. In some embodiments, the ALA and/or
PBG level of the subject (e.g., the urine and/or plasma ALA and/or
PBG level) is monitored at intervals, a further dose of the iRNA
agent is administered when the level increases above the reference
value
[0653] Administration of the iRNA may reduce ALAS1 mRNA or protein
levels, e.g., in a cell, tissue, blood, urine or other compartment
of the patient by at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80% or at least 90% or more. Administration
of the iRNA may reduce levels of products associated with ALAS1
gene expression, e.g., levels of one or more porphyrins or
porphyrin precursors (e.g., the level of ALA and/or PBG).
Administration of the iRNA agent may also inhibit or prevent the
upregulation of ALAS1 mRNA or protein levels during an acute attack
of AIP.
[0654] Before administration of a full dose of the iRNA, patients
can be administered a smaller dose, such as a 5% infusion dose, and
monitored for adverse effects, such as an allergic reaction, or for
elevated lipid levels or blood pressure. In another example, the
patient can be monitored for unwanted effects.
[0655] Methods for Modulating Expression of an ALAS1 Gene
[0656] In yet another aspect, the invention provides a method for
modulating (e.g., inhibiting or activating) the expression of an
ALAS1 gene, e.g., in a cell or in a subject. In some embodiments,
the cell is ex vivo, in vitro, or in vivo. In some embodiments, the
cell is an erythroid cell or a hepatocyte. In some embodiments, the
cell is in a subject (e.g., a mammal, such as, for example, a
human). In some embodiments, the subject (e.g., the human) is at
risk, or is diagnosed with a disease related to ALAS1 expression,
as described above.
[0657] In one embodiment, the method includes contacting the cell
with an iRNA as described herein, in an amount effective to
decrease the expression of an ALAS1 gene in the cell. "Contacting,"
as used herein, includes directly contacting a cell, as well as
indirectly contacting a cell. For example, a cell within a subject
(e.g., an erythroid cell or a liver cell, such as a hepatocyte) may
be contacted when a composition comprising an iRNA is administered
(e.g., intravenously or subcutaneously) to the subject.
[0658] The expression of an ALAS1 gene may be assessed based on the
level of expression of an ALAS1 mRNA, an ALAS1 protein, or the
level of a parameter functionally linked to the level of expression
of an ALAS1 gene (e.g., the level of a porphyrin or the incidence
or severity of a symptom related to a porphyria). In some
embodiments, the expression of ALAS1 is inhibited by at least 5%,
at least 10%, at least 15%, at least 20%, at least 25%, at least
30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%,
at least 80%, at least 85%, at least 90%, or at least 95%. In some
embodiments, the iRNA has an IC.sub.50 in the range of 0.001-0.01
nM, 0.001-0.10 nM, 0.001-1.0 nM, 0.001-10 nM, 0.01-0.05 nM,
0.01-0.50 nM, 0.02-0.60 nM, 0.01-1.0 nM, 0.01-1.5 nM, 0.01-10 nM.
The IC.sub.50 value may be normalized relative to an appropriate
control value, e.g., the IC.sub.50 of a non-targeting iRNA.
[0659] In some embodiments, the method includes introducing into
the cell an iRNA as described herein and maintaining the cell for a
time sufficient to obtain degradation of the mRNA transcript of an
ALAS1 gene, thereby inhibiting the expression of the ALAS1 gene in
the cell.
[0660] In one embodiment, the method includes administering a
composition described herein, e.g., a composition comprising an
iRNA that targets ALAS1, to the mammal such that expression of the
target ALAS1 gene is decreased, such as for an extended duration,
e.g., at least two, three, four days or more, e.g., one week, two
weeks, three weeks, or four weeks or longer. In some embodiments,
the decrease in expression of ALAS1 is detectable within 1 hour, 2
hours, 4 hours, 8 hours, 12 hours, or 24 hours of the first
administration.
[0661] In another embodiment, the method includes administering a
composition as described herein to a mammal such that expression of
the target ALAS1 gene is increased by e.g., at least 10% compared
to an untreated animal. In some embodiments, the activation of
ALAS1 occurs over an extended duration, e.g., at least two, three,
four days or more, e.g., one week, two weeks, three weeks, four
weeks, or more. Without wishing to be bound by theory, an iRNA can
activate ALAS1 expression by stabilizing the ALAS1 mRNA transcript,
interacting with a promoter in the genome, and/or inhibiting an
inhibitor of ALAS1 expression.
[0662] The iRNAs useful for the methods and compositions featured
in the invention specifically target RNAs (primary or processed) of
an ALAS1 gene. Compositions and methods for inhibiting the
expression of an ALAS1 gene using iRNAs can be prepared and
performed as described elsewhere herein.
[0663] In one embodiment, the method includes administering a
composition containing an iRNA, where the iRNA includes a
nucleotide sequence that is complementary to at least a part of an
RNA transcript of the ALAS1 gene of the mammal to be treated. When
the organism to be treated is a mammal such as a human, the
composition may be administered by any means known in the art
including, but not limited to oral, intraperitoneal, or parenteral
routes, including intracranial (e.g., intraventricular,
intraparenchymal and intrathecal), intravenous, intramuscular,
subcutaneous, transdermal, airway (aerosol), nasal, rectal, and
topical (including buccal and sublingual) administration.
[0664] In certain embodiments, the compositions are administered by
intravenous infusion or injection. In some such embodiments, the
compositions comprise a lipid formulated siRNA (e.g., an LNP
formulation, such as an LNP11 formulation) for intravenous
infusion. In particular embodiments, such compositions may be used
to treat acute attacks of porphyria and/or for prophylaxis (e.g.,
to decrease the severity or frequency of attacks).
[0665] In other embodiments, the compositions are administered
subcutaneously. In some such embodiments, the compositions comprise
an iRNA conjugated to a GalNAc ligand. In particular embodiments,
such compositions may be used to treat acute attacks of porphyria
or for prophylaxis (e.g., to decrease the severity or frequency of
attacks).
[0666] Methods for Decreasing a Level of a Porphyrin or Porphyrin
Precursor
[0667] In another aspect, the invention provides a method for
decreasing a level of a porphyrin or a porphyrin precursor, e.g.,
in a cell or in a subject.
[0668] In some embodiments, the cell is ex vivo, in vitro, or in
vivo. In some embodiments, the cell is an erythroid cell or a
hepatocyte. In some embodiments, the cell is a hepatocyte. In some
embodiments, the cell is in a subject (e.g., a mammal, such as, for
example, a human).
[0669] In some embodiments, the subject (e.g., the human) is at
risk, or is diagnosed with a porphyria, as described herein. In
some embodiments, the method is effective to treat a porphyria as
described herein (e.g., by ameliorating one or more symptoms
associated with a porphyria, reducing the frequency of attacks
associated with a porphyria, reducing the likelihood that an attack
of one or more symptoms associated with porphyria will occur upon
exposure to a precipitating factor, or reducing the risk of
developing conditions associated with a porphyria (e.g., neuropathy
(e.g., progressive neuropathy), hepatocellular cancer). In one
embodiment, the method includes contacting the cell with an RNAi,
as described herein, in an amount sufficient to decrease the level
of the porphyrin or porphyrin precursor (e.g., ALA or PBG) in the
cell, or in another related cell or group of cells, or in the
subject. "Contacting," as used herein, includes directly contacting
a cell, as well as indirectly contacting a cell. For example, a
cell within a subject (e.g., an erythroid cell or a liver cell,
such as a hepatocyte) may be contacted when a composition
comprising an RNAi is administered (e.g., intravenously or
subcutaneously) to the subject. "Another related cell or group of
cells," as used herein, includes any cell or group of cells in
which the level of the porphyrin or porphyrin precursor decreases
as a result of the contacting. For example, the cell may be part of
a tissue present within a subject (e.g., a liver cell present
within a subject), and contacting the cell within the subject
(e.g., contacting one or more liver cells present within a subject)
with the RNAi may result in a decrease in the level of the
porphyrin or porphyrin precursor in another related cell or group
of cells (e.g., nerve cells of the subject), or in a tissue or
fluid of the subject (e.g., in the urine, blood, plasma, or
cerebrospinal fluid of the subject).
[0670] In some embodiments, the porphyrin or porphyrin precursor is
selected from the group consisting of .delta.-aminolevulinic acid
(ALA), porphopilinogen (PBG), hydroxymethylbilane (INMB),
uroporphyrinogen III, coproporphyrinogen III, protoporphrinogen IX,
and protoporphyrin IX In some embodiments the porphyrin precursor
is ALA. In some embodiments, the porphyrin precursor is PBG. In
some embodiments, the method decreases the level of ALA and PBG.
The level of a porphyrin or a porphyrin precursor may be measured
as described herein and as known in the art.
[0671] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the iRNAs and
methods featured in the invention, suitable methods and materials
are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and not
intended to be limiting.
EXAMPLES
Example 1. siRNA Synthesis
[0672] Source of Reagents
[0673] Where the source of a reagent is not specifically given
herein, such reagent may be obtained from any supplier of reagents
for molecular biology at a quality/purity standard for application
in molecular biology.
Oligonucleotide Synthesis.
[0674] All oligonucleotides are synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, 500{acute over (.ANG.)}, Prime Synthesis) and RNA
phosphoramidites with standard protecting groups,
5'-O-dimethoxytrityl
N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3'-O--N,N'-diisopropyl-2-cya-
noethylphosphoramidite,
5'-O-dimethoxytrityl-N4-acetyl-2'-t-butyldimethylsilyl-cytidine-3'-O--N,N-
'-diisopropyl-2-cyanoethylphosphoramidite,
5'-O-dimethoxytrityl-N2-isobutryl-2'-t-butyldimethylsilyl-guanosine-3'-O--
-N,N'-diisopropyl-2-cyanoethylphosphoramidite, and
5'-O-dimethoxytrityl-2'-t-butyldimethylsilyl-uridine-3'-O--N,N'-diisoprop-
yl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies)
were used for the oligonucleotide synthesis. The 2'-F
phosphoramidites,
5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3'-O--N,N'-diisopropyl-2-
-cyanoethyl-phosphoramidite and
5'-O-dimethoxytrityl-2'-fluro-uridine-3'-O--N,N'-diisopropyl-2-cyanoethyl-
-phosphoramidite are purchased from (Promega). All phosphoramidites
are used at a concentration of 0.2M in acetonitrile (CH.sub.3CN)
except for guanosine which is used at 0.2M concentration in 10%
THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The
activator is 5-ethyl thiotetrazole (0.75M, American International
Chemicals); for the PO-oxidation iodine/water/pyridine is used and
for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is
used.
[0675] 3'-ligand conjugated strands are synthesized using solid
support containing the corresponding ligand. For example, the
introduction of cholesterol unit in the sequence is performed from
a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is
tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage
to obtain a hydroxyprolinol-cholesterol moiety. 5'-end Cy-3 and
Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the
corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from
Biosearch Technologies. Conjugation of ligands to 5'-end and or
internal position is achieved by using appropriately protected
ligand-phosphoramidite building block. An extended 15 min coupling
of 0.1 M solution of phosphoramidite in anhydrous CH.sub.3CN in the
presence of 5-(ethylthio)-1H-tetrazole activator to a
solid-support-bound oligonucleotide. Oxidation of the
internucleotide phosphite to the phosphate is carried out using
standard iodine-water as reported (1) or by treatment with
tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min
oxidation wait time conjugated oligonucleotide. Phosphorothioate is
introduced by the oxidation of phosphite to phosphorothioate by
using a sulfur transfer reagent such as DDTT (purchased from AM
Chemicals), PADS and or Beaucage reagent. The cholesterol
phosphoramidite is synthesized in house and used at a concentration
of 0.1 M in dichloromethane. Coupling time for the cholesterol
phosphoramidite is 16 minutes.
Deprotection I (Nucleobase Deprotection)
[0676] After completion of synthesis, the support is transferred to
a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from
the support with simultaneous deprotection of base and 30 phosphate
groups with 80 mL of a mixture of ethanolic ammonia [ammonia:
ethanol (3:1)] for 6.5 h at 55.degree. C. The bottle is cooled
briefly on ice and then the ethanolic ammonia mixture is filtered
into a new 250-mL bottle. The CPG is washed with 2.times.40 mL
portions of ethanol/water (1:1 v/v). The volume of the mixture is
then reduced to .about.30 mL by roto-vap. The mixture is then
frozen on dry ice and dried under vacuum on a speed vac.
Deprotection II (Removal of 2'-TBDMS Group)
[0677] The dried residue is resuspended in 26 mL of triethylamine,
triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO
(3:4:6) and heated at 60.degree. C. for 90 minutes to remove the
tert-butyldimethylsilyl (TBDMS) groups at the 2' position. The
reaction is then quenched with 50 mL of 20 mM sodium acetate and
the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer
until purification.
Analysis
[0678] The oligonucleotides are analyzed by high-performance liquid
chromatography (HPLC) prior to purification and selection of buffer
and column depends on nature of the sequence and or conjugated
ligand.
HPLC Purification
[0679] The ligand-conjugated oligonucleotides are purified by
reverse-phase preparative HPLC. The unconjugated oligonucleotides
are purified by anion-exchange HPLC on a TSK gel column packed in
house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10%
CH.sub.3CN, 1M NaBr (buffer B). Fractions containing full-length
oligonucleotides are pooled, desalted, and lyophilized.
Approximately 0.15 OD of desalted oligonucleotides are diluted in
water to 150 .mu.L and then pipetted into special vials for CGE and
LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
siRNA Preparation
[0680] For the general preparation of siRNA, equimolar amounts of
sense and antisense strand are heated in 1.times.PBS at 95.degree.
C. for 5 min and slowly cooled to room temperature. Integrity of
the duplex is confirmed by HPLC analysis.
Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 1.
TABLE-US-00012 TABLE 1 Abbreviations of nucleotide monomers used in
nucleic acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are mutually
linked by 5'-3'-phosphodiester bonds. Abbreviation Nucleotide(s) A
Adenosine-3'-phosphate Ab beta-L-adenosine-3'-phosphate Abs
beta-L-adenosine-3'-phosphorothioate Af
2'-fluoroadenosine-3'-phosphate Afs
2'-fluoroadenosine-3'-phosphorothioate As
adenosine-3'-phosphorothioate C cytidine-3'-phosphate Cb
beta-L-cytidine-3'-phosphate Cbs
beta-L-cytidine-3'-phosphorothioate Cf
2'-fluorocytidine-3'-phosphate Cfs
2'-fluorocytidine-3'-phosphorothioate (Chd)
2'-O-hexadecyl-cytidine-3'-phosphate (Chds)
2'-O-hexadecyl-cytidine-3'-phosphorothioate Cs
cytidine-3'-phosphorothioate G guanosine-3'-phosphate Gb
beta-L-guanosine-3'-phosphate Gbs
beta-L-guanosine-3'-phosphorothioate Gf
2'-fluoroguanosine-3'-phosphate Gfs
2'-fluoroguanosine-3'-phosphorothioate Gs
guanosine-3'-phosphorothioate T 5'-methyluridine-3'-phosphate Tb
beta-L-thymidine-3'-phosphate Tbs
beta-L-thymidine-3'-phosphorothioate Tf
2'-fluoro-5-methyluridine-3'-phosphate Tfs
2'-fluoro-5-methyluridine-3'-phosphorothioate Ts
5-methyluridine-3'-phosphorothioate U Uridine-3'-phosphate Ub
beta-L-uridine-3'-phosphate Ubs beta-L-uridine-3'-phosphorothioate
Uf 2'-fluorouridine-3'-phosphate Ufs
2'-fluorouridine-3'-phosphorothioate (Uhd)
2'-O-hexadecyl-uridine-3'-phosphate (Uhds)
2'-O-hexadecyl-uridine-3'-phosphorothioate Us
uridine-3'-phosphorothioate N any nucleotide (G, A, C, T or U) a
2'-O-methyladenosine-3'-phosphate as
2'-O-methyladenosine-3'-phosphorothioate c
2'-O-methylcytidine-3'-phosphate cs
2'-O-methylcytidine-3'-phosphorothioate g
2'-O-methylguanosine-3'-phosphate gs
2'-O-methylguanosine-3'-phosphorothioate t 2'-O-methyl-5-methyluri
dine-3'-phosphate ts
2'-O-methyl-5-methyluridine-3'-phosphorothioate u
2'-O-methyluridine-3'-phosphate us
2'-O-methyluridine-3'-phosphorothioate dA
2'-deoxyadenosine-3'-phosphate dAs
2'-deoxyadenosine-3'-phosphorothioate dC
2'-deoxycytidine-3'-phosphate dCs
2'-deoxycytidine-3'-phosphorothioate dG
2'-deoxyguanosine-3'-phosphate dGs
2'-deoxyguanosine-3'-phosphorothioate dT 2'-deoxythymidine dTs
2'-deoxythymidine-3'-phosphorothioate dU 2'-deoxyuridine s
phosphorothioate linkage L96.sup.1
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4- hydroxyprolinol
Hyp-(GalNAc-alkyl)3 (Aeo) 2'-O-methoxyethyladenosine-3'-phosphate
(Aeos) 2'-O-methoxyethyladenosine-3'-phosphorothioate (Geo)
2'-O-methoxyethylguanosine-3'-phosphate (Geos)
2'-O-methoxyethylguanosine-3'-phosphorothioate (Teo)
2'-O-methoxyethyl-5-methyluridine-3'-phosphate (Teos)
2'-O-methoxyethyl-5-methyluridine-3'-phosphorothioate (m5Ceo)
2'-O-methoxyethyl-5-methylcytidine-3'-phosphate (m5Ceos)
2'-O-methoxyethyl-5-methylcytidine-3'-phosphorothioate .sup.1The
chemical structure of L96 is as follows:
##STR00025##
Example 2. ALAS1 siRNA Design and Synthesis
Experimental Methods
Bioinformatics
Transcripts
[0681] siRNA design was carried out to identify siRNAs targeting
human, rhesus (Macaca mulatta), mouse, and rat ALAS1 transcripts
annotated in the NCBI Gene database
(http://www.ncbi.nlm.nih.gov/gene/). Design used the following
transcripts from the NCBI RefSeq collection: Human--NM_000688.4
(see FIG. 3), NM_199166.1; Rhesus--XM_001090440.2, XM_001090675.2;
Mouse--NM_020559.2; Rat--NM_024484.2. Due to high primate/rodent
sequence divergence, siRNA duplexes were designed in several
separate batches, including but not limited to batches containing
duplexes matching human and rhesus transcripts only; human, rhesus,
mouse, and rat transcripts only; and mouse and rat transcripts
only. Most siRNA duplexes were designed that shared 100% identity
the listed human transcript and other species transcripts
considered in each design batch (above). In some instances, (see
Table 8) mismatches between duplex and mRNA target were allowed at
the first antisense (last sense) position when the antisense
strand:target mRNA complementary basepair was a GC or CG pair. In
these cases, duplexes were designed with UA or AU pairs at the
first antisense:last sense pair. Thus the duplexes maintained
complementarity but were mismatched with respect to target (U:C,
U:G, A:C, or A:G). Eighteen of these "UA-swap" duplexes were
designed as part of the human/rhesus/mouse/rat set (see duplexes in
Table 8 with "C19U", "G19U", "C19A", or "G19A" labels in the
Position column).
[0682] siRNA Design, Specificity, and Efficacy Prediction
[0683] The predicted specificity of all possible 19mers was
predicted from each sequence. Candidate 19mers were then selected
that lacked repeats longer than 7 nucleotides. These 1510 candidate
human/rhesus, 114 human/rhesus/mouse/rat, and 717 mouse/rat siRNAs
were used in comprehensive searches against the appropriate
transcriptomes (defined as the set of NM_ and XM_records within the
human, rhesus, dog, mouse, or rat NCBI Refseq sets) using an
exhaustive "brute-force" algorithm implemented in the python script
`BruteForce.py`. The script next parsed the transcript-oligo
alignments to generate a score based on the position and number of
mismatches between the siRNA and any potential `off-target`
transcript. The off-target score is weighted to emphasize
differences in the `seed` region of siRNAs, in positions 2-9 from
the 5' end of the molecule. Each oligo-transcript pair from the
brute-force search was given a mismatch score by summing the
individual mismatch scores; mismatches in the position 2-9 were
counted as 2.8, mismatches in the cleavage site positions 10-11
were counted as 1.2, and mismatches in region 12-19 counted as 1.0.
An additional off-target prediction was carried out by comparing
the frequency of heptamers and octomers derived from 3 distinct,
seed-derived hexamers of each oligo. The hexamers from positions
2-7 relative to the 5' start is used to create 2 heptamers and one
octomer. We create `heptamer1` by adding a 3' A to the hexamer; we
create heptamer2 by adding a 5' A to the hexamer; we create the
octomer by adding an A to both 5' and 3' ends of the hexamer. The
frequency of octomers and heptamers in the human, rhesus, mouse, or
rat 3'UTRome (defined as the subsequence of the transcriptome from
NCBI's Refseq database where the end of the coding region, the
`CDS`, is clearly defined) was pre-calculated. The octomer
frequency was normalized to the heptamer frequency using the median
value from the range of octomer frequencies. A `mirSeedScore` was
then calculated by calculating the sum of ((3.times.normalized
octomer count)+(2.times.heptamer2 count)+(1.times.heptamer1
count)).
[0684] Both siRNAs strands were assigned to a category of
specificity according to the calculated scores: a score above 3
qualifies as highly specific, equal to 3 as specific and between
2.2 and 2.8 as moderately specific. We sorted by the specificity of
the antisense strand. We then selected duplexes whose antisense
oligos lacked GC at the first position, lacked G at both positions
13 and 14, and had 3 or more Us or As in the seed region
(characteristics of duplexes with high predicted efficacy)
[0685] Candidate GalNac-conjugated duplexes, 21 and 23 nucleotides
long on the sense and antisense strands respectively, were designed
by extending antisense 19mers 4 additional nucleotides in the 3'
direction (preserving perfect complementarity with the target
transcript). The sense strand was specified as the reverse
complement of the first 21 nucleotides of the antisense 23mer.
Duplexes were selected that maintained perfect matches to all
selected species transcripts across all 23 nucleotides.
[0686] siRNA Sequence Selection
[0687] A total of 90 sense and 90 antisense derived human/rhesus,
40 sense and 40 antisense derived human/rhesus/mouse/mouse/rat, and
40 sense and 40 antisense derived mouse/rat siRNA 19mer oligos were
synthesized and formed into duplexes. A total of 45 sense and 45
antisense derived human/rhesus 21/23mer oligos were synthesized to
yield 45 GalNac-conjugated duplexes.
[0688] The sequences of the sense and antisense strands of the
modified duplexes are shown in Table 2, and the sequences of the
sense and antisense strands of the unmodified duplexes are shown in
Table 3.
Synthesis of ALAS1 Sequences
[0689] ALAS1 sequences were synthesized on MerMade 192 synthesizer
at either 1 or 0.2 umol scale. Single strands were made with
2'O-methyl modifications for in vitro screening using transfection
reagents. 3' GalNAc conjugates were made with sequences containing
2'F and 2'-O-methyl modifications on the sense strand in the 21-23
mer designs for free uptake in cells. For all the 21mer sequences
in the list, `endolight` chemistry was applied as detailed below.
[0690] All pyrimidines (cytosine and uridine) in the sense strand
contained 2'-O-Methyl bases (2' O-Methyl C and 2'-O-Methyl U)
[0691] In the antisense strand, pyrimidines adjacent to (towards 5'
position) ribo A nucleoside were replaced with their corresponding
2-O-Methyl nucleosides [0692] A two base dTsdT extension at 3' end
of both sense and anti sense sequences was introduced [0693] The
sequence file was converted to a text file to make it compatible
for loading in the MerMade 192 synthesis software
[0694] For GalNAc conjugated sense strands and complementary
antisense sequences, 2'F and other modified nucleosides were
introduced in combination with ribo with 2'O-Methyl nucleosides.
The synthesis was performed on a GalNAc modified CPG support for
the sense strand and CPG modified with universal support on the
antisense sequence.
[0695] Synthesis, Cleavage and Deprotection:
[0696] The synthesis of ALAS1 sequences used solid supported
oligonucleotide synthesis using phosphoramidite chemistry. For 21
mer endolight sequences, a deoxy thymidine CPG was used as the
solid support while for the GalNAc conjugates, GalNAc solid support
for sense strand and an universal CPG for the antisense strand were
used.
[0697] The synthesis of the above sequences was performed at either
1 or 0.2 um scale in 96 well plates. The amidite solutions were
prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in
Acetonitrile) was used as activator.
[0698] The synthesized sequences were cleaved and deprotected in 96
well plates, using methylamine in the first step and fluoride
reagent in the second step. For GalNAc and 2'F nucleoside
containing sequences, deprotection conditions were modified.
Sequences after cleavage and deprotection were precipitated using
acetone:ethanol (80:20) mix and the pellet were re-suspended in
0.2M sodium acetate buffer. Samples from each sequence were
analyzed by LC-MS to confirm the identity, UV for quantification
and a selected set of samples by IEX chromatography to determine
purity.
[0699] PURIFICATION and desalting:
[0700] ALAS1 sequences were precipitated and purified on AKTA
Purifier system using Sephadex column. The ALAS1ess was run at
ambient temperature. Sample injection and collection was performed
in 96 well (1.8 mL-deep well) plates. A single peak corresponding
to the full length sequence was collected in the eluent. The
desalted ALAS1 sequences were analyzed for concentration (by UV
measurement at A260) and purity (by ion exchange HPLC). The
complementary single strands were then combined in a 1:1
stoichiometric ratio to form siRNA duplexes.
TABLE-US-00013 TABLE 2 Human ALAS1 Modified Single Strands and
Duplex Sequences SEQ ID SEQ ID NO: Position on NO: (anti-
transcript Duplex Sense Sequence Antisense Sequence (sense) sense)
NM_000688.4 Name (5'-3') (5'-3') 2 3 522-540 AD-55078.2
cuccGGccAGuGAGAAAGAdTsdT UCUUUCUcACUGGCCGGAGdTsdT 4 5 669-687
AD-55084.2 uGGcAGcAcAGAuGAAucAdTsdT UGAUUcAUCUGUGCUGCcAdTsdT 6 7
790-808 AD-55090.2 cAGuGuGGuuAGuGuGAAAdTsdT
UUUcAcACuAACcAcACUGdTsdT 8 9 853-871 AD-55096.2
cAucAuGcAAAAGcAAAGAdTsdT UCUUUGCUUUUGcAUGAUGdTsdT 10 11 876-894
AD-55102.2 AAAGAGuGucucAucuucudTsdT AGAAGAUGAGAcACUCUUUdTsdT 12 13
877-895 AD-55106.2 AAGAGuGucucAucuucuudTsdT
AAGAAGAUGAGAcACUCUUdTsdT 14 15 914-932 AD-55111.2
ucuGuuuccAcuuuucAGudTsdT ACUGAAAAGUGGAAAcAGAdTsdT 16 17 923-941
AD-55073.2 AcuuuucAGuAuGAucGuudTsdT AACGAUcAuACUGAAAAGUdTsdT 18 19
926-944 AD-55079.2 uuucAGuAuGAucGuuucudTsdT
AGAAACGAUcAuACUGAAAdTsdT 20 21 927-945 AD-55085.2
uucAGuAuGAucGuuucuudTsdT AAGAAACGAUcAuACUGAAdTsdT 22 23 928-946
AD-55091.2 ucAGuAuGAucGuuucuuudTsdT AAAGAAACGAUcAuACUGAdTsdT 24 25
932-950 AD-55097.2 uAuGAucGuuucuuuGAGAdTsdT
UCUcAAAGAAACGAUcAuAdTsdT 26 27 973-991 AD-55103.2
uGAccAcAccuAucGAGuudTsdT AACUCGAuAGGUGUGGUcAdTsdT 28 29 975-993
AD-55107.2 AccAcAccuAucGAGuuuudTsdT AAAACUCGAuAGGUGUGGUdTsdT 30 31
1029-1047 AD-55112.2 uGGcAGAuGAcuAuucAGAdTsdT
UCUGAAuAGUcAUCUGCcAdTsdT 32 33 1077-1095 AD-55074.2
ucuGGuGcAGuAAuGAcuAdTsdT uAGUcAUuACUGcACcAGAdTsdT 34 35 1124-1142
AD-55080.2 uGuGGGGcAGuuAuGGAcAdTsdT UGUCcAuAACUGCCCcAcAdTsdT 36 37
1137-1155 AD-55086.2 uGGAcAcuuuGAAAcAAcAdTsdT
UGUUGUUUcAAAGUGUCcAdTsdT 38 39 1182-1200 AD-55098.2
AuAuuucuGGAAcuAGuAAdTsdT UuACuAGUUCcAGAAAuAUdTsdT 40 41 1184-1202
AD-55104.2 AuuucuGGAAcuAGuAAAudTsdT AUUuACuAGUUCcAGAAAUdTsdT 42 43
1185-1203 AD-55108.2 uuucuGGAAcuAGuAAAuudTsdT
AAUUuACuAGUUCcAGAAAdTsdT 44 45 1188-1206 AD-55113.2
cuGGAAcuAGuAAAuuccAdTsdT UGGAAUUuACuAGUUCcAGdTsdT 46 47 1325-1343
AD-55075.2 uGuGAGAuuuAcucuGAuudTsdT AAUcAGAGuAAAUCUcAcAdTsdT 48 49
1364-1382 AD-55081.2 AuccAAGGGAuucGAAAcAdTsdT
UGUUUCGAAUCCCUUGGAUdTsdT 50 51 1382-1400 AD-55087.2
AGccGAGuGccAAAGuAcAdTsdT UGuACUUUGGcACUCGGCUdTsdT 52 53 1478-1496
AD-55093.2 uuuGAAAcuGuccAuucAAdTsdT UUGAAUGGAcAGUUUcAAAdTsdT 54 55
1531-1549 AD-55099.2 uGAuGuGGcccAuGAGuuudTsdT
AAACUcAUGGGCcAcAUcAdTsdT 56 57 1631-1649 AD-53573.3
GucAuGccAAAAAuGGAcAdTsdT UGUCcAUUUUUGGcAUGACdTsdT 58 59 1637-1655
AD-55109.2 ccAAAAAuGGAcAucAuuudTsdT AAAUGAUGUCcAUUUUUGGdTsdT 60 61
1706-1724 AD-55114.2 AcGAGuucucuGAuuGAcAdTsdT
UGUcAAUcAGAGAACUCGUdTsdT 62 63 1962-1980 AD-55076.2
AAGucuGuGAuGAAcuAAudTsdT AUuAGUUcAUcAcAGACUUdTsdT 64 65 1967-1985
AD-55082.2 uGuGAuGAAcuAAuGAGcAdTsdT UGCUcAUuAGUUcAUcAcAdTsdT 66 67
1977-1995 AD-55088.2 uAAuGAGcAGAcAuAAcAudTsdT
AUGUuAUGUCUGCUcAUuAdTsdT 68 69 2189-2207 AD-55094.2
uuuGAAGuGAuGAGuGAAAdTsdT UUUcACUcAUcACUUcAAAdTsdT 70 71 2227-2245
AD-55100.2 AGGcuuGAGcAAGuuGGuAdTsdT uACcAACUUGCUcAAGCCUdTsdT 72 73
2313-2331 AD-55105.2 ucuucAGAGuuGucuuuAudTsdT
AuAAAGAcAACUCUGAAGAdTsdT 74 75 2317-2335 AD-55110.2
cAGAGuuGucuuuAuAuGudTsdT AcAuAuAAAGAcAACUCUGdTsdT 76 77 2319-2337
AD-55115.2 GAGuuGucuuuAuAuGuGAdTsdT UcAcAuAuAAAGAcAACUCdTsdT 78 79
2320-2338 AD-55077.2 AGuuGucuuuAuAuGuGAAdTsdT
UUcAcAuAuAAAGAcAACUdTsdT 80 81 2344-2362 AD-55083.2
uuAuAuuAAAuuuuAAucudTsdT AGAUuAAAAUUuAAuAuAAdTsdT 82 83 2352-2370
AD-55089.2 AAuuuuAAucuAuAGuAAAdTsdT UUuACuAuAGAUuAAAAUUdTsdT 84 85
2353-2371 AD-55095.2 AuuuuAAucuAuAGuAAAAdTsdT
UUUuACuAuAGAUuAAAAUdTsdT 86 87 2376-2394 AD-55101.2
AGuccuGGAAAuAAAuucudTsdT AGAAUUuAUUUCcAGGACUdTsdT 88 89 358-376
AD-53511.1 cuGcccAuucuuAucccGAdTsdT UCGGGAuAAGAAUGGGcAGdTsdT 90 91
789-807 AD-53512.1 ccAGuGuGGuuAGuGuGAAdTsdT
UUcAcACuAACcAcACUGGdTsdT 92 93 1076-1094 AD-53513.1
GucuGGuGcAGuAAuGAcudTsdT AGUcAUuACUGcACcAGACdTsdT 94 95 1253-1271
AD-53514.1 GcAcucuuGuuuuccucGudTsdT ACGAGGAAAAcAAGAGUGCdTsdT 96 97
1544-1562 AD-53515.1 GAGuuuGGAGcAAucAccudTsdT
AGGUGAUUGCUCcAAACUCdTsdT 98 99 2228-2246 AD-53516.1
GGcuuGAGcAAGuuGGuAudTsdT AuACcAACUUGCUcAAGCCdTsdT 100 101 404-422
AD-53517.1 GGcAAAucucuGuuGuucudTsdT AGAAcAAcAGAGAUUUGCCdTsdT 102
103 404-422 AD-53517.1 GGcAAAucucuGuuGuucudTsdT
AGAAcAAcAGAGAUUUGCCdTsdT 104 105 866-884 AD-53518.1
cAAAGAccAGAAAGAGuGudTsdT AcACUCUUUCUGGUCUUUGdTsdT 106 107 1080-1098
AD-53519.1 GGuGcAGuAAuGAcuAccudTsdT AGGuAGUcAUuACUGcACCdTsdT 108
109 1258-1276 AD-53520.1 cuuGuuuuccucGuGcuuudTsdT
AAAGcACGAGGAAAAcAAGdTsdT 110 111 1616-1634 AD-53521.1
GGGGAucGGGAuGGAGucAdTsdT UGACUCcAUCCCGAUCCCCdTsdT 112 113 2230-2248
AD-53522.1 cuuGAGcAAGuuGGuAucudTsdT AGAuACcAACUUGCUcAAGdTsdT 114
115 436-454 AD-53523.1 ccccAAGAuGAuGGAAGuudTsdT
AACUUCcAUcAUCUUGGGGdTsdT 116 117 436-454 AD-53523.1
ccccAAGAuGAuGGAAGuudTsdT AACUUCcAUcAUCUUGGGGdTsdT 118 119 885-903
AD-53524.1 cucAucuucuucAAGAuAAdTsdT UuAUCUUGAAGAAGAUGAGdTsdT 120
121 1127-1145 AD-53525.1 GGGGcAGuuAuGGAcAcuudTsdT
AAGUGUCcAuAACUGCCCCdTsdT 122 123 1315-1333 AD-53526.1
GAuGccAGGcuGuGAGAuudTsdT AAUCUcAcAGCCUGGcAUCdTsdT 124 125 1870-1888
AD-53527.1 GAGAcAGAuGcuAAuGGAudTsdT AUCcAUuAGcAUCUGUCUCdTsdT 126
127 2286-2304 AD-53528.1 ccccAGGccAuuAucAuAudTsdT
AuAUGAuAAUGGCCUGGGGdTsdT 128 129 489-507 AD-53529.1
cAGcAGuAcAcuAccAAcAdTsdT UGUUGGuAGUGuACUGCUGdTsdT 130 131 489-507
AD-53529.1 cAGcAGuAcAcuAccAAcAdTsdT UGUUGGuAGUGuACUGCUGdTsdT 132
133 915-933 AD-53530.1 cuGuuuccAcuuuucAGuAdTsdT
uACUGAAAAGUGGAAAcAGdTsdT 134 135 1138-1156 AD-53531.1
GGAcAcuuuGAAAcAAcAudTsdT AUGUUGUUUcAAAGUGUCCdTsdT 136 137 1324-1342
AD-53532.1 cuGuGAGAuuuAcucuGAudTsdT AUcAGAGuAAAUCUcAcAGdTsdT 138
139 1927-1945 AD-53533.1 cccuGuGcGGGuuGcAGAudTsdT
AUCUGcAACCCGcAcAGGGdTsdT 140 141 2312-2330 AD-53534.1
GucuucAGAGuuGucuuuAdTsdT uAAAGAcAACUCUGAAGACdTsdT 142 143 646-664
AD-53535.1 cAcuGcAAGcAAAuGcccudTsdT AGGGcAUUUGCUUGcAGUGdTsdT 144
145 922-940 AD-53536.1 cAcuuuucAGuAuGAucGudTsdT
ACGAUcAuACUGAAAAGUGdTsdT 146 147 1163-1181 AD-53537.1
GGGGcAGGuGGuAcuAGAAdTsdT UUCuAGuACcACCUGCCCCdTsdT 148 149 1347-1365
AD-53538.1 GGAAccAuGccuccAuGAudTsdT AUcAUGGAGGcAUGGUUCCdTsdT 150
151 1964-1982 AD-53539.1 GucuGuGAuGAAcuAAuGAdTsdT
UcAUuAGUUcAUcAcAGACdTsdT 152 153 2321-2339 AD-53540.1
GuuGucuuuAuAuGuGAAudTsdT AUUcAcAuAuAAAGAcAACdTsdT 154 155 671-689
AD-53541.1 GcAGcAcAGAuGAAucAGAdTsdT UCUGAUUcAUCUGUGCUGCdTsdT 156
157 924-942 AD-53542.1 cuuuucAGuAuGAucGuuudTsdT
AAACGAUcAuACUGAAAAGdTsdT 158 159 1164-1182 AD-53543.1
GGGcAGGuGGuAcuAGAAAdTsdT UUUCuAGuACcACCUGCCCdTsdT 160 161 1460-1478
AD-53544.1 GuccccAAGAuuGuGGcAudTsdT AUGCcAcAAUCUUGGGGACdTsdT 162
163 1976-1994 AD-53545.1 cuAAuGAGcAGAcAuAAcAdTsdT
UGUuAUGUCUGCUcAUuAGdTsdT 164 165 786-804 AD-53546.1
GccccAGuGuGGuuAGuGudTsdT AcACuAACcAcACUGGGGCdTsdT
166 167 935-953 AD-53547.1 GAucGuuucuuuGAGAAAAdTsdT
UUUUCUcAAAGAAACGAUCdTsdT 168 169 1165-1183 AD-53548.1
GGcAGGuGGuAcuAGAAAudTsdT AUUUCuAGuACcACCUGCCdTsdT 170 171 1530-1548
AD-53549.1 GuGAuGuGGcccAuGAGuudTsdT AACUcAUGGGCcAcAUcACdTsdT 172
173 2003-2021 AD-53550.1 cAAGcAAucAAuuAcccuAdTsdT
uAGGGuAAUUGAUUGCUUGdTsdT 174 175 788-806 AD-53551.1
cccAGuGuGGuuAGuGuGAdTsdT UcAcACuAACcAcACUGGGdTsdT 176 177 974-992
AD-53552.1 GAccAcAccuAucGAGuuudTsdT AAACUCGAuAGGUGUGGUCdTsdT 178
179 1191-1209 AD-53553.1 GAAcuAGuAAAuuccAuGudTsdT
AcAUGGAAUUuACuAGUUCdTsdT 180 181 1541-1559 AD-53554.1
cAuGAGuuuGGAGcAAucAdTsdT UGAUUGCUCcAAACUcAUGdTsdT 182 183 2075-2093
AD-53555.1 ccccAGAuGAuGAAcuAcudTsdT AGuAGUUcAUcAUCUGGGGdTsdT 184
185 360-378 AD-53561.1 GcccAuucuuAucccGAGudTsdT
ACUCGGGAuAAGAAUGGGCdTsdT 186 187 1356-1374 AD-53567.1
ccuccAuGAuccAAGGGAudTsdT AUCCCUUGGAUcAUGGAGGdTsdT 188 189 1631-1649
AD-53573.1 GucAuGccAAAAAuGGAcAdTsdT UGUCcAUUUUUGGcAUGACdTsdT 190
191 1634-1652 AD-53579.1 AuGccAAAAAuGGAcAucAdTsdT
UGAUGUCcAUUUUUGGcAUdTsdT
TABLE-US-00014 TABLE 3 Human ALAS1 Unmodified Single Strands and
Duplex Sequences SEQ ID SEQ ID NO: Position on NO: (anti-
transcript Duplex Sense Sequence Antisense Sequence (sense) sense)
NM_000688.4 Name (5'-3') (5'-3') 192 193 522-540 AD-55078.2
CUCCGGCCAGUGAGAAAGA UCUUUCUCACUGGCCGGAG 194 195 669-687 AD-55084.2
UGGCAGCACAGAUGAAUCA UGAUUCAUCUGUGCUGCCA 196 197 790-808 AD-55090.2
CAGUGUGGUUAGUGUGAAA UUUCACACUAACCACACUG 198 199 853-871 AD-55096.2
CAUCAUGCAAAAGCAAAGA UCUUUGCUUUUGCAUGAUG 200 201 876-894 AD-55102.2
AAAGAGUGUCUCAUCUUCU AGAAGAUGAGACACUCUUU 202 203 877-895 AD-55106.2
AAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUU 204 205 914-932 AD-55111.2
UCUGUUUCCACUUUUCAGU ACUGAAAAGUGGAAACAGA 206 207 923-941 AD-55073.2
ACUUUUCAGUAUGAUCGUU AACGAUCAUACUGAAAAGU 208 209 926-944 AD-55079.2
UUUCAGUAUGAUCGUUUCU AGAAACGAUCAUACUGAAA 210 211 927-945 AD-55085.2
UUCAGUAUGAUCGUUUCUU AAGAAACGAUCAUACUGAA 212 213 928-946 AD-55091.2
UCAGUAUGAUCGUUUCUUU AAAGAAACGAUCAUACUGA 214 215 932-950 AD-55097.2
UAUGAUCGUUUCUUUGAGA UCUCAAAGAAACGAUCAUA 216 217 973-991 AD-55103.2
UGACCACACCUAUCGAGUU AACUCGAUAGGUGUGGUCA 218 219 975-993 AD-55107.2
ACCACACCUAUCGAGUUUU AAAACUCGAUAGGUGUGGU 220 221 1029-1047
AD-55112.2 UGGCAGAUGACUAUUCAGA UCUGAAUAGUCAUCUGCCA 222 223
1077-1095 AD-55074.2 UCUGGUGCAGUAAUGACUA UAGUCAUUACUGCACCAGA 224
225 1124-1142 AD-55080.2 UGUGGGGCAGUUAUGGACA UGUCCAUAACUGCCCCACA
226 227 1137-1155 AD-55086.2 UGGACACUUUGAAACAACA
UGUUGUUUCAAAGUGUCCA 228 229 1182-1200 AD-55098.2
AUAUUUCUGGAACUAGUAA UUACUAGUUCCAGAAAUAU 230 231 1184-1202
AD-55104.2 AUUUCUGGAACUAGUAAAU AUUUACUAGUUCCAGAAAU 232 233
1185-1203 AD-55108.2 UUUCUGGAACUAGUAAAUU AAUUUACUAGUUCCAGAAA 234
235 1188-1206 AD-55113.2 CUGGAACUAGUAAAUUCCA UGGAAUUUACUAGUUCCAG
236 237 1325-1343 AD-55075.2 UGUGAGAUUUACUCUGAUU
AAUCAGAGUAAAUCUCACA 238 239 1364-1382 AD-55081.2
AUCCAAGGGAUUCGAAACA UGUUUCGAAUCCCUUGGAU 240 241 1382-1400
AD-55087.2 AGCCGAGUGCCAAAGUACA UGUACUUUGGCACUCGGCU 242 243
1478-1496 AD-55093.2 UUUGAAACUGUCCAUUCAA UUGAAUGGACAGUUUCAAA 244
245 1531-1549 AD-55099.2 UGAUGUGGCCCAUGAGUUU AAACUCAUGGGCCACAUCA
246 247 1631-1649 AD-53573.3 GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC 248 249 1637-1655 AD-55109.2
CCAAAAAUGGACAUCAUUU AAAUGAUGUCCAUUUUUGG 250 251 1706-1724
AD-55114.2 ACGAGUUCUCUGAUUGACA UGUCAAUCAGAGAACUCGU 252 253
1962-1980 AD-55076.2 AAGUCUGUGAUGAACUAAU AUUAGUUCAUCACAGACUU 254
255 1967-1985 AD-55082.2 UGUGAUGAACUAAUGAGCA UGCUCAUUAGUUCAUCACA
256 257 1977-1995 AD-55088.2 UAAUGAGCAGACAUAACAU
AUGUUAUGUCUGCUCAUUA 258 259 2189-2207 AD-55094.2
UUUGAAGUGAUGAGUGAAA UUUCACUCAUCACUUCAAA 260 261 2227-2245
AD-55100.2 AGGCUUGAGCAAGUUGGUA UACCAACUUGCUCAAGCCU 262 263
2313-2331 AD-55105.2 UCUUCAGAGUUGUCUUUAU AUAAAGACAACUCUGAAGA 264
265 2317-2335 AD-55110.2 CAGAGUUGUCUUUAUAUGU ACAUAUAAAGACAACUCUG
266 267 2319-2337 AD-55115.2 GAGUUGUCUUUAUAUGUGA
UCACAUAUAAAGACAACUC 268 269 2320-2338 AD-55077.2
AGUUGUCUUUAUAUGUGAA UUCACAUAUAAAGACAACU 270 271 2344-2362
AD-55083.2 UUAUAUUAAAUUUUAAUCU AGAUUAAAAUUUAAUAUAA 272 273
2352-2370 AD-55089.2 AAUUUUAAUCUAUAGUAAA UUUACUAUAGAUUAAAAUU 274
275 2353-2371 AD-55095.2 AUUUUAAUCUAUAGUAAAA UUUUACUAUAGAUUAAAAU
276 277 2376-2394 AD-55101.2 AGUCCUGGAAAUAAAUUCU
AGAAUUUAUUUCCAGGACU 278 279 358-376 AD-53511.1 CUGCCCAUUCUUAUCCCGA
UCGGGAUAAGAAUGGGCAG 280 281 789-807 AD-53512.1 CCAGUGUGGUUAGUGUGAA
UUCACACUAACCACACUGG 282 283 1076-1094 AD-53513.1
GUCUGGUGCAGUAAUGACU AGUCAUUACUGCACCAGAC 284 285 1253-1271
AD-53514.1 GCACUCUUGUUUUCCUCGU ACGAGGAAAACAAGAGUGC 286 287
1544-1562 AD-53515.1 GAGUUUGGAGCAAUCACCU AGGUGAUUGCUCCAAACUC 288
289 2228-2246 AD-53516.1 GGCUUGAGCAAGUUGGUAU AUACCAACUUGCUCAAGCC
290 291 404-422 AD-53517.1 GGCAAAUCUCUGUUGUUCU AGAACAACAGAGAUUUGCC
292 293 404-422 AD-53517.1 GGCAAAUCUCUGUUGUUCU AGAACAACAGAGAUUUGCC
294 295 866-884 AD-53518.1 CAAAGACCAGAAAGAGUGU ACACUCUUUCUGGUCUUUG
296 297 1080-1098 AD-53519.1 GGUGCAGUAAUGACUACCU
AGGUAGUCAUUACUGCACC 298 299 1258-1276 AD-53520.1
CUUGUUUUCCUCGUGCUUU AAAGCACGAGGAAAACAAG 300 301 1616-1634
AD-53521.1 GGGGAUCGGGAUGGAGUCA UGACUCCAUCCCGAUCCCC 302 303
2230-2248 AD-53522.1 CUUGAGCAAGUUGGUAUCU AGAUACCAACUUGCUCAAG 304
305 436-454 AD-53523.1 CCCCAAGAUGAUGGAAGUU AACUUCCAUCAUCUUGGGG 306
307 436-454 AD-53523.1 CCCCAAGAUGAUGGAAGUU AACUUCCAUCAUCUUGGGG 308
309 885-903 AD-53524.1 CUCAUCUUCUUCAAGAUAA UUAUCUUGAAGAAGAUGAG 310
311 1127-1145 AD-53525.1 GGGGCAGUUAUGGACACUU AAGUGUCCAUAACUGCCCC
312 313 1315-1333 AD-53526.1 GAUGCCAGGCUGUGAGAUU
AAUCUCACAGCCUGGCAUC 314 315 1870-1888 AD-53527.1
GAGACAGAUGCUAAUGGAU AUCCAUUAGCAUCUGUCUC 316 317 2286-2304
AD-53528.1 CCCCAGGCCAUUAUCAUAU AUAUGAUAAUGGCCUGGGG 318 319 489-507
AD-53529.1 CAGCAGUACACUACCAACA UGUUGGUAGUGUACUGCUG 320 321 489-507
AD-53529.1 CAGCAGUACACUACCAACA UGUUGGUAGUGUACUGCUG 322 323 915-933
AD-53530.1 CUGUUUCCACUUUUCAGUA UACUGAAAAGUGGAAACAG 324 325
1138-1156 AD-53531.1 GGACACUUUGAAACAACAU AUGUUGUUUCAAAGUGUCC 326
327 1324-1342 AD-53532.1 CUGUGAGAUUUACUCUGAU AUCAGAGUAAAUCUCACAG
328 329 1927-1945 AD-53533.1 CCCUGUGCGGGUUGCAGAU
AUCUGCAACCCGCACAGGG 330 331 2312-2330 AD-53534.1
GUCUUCAGAGUUGUCUUUA UAAAGACAACUCUGAAGAC 332 333 646-664 AD-53535.1
CACUGCAAGCAAAUGCCCU AGGGCAUUUGCUUGCAGUG 334 335 922-940 AD-53536.1
CACUUUUCAGUAUGAUCGU ACGAUCAUACUGAAAAGUG 336 337 1163-1181
AD-53537.1 GGGGCAGGUGGUACUAGAA UUCUAGUACCACCUGCCCC 338 339
1347-1365 AD-53538.1 GGAACCAUGCCUCCAUGAU AUCAUGGAGGCAUGGUUCC 340
341 1964-1982 AD-53539.1 GUCUGUGAUGAACUAAUGA UCAUUAGUUCAUCACAGAC
342 343 2321-2339 AD-53540.1 GUUGUCUUUAUAUGUGAAU
AUUCACAUAUAAAGACAAC 344 345 671-689 AD-53541.1 GCAGCACAGAUGAAUCAGA
UCUGAUUCAUCUGUGCUGC 346 347 924-942 AD-53542.1 CUUUUCAGUAUGAUCGUUU
AAACGAUCAUACUGAAAAG 348 349 1164-1182 AD-53543.1
GGGCAGGUGGUACUAGAAA UUUCUAGUACCACCUGCCC 350 351 1460-1478
AD-53544.1 GUCCCCAAGAUUGUGGCAU AUGCCACAAUCUUGGGGAC 352 353
1976-1994 AD-53545.1 CUAAUGAGCAGACAUAACA UGUUAUGUCUGCUCAUUAG 354
355 786-804 AD-53546.1 GCCCCAGUGUGGUUAGUGU ACACUAACCACACUGGGGC 356
357 935-953 AD-53547.1 GAUCGUUUCUUUGAGAAAA UUUUCUCAAAGAAACGAUC 358
359 1165-1183 AD-53548.1 GGCAGGUGGUACUAGAAAU AUUUCUAGUACCACCUGCC
360 361 1530-1548 AD-53549.1 GUGAUGUGGCCCAUGAGUU
AACUCAUGGGCCACAUCAC 362 363 2003-2021 AD-53550.1
CAAGCAAUCAAUUACCCUA UAGGGUAAUUGAUUGCUUG 364 365 788-806 AD-53551.1
CCCAGUGUGGUUAGUGUGA UCACACUAACCACACUGGG 366 367 974-992 AD-53552.1
GACCACACCUAUCGAGUUU AAACUCGAUAGGUGUGGUC 368 369 1191-1209
AD-53553.1 GAACUAGUAAAUUCCAUGU ACAUGGAAUUUACUAGUUC 370 371
1541-1559 AD-53554.1 CAUGAGUUUGGAGCAAUCA UGAUUGCUCCAAACUCAUG 372
373 2075-2093 AD-53555.1 CCCCAGAUGAUGAACUACU AGUAGUUCAUCAUCUGGGG
374 375 360-378 AD-53561.1 GCCCAUUCUUAUCCCGAGU ACUCGGGAUAAGAAUGGGC
376 377 1356-1374 AD-53567.1 CCUCCAUGAUCCAAGGGAU
AUCCCUUGGAUCAUGGAGG 378 379 1631-1649 AD-53573.1
GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC 380 381 1634-1652
AD-53579.1 AUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAU
Example 3. In Vitro Screening of ALAS1 siRNA Duplexes for ALAS1
Knockdown Activity
[0701] ALAS1 siRNA duplexes were screened for the ability to
knockdown ALAS1 expression in vitro.
In Vitro Screening
[0702] Cell Culture and Transfections
[0703] Hep3B cells (ATCC, Manassas, Va.) were grown to near
confluence at 37.degree. C. in an atmosphere of 5% CO.sub.2 in MEM
(ATCC) supplemented with 10% FBS, before being released from the
plate by trypsinization. Transfection was carried out by adding
14.8 .mu.l of Opti-MEM plus 0.2 .mu.l of Lipofectamine RNAiMax per
well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 .mu.l of
siRNA duplexes per well into a 96-well plate and incubated at room
temperature for 15 minutes. 80 .mu.l of complete growth media
containing .about.2.times.10.sup.4 Hep3B cells were then added to
the siRNA mixture. Cells were incubated for either 24 or 120 hours
prior to RNA purification. Single dose experiments were performed
at 10 nM and 0.1 nM final duplex concentration and dose response
experiments were done at 10, 1.67, 0.27, 0.046, 0.0077, 0.0013,
0.00021, 0.00004 nM final duplex concentration.
[0704] Total RNA Isolation Using DYNABEADS mRNA Isolation Kit
(Invitrogen, Part #: 610-12)
[0705] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding
Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf
Thermomixer (the mixing speed was the same throughout the process).
Ten microliters of magnetic beads and 80 .mu.l Lysis/Binding Buffer
mixture were added to a round bottom plate and mixed for 1 minute.
Magnetic beads were captured using magnetic stand and the
supernatant was removed without disturbing the beads. After
removing supernatant, the lysed cells were added to the remaining
beads and mixed for 5 minutes. After removing supernatant, magnetic
beads were washed 2 times with 150 .mu.l Wash Buffer A and mixed
for 1 minute. Beads were captured again and supernatant removed.
Beads were then washed with 150 .mu.l Wash Buffer B, captured and
supernatant was removed. Beads were next washed with 150 .mu.l
Elution Buffer, captured and supernatant removed. Beads were
allowed to dry for 2 minutes. After drying, 50 .mu.l of Elution
Buffer was added and mixed for 5 minutes at 70.degree. C. Beads
were captured on magnet for 5 minutes. 40 .mu.l of supernatant was
removed and added to another 96 well plate.
[0706] cDNA Synthesis Using ABI High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, Calif., Cat
#4368813)
[0707] A master mix of 2 .mu.l 10.times. Buffer, 0.8 .mu.l
25.times.dNTPs, 2 .mu.l Random primers, 1 .mu.l Reverse
Transcriptase, 1 .mu.l RNase inhibitor and 3.2 .mu.l of H.sub.2O
per reaction were added into 10 .mu.l total RNA. cDNA was generated
using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.)
through the following steps: 25.degree. C. 10 min, 37.degree. C.
120 min, 85.degree. C. 5 sec, 4.degree. C. hold.
[0708] Real Time PCR
[0709] 2 .mu.l of cDNA were added to a master mix containing 0.5
.mu.l GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5
.mu.l ALAS1 TaqMan probe (Applied Biosystems cat #Hs00167441_m1)
and 5 .mu.l Lightcycler 480 probe master mix (Roche Cat
#04887301001) per well in a 384 well plates (Roche cat
#04887301001). Real time PCR was done in a Roche LC480 Real Time
PCR system (Roche) using the .DELTA..DELTA.Ct(RQ) assay. Each
duplex was tested in two independent transfections with two
biological replicates each, and each transfection was assayed in
duplicate, unless otherwise noted in the summary tables.
[0710] To calculate relative fold change, real time data were
analyzed using the .DELTA..DELTA.Ct method and normalized to assays
performed with cells transfected with 10 nM AD-1955, or mock
transfected cells. IC50s were calculated using a 4 parameter fit
model using XLFit and normalized to cells transfected with AD-1955
or naive cells over the same dose range, or to its own lowest
dose.
In Vitro Knockdown of Endogenous ALAS1 Expression by ALAS1 siRNA
Duplexes
[0711] Table 4 illustrates the knockdown of ALAS1 in Hep3B cells by
ALAS1 modified siRNA duplexes (See Table 2). Silencing is expressed
as the fraction RNA message remaining relative to the negative
(luciferase) control siRNA AD-1955. Data were generated as
described above following transfection of 10 nM or 0.1 nM of each
siRNA. qPCR was run using the ALAS1 TaqMan probe Hs00167441_m1.
TABLE-US-00015 TABLE 4 ALAS1 expression in Hep3B cells following
transfection with ALAS1 siRNA Duplex ID 10 nM Avg 0.1 nM Avg 10 nM
STDEV 0.1 nM STDEV AD-55078.2 0.7 0.87 0.001 0.089 AD-55084.2 0.08
0.3 0 0.04 AD-55090.2 0.06 0.08 0.002 0.003 AD-55096.2 0.61 0.92
0.171 0.34 AD-55102.2 0.63 0.62 0.005 0.069 AD-55106.2 0.07 0.08
0.004 0.027 AD-55111.2 0.06 0.23 0.013 0.062 AD-55073.2 0.21 0.4
0.018 0.061 AD-55079.2 0.17 0.43 0.033 0.089 AD-55085.2 0.13 0.21
0.011 0.019 AD-55091.2 0.27 0.55 0.033 0.009 AD-55097.2 0.31 0.38
0.051 0.059 AD-55103.2 0.05 0.11 0.017 0.006 AD-55107.2 0.12 0.24
0.007 0.008 AD-55112.2 0.15 0.2 0.036 0.025 AD-55074.2 0.16 0.45
0.008 0.002 AD-55080.2 0.79 0.99 0.095 0.304 AD-55086.2 0.09 0.22
0.005 0.035 AD-55098.2 0.25 0.51 0.03 0.07 AD-55104.2 0.06 0.1
0.017 0.001 AD-55108.2 0.47 0.65 0.03 0.015 AD-55113.2 0.38 0.62
0.068 0.039 AD-55075.2 0.12 0.28 0.007 0.051 AD-55081.2 0.21 0.51
0.036 0.066 AD-55087.2 0.1 0.19 0.017 0.02 AD-55093.2 0.24 0.56
0.029 0.053 AD-55099.2 0.05 0.18 0.001 0.038 AD-53573.3 0.67 1.07
0.16 0.153 AD-55109.2 0.07 0.23 0.006 0.052 AD-55114.2 0.08 0.16
0.004 0.017 AD-55076.2 0.05 0.14 0.007 0.035 AD-55082.2 0.08 0.3
0.019 0.016 AD-55088.2 0.06 0.12 0.008 0.02 AD-55094.2 0.06 0.18
0.005 0.023 AD-55100.2 0.45 0.83 0.02 0.05 AD-55105.2 0.02 0.05
0.005 0.004 AD-55110.2 0.15 0.19 0.031 0.016 AD-55115.2 0.35 0.58
0.045 0.052 AD-55077.2 0.14 0.14 0.006 0.019 AD-55083.2 0.56 0.98
0.24 0.188 AD-55089.2 0.62 0.79 0.036 0.094 AD-55095.2 0.59 0.92
0.12 0.079 AD-55101.2 0.71 0.97 0.074 0.097 AD-1955 1.00 1.01 0.03
0.04 AD-53511.1 0.84 1.08 0.028 0.0515 AD-53512.1 0.15 0.65 0.062
0.023 AD-53513.1 0.34 0.86 0.055 0.011 AD-53514.1 0.12 0.61 0.003
0.008 AD-53515.1 0.25 0.66 0.005 0.004 AD-53516.1 1.05 1.02 0.032
0.011 AD-53517.1 0.145 0.725 0.025 0.0155 AD-53518.1 0.72 0.85
0.045 0.028 AD-53519.1 0.18 0.66 0.061 0.004 AD-53520.1 0.18 0.9
0.041 0.001 AD-53521.1 0.97 1.07 0.01 0.003 AD-53522.1 0.87 1.1
0.065 0.112 AD-53523.1 0.48 0.96 0.0305 0.0255 AD-53524.1 0.11 0.66
0.02 0.006 AD-53525.1 0.71 1.03 0.016 0.01 AD-53526.1 0.23 0.85
0.075 0.01 AD-53527.1 0.25 0.83 0.015 0.017 AD-53528.1 0.44 0.93
0.037 0.006 AD-53529.1 0.185 0.73 0.015 0.014 AD-53530.1 0.1 0.62
0.02 0.003 AD-53531.1 0.48 0.93 0.019 0.045 AD-53532.1 0.06 0.17 0
0.003 AD-53533.1 0.36 0.93 0.025 0.034 AD-53534.1 0.1 0.36 0.014
0.012 AD-53535.1 0.58 1.05 0.036 0.071 AD-53536.1 0.12 0.45 0.009
0.026 AD-53537.1 0.73 0.96 0.101 0.015 AD-53538.1 0.74 1.07 0 0.046
AD-53539.1 0.52 0.97 0.057 0.032 AD-53540.1 0.1 0.47 0.017 0.012
AD-53541.1 0.11 0.29 0.026 0.015 AD-53542.1 0.08 0.23 0.008 0.006
AD-53543.1 0.62 1.01 0.027 0.014 AD-53544.1 0.8 1.04 0.002 0.001
AD-53545.1 0.17 0.73 0.007 0.007 AD-53546.1 0.27 0.93 0.058 0.019
AD-53547.1 0.12 0.28 0.008 0.01 AD-53548.1 0.1 0.34 0.022 0.002
AD-53549.1 0.8 1.04 0.011 0.026 AD-53550.1 0.05 0.54 0.02 0.003
AD-53551.1 0.96 1.16 0.029 0.044 AD-53552.1 0.13 0.5 0.002 0.009
AD-53553.1 0.92 1.1 0.027 0.02 AD-53554.1 0.76 0.67 0.005 0.004
AD-53555.1 0.11 0.53 0.009 0.007 AD-53561.1 0.72 0.94 0.014 0.001
AD-53567.1 0.16 0.66 0.019 0.003 AD-53573.1 1.06 1.10 0.019 0.037
AD-53579.1 0.19 0.76 0.036 0.019
IC.sub.50s of Select ALAS1 siRNA Duplexes in In Vitro Screen
[0712] Table 5 illustrates the IC.sub.50s of select ALAS1 siRNA
duplexes determined from the knockdown of endogenously expressed
ALAS1 in the Hep3B cell line, by ALAS1 modified siRNA duplexes (see
Table 2). Data were generated as described above, at 24 or 120
hours following transfection of each siRNA duplex. Silencing of
ALAS1 is expressed as the fraction mRNA message remaining relative
to the siRNA AD-1955, a non-targeting siRNA that was used as a
negative control. Data from replicate transfection experiments were
used to fit a single line to determine the IC.sub.50. Several of
the duplexes (e.g., AD-53541.1, AD-53542.1, and AD-53547.1) had an
IC.sub.50 as low as about 0.03 nM at 24 hours. Numerous duplexes
had an IC.sub.50 of less than 0.1 nM (e.g., AD-53534.1, AD-53536.1,
AD-53540.1, AD-53541.1, AD-53542.1, AD-53547.1, AD-53548.1,
AD-53550.1, AD-53552.1) at 24 hours, and some of these also had an
IC.sub.50 of less than 0.1 nM (e.g., AD-53534.1, AD-53540.1,
AD-53541.1, AD-53542.1, AD-53547.1, AD-53552.1) at 120 hours.
TABLE-US-00016 TABLE 5 IC.sub.50S of select ALAS1 siRNA duplexes
normalized to AD-1955 IC.sub.50 (nM) DUPLEX ID 24 hrs 120 hrs
AD-53534.1 0.045 0.076 AD-53536.1 0.049 0.105 AD-53540.1 0.054
0.077 AD-53541.1 0.032 0.062 AD-53542.1 0.028 0.093 AD-53547.1 0.03
0.062 AD-53548.1 0.044 0.101 AD-53550.1 0.085 0.152 AD-53552.1
0.077 0.063 AD-53567.1 0.219 0.357 AD-53579.1 0.217 0.566
Example 4. In Vivo Silencing Using a Mouse/Rat ALAS1 siRNA
Formulated as a LNP
[0713] The sequences of the modified duplex AD-53558 are shown in
Table 6 below.
TABLE-US-00017 TABLE 6 Sequences of ALAS1 siRNA Duplex AD-53558.4
SEQ ID Start SEQ ID NO: Position on NO: (anti- transcript of Duplex
Sense Sequence Antisense Sequence (sense) sense) NM_020559.2 Name
(5'-3') (5'-3') 383 384 1184 AD-53558 cuGuGAAAuuuAcucuGAudTsdT
AUcAGAGuAAAUUUcAcAGdTsdT
[0714] This duplex was formulated as a LNP11 formulation (see Table
10 above). The LNP-formulated AD-53558 siRNA was tested in in vivo
in mice (N=25 animals; 5 animals per group) and rats (N=20 animals;
4 animals per group) and was confirmed to silence ALAS1 mRNA in
vivo. The results are shown in FIG. 5 and FIG. 6.
[0715] FIG. 5 shows that the siRNA demonstrated a dose-response
effect in mice. The expression of mouse ALAS1 (mALAS1) mRNA was
reduced by about 78% when the siRNA was administered at 1 mg/kg;
mouse ALAS1 mRNA was reduced by about 60% when the siRNA was
administered at 0.3 mg/kg; and mouse ALAS1 mRNA was reduced by
about 49% when the siRNA was administered at 0.1 mg/kg. These
reductions are expressed relative to a PBS control. An AD-1955 LUC
control was also employed, as shown in FIG. 5.
[0716] Similarly, FIG. 6 shows that the siRNA demonstrated a
dose-response effect in rats. The expression of ALAS1 RNA was
reduced by about 70% when the when the siRNA was administered at 1
mg/kg; ALAS1 mRNA was reduced by about 62% when the siRNA was
administered at 0.3 mg/kg; and ALAS1 mRNA was reduced by about 34%
when the siRNA was administered at 0.1 mg/kg.
[0717] The durability of silencing was also tested in mice (N=15; 3
animals per timepoint. The results are shown in FIG. 7, which shows
that AD-53558 suppressed mALAS1 mRNA by about 80% for at least 9
days. Suppression of at least about 50% persisted for at least 14
days.
Example 5. Efficacy of ALAS1 siRNA in an Animal Model of AIP
[0718] The effects of the AD-53558 LNP11 formulation (a mouse/rat
ALAS1 siRNA described in the previous example) were investigated in
a mouse model of AP. The PBGD knockout is not viable (-/-, 0%
activity). Heterozygous PBGD knockout mice (+/-, 50% activity) are
available but do not have the full biochemical phenotype and thus
do not recapitulate the human disease phenotype. Thus, a mouse
model of AIP has been developed that is a compound heterozygote
with T1/T2 alleles, including T1 (+/-) promoter disruption and T2
(-/-) splice-site alteration. These mice have been shown to have
hepatic residual PBGD activity that is about .about.30% of the
wild-type level and normal or slightly elevated baseline plasma ALA
and PBG levels. The mice have been found to appear normal early in
life and to become slightly slower and ataxic with age. By six
months of age, the mice have been documented to develop impaired
motor coordination and muscular performance and axonal degeneration
on pathological examination. Investigation of the pathology of the
mouse model has shown axonal degeneration, impaired motor
coordination and muscular performance in older mice. Urinary and
plasma ALA and PBG have been found to markedly increase with serial
i.p. administration of phenobarbital (see Lindberg et al., (1996),
Nature Genetics, 12:195-219 and Lindberg et al., (1999), Journal of
Clinical Investigation, 103:1127-34). The mice were rescued by
AAV-mediated expression of PBGD in the liver (Yasuda et al. (2010),
Molecular Medicine, 1:17-22 and Unzu et al. (2011), Molecular
Medicine, 2:243-50).
[0719] On day 1, the mice were administered 1 mg/kg ALAS1 siRNA
(n=5) or LUC AD-1955 control (n=3) by i.v. injection. Three
phenobarbitol injections were given (1 injection per day on days 2,
3, and 4) to induce hepatic ALAS1 ande the porphyrin precursors,
ALA and PBG. Plasma and overnight urine specimens were collected on
day 5 and metabolite levels were measured by LC-MS. Metabolite
levels were measured in plasma by LC-MS and were also measured in
urine. Baseline levels of metabolites were measured prior to the
first treatment on day 1. The results are shown in FIGS. 8-12 and
in Tables 12 and 13.
[0720] FIG. 8 and FIG. 9 show the plasma ALA levels in .mu.M.
Baseline ALA levels were low, (n=4), and phenobarbitol treatment
induced significant increases in plasma ALA levels in the control
LUC siRNA treated animals (n=3). Treatment with ALAS1 siRNA
inhibited the induction of plasma ALA (n=5), as shown in FIG. 8.
The ALAS1 siRNA was consistently effective in blocking the
induction of plasma ALA in each of the individual animals studied
(see FIG. 9). These results indicate that ALAS1 siRNA treatment was
effective in preventing the increases in plasma ALA associated with
the phenobarbital-induced acute attacks in this AIP animal
model.
[0721] FIG. 10 and FIG. 11 show the plasma PBG levels in .mu.M.
Baseline PBG levels were low (n=4), and phenobarbitol treatment
induced significant increases in plasma PBG levels in the control
LUC siRNA treated animals (n=3) Treatment with ALAS1 siRNA
inhibited the induction of plasma PBG (n=5), as shown in FIG. 10.
The ALAS1 siRNA was consistently effective in blocking the
induction of plasma PBG in each of the individual animals studied
(see FIG. 11). These results indicate that ALAS1 siRNA treatment
was effective in preventing the increases in plasma PBG associated
with the phenobarbital-induced acute attacks in this AP animal
model.
[0722] Tables 12 and 13 shows urine ALA and PBG levels at baseline
and after phenobarbitol treatment in LUC siRNA (n=2) control (CTR,
which refers to a PBS buffer treated animal, n=1) and ALAS1 siRNA
(n=5) treated animals.
TABLE-US-00018 TABLE 12 Urine data from individual animals showing
prevention of induced acute attack ALA PBG ALA PBG (micro (micro
Creatinine (microM/mg (microM/mg Mouse ID M/I) M/L) (mg/dl)
creatinine) creatinine) siRNA PB Ha-17-4-6 29.7 7.9 Baseline -
Ha-19-5-4/2 15.7 5.1 Baseline - Ha-20-39- 28.6 6.7 Baseline - 4/3
Ha-20-38-4 21.4 4.7 Baseline - Ha-21-33-4 934.92 483.71 0.4205
222.33 115.03 Luc + Ha-21-36-9 944.08 563.53 0.5055 186.76 111.48
Luc + Ha-21-18-8 32.88 8.69 0.133 24.72 6.53 ALAS1; + 1 mg/kg
Ha-21-33-7 83.07 23.28 0.426 19.50 5.46 ALAS1; + 1 mg/kg Ha-21-34-5
59.15 18.41 0.263 22.49 7.00 ALAS1; + 1 mg/kg PB stands for
phenobarbitol. A ''+'' indicates that phenobarbitol was
administered.
TABLE-US-00019 TABLE 13 Average Urine Data Mean ALA Mean PBG
(microM/mg creatinine) (microM/mg creatinine) 23.8 6.1 AIP Baseline
204.55 113.26 Luc-siRNA 22.24 6.33 ALAS1-siRNA
[0723] Phenobarbitol treatment induced strong increases
(.about.25-30 fold increases) in urine ALA (.about.9-fold over
baseline levels) and PBG (.about.19-fold over baseline levels) in
the LUC siRNA treated mice, control, whereas such increases were
not observed in the ALAS1 siRNA treated animals. Thus, ALAS1 siRNA
blocked phenobarbitol-induced increases in urinary ALA and PBG.
These results are consistent with the plasma measurements and show
that ALAS1 siRNA treatment was effective in preventing increases in
urinary metabolites (ALA and PBG) associated with the
phenobarbital-induced acute attacks in this AIP animal model.
[0724] In further experiments (FIG. 12), it was found that
phenobarbitol treatment induced large increases (.about.25 fold) in
ALAS1 mRNA expression in the liver of the mouse model.
Administration of ALAS1 siRNA completely blocked this ALAS1 mRNA
induction. These results provide further evidence that ALAS1 siRNA
is effective in an animal model of AIP.
[0725] Collectively, the results provided in this Example show that
ALAS1 siRNA was effective in treating acute attacks in an animal
model of the acute hepatic porphyria AIP. Multiple outcome measures
support this conclusion, including plasma ALA levels, plasma PBG
levels, urine ALA levels, urine PBG levels, and liver ALAS1 mRNA
expression levels.
Example 6. In Vivo Silencing Using GalNAc-Conjugated Mouse ALAS1
siRNA
[0726] The experiments described in this example investigated the
in vivo efficacy of three GalNAc-conjugated siRNAs (see Table 7).
These siRNAs were designed and produced with methods such as those
described in Example 2.
TABLE-US-00020 TABLE 7 Sequences AD-57929 Position SEQ Position of
SEQ ID of sense antisense ID NO: seq. on seq. on NO: (anti-
transcript Duplex Sense Sequence Antisense Sequence transcript
(sense) sense) NM_ 020559.2 Name (5'-3') (5'-3') NM_020559.2 385
386 775-795 AD- AfaGfuCfuGfuUfUfCf uUfgAfaAfaGfuGfgaa 773-795 56211
cAfcUfuUfuCfaAfL96 AfcAfgAfcUfusUfsg 387 388 2168-2188 AD-
AfcAfuAfgUfaGfCfCf aGfaCfaAfuUfcUfggc 2166-2188 56173
aGfaAfuUfgUfcUfL96 UfaCfuAfuGfusGfsg 389 390 775-795 AD-
AfsasGfuCfuGfuUfUf usUfsgAfaAfaGfuGfg 773-795 57929
CfcAfcUfuUfuCfaAfL aaAfcAfgAfcUfususg 96
[0727] The mice (n=40; n=4 per experimental condition) were divided
into groups that received PBS or doses of 3 mg/kg, 10 mg/kg, or 30
mg/kg of siRNA administered subcutaneously. The level of
mALAS1/mGAPDH mRNA, relative to the PBS control, was determined in
liver cells at 72 hours post-administration. The results are shown
in FIG. 13. There was not a clear dose-response effect for the
siRNAs AD-56211 and AD-56173. In contrast, the ALAS1 siRNA AD-57929
showed a dose-response effect in inhibiting mALAS1 expression.
These results demonstrate that an ALAS1 GalNAc conjugate was
effective in inhibiting expression of ALAS1 mRNA in vivo and showed
a dose-response effect.
Example 7. Human siRNAs
[0728] Additional human siRNAs were designed and produced as
described in Example 2. The top 45 siRNAs were selected based on
their predicted efficacy. The sequences of these 45 siRNAs are
provided in Table 8.
TABLE-US-00021 TABLE 8 Human ALAS1 siRNA Sense and Antisense
Sequences SEQ ID Position SEQ ID NO: on NO: (anti- transcript Sense
Sequence Antisense Sequence (sense) sense) NM_000688.4 (5'-3')
(5'-3') 391 392 1635-1657 CAUGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCAUGAC 393 394 2352-2374 UAAAUUUUAAUCUAUAGUAAA
UUUACUAUAGAUUAAAAUUUAAU 395 396 1324-1346 GGCUGUGAGAUUUACUCUGAU
AUCAGAGUAAAUCUCACAGCCUG 397 398 1637-1659 UGCCAAAAAUGGACAUCAUUU
AAAUGAUGUCCAUUUUUGGCAUG 399 400 1363-1385 AUGAUCCAAGGGAUUCGAAAC
GUUUCGAAUCCCUUGGAUCAUGG 401 402 925-947 ACUUUUCAGUAUGAUCGUUUC
GAAACGAUCAUACUGAAAAGUGG 403 404 790-812 CCCAGUGUGGUUAGUGUGAAA
UUUCACACUAACCACACUGGGGC 405 406 1531-1553 UGUGAUGUGGCCCAUGAGUUU
AAACUCAUGGGCCACAUCACACA 407 408 2189-2211 AUUUUGAAGUGAUGAGUGAAA
UUUCACUCAUCACUUCAAAAUGC 409 410 929-951 UUCAGUAUGAUCGUUUCUUUG
CAAAGAAACGAUCAUACUGAAAA 411 412 872-894 GACCAGAAAGAGUGUCUCAUC
GAUGAGACACUCUUUCUGGUCUU 413 414 706-728 UUCUGCAAAGCCAGUCUUGAG
CUCAAGACUGGCUUUGCAGAAGA 415 416 1362-1384 CAUGAUCCAAGGGAUUCGAAA
UUUCGAAUCCCUUGGAUCAUGGA 417 418 1634-1656 UCAUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAUGACU 419 420 1325-1347 GCUGUGAGAUUUACUCUGAUU
AAUCAGAGUAAAUCUCACAGCCU 421 422 2208-2230 AAGAGAGAAGUCCUAUUUCUC
GAGAAAUAGGACUUCUCUCUUUC 423 424 2344-2366 AGUUAUAUUAAAUUUUAAUCU
AGAUUAAAAUUUAAUAUAACUUA 425 426 924-946 CACUUUUCAGUAUGAUCGUUU
AAACGAUCAUACUGAAAAGUGGA 427 428 873-895 ACCAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUGGUCU 429 430 759-781 GAGGAAAGAGGUUGCUGAAAC
GUUUCAGCAACCUCUUUCCUCAC 431 432 871-893 AGACCAGAAAGAGUGUCUCAU
AUGAGACACUCUUUCUGGUCUUU 433 434 1183-1205 AAUAUUUCUGGAACUAGUAAA
UUUACUAGUUCCAGAAAUAUUUC 435 436 2229-2251 AGGCUUGAGCAAGUUGGUAUC
GAUACCAACUUGCUCAAGCCUGA 437 438 671-693 UGGCAGCACAGAUGAAUCAGA
UCUGAUUCAUCUGUGCUGCCAGG 439 440 2187-2209 GCAUUUUGAAGUGAUGAGUGA
UCACUCAUCACUUCAAAAUGCAG 441 442 913-935 AAAUCUGUUUCCACUUUUCAG
CUGAAAAGUGGAAACAGAUUUUG 443 444 1977-1999 ACUAAUGAGCAGACAUAACAU
AUGUUAUGUCUGCUCAUUAGUUC 445 446 1174-1196 GGUACUAGAAAUAUUUCUGGA
UCCAGAAAUAUUUCUAGUACCAC 447 448 1810-1832 AUCCUGAAGAGCGCUGAGGGA
UCCCUCAGCGCUCUUCAGGAUCC 449 450 892-914 CUUCUUCAAGAUAACUUGCCA
UGGCAAGUUAUCUUGAAGAAGAU 451 452 877-899 GAAAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUUUCUG 453 454 935-957 AUGAUCGUUUCUUUGAGAAAA
UUUUCUCAAAGAAACGAUCAUAC 455 456 1975-1997 GAACUAAUGAGCAGACAUAAC
GUUAUGUCUGCUCAUUAGUUCAU 457 458 1478-1500 CAUUUGAAACUGUCCAUUCAA
UUGAAUGGACAGUUUCAAAUGCC 459 460 2366-2388 UAGUAAAAACAUAGUCCUGGA
UCCAGGACUAUGUUUUUACUAUA 461 462 853-875 GACAUCAUGCAAAAGCAAAGA
UCUUUGCUUUUGCAUGAUGUCCU 463 464 1966-1988 GUCUGUGAUGAACUAAUGAGC
GCUCAUUAGUUCAUCACAGACUU 465 466 928-950 UUUCAGUAUGAUCGUUUCUUU
AAAGAAACGAUCAUACUGAAAAG 467 468 1186-1208 AUUUCUGGAACUAGUAAAUUC
GAAUUUACUAGUUCCAGAAAUAU 469 470 1189-1211 UCUGGAACUAGUAAAUUCCAU
AUGGAAUUUACUAGUUCCAGAAA 471 472 973-995 AAUGACCACACCUAUCGAGUU
AACUCGAUAGGUGUGGUCAUUCU 473 474 983-1005 CCUAUCGAGUUUUUAAAACUG
CAGUUUUAAAAACUCGAUAGGUG 475 476 1185-1207 UAUUUCUGGAACUAGUAAAUU
AAUUUACUAGUUCCAGAAAUAUU 477 478 2353-2375 AAAUUUUAAUCUAUAGUAAAA
UUUUACUAUAGAUUAAAAUUUAA 479 480 875-897 CAGAAAGAGUGUCUCAUCUUC
GAAGAUGAGACACUCUUUCUGGU 481 482 360-378 GCCCAUUCUUAUCCCGAGU
ACUCGGGAUAAGAAUGGGC 483 484 428-446 CAAAACUGCCCCAAGAUGA
UCAUCUUGGGGCAGUUUUG 485 486 873-891 CAGAAAGAGUGUCUCAUCU
AGAUGAGACACUCUUUCUG 487 488 874-892 AGAAAGAGUGUCUCAUCUU
AAGAUGAGACACUCUUUCU 489 490 877-895 AAGAGUGUCUCAUCUUCUU
AAGAAGAUGAGACACUCUU 491 492 1295-1313 CUCUUCACCCUGGCUAAGA
UCUUAGCCAGGGUGAAGAG 493 494 1296-1314 UCUUCACCCUGGCUAAGAU
AUCUUAGCCAGGGUGAAGA 495 496 1299-1317 UCACCCUGGCUAAGAUGAU
AUCAUCUUAGCCAGGGUGA 497 498 1347-1365 GGAACCAUGCCUCCAUGAU
AUCAUGGAGGCAUGGUUCC 499 500 1355-1373 GCCUCCAUGAUCCAAGGGA
UCCCUUGGAUCAUGGAGGC 501 502 1356-1374 CCUCCAUGAUCCAAGGGAU
AUCCCUUGGAUCAUGGAGG 503 504 1357-1375 CUCCAUGAUCCAAGGGAUU
AAUCCCUUGGAUCAUGGAG 505 506 1631-1649 GUCAUGCCAAAAAUGGACA
UGUCCAUUUUUGGCAUGAC 507 508 1634-1652 AUGCCAAAAAUGGACAUCA
UGAUGUCCAUUUUUGGCAU 509 510 1635-1653 UGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCA 511 512 1791-1809 CCCUGGAGUCUGUGCGGAU
AUCCGCACAGACUCCAGGG 513 514 1794-1812 UGGAGUCUGUGCGGAUCCU
AGGAUCCGCACAGACUCCA 515 516 1921-1939 CAUCAUCCCUGUGCGGGUU
AACCCGCACAGGGAUGAUG 517 518 359-377 UGCCCAUUCUUAUCCCGAA
UUCGGGAUAAGAAUGGGCA 519 520 362-380 CCAUUCUUAUCCCGAGUCA
UGACUCGGGAUAAGAAUGG 521 522 363-381 CAUUCUUAUCCCGAGUCCA
UGGACUCGGGAUAAGAAUG 523 524 434-452 UGCCCCAAGAUGAUGGAAU
AUUCCAUCAUCUUGGGGCA 525 526 872-890 CCAGAAAGAGUGUCUCAUA
UAUGAGACACUCUUUCUGG 527 528 875-893 GAAAGAGUGUCUCAUCUUA
UAAGAUGAGACACUCUUUC 529 530 1112-1130 CACCCACGGGUGUGUGGGA
UCCCACACACCCGUGGGUG 531 532 1113-1131 ACCCACGGGUGUGUGGGGA
UCCCCACACACCCGUGGGU 533 534 1297-1315 CUUCACCCUGGCUAAGAUA
UAUCUUAGCCAGGGUGAAG 535 536 1300-1318 CACCCUGGCUAAGAUGAUA
UAUCAUCUUAGCCAGGGUG 537 538 1301-1319 ACCCUGGCUAAGAUGAUGA
UCAUCAUCUUAGCCAGGGU 539 540 1348-1366 GAACCAUGCCUCCAUGAUA
UAUCAUGGAGGCAUGGUUC 541 542 1481-1499 GAAACUGUCCAUUCAAUGA
UCAUUGAAUGGACAGUUUC 543 544 1786-1804 UGGAGCCCUGGAGUCUGUA
UACAGACUCCAGGGCUCCA 545 546 1795-1813 GGAGUCUGUGCGGAUCCUA
UAGGAUCCGCACAGACUCC 547 548 1919-1937 CACAUCAUCCCUGUGCGGA
UCCGCACAGGGAUGAUGUG 549 550 1922-1940 AUCAUCCCUGUGCGGGUUA
UAACCCGCACAGGGAUGAU 551 552 1923-1941 UCAUCCCUGUGCGGGUUGA
UCAACCCGCACAGGGAUGA
Example 8. Human siRNAs
[0729] Additional 19mer human siRNAs were generated. The sequences
of these siRNAs are provided in Table 9. These siRNAs can be tested
for efficacy using methods described herein and/or methods known in
the art.
TABLE-US-00022 TABLE 9 Human ALAS1 siRNA Sense and Antisense
Sequences SEQ SEQ ID ID NO: Position on NO: (anti- transcript Sense
Sequence Antisense Sequence (sense) sense) NM_000688.4 (5'-3')
(5'-3') 553 554 4-22 UAUAUUAAGGCGCCGGCGA UCGCCGGCGCCUUAAUAUA 555
556 5-23 AUAUUAAGGCGCCGGCGAU AUCGCCGGCGCCUUAAUAU 557 558 6-24
UAUUAAGGCGCCGGCGAUC GAUCGCCGGCGCCUUAAUA 559 560 7-25
AUUAAGGCGCCGGCGAUCG CGAUCGCCGGCGCCUUAAU 561 562 8-26
UUAAGGCGCCGGCGAUCGC GCGAUCGCCGGCGCCUUAA 563 564 9-27
UAAGGCGCCGGCGAUCGCG CGCGAUCGCCGGCGCCUUA 565 566 10-28
AAGGCGCCGGCGAUCGCGG CCGCGAUCGCCGGCGCCUU 567 568 11-29
AGGCGCCGGCGAUCGCGGC GCCGCGAUCGCCGGCGCCU 569 570 12-30
GGCGCCGGCGAUCGCGGCC GGCCGCGAUCGCCGGCGCC 571 572 13-31
GCGCCGGCGAUCGCGGCCU AGGCCGCGAUCGCCGGCGC 573 574 14-32
CGCCGGCGAUCGCGGCCUG CAGGCCGCGAUCGCCGGCG 575 576 81-99
CUUGAGUGCCCGCCUCCUU AAGGAGGCGGGCACUCAAG 577 578 82-100
UUGAGUGCCCGCCUCCUUC GAAGGAGGCGGGCACUCAA 579 580 83-101
UGAGUGCCCGCCUCCUUCG CGAAGGAGGCGGGCACUCA 581 582 84-102
GAGUGCCCGCCUCCUUCGC GCGAAGGAGGCGGGCACUC 583 584 85-103
AGUGCCCGCCUCCUUCGCC GGCGAAGGAGGCGGGCACU 585 586 86-104
GUGCCCGCCUCCUUCGCCG CGGCGAAGGAGGCGGGCAC 587 588 87-105
UGCCCGCCUCCUUCGCCGC GCGGCGAAGGAGGCGGGCA 589 590 88-106
GCCCGCCUCCUUCGCCGCC GGCGGCGAAGGAGGCGGGC 591 592 89-107
CCCGCCUCCUUCGCCGCCG CGGCGGCGAAGGAGGCGGG 593 594 90-108
CCGCCUCCUUCGCCGCCGC GCGGCGGCGAAGGAGGCGG 595 596 91-109
CGCCUCCUUCGCCGCCGCC GGCGGCGGCGAAGGAGGCG 597 598 92-110
GCCUCCUUCGCCGCCGCCU AGGCGGCGGCGAAGGAGGC 599 600 93-111
CCUCCUUCGCCGCCGCCUC GAGGCGGCGGCGAAGGAGG 601 602 356-374
CGCUGCCCAUUCUUAUCCC GGGAUAAGAAUGGGCAGCG 603 604 357-375
GCUGCCCAUUCUUAUCCCG CGGGAUAAGAAUGGGCAGC 605 606 359-377
UGCCCAUUCUUAUCCCGAG CUCGGGAUAAGAAUGGGCA 607 608 361-379
CCCAUUCUUAUCCCGAGUC GACUCGGGAUAAGAAUGGG 609 610 362-380
CCAUUCUUAUCCCGAGUCC GGACUCGGGAUAAGAAUGG 611 612 363-381
CAUUCUUAUCCCGAGUCCC GGGACUCGGGAUAAGAAUG 613 614 364-382
AUUCUUAUCCCGAGUCCCC GGGGACUCGGGAUAAGAAU 615 616 365-383
UUCUUAUCCCGAGUCCCCC GGGGGACUCGGGAUAAGAA 617 618 366-384
UCUUAUCCCGAGUCCCCCA UGGGGGACUCGGGAUAAGA 619 620 367-385
CUUAUCCCGAGUCCCCCAG CUGGGGGACUCGGGAUAAG 621 622 368-386
UUAUCCCGAGUCCCCCAGG CCUGGGGGACUCGGGAUAA 623 624 369-387
UAUCCCGAGUCCCCCAGGC GCCUGGGGGACUCGGGAUA 625 626 370-388
AUCCCGAGUCCCCCAGGCC GGCCUGGGGGACUCGGGAU 627 628 371-389
UCCCGAGUCCCCCAGGCCU AGGCCUGGGGGACUCGGGA 629 630 372-390
CCCGAGUCCCCCAGGCCUU AAGGCCUGGGGGACUCGGG 631 632 373-391
CCGAGUCCCCCAGGCCUUU AAAGGCCUGGGGGACUCGG 633 634 374-392
CGAGUCCCCCAGGCCUUUC GAAAGGCCUGGGGGACUCG 635 636 375-393
GAGUCCCCCAGGCCUUUCU AGAAAGGCCUGGGGGACUC 637 638 376-394
AGUCCCCCAGGCCUUUCUG CAGAAAGGCCUGGGGGACU 639 640 377-395
GUCCCCCAGGCCUUUCUGC GCAGAAAGGCCUGGGGGAC 641 642 378-396
UCCCCCAGGCCUUUCUGCA UGCAGAAAGGCCUGGGGGA 643 644 379-397
CCCCCAGGCCUUUCUGCAG CUGCAGAAAGGCCUGGGGG 645 646 380-398
CCCCAGGCCUUUCUGCAGA UCUGCAGAAAGGCCUGGGG 647 648 381-399
CCCAGGCCUUUCUGCAGAA UUCUGCAGAAAGGCCUGGG 649 650 382-400
CCAGGCCUUUCUGCAGAAA UUUCUGCAGAAAGGCCUGG 651 652 383-401
CAGGCCUUUCUGCAGAAAG CUUUCUGCAGAAAGGCCUG 653 654 384-402
AGGCCUUUCUGCAGAAAGC GCUUUCUGCAGAAAGGCCU 655 656 385-403
GGCCUUUCUGCAGAAAGCA UGCUUUCUGCAGAAAGGCC 657 658 386-404
GCCUUUCUGCAGAAAGCAG CUGCUUUCUGCAGAAAGGC 659 660 387-405
CCUUUCUGCAGAAAGCAGG CCUGCUUUCUGCAGAAAGG 661 662 388-406
CUUUCUGCAGAAAGCAGGC GCCUGCUUUCUGCAGAAAG 663 664 389-407
UUUCUGCAGAAAGCAGGCA UGCCUGCUUUCUGCAGAAA 665 666 390-408
UUCUGCAGAAAGCAGGCAA UUGCCUGCUUUCUGCAGAA 667 668 391-409
UCUGCAGAAAGCAGGCAAA UUUGCCUGCUUUCUGCAGA 669 670 392-410
CUGCAGAAAGCAGGCAAAU AUUUGCCUGCUUUCUGCAG 671 672 393-411
UGCAGAAAGCAGGCAAAUC GAUUUGCCUGCUUUCUGCA 673 674 394-412
GCAGAAAGCAGGCAAAUCU AGAUUUGCCUGCUUUCUGC 675 676 395-413
CAGAAAGCAGGCAAAUCUC GAGAUUUGCCUGCUUUCUG 677 678 396-414
AGAAAGCAGGCAAAUCUCU AGAGAUUUGCCUGCUUUCU 679 680 397-415
GAAAGCAGGCAAAUCUCUG CAGAGAUUUGCCUGCUUUC 681 682 398-416
AAAGCAGGCAAAUCUCUGU ACAGAGAUUUGCCUGCUUU 683 684 399-417
AAGCAGGCAAAUCUCUGUU AACAGAGAUUUGCCUGCUU 685 686 400-418
AGCAGGCAAAUCUCUGUUG CAACAGAGAUUUGCCUGCU 687 688 401-419
GCAGGCAAAUCUCUGUUGU ACAACAGAGAUUUGCCUGC 689 690 402-420
CAGGCAAAUCUCUGUUGUU AACAACAGAGAUUUGCCUG 691 692 403-421
AGGCAAAUCUCUGUUGUUC GAACAACAGAGAUUUGCCU 693 694 405-423
GCAAAUCUCUGUUGUUCUA UAGAACAACAGAGAUUUGC 695 696 406-424
CAAAUCUCUGUUGUUCUAU AUAGAACAACAGAGAUUUG 697 698 407-425
AAAUCUCUGUUGUUCUAUG CAUAGAACAACAGAGAUUU 699 700 408-426
AAUCUCUGUUGUUCUAUGC GCAUAGAACAACAGAGAUU 701 702 409-427
AUCUCUGUUGUUCUAUGCC GGCAUAGAACAACAGAGAU 703 704 410-428
UCUCUGUUGUUCUAUGCCC GGGCAUAGAACAACAGAGA 705 706 411-429
CUCUGUUGUUCUAUGCCCA UGGGCAUAGAACAACAGAG 707 708 412-430
UCUGUUGUUCUAUGCCCAA UUGGGCAUAGAACAACAGA 709 710 413-431
CUGUUGUUCUAUGCCCAAA UUUGGGCAUAGAACAACAG 711 712 414-432
UGUUGUUCUAUGCCCAAAA UUUUGGGCAUAGAACAACA 713 714 415-433
GUUGUUCUAUGCCCAAAAC GUUUUGGGCAUAGAACAAC 715 716 416-434
UUGUUCUAUGCCCAAAACU AGUUUUGGGCAUAGAACAA 717 718 417-435
UGUUCUAUGCCCAAAACUG CAGUUUUGGGCAUAGAACA 719 720 418-436
GUUCUAUGCCCAAAACUGC GCAGUUUUGGGCAUAGAAC 721 722 419-437
UUCUAUGCCCAAAACUGCC GGCAGUUUUGGGCAUAGAA 723 724 420-438
UCUAUGCCCAAAACUGCCC GGGCAGUUUUGGGCAUAGA 725 726 421-439
CUAUGCCCAAAACUGCCCC GGGGCAGUUUUGGGCAUAG 727 728 422-440
UAUGCCCAAAACUGCCCCA UGGGGCAGUUUUGGGCAUA 729 730 423-441
AUGCCCAAAACUGCCCCAA UUGGGGCAGUUUUGGGCAU 731 732 424-442
UGCCCAAAACUGCCCCAAG CUUGGGGCAGUUUUGGGCA 733 734 425-443
GCCCAAAACUGCCCCAAGA UCUUGGGGCAGUUUUGGGC 735 736 426-444
CCCAAAACUGCCCCAAGAU AUCUUGGGGCAGUUUUGGG 737 738 427-445
CCAAAACUGCCCCAAGAUG CAUCUUGGGGCAGUUUUGG 739 740 429-447
AAAACUGCCCCAAGAUGAU AUCAUCUUGGGGCAGUUUU 741 742 430-448
AAACUGCCCCAAGAUGAUG CAUCAUCUUGGGGCAGUUU 743 744 431-449
AACUGCCCCAAGAUGAUGG CCAUCAUCUUGGGGCAGUU 745 746 432-450
ACUGCCCCAAGAUGAUGGA UCCAUCAUCUUGGGGCAGU 747 748 433-451
CUGCCCCAAGAUGAUGGAA UUCCAUCAUCUUGGGGCAG 749 750 434-452
UGCCCCAAGAUGAUGGAAG CUUCCAUCAUCUUGGGGCA 751 752 435-453
GCCCCAAGAUGAUGGAAGU ACUUCCAUCAUCUUGGGGC 753 754 437-455
CCCAAGAUGAUGGAAGUUG CAACUUCCAUCAUCUUGGG 755 756 438-456
CCAAGAUGAUGGAAGUUGG CCAACUUCCAUCAUCUUGG 757 758 439-457
CAAGAUGAUGGAAGUUGGG CCCAACUUCCAUCAUCUUG 759 760 440-458
AAGAUGAUGGAAGUUGGGG CCCCAACUUCCAUCAUCUU 761 762 441-459
AGAUGAUGGAAGUUGGGGC GCCCCAACUUCCAUCAUCU 763 764 442-460
GAUGAUGGAAGUUGGGGCC GGCCCCAACUUCCAUCAUC 765 766 443-461
AUGAUGGAAGUUGGGGCCA UGGCCCCAACUUCCAUCAU 767 768 444-462
UGAUGGAAGUUGGGGCCAA UUGGCCCCAACUUCCAUCA 769 770 445-463
GAUGGAAGUUGGGGCCAAG CUUGGCCCCAACUUCCAUC 771 772 446-464
AUGGAAGUUGGGGCCAAGC GCUUGGCCCCAACUUCCAU 773 774 447-465
UGGAAGUUGGGGCCAAGCC GGCUUGGCCCCAACUUCCA 775 776 448-466
GGAAGUUGGGGCCAAGCCA UGGCUUGGCCCCAACUUCC 777 778 449-467
GAAGUUGGGGCCAAGCCAG CUGGCUUGGCCCCAACUUC 779 780 450-468
AAGUUGGGGCCAAGCCAGC GCUGGCUUGGCCCCAACUU 781 782 451-469
AGUUGGGGCCAAGCCAGCC GGCUGGCUUGGCCCCAACU 783 784 452-470
GUUGGGGCCAAGCCAGCCC GGGCUGGCUUGGCCCCAAC 785 786 453-471
UUGGGGCCAAGCCAGCCCC GGGGCUGGCUUGGCCCCAA 787 788 454-472
UGGGGCCAAGCCAGCCCCU AGGGGCUGGCUUGGCCCCA 789 790 455-473
GGGGCCAAGCCAGCCCCUC GAGGGGCUGGCUUGGCCCC 791 792 456-474
GGGCCAAGCCAGCCCCUCG CGAGGGGCUGGCUUGGCCC 793 794 457-475
GGCCAAGCCAGCCCCUCGG CCGAGGGGCUGGCUUGGCC
795 796 458-476 GCCAAGCCAGCCCCUCGGG CCCGAGGGGCUGGCUUGGC 797 798
459-477 CCAAGCCAGCCCCUCGGGC GCCCGAGGGGCUGGCUUGG 799 800 460-478
CAAGCCAGCCCCUCGGGCA UGCCCGAGGGGCUGGCUUG 801 802 461-479
AAGCCAGCCCCUCGGGCAU AUGCCCGAGGGGCUGGCUU 803 804 462-480
AGCCAGCCCCUCGGGCAUU AAUGCCCGAGGGGCUGGCU 805 806 463-481
GCCAGCCCCUCGGGCAUUG CAAUGCCCGAGGGGCUGGC 807 808 464-482
CCAGCCCCUCGGGCAUUGU ACAAUGCCCGAGGGGCUGG 809 810 465-483
CAGCCCCUCGGGCAUUGUC GACAAUGCCCGAGGGGCUG 811 812 466-484
AGCCCCUCGGGCAUUGUCC GGACAAUGCCCGAGGGGCU 813 814 467-485
GCCCCUCGGGCAUUGUCCA UGGACAAUGCCCGAGGGGC 815 816 468-486
CCCCUCGGGCAUUGUCCAC GUGGACAAUGCCCGAGGGG 817 818 469-487
CCCUCGGGCAUUGUCCACU AGUGGACAAUGCCCGAGGG 819 820 470-488
CCUCGGGCAUUGUCCACUG CAGUGGACAAUGCCCGAGG 821 822 471-489
CUCGGGCAUUGUCCACUGC GCAGUGGACAAUGCCCGAG 823 824 472-490
UCGGGCAUUGUCCACUGCA UGCAGUGGACAAUGCCCGA 825 826 473-491
CGGGCAUUGUCCACUGCAG CUGCAGUGGACAAUGCCCG 827 828 474-492
GGGCAUUGUCCACUGCAGC GCUGCAGUGGACAAUGCCC 829 830 475-493
GGCAUUGUCCACUGCAGCA UGCUGCAGUGGACAAUGCC 831 832 476-494
GCAUUGUCCACUGCAGCAG CUGCUGCAGUGGACAAUGC 833 834 477-495
CAUUGUCCACUGCAGCAGU ACUGCUGCAGUGGACAAUG 835 836 478-496
AUUGUCCACUGCAGCAGUA UACUGCUGCAGUGGACAAU 837 838 479-497
UUGUCCACUGCAGCAGUAC GUACUGCUGCAGUGGACAA 839 840 480-498
UGUCCACUGCAGCAGUACA UGUACUGCUGCAGUGGACA 841 842 481-499
GUCCACUGCAGCAGUACAC GUGUACUGCUGCAGUGGAC 843 844 482-500
UCCACUGCAGCAGUACACU AGUGUACUGCUGCAGUGGA 845 846 483-501
CCACUGCAGCAGUACACUA UAGUGUACUGCUGCAGUGG 847 848 484-502
CACUGCAGCAGUACACUAC GUAGUGUACUGCUGCAGUG 849 850 485-503
ACUGCAGCAGUACACUACC GGUAGUGUACUGCUGCAGU 851 852 486-504
CUGCAGCAGUACACUACCA UGGUAGUGUACUGCUGCAG 853 854 487-505
UGCAGCAGUACACUACCAA UUGGUAGUGUACUGCUGCA 855 856 488-506
GCAGCAGUACACUACCAAC GUUGGUAGUGUACUGCUGC 857 858 490-508
AGCAGUACACUACCAACAG CUGUUGGUAGUGUACUGCU 859 860 491-509
GCAGUACACUACCAACAGA UCUGUUGGUAGUGUACUGC 861 862 492-510
CAGUACACUACCAACAGAU AUCUGUUGGUAGUGUACUG 863 864 493-511
AGUACACUACCAACAGAUC GAUCUGUUGGUAGUGUACU 865 866 494-512
GUACACUACCAACAGAUCA UGAUCUGUUGGUAGUGUAC 867 868 495-513
UACACUACCAACAGAUCAA UUGAUCUGUUGGUAGUGUA 869 870 496-514
ACACUACCAACAGAUCAAA UUUGAUCUGUUGGUAGUGU 871 872 497-515
CACUACCAACAGAUCAAAG CUUUGAUCUGUUGGUAGUG 873 874 498-516
ACUACCAACAGAUCAAAGA UCUUUGAUCUGUUGGUAGU 875 876 499-517
CUACCAACAGAUCAAAGAA UUCUUUGAUCUGUUGGUAG 877 878 500-518
UACCAACAGAUCAAAGAAA UUUCUUUGAUCUGUUGGUA 879 880 501-519
ACCAACAGAUCAAAGAAAC GUUUCUUUGAUCUGUUGGU 881 882 502-520
CCAACAGAUCAAAGAAACC GGUUUCUUUGAUCUGUUGG 883 884 523-541
UCCGGCCAGUGAGAAAGAC GUCUUUCUCACUGGCCGGA 885 886 524-542
CCGGCCAGUGAGAAAGACA UGUCUUUCUCACUGGCCGG 887 888 525-543
CGGCCAGUGAGAAAGACAA UUGUCUUUCUCACUGGCCG 889 890 526-544
GGCCAGUGAGAAAGACAAA UUUGUCUUUCUCACUGGCC 891 892 527-545
GCCAGUGAGAAAGACAAAA UUUUGUCUUUCUCACUGGC 893 894 528-546
CCAGUGAGAAAGACAAAAC GUUUUGUCUUUCUCACUGG 895 896 529-547
CAGUGAGAAAGACAAAACU AGUUUUGUCUUUCUCACUG 897 898 530-548
AGUGAGAAAGACAAAACUG CAGUUUUGUCUUUCUCACU 899 900 531-549
GUGAGAAAGACAAAACUGC GCAGUUUUGUCUUUCUCAC 901 902 570-588
CUCCUGAUGGAUCCCAGCA UGCUGGGAUCCAUCAGGAG 903 904 571-589
UCCUGAUGGAUCCCAGCAG CUGCUGGGAUCCAUCAGGA 905 906 572-590
CCUGAUGGAUCCCAGCAGA UCUGCUGGGAUCCAUCAGG 907 908 573-591
CUGAUGGAUCCCAGCAGAG CUCUGCUGGGAUCCAUCAG 909 910 574-592
UGAUGGAUCCCAGCAGAGU ACUCUGCUGGGAUCCAUCA 911 912 575-593
GAUGGAUCCCAGCAGAGUC GACUCUGCUGGGAUCCAUC 913 914 576-594
AUGGAUCCCAGCAGAGUCC GGACUCUGCUGGGAUCCAU 915 916 577-595
UGGAUCCCAGCAGAGUCCA UGGACUCUGCUGGGAUCCA 917 918 578-596
GGAUCCCAGCAGAGUCCAG CUGGACUCUGCUGGGAUCC 919 920 579-597
GAUCCCAGCAGAGUCCAGA UCUGGACUCUGCUGGGAUC 921 922 580-598
AUCCCAGCAGAGUCCAGAU AUCUGGACUCUGCUGGGAU 923 924 581-599
UCCCAGCAGAGUCCAGAUG CAUCUGGACUCUGCUGGGA 925 926 582-600
CCCAGCAGAGUCCAGAUGG CCAUCUGGACUCUGCUGGG 927 928 583-601
CCAGCAGAGUCCAGAUGGC GCCAUCUGGACUCUGCUGG 929 930 584-602
CAGCAGAGUCCAGAUGGCA UGCCAUCUGGACUCUGCUG 931 932 585-603
AGCAGAGUCCAGAUGGCAC GUGCCAUCUGGACUCUGCU 933 934 586-604
GCAGAGUCCAGAUGGCACA UGUGCCAUCUGGACUCUGC 935 936 587-605
CAGAGUCCAGAUGGCACAC GUGUGCCAUCUGGACUCUG 937 938 588-606
AGAGUCCAGAUGGCACACA UGUGUGCCAUCUGGACUCU 939 940 589-607
GAGUCCAGAUGGCACACAG CUGUGUGCCAUCUGGACUC 941 942 590-608
AGUCCAGAUGGCACACAGC GCUGUGUGCCAUCUGGACU 943 944 591-609
GUCCAGAUGGCACACAGCU AGCUGUGUGCCAUCUGGAC 945 946 592-610
UCCAGAUGGCACACAGCUU AAGCUGUGUGCCAUCUGGA 947 948 593-611
CCAGAUGGCACACAGCUUC GAAGCUGUGUGCCAUCUGG 949 950 594-612
CAGAUGGCACACAGCUUCC GGAAGCUGUGUGCCAUCUG 951 952 595-613
AGAUGGCACACAGCUUCCG CGGAAGCUGUGUGCCAUCU 953 954 596-614
GAUGGCACACAGCUUCCGU ACGGAAGCUGUGUGCCAUC 955 956 597-615
AUGGCACACAGCUUCCGUC GACGGAAGCUGUGUGCCAU 957 958 598-616
UGGCACACAGCUUCCGUCU AGACGGAAGCUGUGUGCCA 959 960 599-617
GGCACACAGCUUCCGUCUG CAGACGGAAGCUGUGUGCC 961 962 600-618
GCACACAGCUUCCGUCUGG CCAGACGGAAGCUGUGUGC 963 964 601-619
CACACAGCUUCCGUCUGGA UCCAGACGGAAGCUGUGUG 965 966 602-620
ACACAGCUUCCGUCUGGAC GUCCAGACGGAAGCUGUGU 967 968 603-621
CACAGCUUCCGUCUGGACA UGUCCAGACGGAAGCUGUG 969 970 604-622
ACAGCUUCCGUCUGGACAC GUGUCCAGACGGAAGCUGU 971 972 605-623
CAGCUUCCGUCUGGACACC GGUGUCCAGACGGAAGCUG 973 974 606-624
AGCUUCCGUCUGGACACCC GGGUGUCCAGACGGAAGCU 975 976 607-625
GCUUCCGUCUGGACACCCC GGGGUGUCCAGACGGAAGC 977 978 608-626
CUUCCGUCUGGACACCCCU AGGGGUGUCCAGACGGAAG 979 980 609-627
UUCCGUCUGGACACCCCUU AAGGGGUGUCCAGACGGAA 981 982 610-628
UCCGUCUGGACACCCCUUG CAAGGGGUGUCCAGACGGA 983 984 611-629
CCGUCUGGACACCCCUUGC GCAAGGGGUGUCCAGACGG 985 986 612-630
CGUCUGGACACCCCUUGCC GGCAAGGGGUGUCCAGACG 987 988 613-631
GUCUGGACACCCCUUGCCU AGGCAAGGGGUGUCCAGAC 989 990 614-632
UCUGGACACCCCUUGCCUG CAGGCAAGGGGUGUCCAGA 991 992 615-633
CUGGACACCCCUUGCCUGC GCAGGCAAGGGGUGUCCAG 993 994 616-634
UGGACACCCCUUGCCUGCC GGCAGGCAAGGGGUGUCCA 995 996 617-635
GGACACCCCUUGCCUGCCA UGGCAGGCAAGGGGUGUCC 997 998 618-636
GACACCCCUUGCCUGCCAC GUGGCAGGCAAGGGGUGUC 999 1000 619-637
ACACCCCUUGCCUGCCACA UGUGGCAGGCAAGGGGUGU 1001 1002 620-638
CACCCCUUGCCUGCCACAA UUGUGGCAGGCAAGGGGUG 1003 1004 621-639
ACCCCUUGCCUGCCACAAG CUUGUGGCAGGCAAGGGGU 1005 1006 622-640
CCCCUUGCCUGCCACAAGC GCUUGUGGCAGGCAAGGGG 1007 1008 623-641
CCCUUGCCUGCCACAAGCC GGCUUGUGGCAGGCAAGGG 1009 1010 624-642
CCUUGCCUGCCACAAGCCA UGGCUUGUGGCAGGCAAGG 1011 1012 625-643
CUUGCCUGCCACAAGCCAG CUGGCUUGUGGCAGGCAAG 1013 1014 626-644
UUGCCUGCCACAAGCCAGG CCUGGCUUGUGGCAGGCAA 1015 1016 627-645
UGCCUGCCACAAGCCAGGG CCCUGGCUUGUGGCAGGCA 1017 1018 628-646
GCCUGCCACAAGCCAGGGC GCCCUGGCUUGUGGCAGGC 1019 1020 629-647
CCUGCCACAAGCCAGGGCA UGCCCUGGCUUGUGGCAGG 1021 1022 630-648
CUGCCACAAGCCAGGGCAC GUGCCCUGGCUUGUGGCAG 1023 1024 631-649
UGCCACAAGCCAGGGCACU AGUGCCCUGGCUUGUGGCA 1025 1026 632-650
GCCACAAGCCAGGGCACUG CAGUGCCCUGGCUUGUGGC 1027 1028 633-651
CCACAAGCCAGGGCACUGC GCAGUGCCCUGGCUUGUGG 1029 1030 634-652
CACAAGCCAGGGCACUGCA UGCAGUGCCCUGGCUUGUG 1031 1032 635-653
ACAAGCCAGGGCACUGCAA UUGCAGUGCCCUGGCUUGU 1033 1034 636-654
CAAGCCAGGGCACUGCAAG CUUGCAGUGCCCUGGCUUG 1035 1036 637-655
AAGCCAGGGCACUGCAAGC GCUUGCAGUGCCCUGGCUU 1037 1038 638-656
AGCCAGGGCACUGCAAGCA UGCUUGCAGUGCCCUGGCU 1039 1040 639-657
GCCAGGGCACUGCAAGCAA UUGCUUGCAGUGCCCUGGC 1041 1042 640-658
CCAGGGCACUGCAAGCAAA UUUGCUUGCAGUGCCCUGG 1043 1044 641-659
CAGGGCACUGCAAGCAAAU AUUUGCUUGCAGUGCCCUG
1045 1046 642-660 AGGGCACUGCAAGCAAAUG CAUUUGCUUGCAGUGCCCU 1047 1048
643-661 GGGCACUGCAAGCAAAUGC GCAUUUGCUUGCAGUGCCC 1049 1050 644-662
GGCACUGCAAGCAAAUGCC GGCAUUUGCUUGCAGUGCC 1051 1052 645-663
GCACUGCAAGCAAAUGCCC GGGCAUUUGCUUGCAGUGC 1053 1054 647-665
ACUGCAAGCAAAUGCCCUU AAGGGCAUUUGCUUGCAGU 1055 1056 648-666
CUGCAAGCAAAUGCCCUUU AAAGGGCAUUUGCUUGCAG 1057 1058 649-667
UGCAAGCAAAUGCCCUUUC GAAAGGGCAUUUGCUUGCA 1059 1060 650-668
GCAAGCAAAUGCCCUUUCC GGAAAGGGCAUUUGCUUGC 1061 1062 651-669
CAAGCAAAUGCCCUUUCCU AGGAAAGGGCAUUUGCUUG 1063 1064 652-670
AAGCAAAUGCCCUUUCCUG CAGGAAAGGGCAUUUGCUU 1065 1066 653-671
AGCAAAUGCCCUUUCCUGG CCAGGAAAGGGCAUUUGCU 1067 1068 654-672
GCAAAUGCCCUUUCCUGGC GCCAGGAAAGGGCAUUUGC 1069 1070 655-673
CAAAUGCCCUUUCCUGGCA UGCCAGGAAAGGGCAUUUG 1071 1072 656-674
AAAUGCCCUUUCCUGGCAG CUGCCAGGAAAGGGCAUUU 1073 1074 657-675
AAUGCCCUUUCCUGGCAGC GCUGCCAGGAAAGGGCAUU 1075 1076 658-676
AUGCCCUUUCCUGGCAGCA UGCUGCCAGGAAAGGGCAU 1077 1078 659-677
UGCCCUUUCCUGGCAGCAC GUGCUGCCAGGAAAGGGCA 1079 1080 660-678
GCCCUUUCCUGGCAGCACA UGUGCUGCCAGGAAAGGGC 1081 1082 661-679
CCCUUUCCUGGCAGCACAG CUGUGCUGCCAGGAAAGGG 1083 1084 662-680
CCUUUCCUGGCAGCACAGA UCUGUGCUGCCAGGAAAGG 1085 1086 663-681
CUUUCCUGGCAGCACAGAU AUCUGUGCUGCCAGGAAAG 1087 1088 664-682
UUUCCUGGCAGCACAGAUG CAUCUGUGCUGCCAGGAAA 1089 1090 665-683
UUCCUGGCAGCACAGAUGA UCAUCUGUGCUGCCAGGAA 1091 1092 666-684
UCCUGGCAGCACAGAUGAA UUCAUCUGUGCUGCCAGGA 1093 1094 667-685
CCUGGCAGCACAGAUGAAU AUUCAUCUGUGCUGCCAGG 1095 1096 668-686
CUGGCAGCACAGAUGAAUC GAUUCAUCUGUGCUGCCAG 1097 1098 670-688
GGCAGCACAGAUGAAUCAG CUGAUUCAUCUGUGCUGCC 1099 1100 672-690
CAGCACAGAUGAAUCAGAG CUCUGAUUCAUCUGUGCUG 1101 1102 692-710
GGCAGCAGUGUCUUCUGCA UGCAGAAGACACUGCUGCC 1103 1104 693-711
GCAGCAGUGUCUUCUGCAA UUGCAGAAGACACUGCUGC 1105 1106 694-712
CAGCAGUGUCUUCUGCAAA UUUGCAGAAGACACUGCUG 1107 1108 695-713
AGCAGUGUCUUCUGCAAAG CUUUGCAGAAGACACUGCU 1109 1110 696-714
GCAGUGUCUUCUGCAAAGC GCUUUGCAGAAGACACUGC 1111 1112 697-715
CAGUGUCUUCUGCAAAGCC GGCUUUGCAGAAGACACUG 1113 1114 698-716
AGUGUCUUCUGCAAAGCCA UGGCUUUGCAGAAGACACU 1115 1116 699-717
GUGUCUUCUGCAAAGCCAG CUGGCUUUGCAGAAGACAC 1117 1118 700-718
UGUCUUCUGCAAAGCCAGU ACUGGCUUUGCAGAAGACA 1119 1120 701-719
GUCUUCUGCAAAGCCAGUC GACUGGCUUUGCAGAAGAC 1121 1122 702-720
UCUUCUGCAAAGCCAGUCU AGACUGGCUUUGCAGAAGA 1123 1124 703-721
CUUCUGCAAAGCCAGUCUU AAGACUGGCUUUGCAGAAG 1125 1126 704-722
UUCUGCAAAGCCAGUCUUG CAAGACUGGCUUUGCAGAA 1127 1128 705-723
UCUGCAAAGCCAGUCUUGA UCAAGACUGGCUUUGCAGA 1129 1130 706-724
CUGCAAAGCCAGUCUUGAG CUCAAGACUGGCUUUGCAG 1131 1132 707-725
UGCAAAGCCAGUCUUGAGC GCUCAAGACUGGCUUUGCA 1133 1134 708-726
GCAAAGCCAGUCUUGAGCU AGCUCAAGACUGGCUUUGC 1135 1136 709-727
CAAAGCCAGUCUUGAGCUU AAGCUCAAGACUGGCUUUG 1137 1138 710-728
AAAGCCAGUCUUGAGCUUC GAAGCUCAAGACUGGCUUU 1139 1140 711-729
AAGCCAGUCUUGAGCUUCA UGAAGCUCAAGACUGGCUU 1141 1142 712-730
AGCCAGUCUUGAGCUUCAG CUGAAGCUCAAGACUGGCU 1143 1144 713-731
GCCAGUCUUGAGCUUCAGG CCUGAAGCUCAAGACUGGC 1145 1146 714-732
CCAGUCUUGAGCUUCAGGA UCCUGAAGCUCAAGACUGG 1147 1148 715-733
CAGUCUUGAGCUUCAGGAG CUCCUGAAGCUCAAGACUG 1149 1150 716-734
AGUCUUGAGCUUCAGGAGG CCUCCUGAAGCUCAAGACU 1151 1152 717-735
GUCUUGAGCUUCAGGAGGA UCCUCCUGAAGCUCAAGAC 1153 1154 718-736
UCUUGAGCUUCAGGAGGAU AUCCUCCUGAAGCUCAAGA 1155 1156 719-737
CUUGAGCUUCAGGAGGAUG CAUCCUCCUGAAGCUCAAG 1157 1158 720-738
UUGAGCUUCAGGAGGAUGU ACAUCCUCCUGAAGCUCAA 1159 1160 721-739
UGAGCUUCAGGAGGAUGUG CACAUCCUCCUGAAGCUCA 1161 1162 722-740
GAGCUUCAGGAGGAUGUGC GCACAUCCUCCUGAAGCUC 1163 1164 723-741
AGCUUCAGGAGGAUGUGCA UGCACAUCCUCCUGAAGCU 1165 1166 724-742
GCUUCAGGAGGAUGUGCAG CUGCACAUCCUCCUGAAGC 1167 1168 725-743
CUUCAGGAGGAUGUGCAGG CCUGCACAUCCUCCUGAAG 1169 1170 726-744
UUCAGGAGGAUGUGCAGGA UCCUGCACAUCCUCCUGAA 1171 1172 727-745
UCAGGAGGAUGUGCAGGAA UUCCUGCACAUCCUCCUGA 1173 1174 728-746
CAGGAGGAUGUGCAGGAAA UUUCCUGCACAUCCUCCUG 1175 1176 729-747
AGGAGGAUGUGCAGGAAAU AUUUCCUGCACAUCCUCCU 1177 1178 730-748
GGAGGAUGUGCAGGAAAUG CAUUUCCUGCACAUCCUCC 1179 1180 731-749
GAGGAUGUGCAGGAAAUGA UCAUUUCCUGCACAUCCUC 1181 1182 732-750
AGGAUGUGCAGGAAAUGAA UUCAUUUCCUGCACAUCCU 1183 1184 733-751
GGAUGUGCAGGAAAUGAAU AUUCAUUUCCUGCACAUCC 1185 1186 734-752
GAUGUGCAGGAAAUGAAUG CAUUCAUUUCCUGCACAUC 1187 1188 735-753
AUGUGCAGGAAAUGAAUGC GCAUUCAUUUCCUGCACAU 1189 1190 755-773
GUGAGGAAAGAGGUUGCUG CAGCAACCUCUUUCCUCAC 1191 1192 756-774
UGAGGAAAGAGGUUGCUGA UCAGCAACCUCUUUCCUCA 1193 1194 757-775
GAGGAAAGAGGUUGCUGAA UUCAGCAACCUCUUUCCUC 1195 1196 758-776
AGGAAAGAGGUUGCUGAAA UUUCAGCAACCUCUUUCCU 1197 1198 759-777
GGAAAGAGGUUGCUGAAAC GUUUCAGCAACCUCUUUCC 1199 1200 760-778
GAAAGAGGUUGCUGAAACC GGUUUCAGCAACCUCUUUC 1201 1202 761-779
AAAGAGGUUGCUGAAACCU AGGUUUCAGCAACCUCUUU 1203 1204 762-780
AAGAGGUUGCUGAAACCUC GAGGUUUCAGCAACCUCUU 1205 1206 763-781
AGAGGUUGCUGAAACCUCA UGAGGUUUCAGCAACCUCU 1207 1208 764-782
GAGGUUGCUGAAACCUCAG CUGAGGUUUCAGCAACCUC 1209 1210 765-783
AGGUUGCUGAAACCUCAGC GCUGAGGUUUCAGCAACCU 1211 1212 766-784
GGUUGCUGAAACCUCAGCA UGCUGAGGUUUCAGCAACC 1213 1214 787-805
CCCCAGUGUGGUUAGUGUG CACACUAACCACACUGGGG 1215 1216 791-809
AGUGUGGUUAGUGUGAAAA UUUUCACACUAACCACACU 1217 1218 792-810
GUGUGGUUAGUGUGAAAAC GUUUUCACACUAACCACAC 1219 1220 812-830
GAUGGAGGGGAUCCCAGUG CACUGGGAUCCCCUCCAUC 1221 1222 813-831
AUGGAGGGGAUCCCAGUGG CCACUGGGAUCCCCUCCAU 1223 1224 833-851
CUGCUGAAGAACUUCCAGG CCUGGAAGUUCUUCAGCAG 1225 1226 834-852
UGCUGAAGAACUUCCAGGA UCCUGGAAGUUCUUCAGCA 1227 1228 835-853
GCUGAAGAACUUCCAGGAC GUCCUGGAAGUUCUUCAGC 1229 1230 836-854
CUGAAGAACUUCCAGGACA UGUCCUGGAAGUUCUUCAG 1231 1232 837-855
UGAAGAACUUCCAGGACAU AUGUCCUGGAAGUUCUUCA 1233 1234 838-856
GAAGAACUUCCAGGACAUC GAUGUCCUGGAAGUUCUUC 1235 1236 839-857
AAGAACUUCCAGGACAUCA UGAUGUCCUGGAAGUUCUU 1237 1238 840-858
AGAACUUCCAGGACAUCAU AUGAUGUCCUGGAAGUUCU 1239 1240 841-859
GAACUUCCAGGACAUCAUG CAUGAUGUCCUGGAAGUUC 1241 1242 842-860
AACUUCCAGGACAUCAUGC GCAUGAUGUCCUGGAAGUU 1243 1244 843-861
ACUUCCAGGACAUCAUGCA UGCAUGAUGUCCUGGAAGU 1245 1246 844-862
CUUCCAGGACAUCAUGCAA UUGCAUGAUGUCCUGGAAG 1247 1248 845-863
UUCCAGGACAUCAUGCAAA UUUGCAUGAUGUCCUGGAA 1249 1250 846-864
UCCAGGACAUCAUGCAAAA UUUUGCAUGAUGUCCUGGA 1251 1252 847-865
CCAGGACAUCAUGCAAAAG CUUUUGCAUGAUGUCCUGG 1253 1254 848-866
CAGGACAUCAUGCAAAAGC GCUUUUGCAUGAUGUCCUG 1255 1256 849-867
AGGACAUCAUGCAAAAGCA UGCUUUUGCAUGAUGUCCU 1257 1258 850-868
GGACAUCAUGCAAAAGCAA UUGCUUUUGCAUGAUGUCC 1259 1260 851-869
GACAUCAUGCAAAAGCAAA UUUGCUUUUGCAUGAUGUC 1261 1262 852-870
ACAUCAUGCAAAAGCAAAG CUUUGCUUUUGCAUGAUGU 1263 1264 854-872
AUCAUGCAAAAGCAAAGAC GUCUUUGCUUUUGCAUGAU 1265 1266 855-873
UCAUGCAAAAGCAAAGACC GGUCUUUGCUUUUGCAUGA 1267 1268 856-874
CAUGCAAAAGCAAAGACCA UGGUCUUUGCUUUUGCAUG 1269 1270 857-875
AUGCAAAAGCAAAGACCAG CUGGUCUUUGCUUUUGCAU 1271 1272 858-876
UGCAAAAGCAAAGACCAGA UCUGGUCUUUGCUUUUGCA 1273 1274 859-877
GCAAAAGCAAAGACCAGAA UUCUGGUCUUUGCUUUUGC 1275 1276 860-878
CAAAAGCAAAGACCAGAAA UUUCUGGUCUUUGCUUUUG 1277 1278 861-879
AAAAGCAAAGACCAGAAAG CUUUCUGGUCUUUGCUUUU 1279 1280 862-880
AAAGCAAAGACCAGAAAGA UCUUUCUGGUCUUUGCUUU 1281 1282 863-881
AAGCAAAGACCAGAAAGAG CUCUUUCUGGUCUUUGCUU 1283 1284 864-882
AGCAAAGACCAGAAAGAGU ACUCUUUCUGGUCUUUGCU 1285 1286 865-883
GCAAAGACCAGAAAGAGUG CACUCUUUCUGGUCUUUGC 1287 1288 867-885
AAAGACCAGAAAGAGUGUC GACACUCUUUCUGGUCUUU 1289 1290 868-886
AAGACCAGAAAGAGUGUCU AGACACUCUUUCUGGUCUU 1291 1292 869-887
AGACCAGAAAGAGUGUCUC GAGACACUCUUUCUGGUCU 1293 1294 870-888
GACCAGAAAGAGUGUCUCA UGAGACACUCUUUCUGGUC 1295 1296 871-889
ACCAGAAAGAGUGUCUCAU AUGAGACACUCUUUCUGGU
1297 1298 872-890 CCAGAAAGAGUGUCUCAUC GAUGAGACACUCUUUCUGG 1299 1300
875-893 GAAAGAGUGUCUCAUCUUC GAAGAUGAGACACUCUUUC 1301 1302 878-896
AGAGUGUCUCAUCUUCUUC GAAGAAGAUGAGACACUCU 1303 1304 879-897
GAGUGUCUCAUCUUCUUCA UGAAGAAGAUGAGACACUC 1305 1306 880-898
AGUGUCUCAUCUUCUUCAA UUGAAGAAGAUGAGACACU 1307 1308 881-899
GUGUCUCAUCUUCUUCAAG CUUGAAGAAGAUGAGACAC 1309 1310 882-900
UGUCUCAUCUUCUUCAAGA UCUUGAAGAAGAUGAGACA 1311 1312 883-901
GUCUCAUCUUCUUCAAGAU AUCUUGAAGAAGAUGAGAC 1313 1314 884-902
UCUCAUCUUCUUCAAGAUA UAUCUUGAAGAAGAUGAGA 1315 1316 886-904
UCAUCUUCUUCAAGAUAAC GUUAUCUUGAAGAAGAUGA 1317 1318 887-905
CAUCUUCUUCAAGAUAACU AGUUAUCUUGAAGAAGAUG 1319 1320 888-906
AUCUUCUUCAAGAUAACUU AAGUUAUCUUGAAGAAGAU 1321 1322 889-907
UCUUCUUCAAGAUAACUUG CAAGUUAUCUUGAAGAAGA 1323 1324 890-908
CUUCUUCAAGAUAACUUGC GCAAGUUAUCUUGAAGAAG 1325 1326 891-909
UUCUUCAAGAUAACUUGCC GGCAAGUUAUCUUGAAGAA 1327 1328 892-910
UCUUCAAGAUAACUUGCCA UGGCAAGUUAUCUUGAAGA 1329 1330 893-911
CUUCAAGAUAACUUGCCAA UUGGCAAGUUAUCUUGAAG 1331 1332 894-912
UUCAAGAUAACUUGCCAAA UUUGGCAAGUUAUCUUGAA 1333 1334 895-913
UCAAGAUAACUUGCCAAAA UUUUGGCAAGUUAUCUUGA 1335 1336 896-914
CAAGAUAACUUGCCAAAAU AUUUUGGCAAGUUAUCUUG 1337 1338 897-915
AAGAUAACUUGCCAAAAUC GAUUUUGGCAAGUUAUCUU 1339 1340 898-916
AGAUAACUUGCCAAAAUCU AGAUUUUGGCAAGUUAUCU 1341 1342 899-917
GAUAACUUGCCAAAAUCUG CAGAUUUUGGCAAGUUAUC 1343 1344 900-918
AUAACUUGCCAAAAUCUGU ACAGAUUUUGGCAAGUUAU 1345 1346 901-919
UAACUUGCCAAAAUCUGUU AACAGAUUUUGGCAAGUUA 1347 1348 902-920
AACUUGCCAAAAUCUGUUU AAACAGAUUUUGGCAAGUU 1349 1350 903-921
ACUUGCCAAAAUCUGUUUC GAAACAGAUUUUGGCAAGU 1351 1352 904-922
CUUGCCAAAAUCUGUUUCC GGAAACAGAUUUUGGCAAG 1353 1354 905-923
UUGCCAAAAUCUGUUUCCA UGGAAACAGAUUUUGGCAA 1355 1356 906-924
UGCCAAAAUCUGUUUCCAC GUGGAAACAGAUUUUGGCA 1357 1358 907-925
GCCAAAAUCUGUUUCCACU AGUGGAAACAGAUUUUGGC 1359 1360 908-926
CCAAAAUCUGUUUCCACUU AAGUGGAAACAGAUUUUGG 1361 1362 909-927
CAAAAUCUGUUUCCACUUU AAAGUGGAAACAGAUUUUG 1363 1364 910-928
AAAAUCUGUUUCCACUUUU AAAAGUGGAAACAGAUUUU 1365 1366 911-929
AAAUCUGUUUCCACUUUUC GAAAAGUGGAAACAGAUUU 1367 1368 912-930
AAUCUGUUUCCACUUUUCA UGAAAAGUGGAAACAGAUU 1369 1370 913-931
AUCUGUUUCCACUUUUCAG CUGAAAAGUGGAAACAGAU 1371 1372 916-934
UGUUUCCACUUUUCAGUAU AUACUGAAAAGUGGAAACA 1373 1374 917-935
GUUUCCACUUUUCAGUAUG CAUACUGAAAAGUGGAAAC 1375 1376 918-936
UUUCCACUUUUCAGUAUGA UCAUACUGAAAAGUGGAAA 1377 1378 919-937
UUCCACUUUUCAGUAUGAU AUCAUACUGAAAAGUGGAA 1379 1380 920-938
UCCACUUUUCAGUAUGAUC GAUCAUACUGAAAAGUGGA 1381 1382 921-939
CCACUUUUCAGUAUGAUCG CGAUCAUACUGAAAAGUGG 1383 1384 925-943
UUUUCAGUAUGAUCGUUUC GAAACGAUCAUACUGAAAA 1385 1386 929-947
CAGUAUGAUCGUUUCUUUG CAAAGAAACGAUCAUACUG 1387 1388 930-948
AGUAUGAUCGUUUCUUUGA UCAAAGAAACGAUCAUACU 1389 1390 931-949
GUAUGAUCGUUUCUUUGAG CUCAAAGAAACGAUCAUAC 1391 1392 933-951
AUGAUCGUUUCUUUGAGAA UUCUCAAAGAAACGAUCAU 1393 1394 934-952
UGAUCGUUUCUUUGAGAAA UUUCUCAAAGAAACGAUCA 1395 1396 936-954
AUCGUUUCUUUGAGAAAAA UUUUUCUCAAAGAAACGAU 1397 1398 937-955
UCGUUUCUUUGAGAAAAAA UUUUUUCUCAAAGAAACGA 1399 1400 938-956
CGUUUCUUUGAGAAAAAAA UUUUUUUCUCAAAGAAACG 1401 1402 939-957
GUUUCUUUGAGAAAAAAAU AUUUUUUUCUCAAAGAAAC 1403 1404 940-958
UUUCUUUGAGAAAAAAAUU AAUUUUUUUCUCAAAGAAA 1405 1406 941-959
UUCUUUGAGAAAAAAAUUG CAAUUUUUUUCUCAAAGAA 1407 1408 942-960
UCUUUGAGAAAAAAAUUGA UCAAUUUUUUUCUCAAAGA 1409 1410 943-961
CUUUGAGAAAAAAAUUGAU AUCAAUUUUUUUCUCAAAG 1411 1412 944-962
UUUGAGAAAAAAAUUGAUG CAUCAAUUUUUUUCUCAAA 1413 1414 945-963
UUGAGAAAAAAAUUGAUGA UCAUCAAUUUUUUUCUCAA 1415 1416 946-964
UGAGAAAAAAAUUGAUGAG CUCAUCAAUUUUUUUCUCA 1417 1418 947-965
GAGAAAAAAAUUGAUGAGA UCUCAUCAAUUUUUUUCUC 1419 1420 948-966
AGAAAAAAAUUGAUGAGAA UUCUCAUCAAUUUUUUUCU 1421 1422 949-967
GAAAAAAAUUGAUGAGAAA UUUCUCAUCAAUUUUUUUC 1423 1424 950-968
AAAAAAAUUGAUGAGAAAA UUUUCUCAUCAAUUUUUUU 1425 1426 951-969
AAAAAAUUGAUGAGAAAAA UUUUUCUCAUCAAUUUUUU 1427 1428 952-970
AAAAAUUGAUGAGAAAAAG CUUUUUCUCAUCAAUUUUU 1429 1430 953-971
AAAAUUGAUGAGAAAAAGA UCUUUUUCUCAUCAAUUUU 1431 1432 954-972
AAAUUGAUGAGAAAAAGAA UUCUUUUUCUCAUCAAUUU 1433 1434 955-973
AAUUGAUGAGAAAAAGAAU AUUCUUUUUCUCAUCAAUU 1435 1436 956-974
AUUGAUGAGAAAAAGAAUG CAUUCUUUUUCUCAUCAAU 1437 1438 957-975
UUGAUGAGAAAAAGAAUGA UCAUUCUUUUUCUCAUCAA 1439 1440 958-976
UGAUGAGAAAAAGAAUGAC GUCAUUCUUUUUCUCAUCA 1441 1442 959-977
GAUGAGAAAAAGAAUGACC GGUCAUUCUUUUUCUCAUC 1443 1444 960-978
AUGAGAAAAAGAAUGACCA UGGUCAUUCUUUUUCUCAU 1445 1446 961-979
UGAGAAAAAGAAUGACCAC GUGGUCAUUCUUUUUCUCA 1447 1448 962-980
GAGAAAAAGAAUGACCACA UGUGGUCAUUCUUUUUCUC 1449 1450 963-981
AGAAAAAGAAUGACCACAC GUGUGGUCAUUCUUUUUCU 1451 1452 964-982
GAAAAAGAAUGACCACACC GGUGUGGUCAUUCUUUUUC 1453 1454 965-983
AAAAAGAAUGACCACACCU AGGUGUGGUCAUUCUUUUU 1455 1456 966-984
AAAAGAAUGACCACACCUA UAGGUGUGGUCAUUCUUUU 1457 1458 967-985
AAAGAAUGACCACACCUAU AUAGGUGUGGUCAUUCUUU 1459 1460 968-986
AAGAAUGACCACACCUAUC GAUAGGUGUGGUCAUUCUU 1461 1462 969-987
AGAAUGACCACACCUAUCG CGAUAGGUGUGGUCAUUCU 1463 1464 970-988
GAAUGACCACACCUAUCGA UCGAUAGGUGUGGUCAUUC 1465 1466 971-989
AAUGACCACACCUAUCGAG CUCGAUAGGUGUGGUCAUU 1467 1468 972-990
AUGACCACACCUAUCGAGU ACUCGAUAGGUGUGGUCAU 1469 1470 976-994
CCACACCUAUCGAGUUUUU AAAAACUCGAUAGGUGUGG 1471 1472 977-995
CACACCUAUCGAGUUUUUA UAAAAACUCGAUAGGUGUG 1473 1474 978-996
ACACCUAUCGAGUUUUUAA UUAAAAACUCGAUAGGUGU 1475 1476 979-997
CACCUAUCGAGUUUUUAAA UUUAAAAACUCGAUAGGUG 1477 1478 980-998
ACCUAUCGAGUUUUUAAAA UUUUAAAAACUCGAUAGGU 1479 1480 981-999
CCUAUCGAGUUUUUAAAAC GUUUUAAAAACUCGAUAGG 1481 1482 982-1000
CUAUCGAGUUUUUAAAACU AGUUUUAAAAACUCGAUAG 1483 1484 983-1001
UAUCGAGUUUUUAAAACUG CAGUUUUAAAAACUCGAUA 1485 1486 984-1002
AUCGAGUUUUUAAAACUGU ACAGUUUUAAAAACUCGAU 1487 1488 985-1003
UCGAGUUUUUAAAACUGUG CACAGUUUUAAAAACUCGA 1489 1490 986-1004
CGAGUUUUUAAAACUGUGA UCACAGUUUUAAAAACUCG 1491 1492 987-1005
GAGUUUUUAAAACUGUGAA UUCACAGUUUUAAAAACUC 1493 1494 988-1006
AGUUUUUAAAACUGUGAAC GUUCACAGUUUUAAAAACU 1495 1496 989-1007
GUUUUUAAAACUGUGAACC GGUUCACAGUUUUAAAAAC 1497 1498 990-1008
UUUUUAAAACUGUGAACCG CGGUUCACAGUUUUAAAAA 1499 1500 991-1009
UUUUAAAACUGUGAACCGG CCGGUUCACAGUUUUAAAA 1501 1502 992-1010
UUUAAAACUGUGAACCGGC GCCGGUUCACAGUUUUAAA 1503 1504 993-1011
UUAAAACUGUGAACCGGCG CGCCGGUUCACAGUUUUAA 1505 1506 994-1012
UAAAACUGUGAACCGGCGA UCGCCGGUUCACAGUUUUA 1507 1508 995-1013
AAAACUGUGAACCGGCGAG CUCGCCGGUUCACAGUUUU 1509 1510 996-1014
AAACUGUGAACCGGCGAGC GCUCGCCGGUUCACAGUUU 1511 1512 997-1015
AACUGUGAACCGGCGAGCA UGCUCGCCGGUUCACAGUU 1513 1514 998-1016
ACUGUGAACCGGCGAGCAC GUGCUCGCCGGUUCACAGU 1515 1516 999-1017
CUGUGAACCGGCGAGCACA UGUGCUCGCCGGUUCACAG 1517 1518 1000-1018
UGUGAACCGGCGAGCACAC GUGUGCUCGCCGGUUCACA 1519 1520 1001-1019
GUGAACCGGCGAGCACACA UGUGUGCUCGCCGGUUCAC 1521 1522 1002-1020
UGAACCGGCGAGCACACAU AUGUGUGCUCGCCGGUUCA 1523 1524 1003-1021
GAACCGGCGAGCACACAUC GAUGUGUGCUCGCCGGUUC 1525 1526 1004-1022
AACCGGCGAGCACACAUCU AGAUGUGUGCUCGCCGGUU 1527 1528 1005-1023
ACCGGCGAGCACACAUCUU AAGAUGUGUGCUCGCCGGU 1529 1530 1006-1024
CCGGCGAGCACACAUCUUC GAAGAUGUGUGCUCGCCGG 1531 1532 1007-1025
CGGCGAGCACACAUCUUCC GGAAGAUGUGUGCUCGCCG 1533 1534 1008-1026
GGCGAGCACACAUCUUCCC GGGAAGAUGUGUGCUCGCC 1535 1536 1028-1046
AUGGCAGAUGACUAUUCAG CUGAAUAGUCAUCUGCCAU 1537 1538 1030-1048
GGCAGAUGACUAUUCAGAC GUCUGAAUAGUCAUCUGCC 1539 1540 1031-1049
GCAGAUGACUAUUCAGACU AGUCUGAAUAGUCAUCUGC 1541 1542 1032-1050
CAGAUGACUAUUCAGACUC GAGUCUGAAUAGUCAUCUG 1543 1544 1033-1051
AGAUGACUAUUCAGACUCC GGAGUCUGAAUAGUCAUCU 1545 1546 1034-1052
GAUGACUAUUCAGACUCCC GGGAGUCUGAAUAGUCAUC
1547 1548 1035-1053 AUGACUAUUCAGACUCCCU AGGGAGUCUGAAUAGUCAU 1549
1550 1036-1054 UGACUAUUCAGACUCCCUC GAGGGAGUCUGAAUAGUCA 1551 1552
1037-1055 GACUAUUCAGACUCCCUCA UGAGGGAGUCUGAAUAGUC 1553 1554
1038-1056 ACUAUUCAGACUCCCUCAU AUGAGGGAGUCUGAAUAGU 1555 1556
1039-1057 CUAUUCAGACUCCCUCAUC GAUGAGGGAGUCUGAAUAG 1557 1558
1040-1058 UAUUCAGACUCCCUCAUCA UGAUGAGGGAGUCUGAAUA 1559 1560
1041-1059 AUUCAGACUCCCUCAUCAC GUGAUGAGGGAGUCUGAAU 1561 1562
1042-1060 UUCAGACUCCCUCAUCACC GGUGAUGAGGGAGUCUGAA 1563 1564
1043-1061 UCAGACUCCCUCAUCACCA UGGUGAUGAGGGAGUCUGA 1565 1566
1044-1062 CAGACUCCCUCAUCACCAA UUGGUGAUGAGGGAGUCUG 1567 1568
1045-1063 AGACUCCCUCAUCACCAAA UUUGGUGAUGAGGGAGUCU 1569 1570
1046-1064 GACUCCCUCAUCACCAAAA UUUUGGUGAUGAGGGAGUC 1571 1572
1047-1065 ACUCCCUCAUCACCAAAAA UUUUUGGUGAUGAGGGAGU 1573 1574
1048-1066 CUCCCUCAUCACCAAAAAG CUUUUUGGUGAUGAGGGAG 1575 1576
1049-1067 UCCCUCAUCACCAAAAAGC GCUUUUUGGUGAUGAGGGA 1577 1578
1050-1068 CCCUCAUCACCAAAAAGCA UGCUUUUUGGUGAUGAGGG 1579 1580
1070-1088 GUGUCAGUCUGGUGCAGUA UACUGCACCAGACUGACAC 1581 1582
1071-1089 UGUCAGUCUGGUGCAGUAA UUACUGCACCAGACUGACA 1583 1584
1072-1090 GUCAGUCUGGUGCAGUAAU AUUACUGCACCAGACUGAC 1585 1586
1073-1091 UCAGUCUGGUGCAGUAAUG CAUUACUGCACCAGACUGA 1587 1588
1074-1092 CAGUCUGGUGCAGUAAUGA UCAUUACUGCACCAGACUG 1589 1590
1075-1093 AGUCUGGUGCAGUAAUGAC GUCAUUACUGCACCAGACU 1591 1592
1078-1096 CUGGUGCAGUAAUGACUAC GUAGUCAUUACUGCACCAG 1593 1594
1079-1097 UGGUGCAGUAAUGACUACC GGUAGUCAUUACUGCACCA 1595 1596
1081-1099 GUGCAGUAAUGACUACCUA UAGGUAGUCAUUACUGCAC 1597 1598
1082-1100 UGCAGUAAUGACUACCUAG CUAGGUAGUCAUUACUGCA 1599 1600
1083-1101 GCAGUAAUGACUACCUAGG CCUAGGUAGUCAUUACUGC 1601 1602
1084-1102 CAGUAAUGACUACCUAGGA UCCUAGGUAGUCAUUACUG 1603 1604
1085-1103 AGUAAUGACUACCUAGGAA UUCCUAGGUAGUCAUUACU 1605 1606
1086-1104 GUAAUGACUACCUAGGAAU AUUCCUAGGUAGUCAUUAC 1607 1608
1087-1105 UAAUGACUACCUAGGAAUG CAUUCCUAGGUAGUCAUUA 1609 1610
1088-1106 AAUGACUACCUAGGAAUGA UCAUUCCUAGGUAGUCAUU 1611 1612
1089-1107 AUGACUACCUAGGAAUGAG CUCAUUCCUAGGUAGUCAU 1613 1614
1090-1108 UGACUACCUAGGAAUGAGU ACUCAUUCCUAGGUAGUCA 1615 1616
1091-1109 GACUACCUAGGAAUGAGUC GACUCAUUCCUAGGUAGUC 1617 1618
1092-1110 ACUACCUAGGAAUGAGUCG CGACUCAUUCCUAGGUAGU 1619 1620
1093-1111 CUACCUAGGAAUGAGUCGC GCGACUCAUUCCUAGGUAG 1621 1622
1094-1112 UACCUAGGAAUGAGUCGCC GGCGACUCAUUCCUAGGUA 1623 1624
1095-1113 ACCUAGGAAUGAGUCGCCA UGGCGACUCAUUCCUAGGU 1625 1626
1096-1114 CCUAGGAAUGAGUCGCCAC GUGGCGACUCAUUCCUAGG 1627 1628
1097-1115 CUAGGAAUGAGUCGCCACC GGUGGCGACUCAUUCCUAG 1629 1630
1098-1116 UAGGAAUGAGUCGCCACCC GGGUGGCGACUCAUUCCUA 1631 1632
1099-1117 AGGAAUGAGUCGCCACCCA UGGGUGGCGACUCAUUCCU 1633 1634
1100-1118 GGAAUGAGUCGCCACCCAC GUGGGUGGCGACUCAUUCC 1635 1636
1101-1119 GAAUGAGUCGCCACCCACG CGUGGGUGGCGACUCAUUC 1637 1638
1102-1120 AAUGAGUCGCCACCCACGG CCGUGGGUGGCGACUCAUU 1639 1640
1103-1121 AUGAGUCGCCACCCACGGG CCCGUGGGUGGCGACUCAU 1641 1642
1104-1122 UGAGUCGCCACCCACGGGU ACCCGUGGGUGGCGACUCA 1643 1644
1105-1123 GAGUCGCCACCCACGGGUG CACCCGUGGGUGGCGACUC 1645 1646
1106-1124 AGUCGCCACCCACGGGUGU ACACCCGUGGGUGGCGACU 1647 1648
1107-1125 GUCGCCACCCACGGGUGUG CACACCCGUGGGUGGCGAC 1649 1650
1108-1126 UCGCCACCCACGGGUGUGU ACACACCCGUGGGUGGCGA 1651 1652
1109-1127 CGCCACCCACGGGUGUGUG CACACACCCGUGGGUGGCG 1653 1654
1110-1128 GCCACCCACGGGUGUGUGG CCACACACCCGUGGGUGGC 1655 1656
1111-1129 CCACCCACGGGUGUGUGGG CCCACACACCCGUGGGUGG 1657 1658
1112-1130 CACCCACGGGUGUGUGGGG CCCCACACACCCGUGGGUG 1659 1660
1113-1131 ACCCACGGGUGUGUGGGGC GCCCCACACACCCGUGGGU 1661 1662
1114-1132 CCCACGGGUGUGUGGGGCA UGCCCCACACACCCGUGGG 1663 1664
1115-1133 CCACGGGUGUGUGGGGCAG CUGCCCCACACACCCGUGG 1665 1666
1116-1134 CACGGGUGUGUGGGGCAGU ACUGCCCCACACACCCGUG 1667 1668
1117-1135 ACGGGUGUGUGGGGCAGUU AACUGCCCCACACACCCGU 1669 1670
1118-1136 CGGGUGUGUGGGGCAGUUA UAACUGCCCCACACACCCG 1671 1672
1119-1137 GGGUGUGUGGGGCAGUUAU AUAACUGCCCCACACACCC 1673 1674
1120-1138 GGUGUGUGGGGCAGUUAUG CAUAACUGCCCCACACACC 1675 1676
1121-1139 GUGUGUGGGGCAGUUAUGG CCAUAACUGCCCCACACAC 1677 1678
1122-1140 UGUGUGGGGCAGUUAUGGA UCCAUAACUGCCCCACACA 1679 1680
1123-1141 GUGUGGGGCAGUUAUGGAC GUCCAUAACUGCCCCACAC 1681 1682
1125-1143 GUGGGGCAGUUAUGGACAC GUGUCCAUAACUGCCCCAC 1683 1684
1126-1144 UGGGGCAGUUAUGGACACU AGUGUCCAUAACUGCCCCA 1685 1686
1128-1146 GGGCAGUUAUGGACACUUU AAAGUGUCCAUAACUGCCC 1687 1688
1129-1147 GGCAGUUAUGGACACUUUG CAAAGUGUCCAUAACUGCC 1689 1690
1130-1148 GCAGUUAUGGACACUUUGA UCAAAGUGUCCAUAACUGC 1691 1692
1131-1149 CAGUUAUGGACACUUUGAA UUCAAAGUGUCCAUAACUG 1693 1694
1132-1150 AGUUAUGGACACUUUGAAA UUUCAAAGUGUCCAUAACU 1695 1696
1133-1151 GUUAUGGACACUUUGAAAC GUUUCAAAGUGUCCAUAAC 1697 1698
1134-1152 UUAUGGACACUUUGAAACA UGUUUCAAAGUGUCCAUAA 1699 1700
1135-1153 UAUGGACACUUUGAAACAA UUGUUUCAAAGUGUCCAUA 1701 1702
1136-1154 AUGGACACUUUGAAACAAC GUUGUUUCAAAGUGUCCAU 1703 1704
1139-1157 GACACUUUGAAACAACAUG CAUGUUGUUUCAAAGUGUC 1705 1706
1140-1158 ACACUUUGAAACAACAUGG CCAUGUUGUUUCAAAGUGU 1707 1708
1141-1159 CACUUUGAAACAACAUGGU ACCAUGUUGUUUCAAAGUG 1709 1710
1142-1160 ACUUUGAAACAACAUGGUG CACCAUGUUGUUUCAAAGU 1711 1712
1143-1161 CUUUGAAACAACAUGGUGC GCACCAUGUUGUUUCAAAG 1713 1714
1144-1162 UUUGAAACAACAUGGUGCU AGCACCAUGUUGUUUCAAA 1715 1716
1145-1163 UUGAAACAACAUGGUGCUG CAGCACCAUGUUGUUUCAA 1717 1718
1146-1164 UGAAACAACAUGGUGCUGG CCAGCACCAUGUUGUUUCA 1719 1720
1147-1165 GAAACAACAUGGUGCUGGG CCCAGCACCAUGUUGUUUC 1721 1722
1148-1166 AAACAACAUGGUGCUGGGG CCCCAGCACCAUGUUGUUU 1723 1724
1149-1167 AACAACAUGGUGCUGGGGC GCCCCAGCACCAUGUUGUU 1725 1726
1150-1168 ACAACAUGGUGCUGGGGCA UGCCCCAGCACCAUGUUGU 1727 1728
1151-1169 CAACAUGGUGCUGGGGCAG CUGCCCCAGCACCAUGUUG 1729 1730
1152-1170 AACAUGGUGCUGGGGCAGG CCUGCCCCAGCACCAUGUU 1731 1732
1153-1171 ACAUGGUGCUGGGGCAGGU ACCUGCCCCAGCACCAUGU 1733 1734
1154-1172 CAUGGUGCUGGGGCAGGUG CACCUGCCCCAGCACCAUG 1735 1736
1155-1173 AUGGUGCUGGGGCAGGUGG CCACCUGCCCCAGCACCAU 1737 1738
1156-1174 UGGUGCUGGGGCAGGUGGU ACCACCUGCCCCAGCACCA 1739 1740
1157-1175 GGUGCUGGGGCAGGUGGUA UACCACCUGCCCCAGCACC 1741 1742
1158-1176 GUGCUGGGGCAGGUGGUAC GUACCACCUGCCCCAGCAC 1743 1744
1159-1177 UGCUGGGGCAGGUGGUACU AGUACCACCUGCCCCAGCA 1745 1746
1160-1178 GCUGGGGCAGGUGGUACUA UAGUACCACCUGCCCCAGC 1747 1748
1161-1179 CUGGGGCAGGUGGUACUAG CUAGUACCACCUGCCCCAG 1749 1750
1162-1180 UGGGGCAGGUGGUACUAGA UCUAGUACCACCUGCCCCA 1751 1752
1166-1184 GCAGGUGGUACUAGAAAUA UAUUUCUAGUACCACCUGC 1753 1754
1167-1185 CAGGUGGUACUAGAAAUAU AUAUUUCUAGUACCACCUG 1755 1756
1168-1186 AGGUGGUACUAGAAAUAUU AAUAUUUCUAGUACCACCU 1757 1758
1169-1187 GGUGGUACUAGAAAUAUUU AAAUAUUUCUAGUACCACC 1759 1760
1170-1188 GUGGUACUAGAAAUAUUUC GAAAUAUUUCUAGUACCAC 1761 1762
1171-1189 UGGUACUAGAAAUAUUUCU AGAAAUAUUUCUAGUACCA 1763 1764
1172-1190 GGUACUAGAAAUAUUUCUG CAGAAAUAUUUCUAGUACC 1765 1766
1173-1191 GUACUAGAAAUAUUUCUGG CCAGAAAUAUUUCUAGUAC 1767 1768
1174-1192 UACUAGAAAUAUUUCUGGA UCCAGAAAUAUUUCUAGUA 1769 1770
1175-1193 ACUAGAAAUAUUUCUGGAA UUCCAGAAAUAUUUCUAGU 1771 1772
1176-1194 CUAGAAAUAUUUCUGGAAC GUUCCAGAAAUAUUUCUAG 1773 1774
1177-1195 UAGAAAUAUUUCUGGAACU AGUUCCAGAAAUAUUUCUA 1775 1776
1178-1196 AGAAAUAUUUCUGGAACUA UAGUUCCAGAAAUAUUUCU 1777 1778
1179-1197 GAAAUAUUUCUGGAACUAG CUAGUUCCAGAAAUAUUUC 1779 1780
1180-1198 AAAUAUUUCUGGAACUAGU ACUAGUUCCAGAAAUAUUU 1781 1782
1181-1199 AAUAUUUCUGGAACUAGUA UACUAGUUCCAGAAAUAUU 1783 1784
1183-1201 UAUUUCUGGAACUAGUAAA UUUACUAGUUCCAGAAAUA 1785 1786
1186-1204 UUCUGGAACUAGUAAAUUC GAAUUUACUAGUUCCAGAA 1787 1788
1187-1205 UCUGGAACUAGUAAAUUCC GGAAUUUACUAGUUCCAGA 1789 1790
1189-1207 UGGAACUAGUAAAUUCCAU AUGGAAUUUACUAGUUCCA 1791 1792
1190-1208 GGAACUAGUAAAUUCCAUG CAUGGAAUUUACUAGUUCC 1793 1794
1192-1210 AACUAGUAAAUUCCAUGUG CACAUGGAAUUUACUAGUU 1795 1796
1193-1211 ACUAGUAAAUUCCAUGUGG CCACAUGGAAUUUACUAGU 1797 1798
1194-1212 CUAGUAAAUUCCAUGUGGA UCCACAUGGAAUUUACUAG
1799 1800 1195-1213 UAGUAAAUUCCAUGUGGAC GUCCACAUGGAAUUUACUA 1801
1802 1196-1214 AGUAAAUUCCAUGUGGACU AGUCCACAUGGAAUUUACU 1803 1804
1197-1215 GUAAAUUCCAUGUGGACUU AAGUCCACAUGGAAUUUAC 1805 1806
1198-1216 UAAAUUCCAUGUGGACUUA UAAGUCCACAUGGAAUUUA 1807 1808
1199-1217 AAAUUCCAUGUGGACUUAG CUAAGUCCACAUGGAAUUU 1809 1810
1200-1218 AAUUCCAUGUGGACUUAGA UCUAAGUCCACAUGGAAUU 1811 1812
1201-1219 AUUCCAUGUGGACUUAGAG CUCUAAGUCCACAUGGAAU 1813 1814
1202-1220 UUCCAUGUGGACUUAGAGC GCUCUAAGUCCACAUGGAA 1815 1816
1222-1240 GGAGCUGGCAGACCUCCAU AUGGAGGUCUGCCAGCUCC 1817 1818
1223-1241 GAGCUGGCAGACCUCCAUG CAUGGAGGUCUGCCAGCUC 1819 1820
1224-1242 AGCUGGCAGACCUCCAUGG CCAUGGAGGUCUGCCAGCU 1821 1822
1225-1243 GCUGGCAGACCUCCAUGGG CCCAUGGAGGUCUGCCAGC 1823 1824
1226-1244 CUGGCAGACCUCCAUGGGA UCCCAUGGAGGUCUGCCAG 1825 1826
1227-1245 UGGCAGACCUCCAUGGGAA UUCCCAUGGAGGUCUGCCA 1827 1828
1228-1246 GGCAGACCUCCAUGGGAAA UUUCCCAUGGAGGUCUGCC 1829 1830
1229-1247 GCAGACCUCCAUGGGAAAG CUUUCCCAUGGAGGUCUGC 1831 1832
1230-1248 CAGACCUCCAUGGGAAAGA UCUUUCCCAUGGAGGUCUG 1833 1834
1231-1249 AGACCUCCAUGGGAAAGAU AUCUUUCCCAUGGAGGUCU 1835 1836
1232-1250 GACCUCCAUGGGAAAGAUG CAUCUUUCCCAUGGAGGUC 1837 1838
1233-1251 ACCUCCAUGGGAAAGAUGC GCAUCUUUCCCAUGGAGGU 1839 1840
1254-1272 CACUCUUGUUUUCCUCGUG CACGAGGAAAACAAGAGUG 1841 1842
1255-1273 ACUCUUGUUUUCCUCGUGC GCACGAGGAAAACAAGAGU 1843 1844
1256-1274 CUCUUGUUUUCCUCGUGCU AGCACGAGGAAAACAAGAG 1845 1846
1257-1275 UCUUGUUUUCCUCGUGCUU AAGCACGAGGAAAACAAGA 1847 1848
1259-1277 UUGUUUUCCUCGUGCUUUG CAAAGCACGAGGAAAACAA 1849 1850
1260-1278 UGUUUUCCUCGUGCUUUGU ACAAAGCACGAGGAAAACA 1851 1852
1261-1279 GUUUUCCUCGUGCUUUGUG CACAAAGCACGAGGAAAAC 1853 1854
1262-1280 UUUUCCUCGUGCUUUGUGG CCACAAAGCACGAGGAAAA 1855 1856
1263-1281 UUUCCUCGUGCUUUGUGGC GCCACAAAGCACGAGGAAA 1857 1858
1264-1282 UUCCUCGUGCUUUGUGGCC GGCCACAAAGCACGAGGAA 1859 1860
1265-1283 UCCUCGUGCUUUGUGGCCA UGGCCACAAAGCACGAGGA 1861 1862
1266-1284 CCUCGUGCUUUGUGGCCAA UUGGCCACAAAGCACGAGG 1863 1864
1267-1285 CUCGUGCUUUGUGGCCAAU AUUGGCCACAAAGCACGAG 1865 1866
1268-1286 UCGUGCUUUGUGGCCAAUG CAUUGGCCACAAAGCACGA 1867 1868
1269-1287 CGUGCUUUGUGGCCAAUGA UCAUUGGCCACAAAGCACG 1869 1870
1270-1288 GUGCUUUGUGGCCAAUGAC GUCAUUGGCCACAAAGCAC 1871 1872
1271-1289 UGCUUUGUGGCCAAUGACU AGUCAUUGGCCACAAAGCA 1873 1874
1272-1290 GCUUUGUGGCCAAUGACUC GAGUCAUUGGCCACAAAGC 1875 1876
1273-1291 CUUUGUGGCCAAUGACUCA UGAGUCAUUGGCCACAAAG 1877 1878
1274-1292 UUUGUGGCCAAUGACUCAA UUGAGUCAUUGGCCACAAA 1879 1880
1275-1293 UUGUGGCCAAUGACUCAAC GUUGAGUCAUUGGCCACAA 1881 1882
1276-1294 UGUGGCCAAUGACUCAACC GGUUGAGUCAUUGGCCACA 1883 1884
1277-1295 GUGGCCAAUGACUCAACCC GGGUUGAGUCAUUGGCCAC 1885 1886
1278-1296 UGGCCAAUGACUCAACCCU AGGGUUGAGUCAUUGGCCA 1887 1888
1279-1297 GGCCAAUGACUCAACCCUC GAGGGUUGAGUCAUUGGCC 1889 1890
1280-1298 GCCAAUGACUCAACCCUCU AGAGGGUUGAGUCAUUGGC 1891 1892
1281-1299 CCAAUGACUCAACCCUCUU AAGAGGGUUGAGUCAUUGG 1893 1894
1282-1300 CAAUGACUCAACCCUCUUC GAAGAGGGUUGAGUCAUUG 1895 1896
1283-1301 AAUGACUCAACCCUCUUCA UGAAGAGGGUUGAGUCAUU 1897 1898
1284-1302 AUGACUCAACCCUCUUCAC GUGAAGAGGGUUGAGUCAU 1899 1900
1285-1303 UGACUCAACCCUCUUCACC GGUGAAGAGGGUUGAGUCA 1901 1902
1286-1304 GACUCAACCCUCUUCACCC GGGUGAAGAGGGUUGAGUC 1903 1904
1287-1305 ACUCAACCCUCUUCACCCU AGGGUGAAGAGGGUUGAGU 1905 1906
1288-1306 CUCAACCCUCUUCACCCUG CAGGGUGAAGAGGGUUGAG 1907 1908
1289-1307 UCAACCCUCUUCACCCUGG CCAGGGUGAAGAGGGUUGA 1909 1910
1290-1308 CAACCCUCUUCACCCUGGC GCCAGGGUGAAGAGGGUUG 1911 1912
1291-1309 AACCCUCUUCACCCUGGCU AGCCAGGGUGAAGAGGGUU 1913 1914
1292-1310 ACCCUCUUCACCCUGGCUA UAGCCAGGGUGAAGAGGGU 1915 1916
1293-1311 CCCUCUUCACCCUGGCUAA UUAGCCAGGGUGAAGAGGG 1917 1918
1294-1312 CCUCUUCACCCUGGCUAAG CUUAGCCAGGGUGAAGAGG 1919 1920
1297-1315 CUUCACCCUGGCUAAGAUG CAUCUUAGCCAGGGUGAAG 1921 1922
1298-1316 UUCACCCUGGCUAAGAUGA UCAUCUUAGCCAGGGUGAA 1923 1924
1300-1318 CACCCUGGCUAAGAUGAUG CAUCAUCUUAGCCAGGGUG 1925 1926
1301-1319 ACCCUGGCUAAGAUGAUGC GCAUCAUCUUAGCCAGGGU 1927 1928
1302-1320 CCCUGGCUAAGAUGAUGCC GGCAUCAUCUUAGCCAGGG 1929 1930
1303-1321 CCUGGCUAAGAUGAUGCCA UGGCAUCAUCUUAGCCAGG 1931 1932
1304-1322 CUGGCUAAGAUGAUGCCAG CUGGCAUCAUCUUAGCCAG 1933 1934
1305-1323 UGGCUAAGAUGAUGCCAGG CCUGGCAUCAUCUUAGCCA 1935 1936
1306-1324 GGCUAAGAUGAUGCCAGGC GCCUGGCAUCAUCUUAGCC 1937 1938
1307-1325 GCUAAGAUGAUGCCAGGCU AGCCUGGCAUCAUCUUAGC 1939 1940
1308-1326 CUAAGAUGAUGCCAGGCUG CAGCCUGGCAUCAUCUUAG 1941 1942
1309-1327 UAAGAUGAUGCCAGGCUGU ACAGCCUGGCAUCAUCUUA 1943 1944
1310-1328 AAGAUGAUGCCAGGCUGUG CACAGCCUGGCAUCAUCUU 1945 1946
1311-1329 AGAUGAUGCCAGGCUGUGA UCACAGCCUGGCAUCAUCU 1947 1948
1312-1330 GAUGAUGCCAGGCUGUGAG CUCACAGCCUGGCAUCAUC 1949 1950
1313-1331 AUGAUGCCAGGCUGUGAGA UCUCACAGCCUGGCAUCAU 1951 1952
1314-1332 UGAUGCCAGGCUGUGAGAU AUCUCACAGCCUGGCAUCA 1953 1954
1316-1334 AUGCCAGGCUGUGAGAUUU AAAUCUCACAGCCUGGCAU 1955 1956
1317-1335 UGCCAGGCUGUGAGAUUUA UAAAUCUCACAGCCUGGCA 1957 1958
1318-1336 GCCAGGCUGUGAGAUUUAC GUAAAUCUCACAGCCUGGC 1959 1960
1319-1337 CCAGGCUGUGAGAUUUACU AGUAAAUCUCACAGCCUGG 1961 1962
1320-1338 CAGGCUGUGAGAUUUACUC GAGUAAAUCUCACAGCCUG 1963 1964
1321-1339 AGGCUGUGAGAUUUACUCU AGAGUAAAUCUCACAGCCU 1965 1966
1322-1340 GGCUGUGAGAUUUACUCUG CAGAGUAAAUCUCACAGCC 1967 1968
1323-1341 GCUGUGAGAUUUACUCUGA UCAGAGUAAAUCUCACAGC 1969 1970
1326-1344 GUGAGAUUUACUCUGAUUC GAAUCAGAGUAAAUCUCAC 1971 1972
1327-1345 UGAGAUUUACUCUGAUUCU AGAAUCAGAGUAAAUCUCA 1973 1974
1328-1346 GAGAUUUACUCUGAUUCUG CAGAAUCAGAGUAAAUCUC 1975 1976
1329-1347 AGAUUUACUCUGAUUCUGG CCAGAAUCAGAGUAAAUCU 1977 1978
1330-1348 GAUUUACUCUGAUUCUGGG CCCAGAAUCAGAGUAAAUC 1979 1980
1331-1349 AUUUACUCUGAUUCUGGGA UCCCAGAAUCAGAGUAAAU 1981 1982
1332-1350 UUUACUCUGAUUCUGGGAA UUCCCAGAAUCAGAGUAAA 1983 1984
1333-1351 UUACUCUGAUUCUGGGAAC GUUCCCAGAAUCAGAGUAA 1985 1986
1334-1352 UACUCUGAUUCUGGGAACC GGUUCCCAGAAUCAGAGUA 1987 1988
1335-1353 ACUCUGAUUCUGGGAACCA UGGUUCCCAGAAUCAGAGU 1989 1990
1336-1354 CUCUGAUUCUGGGAACCAU AUGGUUCCCAGAAUCAGAG 1991 1992
1337-1355 UCUGAUUCUGGGAACCAUG CAUGGUUCCCAGAAUCAGA 1993 1994
1338-1356 CUGAUUCUGGGAACCAUGC GCAUGGUUCCCAGAAUCAG 1995 1996
1339-1357 UGAUUCUGGGAACCAUGCC GGCAUGGUUCCCAGAAUCA 1997 1998
1340-1358 GAUUCUGGGAACCAUGCCU AGGCAUGGUUCCCAGAAUC 1999 2000
1341-1359 AUUCUGGGAACCAUGCCUC GAGGCAUGGUUCCCAGAAU 2001 2002
1342-1360 UUCUGGGAACCAUGCCUCC GGAGGCAUGGUUCCCAGAA 2003 2004
1343-1361 UCUGGGAACCAUGCCUCCA UGGAGGCAUGGUUCCCAGA 2005 2006
1344-1362 CUGGGAACCAUGCCUCCAU AUGGAGGCAUGGUUCCCAG 2007 2008
1345-1363 UGGGAACCAUGCCUCCAUG CAUGGAGGCAUGGUUCCCA 2009 2010
1346-1364 GGGAACCAUGCCUCCAUGA UCAUGGAGGCAUGGUUCCC 2011 2012
1348-1366 GAACCAUGCCUCCAUGAUC GAUCAUGGAGGCAUGGUUC 2013 2014
1349-1367 AACCAUGCCUCCAUGAUCC GGAUCAUGGAGGCAUGGUU 2015 2016
1350-1368 ACCAUGCCUCCAUGAUCCA UGGAUCAUGGAGGCAUGGU 2017 2018
1351-1369 CCAUGCCUCCAUGAUCCAA UUGGAUCAUGGAGGCAUGG 2019 2020
1352-1370 CAUGCCUCCAUGAUCCAAG CUUGGAUCAUGGAGGCAUG 2021 2022
1353-1371 AUGCCUCCAUGAUCCAAGG CCUUGGAUCAUGGAGGCAU 2023 2024
1354-1372 UGCCUCCAUGAUCCAAGGG CCCUUGGAUCAUGGAGGCA 2025 2026
1358-1376 UCCAUGAUCCAAGGGAUUC GAAUCCCUUGGAUCAUGGA 2027 2028
1359-1377 CCAUGAUCCAAGGGAUUCG CGAAUCCCUUGGAUCAUGG 2029 2030
1360-1378 CAUGAUCCAAGGGAUUCGA UCGAAUCCCUUGGAUCAUG 2031 2032
1361-1379 AUGAUCCAAGGGAUUCGAA UUCGAAUCCCUUGGAUCAU 2033 2034
1362-1380 UGAUCCAAGGGAUUCGAAA UUUCGAAUCCCUUGGAUCA 2035 2036
1363-1381 GAUCCAAGGGAUUCGAAAC GUUUCGAAUCCCUUGGAUC 2037 2038
1365-1383 UCCAAGGGAUUCGAAACAG CUGUUUCGAAUCCCUUGGA 2039 2040
1366-1384 CCAAGGGAUUCGAAACAGC GCUGUUUCGAAUCCCUUGG 2041 2042
1367-1385 CAAGGGAUUCGAAACAGCC GGCUGUUUCGAAUCCCUUG 2043 2044
1368-1386 AAGGGAUUCGAAACAGCCG CGGCUGUUUCGAAUCCCUU 2045 2046
1369-1387 AGGGAUUCGAAACAGCCGA UCGGCUGUUUCGAAUCCCU 2047 2048
1370-1388 GGGAUUCGAAACAGCCGAG CUCGGCUGUUUCGAAUCCC
2049 2050 1371-1389 GGAUUCGAAACAGCCGAGU ACUCGGCUGUUUCGAAUCC 2051
2052 1372-1390 GAUUCGAAACAGCCGAGUG CACUCGGCUGUUUCGAAUC 2053 2054
1373-1391 AUUCGAAACAGCCGAGUGC GCACUCGGCUGUUUCGAAU 2055 2056
1374-1392 UUCGAAACAGCCGAGUGCC GGCACUCGGCUGUUUCGAA 2057 2058
1375-1393 UCGAAACAGCCGAGUGCCA UGGCACUCGGCUGUUUCGA 2059 2060
1376-1394 CGAAACAGCCGAGUGCCAA UUGGCACUCGGCUGUUUCG 2061 2062
1377-1395 GAAACAGCCGAGUGCCAAA UUUGGCACUCGGCUGUUUC 2063 2064
1378-1396 AAACAGCCGAGUGCCAAAG CUUUGGCACUCGGCUGUUU 2065 2066
1379-1397 AACAGCCGAGUGCCAAAGU ACUUUGGCACUCGGCUGUU 2067 2068
1380-1398 ACAGCCGAGUGCCAAAGUA UACUUUGGCACUCGGCUGU 2069 2070
1381-1399 CAGCCGAGUGCCAAAGUAC GUACUUUGGCACUCGGCUG 2071 2072
1383-1401 GCCGAGUGCCAAAGUACAU AUGUACUUUGGCACUCGGC 2073 2074
1384-1402 CCGAGUGCCAAAGUACAUC GAUGUACUUUGGCACUCGG 2075 2076
1385-1403 CGAGUGCCAAAGUACAUCU AGAUGUACUUUGGCACUCG 2077 2078
1386-1404 GAGUGCCAAAGUACAUCUU AAGAUGUACUUUGGCACUC 2079 2080
1387-1405 AGUGCCAAAGUACAUCUUC GAAGAUGUACUUUGGCACU 2081 2082
1388-1406 GUGCCAAAGUACAUCUUCC GGAAGAUGUACUUUGGCAC 2083 2084
1389-1407 UGCCAAAGUACAUCUUCCG CGGAAGAUGUACUUUGGCA 2085 2086
1390-1408 GCCAAAGUACAUCUUCCGC GCGGAAGAUGUACUUUGGC 2087 2088
1391-1409 CCAAAGUACAUCUUCCGCC GGCGGAAGAUGUACUUUGG 2089 2090
1392-1410 CAAAGUACAUCUUCCGCCA UGGCGGAAGAUGUACUUUG 2091 2092
1393-1411 AAAGUACAUCUUCCGCCAC GUGGCGGAAGAUGUACUUU 2093 2094
1394-1412 AAGUACAUCUUCCGCCACA UGUGGCGGAAGAUGUACUU 2095 2096
1395-1413 AGUACAUCUUCCGCCACAA UUGUGGCGGAAGAUGUACU 2097 2098
1396-1414 GUACAUCUUCCGCCACAAU AUUGUGGCGGAAGAUGUAC 2099 2100
1397-1415 UACAUCUUCCGCCACAAUG CAUUGUGGCGGAAGAUGUA 2101 2102
1398-1416 ACAUCUUCCGCCACAAUGA UCAUUGUGGCGGAAGAUGU 2103 2104
1399-1417 CAUCUUCCGCCACAAUGAU AUCAUUGUGGCGGAAGAUG 2105 2106
1400-1418 AUCUUCCGCCACAAUGAUG CAUCAUUGUGGCGGAAGAU 2107 2108
1401-1419 UCUUCCGCCACAAUGAUGU ACAUCAUUGUGGCGGAAGA 2109 2110
1402-1420 CUUCCGCCACAAUGAUGUC GACAUCAUUGUGGCGGAAG 2111 2112
1403-1421 UUCCGCCACAAUGAUGUCA UGACAUCAUUGUGGCGGAA 2113 2114
1404-1422 UCCGCCACAAUGAUGUCAG CUGACAUCAUUGUGGCGGA 2115 2116
1405-1423 CCGCCACAAUGAUGUCAGC GCUGACAUCAUUGUGGCGG 2117 2118
1406-1424 CGCCACAAUGAUGUCAGCC GGCUGACAUCAUUGUGGCG 2119 2120
1407-1425 GCCACAAUGAUGUCAGCCA UGGCUGACAUCAUUGUGGC 2121 2122
1427-1445 CUCAGAGAACUGCUGCAAA UUUGCAGCAGUUCUCUGAG 2123 2124
1428-1446 UCAGAGAACUGCUGCAAAG CUUUGCAGCAGUUCUCUGA 2125 2126
1429-1447 CAGAGAACUGCUGCAAAGA UCUUUGCAGCAGUUCUCUG 2127 2128
1430-1448 AGAGAACUGCUGCAAAGAU AUCUUUGCAGCAGUUCUCU 2129 2130
1431-1449 GAGAACUGCUGCAAAGAUC GAUCUUUGCAGCAGUUCUC 2131 2132
1432-1450 AGAACUGCUGCAAAGAUCU AGAUCUUUGCAGCAGUUCU 2133 2134
1433-1451 GAACUGCUGCAAAGAUCUG CAGAUCUUUGCAGCAGUUC 2135 2136
1434-1452 AACUGCUGCAAAGAUCUGA UCAGAUCUUUGCAGCAGUU 2137 2138
1435-1453 ACUGCUGCAAAGAUCUGAC GUCAGAUCUUUGCAGCAGU 2139 2140
1436-1454 CUGCUGCAAAGAUCUGACC GGUCAGAUCUUUGCAGCAG 2141 2142
1437-1455 UGCUGCAAAGAUCUGACCC GGGUCAGAUCUUUGCAGCA 2143 2144
1457-1475 UCAGUCCCCAAGAUUGUGG CCACAAUCUUGGGGACUGA 2145 2146
1458-1476 CAGUCCCCAAGAUUGUGGC GCCACAAUCUUGGGGACUG 2147 2148
1459-1477 AGUCCCCAAGAUUGUGGCA UGCCACAAUCUUGGGGACU 2149 2150
1461-1479 UCCCCAAGAUUGUGGCAUU AAUGCCACAAUCUUGGGGA 2151 2152
1462-1480 CCCCAAGAUUGUGGCAUUU AAAUGCCACAAUCUUGGGG 2153 2154
1463-1481 CCCAAGAUUGUGGCAUUUG CAAAUGCCACAAUCUUGGG 2155 2156
1464-1482 CCAAGAUUGUGGCAUUUGA UCAAAUGCCACAAUCUUGG 2157 2158
1465-1483 CAAGAUUGUGGCAUUUGAA UUCAAAUGCCACAAUCUUG 2159 2160
1466-1484 AAGAUUGUGGCAUUUGAAA UUUCAAAUGCCACAAUCUU 2161 2162
1467-1485 AGAUUGUGGCAUUUGAAAC GUUUCAAAUGCCACAAUCU 2163 2164
1468-1486 GAUUGUGGCAUUUGAAACU AGUUUCAAAUGCCACAAUC 2165 2166
1469-1487 AUUGUGGCAUUUGAAACUG CAGUUUCAAAUGCCACAAU 2167 2168
1470-1488 UUGUGGCAUUUGAAACUGU ACAGUUUCAAAUGCCACAA 2169 2170
1471-1489 UGUGGCAUUUGAAACUGUC GACAGUUUCAAAUGCCACA 2171 2172
1472-1490 GUGGCAUUUGAAACUGUCC GGACAGUUUCAAAUGCCAC 2173 2174
1473-1491 UGGCAUUUGAAACUGUCCA UGGACAGUUUCAAAUGCCA 2175 2176
1474-1492 GGCAUUUGAAACUGUCCAU AUGGACAGUUUCAAAUGCC 2177 2178
1475-1493 GCAUUUGAAACUGUCCAUU AAUGGACAGUUUCAAAUGC 2179 2180
1476-1494 CAUUUGAAACUGUCCAUUC GAAUGGACAGUUUCAAAUG 2181 2182
1477-1495 AUUUGAAACUGUCCAUUCA UGAAUGGACAGUUUCAAAU 2183 2184
1479-1497 UUGAAACUGUCCAUUCAAU AUUGAAUGGACAGUUUCAA 2185 2186
1480-1498 UGAAACUGUCCAUUCAAUG CAUUGAAUGGACAGUUUCA 2187 2188
1481-1499 GAAACUGUCCAUUCAAUGG CCAUUGAAUGGACAGUUUC 2189 2190
1482-1500 AAACUGUCCAUUCAAUGGA UCCAUUGAAUGGACAGUUU 2191 2192
1483-1501 AACUGUCCAUUCAAUGGAU AUCCAUUGAAUGGACAGUU 2193 2194
1484-1502 ACUGUCCAUUCAAUGGAUG CAUCCAUUGAAUGGACAGU 2195 2196
1485-1503 CUGUCCAUUCAAUGGAUGG CCAUCCAUUGAAUGGACAG 2197 2198
1486-1504 UGUCCAUUCAAUGGAUGGG CCCAUCCAUUGAAUGGACA 2199 2200
1487-1505 GUCCAUUCAAUGGAUGGGG CCCCAUCCAUUGAAUGGAC 2201 2202
1488-1506 UCCAUUCAAUGGAUGGGGC GCCCCAUCCAUUGAAUGGA 2203 2204
1508-1526 GUGUGCCCACUGGAAGAGC GCUCUUCCAGUGGGCACAC 2205 2206
1509-1527 UGUGCCCACUGGAAGAGCU AGCUCUUCCAGUGGGCACA 2207 2208
1510-1528 GUGCCCACUGGAAGAGCUG CAGCUCUUCCAGUGGGCAC 2209 2210
1511-1529 UGCCCACUGGAAGAGCUGU ACAGCUCUUCCAGUGGGCA 2211 2212
1512-1530 GCCCACUGGAAGAGCUGUG CACAGCUCUUCCAGUGGGC 2213 2214
1513-1531 CCCACUGGAAGAGCUGUGU ACACAGCUCUUCCAGUGGG 2215 2216
1514-1532 CCACUGGAAGAGCUGUGUG CACACAGCUCUUCCAGUGG 2217 2218
1515-1533 CACUGGAAGAGCUGUGUGA UCACACAGCUCUUCCAGUG 2219 2220
1516-1534 ACUGGAAGAGCUGUGUGAU AUCACACAGCUCUUCCAGU 2221 2222
1517-1535 CUGGAAGAGCUGUGUGAUG CAUCACACAGCUCUUCCAG 2223 2224
1518-1536 UGGAAGAGCUGUGUGAUGU ACAUCACACAGCUCUUCCA 2225 2226
1519-1537 GGAAGAGCUGUGUGAUGUG CACAUCACACAGCUCUUCC 2227 2228
1520-1538 GAAGAGCUGUGUGAUGUGG CCACAUCACACAGCUCUUC 2229 2230
1521-1539 AAGAGCUGUGUGAUGUGGC GCCACAUCACACAGCUCUU 2231 2232
1522-1540 AGAGCUGUGUGAUGUGGCC GGCCACAUCACACAGCUCU 2233 2234
1523-1541 GAGCUGUGUGAUGUGGCCC GGGCCACAUCACACAGCUC 2235 2236
1524-1542 AGCUGUGUGAUGUGGCCCA UGGGCCACAUCACACAGCU 2237 2238
1525-1543 GCUGUGUGAUGUGGCCCAU AUGGGCCACAUCACACAGC 2239 2240
1526-1544 CUGUGUGAUGUGGCCCAUG CAUGGGCCACAUCACACAG 2241 2242
1527-1545 UGUGUGAUGUGGCCCAUGA UCAUGGGCCACAUCACACA 2243 2244
1528-1546 GUGUGAUGUGGCCCAUGAG CUCAUGGGCCACAUCACAC 2245 2246
1529-1547 UGUGAUGUGGCCCAUGAGU ACUCAUGGGCCACAUCACA 2247 2248
1532-1550 GAUGUGGCCCAUGAGUUUG CAAACUCAUGGGCCACAUC 2249 2250
1533-1551 AUGUGGCCCAUGAGUUUGG CCAAACUCAUGGGCCACAU 2251 2252
1534-1552 UGUGGCCCAUGAGUUUGGA UCCAAACUCAUGGGCCACA 2253 2254
1535-1553 GUGGCCCAUGAGUUUGGAG CUCCAAACUCAUGGGCCAC 2255 2256
1536-1554 UGGCCCAUGAGUUUGGAGC GCUCCAAACUCAUGGGCCA 2257 2258
1537-1555 GGCCCAUGAGUUUGGAGCA UGCUCCAAACUCAUGGGCC 2259 2260
1538-1556 GCCCAUGAGUUUGGAGCAA UUGCUCCAAACUCAUGGGC 2261 2262
1539-1557 CCCAUGAGUUUGGAGCAAU AUUGCUCCAAACUCAUGGG 2263 2264
1540-1558 CCAUGAGUUUGGAGCAAUC GAUUGCUCCAAACUCAUGG 2265 2266
1542-1560 AUGAGUUUGGAGCAAUCAC GUGAUUGCUCCAAACUCAU 2267 2268
1543-1561 UGAGUUUGGAGCAAUCACC GGUGAUUGCUCCAAACUCA 2269 2270
1545-1563 AGUUUGGAGCAAUCACCUU AAGGUGAUUGCUCCAAACU 2271 2272
1546-1564 GUUUGGAGCAAUCACCUUC GAAGGUGAUUGCUCCAAAC 2273 2274
1547-1565 UUUGGAGCAAUCACCUUCG CGAAGGUGAUUGCUCCAAA 2275 2276
1548-1566 UUGGAGCAAUCACCUUCGU ACGAAGGUGAUUGCUCCAA 2277 2278
1549-1567 UGGAGCAAUCACCUUCGUG CACGAAGGUGAUUGCUCCA 2279 2280
1550-1568 GGAGCAAUCACCUUCGUGG CCACGAAGGUGAUUGCUCC 2281 2282
1551-1569 GAGCAAUCACCUUCGUGGA UCCACGAAGGUGAUUGCUC 2283 2284
1552-1570 AGCAAUCACCUUCGUGGAU AUCCACGAAGGUGAUUGCU 2285 2286
1553-1571 GCAAUCACCUUCGUGGAUG CAUCCACGAAGGUGAUUGC 2287 2288
1554-1572 CAAUCACCUUCGUGGAUGA UCAUCCACGAAGGUGAUUG 2289 2290
1555-1573 AAUCACCUUCGUGGAUGAG CUCAUCCACGAAGGUGAUU 2291 2292
1556-1574 AUCACCUUCGUGGAUGAGG CCUCAUCCACGAAGGUGAU 2293 2294
1557-1575 UCACCUUCGUGGAUGAGGU ACCUCAUCCACGAAGGUGA 2295 2296
1558-1576 CACCUUCGUGGAUGAGGUC GACCUCAUCCACGAAGGUG 2297 2298
1559-1577 ACCUUCGUGGAUGAGGUCC GGACCUCAUCCACGAAGGU 2299 2300
1560-1578 CCUUCGUGGAUGAGGUCCA UGGACCUCAUCCACGAAGG
2301 2302 1561-1579 CUUCGUGGAUGAGGUCCAC GUGGACCUCAUCCACGAAG 2303
2304 1562-1580 UUCGUGGAUGAGGUCCACG CGUGGACCUCAUCCACGAA 2305 2306
1563-1581 UCGUGGAUGAGGUCCACGC GCGUGGACCUCAUCCACGA 2307 2308
1564-1582 CGUGGAUGAGGUCCACGCA UGCGUGGACCUCAUCCACG 2309 2310
1565-1583 GUGGAUGAGGUCCACGCAG CUGCGUGGACCUCAUCCAC 2311 2312
1566-1584 UGGAUGAGGUCCACGCAGU ACUGCGUGGACCUCAUCCA 2313 2314
1567-1585 GGAUGAGGUCCACGCAGUG CACUGCGUGGACCUCAUCC 2315 2316
1568-1586 GAUGAGGUCCACGCAGUGG CCACUGCGUGGACCUCAUC 2317 2318
1569-1587 AUGAGGUCCACGCAGUGGG CCCACUGCGUGGACCUCAU 2319 2320
1570-1588 UGAGGUCCACGCAGUGGGG CCCCACUGCGUGGACCUCA 2321 2322
1571-1589 GAGGUCCACGCAGUGGGGC GCCCCACUGCGUGGACCUC 2323 2324
1572-1590 AGGUCCACGCAGUGGGGCU AGCCCCACUGCGUGGACCU 2325 2326
1595-1613 GGGGCUCGAGGCGGAGGGA UCCCUCCGCCUCGAGCCCC 2327 2328
1596-1614 GGGCUCGAGGCGGAGGGAU AUCCCUCCGCCUCGAGCCC 2329 2330
1597-1615 GGCUCGAGGCGGAGGGAUU AAUCCCUCCGCCUCGAGCC 2331 2332
1598-1616 GCUCGAGGCGGAGGGAUUG CAAUCCCUCCGCCUCGAGC 2333 2334
1599-1617 CUCGAGGCGGAGGGAUUGG CCAAUCCCUCCGCCUCGAG 2335 2336
1600-1618 UCGAGGCGGAGGGAUUGGG CCCAAUCCCUCCGCCUCGA 2337 2338
1601-1619 CGAGGCGGAGGGAUUGGGG CCCCAAUCCCUCCGCCUCG 2339 2340
1602-1620 GAGGCGGAGGGAUUGGGGA UCCCCAAUCCCUCCGCCUC 2341 2342
1603-1621 AGGCGGAGGGAUUGGGGAU AUCCCCAAUCCCUCCGCCU 2343 2344
1604-1622 GGCGGAGGGAUUGGGGAUC GAUCCCCAAUCCCUCCGCC 2345 2346
1605-1623 GCGGAGGGAUUGGGGAUCG CGAUCCCCAAUCCCUCCGC 2347 2348
1606-1624 CGGAGGGAUUGGGGAUCGG CCGAUCCCCAAUCCCUCCG 2349 2350
1607-1625 GGAGGGAUUGGGGAUCGGG CCCGAUCCCCAAUCCCUCC 2351 2352
1608-1626 GAGGGAUUGGGGAUCGGGA UCCCGAUCCCCAAUCCCUC 2353 2354
1609-1627 AGGGAUUGGGGAUCGGGAU AUCCCGAUCCCCAAUCCCU 2355 2356
1610-1628 GGGAUUGGGGAUCGGGAUG CAUCCCGAUCCCCAAUCCC 2357 2358
1611-1629 GGAUUGGGGAUCGGGAUGG CCAUCCCGAUCCCCAAUCC 2359 2360
1612-1630 GAUUGGGGAUCGGGAUGGA UCCAUCCCGAUCCCCAAUC 2361 2362
1613-1631 AUUGGGGAUCGGGAUGGAG CUCCAUCCCGAUCCCCAAU 2363 2364
1614-1632 UUGGGGAUCGGGAUGGAGU ACUCCAUCCCGAUCCCCAA 2365 2366
1615-1633 UGGGGAUCGGGAUGGAGUC GACUCCAUCCCGAUCCCCA 2367 2368
1617-1635 GGGAUCGGGAUGGAGUCAU AUGACUCCAUCCCGAUCCC 2369 2370
1618-1636 GGAUCGGGAUGGAGUCAUG CAUGACUCCAUCCCGAUCC 2371 2372
1619-1637 GAUCGGGAUGGAGUCAUGC GCAUGACUCCAUCCCGAUC 2373 2374
1620-1638 AUCGGGAUGGAGUCAUGCC GGCAUGACUCCAUCCCGAU 2375 2376
1621-1639 UCGGGAUGGAGUCAUGCCA UGGCAUGACUCCAUCCCGA 2377 2378
1622-1640 CGGGAUGGAGUCAUGCCAA UUGGCAUGACUCCAUCCCG 2379 2380
1623-1641 GGGAUGGAGUCAUGCCAAA UUUGGCAUGACUCCAUCCC 2381 2382
1624-1642 GGAUGGAGUCAUGCCAAAA UUUUGGCAUGACUCCAUCC 2383 2384
1625-1643 GAUGGAGUCAUGCCAAAAA UUUUUGGCAUGACUCCAUC 2385 2386
1626-1644 AUGGAGUCAUGCCAAAAAU AUUUUUGGCAUGACUCCAU 2387 2388
1627-1645 UGGAGUCAUGCCAAAAAUG CAUUUUUGGCAUGACUCCA 2389 2390
1628-1646 GGAGUCAUGCCAAAAAUGG CCAUUUUUGGCAUGACUCC 2391 2392
1629-1647 GAGUCAUGCCAAAAAUGGA UCCAUUUUUGGCAUGACUC 2393 2394
1630-1648 AGUCAUGCCAAAAAUGGAC GUCCAUUUUUGGCAUGACU 2395 2396
1632-1650 UCAUGCCAAAAAUGGACAU AUGUCCAUUUUUGGCAUGA 2397 2398
1633-1651 CAUGCCAAAAAUGGACAUC GAUGUCCAUUUUUGGCAUG 2399 2400
1636-1654 GCCAAAAAUGGACAUCAUU AAUGAUGUCCAUUUUUGGC 2401 2402
1638-1656 CAAAAAUGGACAUCAUUUC GAAAUGAUGUCCAUUUUUG 2403 2404
1639-1657 AAAAAUGGACAUCAUUUCU AGAAAUGAUGUCCAUUUUU 2405 2406
1640-1658 AAAAUGGACAUCAUUUCUG CAGAAAUGAUGUCCAUUUU 2407 2408
1641-1659 AAAUGGACAUCAUUUCUGG CCAGAAAUGAUGUCCAUUU 2409 2410
1642-1660 AAUGGACAUCAUUUCUGGA UCCAGAAAUGAUGUCCAUU 2411 2412
1643-1661 AUGGACAUCAUUUCUGGAA UUCCAGAAAUGAUGUCCAU 2413 2414
1644-1662 UGGACAUCAUUUCUGGAAC GUUCCAGAAAUGAUGUCCA 2415 2416
1645-1663 GGACAUCAUUUCUGGAACA UGUUCCAGAAAUGAUGUCC 2417 2418
1646-1664 GACAUCAUUUCUGGAACAC GUGUUCCAGAAAUGAUGUC 2419 2420
1647-1665 ACAUCAUUUCUGGAACACU AGUGUUCCAGAAAUGAUGU 2421 2422
1648-1666 CAUCAUUUCUGGAACACUU AAGUGUUCCAGAAAUGAUG 2423 2424
1649-1667 AUCAUUUCUGGAACACUUG CAAGUGUUCCAGAAAUGAU 2425 2426
1650-1668 UCAUUUCUGGAACACUUGG CCAAGUGUUCCAGAAAUGA 2427 2428
1651-1669 CAUUUCUGGAACACUUGGC GCCAAGUGUUCCAGAAAUG 2429 2430
1652-1670 AUUUCUGGAACACUUGGCA UGCCAAGUGUUCCAGAAAU 2431 2432
1653-1671 UUUCUGGAACACUUGGCAA UUGCCAAGUGUUCCAGAAA 2433 2434
1654-1672 UUCUGGAACACUUGGCAAA UUUGCCAAGUGUUCCAGAA 2435 2436
1655-1673 UCUGGAACACUUGGCAAAG CUUUGCCAAGUGUUCCAGA 2437 2438
1656-1674 CUGGAACACUUGGCAAAGC GCUUUGCCAAGUGUUCCAG 2439 2440
1657-1675 UGGAACACUUGGCAAAGCC GGCUUUGCCAAGUGUUCCA 2441 2442
1658-1676 GGAACACUUGGCAAAGCCU AGGCUUUGCCAAGUGUUCC 2443 2444
1659-1677 GAACACUUGGCAAAGCCUU AAGGCUUUGCCAAGUGUUC 2445 2446
1660-1678 AACACUUGGCAAAGCCUUU AAAGGCUUUGCCAAGUGUU 2447 2448
1661-1679 ACACUUGGCAAAGCCUUUG CAAAGGCUUUGCCAAGUGU 2449 2450
1662-1680 CACUUGGCAAAGCCUUUGG CCAAAGGCUUUGCCAAGUG 2451 2452
1682-1700 UGUGUUGGAGGGUACAUCG CGAUGUACCCUCCAACACA 2453 2454
1683-1701 GUGUUGGAGGGUACAUCGC GCGAUGUACCCUCCAACAC 2455 2456
1684-1702 UGUUGGAGGGUACAUCGCC GGCGAUGUACCCUCCAACA 2457 2458
1685-1703 GUUGGAGGGUACAUCGCCA UGGCGAUGUACCCUCCAAC 2459 2460
1686-1704 UUGGAGGGUACAUCGCCAG CUGGCGAUGUACCCUCCAA 2461 2462
1687-1705 UGGAGGGUACAUCGCCAGC GCUGGCGAUGUACCCUCCA 2463 2464
1688-1706 GGAGGGUACAUCGCCAGCA UGCUGGCGAUGUACCCUCC 2465 2466
1689-1707 GAGGGUACAUCGCCAGCAC GUGCUGGCGAUGUACCCUC 2467 2468
1690-1708 AGGGUACAUCGCCAGCACG CGUGCUGGCGAUGUACCCU 2469 2470
1691-1709 GGGUACAUCGCCAGCACGA UCGUGCUGGCGAUGUACCC 2471 2472
1692-1710 GGUACAUCGCCAGCACGAG CUCGUGCUGGCGAUGUACC 2473 2474
1693-1711 GUACAUCGCCAGCACGAGU ACUCGUGCUGGCGAUGUAC 2475 2476
1694-1712 UACAUCGCCAGCACGAGUU AACUCGUGCUGGCGAUGUA 2477 2478
1695-1713 ACAUCGCCAGCACGAGUUC GAACUCGUGCUGGCGAUGU 2479 2480
1696-1714 CAUCGCCAGCACGAGUUCU AGAACUCGUGCUGGCGAUG 2481 2482
1697-1715 AUCGCCAGCACGAGUUCUC GAGAACUCGUGCUGGCGAU 2483 2484
1698-1716 UCGCCAGCACGAGUUCUCU AGAGAACUCGUGCUGGCGA 2485 2486
1699-1717 CGCCAGCACGAGUUCUCUG CAGAGAACUCGUGCUGGCG 2487 2488
1700-1718 GCCAGCACGAGUUCUCUGA UCAGAGAACUCGUGCUGGC 2489 2490
1701-1719 CCAGCACGAGUUCUCUGAU AUCAGAGAACUCGUGCUGG 2491 2492
1702-1720 CAGCACGAGUUCUCUGAUU AAUCAGAGAACUCGUGCUG 2493 2494
1703-1721 AGCACGAGUUCUCUGAUUG CAAUCAGAGAACUCGUGCU 2495 2496
1704-1722 GCACGAGUUCUCUGAUUGA UCAAUCAGAGAACUCGUGC 2497 2498
1705-1723 CACGAGUUCUCUGAUUGAC GUCAAUCAGAGAACUCGUG 2499 2500
1707-1725 CGAGUUCUCUGAUUGACAC GUGUCAAUCAGAGAACUCG 2501 2502
1727-1745 GUACGGUCCUAUGCUGCUG CAGCAGCAUAGGACCGUAC 2503 2504
1728-1746 UACGGUCCUAUGCUGCUGG CCAGCAGCAUAGGACCGUA 2505 2506
1729-1747 ACGGUCCUAUGCUGCUGGC GCCAGCAGCAUAGGACCGU 2507 2508
1730-1748 CGGUCCUAUGCUGCUGGCU AGCCAGCAGCAUAGGACCG 2509 2510
1731-1749 GGUCCUAUGCUGCUGGCUU AAGCCAGCAGCAUAGGACC 2511 2512
1732-1750 GUCCUAUGCUGCUGGCUUC GAAGCCAGCAGCAUAGGAC 2513 2514
1733-1751 UCCUAUGCUGCUGGCUUCA UGAAGCCAGCAGCAUAGGA 2515 2516
1734-1752 CCUAUGCUGCUGGCUUCAU AUGAAGCCAGCAGCAUAGG 2517 2518
1735-1753 CUAUGCUGCUGGCUUCAUC GAUGAAGCCAGCAGCAUAG 2519 2520
1736-1754 UAUGCUGCUGGCUUCAUCU AGAUGAAGCCAGCAGCAUA 2521 2522
1737-1755 AUGCUGCUGGCUUCAUCUU AAGAUGAAGCCAGCAGCAU 2523 2524
1738-1756 UGCUGCUGGCUUCAUCUUC GAAGAUGAAGCCAGCAGCA 2525 2526
1739-1757 GCUGCUGGCUUCAUCUUCA UGAAGAUGAAGCCAGCAGC 2527 2528
1740-1758 CUGCUGGCUUCAUCUUCAC GUGAAGAUGAAGCCAGCAG 2529 2530
1741-1759 UGCUGGCUUCAUCUUCACC GGUGAAGAUGAAGCCAGCA 2531 2532
1742-1760 GCUGGCUUCAUCUUCACCA UGGUGAAGAUGAAGCCAGC 2533 2534
1743-1761 CUGGCUUCAUCUUCACCAC GUGGUGAAGAUGAAGCCAG 2535 2536
1744-1762 UGGCUUCAUCUUCACCACC GGUGGUGAAGAUGAAGCCA 2537 2538
1745-1763 GGCUUCAUCUUCACCACCU AGGUGGUGAAGAUGAAGCC 2539 2540
1746-1764 GCUUCAUCUUCACCACCUC GAGGUGGUGAAGAUGAAGC 2541 2542
1747-1765 CUUCAUCUUCACCACCUCU AGAGGUGGUGAAGAUGAAG 2543 2544
1748-1766 UUCAUCUUCACCACCUCUC GAGAGGUGGUGAAGAUGAA 2545 2546
1749-1767 UCAUCUUCACCACCUCUCU AGAGAGGUGGUGAAGAUGA 2547 2548
1750-1768 CAUCUUCACCACCUCUCUG CAGAGAGGUGGUGAAGAUG 2549 2550
1751-1769 AUCUUCACCACCUCUCUGC GCAGAGAGGUGGUGAAGAU
2551 2552 1752-1770 UCUUCACCACCUCUCUGCC GGCAGAGAGGUGGUGAAGA 2553
2554 1753-1771 CUUCACCACCUCUCUGCCA UGGCAGAGAGGUGGUGAAG 2555 2556
1754-1772 UUCACCACCUCUCUGCCAC GUGGCAGAGAGGUGGUGAA 2557 2558
1755-1773 UCACCACCUCUCUGCCACC GGUGGCAGAGAGGUGGUGA 2559 2560
1756-1774 CACCACCUCUCUGCCACCC GGGUGGCAGAGAGGUGGUG 2561 2562
1757-1775 ACCACCUCUCUGCCACCCA UGGGUGGCAGAGAGGUGGU 2563 2564
1758-1776 CCACCUCUCUGCCACCCAU AUGGGUGGCAGAGAGGUGG 2565 2566
1759-1777 CACCUCUCUGCCACCCAUG CAUGGGUGGCAGAGAGGUG 2567 2568
1760-1778 ACCUCUCUGCCACCCAUGC GCAUGGGUGGCAGAGAGGU 2569 2570
1761-1779 CCUCUCUGCCACCCAUGCU AGCAUGGGUGGCAGAGAGG 2571 2572
1762-1780 CUCUCUGCCACCCAUGCUG CAGCAUGGGUGGCAGAGAG 2573 2574
1763-1781 UCUCUGCCACCCAUGCUGC GCAGCAUGGGUGGCAGAGA 2575 2576
1764-1782 CUCUGCCACCCAUGCUGCU AGCAGCAUGGGUGGCAGAG 2577 2578
1765-1783 UCUGCCACCCAUGCUGCUG CAGCAGCAUGGGUGGCAGA 2579 2580
1766-1784 CUGCCACCCAUGCUGCUGG CCAGCAGCAUGGGUGGCAG 2581 2582
1767-1785 UGCCACCCAUGCUGCUGGC GCCAGCAGCAUGGGUGGCA 2583 2584
1768-1786 GCCACCCAUGCUGCUGGCU AGCCAGCAGCAUGGGUGGC 2585 2586
1769-1787 CCACCCAUGCUGCUGGCUG CAGCCAGCAGCAUGGGUGG 2587 2588
1770-1788 CACCCAUGCUGCUGGCUGG CCAGCCAGCAGCAUGGGUG 2589 2590
1771-1789 ACCCAUGCUGCUGGCUGGA UCCAGCCAGCAGCAUGGGU 2591 2592
1772-1790 CCCAUGCUGCUGGCUGGAG CUCCAGCCAGCAGCAUGGG 2593 2594
1773-1791 CCAUGCUGCUGGCUGGAGC GCUCCAGCCAGCAGCAUGG 2595 2596
1774-1792 CAUGCUGCUGGCUGGAGCC GGCUCCAGCCAGCAGCAUG 2597 2598
1775-1793 AUGCUGCUGGCUGGAGCCC GGGCUCCAGCCAGCAGCAU 2599 2600
1776-1794 UGCUGCUGGCUGGAGCCCU AGGGCUCCAGCCAGCAGCA 2601 2602
1777-1795 GCUGCUGGCUGGAGCCCUG CAGGGCUCCAGCCAGCAGC 2603 2604
1778-1796 CUGCUGGCUGGAGCCCUGG CCAGGGCUCCAGCCAGCAG 2605 2606
1779-1797 UGCUGGCUGGAGCCCUGGA UCCAGGGCUCCAGCCAGCA 2607 2608
1780-1798 GCUGGCUGGAGCCCUGGAG CUCCAGGGCUCCAGCCAGC 2609 2610
1781-1799 CUGGCUGGAGCCCUGGAGU ACUCCAGGGCUCCAGCCAG 2611 2612
1782-1800 UGGCUGGAGCCCUGGAGUC GACUCCAGGGCUCCAGCCA 2613 2614
1783-1801 GGCUGGAGCCCUGGAGUCU AGACUCCAGGGCUCCAGCC 2615 2616
1784-1802 GCUGGAGCCCUGGAGUCUG CAGACUCCAGGGCUCCAGC 2617 2618
1785-1803 CUGGAGCCCUGGAGUCUGU ACAGACUCCAGGGCUCCAG 2619 2620
1786-1804 UGGAGCCCUGGAGUCUGUG CACAGACUCCAGGGCUCCA 2621 2622
1787-1805 GGAGCCCUGGAGUCUGUGC GCACAGACUCCAGGGCUCC 2623 2624
1788-1806 GAGCCCUGGAGUCUGUGCG CGCACAGACUCCAGGGCUC 2625 2626
1789-1807 AGCCCUGGAGUCUGUGCGG CCGCACAGACUCCAGGGCU 2627 2628
1790-1808 GCCCUGGAGUCUGUGCGGA UCCGCACAGACUCCAGGGC 2629 2630
1792-1810 CCUGGAGUCUGUGCGGAUC GAUCCGCACAGACUCCAGG 2631 2632
1793-1811 CUGGAGUCUGUGCGGAUCC GGAUCCGCACAGACUCCAG 2633 2634
1795-1813 GGAGUCUGUGCGGAUCCUG CAGGAUCCGCACAGACUCC 2635 2636
1796-1814 GAGUCUGUGCGGAUCCUGA UCAGGAUCCGCACAGACUC 2637 2638
1797-1815 AGUCUGUGCGGAUCCUGAA UUCAGGAUCCGCACAGACU 2639 2640
1798-1816 GUCUGUGCGGAUCCUGAAG CUUCAGGAUCCGCACAGAC 2641 2642
1799-1817 UCUGUGCGGAUCCUGAAGA UCUUCAGGAUCCGCACAGA 2643 2644
1800-1818 CUGUGCGGAUCCUGAAGAG CUCUUCAGGAUCCGCACAG 2645 2646
1801-1819 UGUGCGGAUCCUGAAGAGC GCUCUUCAGGAUCCGCACA 2647 2648
1802-1820 GUGCGGAUCCUGAAGAGCG CGCUCUUCAGGAUCCGCAC 2649 2650
1803-1821 UGCGGAUCCUGAAGAGCGC GCGCUCUUCAGGAUCCGCA 2651 2652
1804-1822 GCGGAUCCUGAAGAGCGCU AGCGCUCUUCAGGAUCCGC 2653 2654
1805-1823 CGGAUCCUGAAGAGCGCUG CAGCGCUCUUCAGGAUCCG 2655 2656
1806-1824 GGAUCCUGAAGAGCGCUGA UCAGCGCUCUUCAGGAUCC 2657 2658
1807-1825 GAUCCUGAAGAGCGCUGAG CUCAGCGCUCUUCAGGAUC 2659 2660
1808-1826 AUCCUGAAGAGCGCUGAGG CCUCAGCGCUCUUCAGGAU 2661 2662
1809-1827 UCCUGAAGAGCGCUGAGGG CCCUCAGCGCUCUUCAGGA 2663 2664
1810-1828 CCUGAAGAGCGCUGAGGGA UCCCUCAGCGCUCUUCAGG 2665 2666
1811-1829 CUGAAGAGCGCUGAGGGAC GUCCCUCAGCGCUCUUCAG 2667 2668
1812-1830 UGAAGAGCGCUGAGGGACG CGUCCCUCAGCGCUCUUCA 2669 2670
1813-1831 GAAGAGCGCUGAGGGACGG CCGUCCCUCAGCGCUCUUC 2671 2672
1814-1832 AAGAGCGCUGAGGGACGGG CCCGUCCCUCAGCGCUCUU 2673 2674
1815-1833 AGAGCGCUGAGGGACGGGU ACCCGUCCCUCAGCGCUCU 2675 2676
1816-1834 GAGCGCUGAGGGACGGGUG CACCCGUCCCUCAGCGCUC 2677 2678
1817-1835 AGCGCUGAGGGACGGGUGC GCACCCGUCCCUCAGCGCU 2679 2680
1818-1836 GCGCUGAGGGACGGGUGCU AGCACCCGUCCCUCAGCGC 2681 2682
1819-1837 CGCUGAGGGACGGGUGCUU AAGCACCCGUCCCUCAGCG 2683 2684
1820-1838 GCUGAGGGACGGGUGCUUC GAAGCACCCGUCCCUCAGC 2685 2686
1821-1839 CUGAGGGACGGGUGCUUCG CGAAGCACCCGUCCCUCAG 2687 2688
1822-1840 UGAGGGACGGGUGCUUCGC GCGAAGCACCCGUCCCUCA 2689 2690
1823-1841 GAGGGACGGGUGCUUCGCC GGCGAAGCACCCGUCCCUC 2691 2692
1824-1842 AGGGACGGGUGCUUCGCCG CGGCGAAGCACCCGUCCCU 2693 2694
1825-1843 GGGACGGGUGCUUCGCCGC GCGGCGAAGCACCCGUCCC 2695 2696
1826-1844 GGACGGGUGCUUCGCCGCC GGCGGCGAAGCACCCGUCC 2697 2698
1827-1845 GACGGGUGCUUCGCCGCCA UGGCGGCGAAGCACCCGUC 2699 2700
1828-1846 ACGGGUGCUUCGCCGCCAG CUGGCGGCGAAGCACCCGU 2701 2702
1829-1847 CGGGUGCUUCGCCGCCAGC GCUGGCGGCGAAGCACCCG 2703 2704
1830-1848 GGGUGCUUCGCCGCCAGCA UGCUGGCGGCGAAGCACCC 2705 2706
1831-1849 GGUGCUUCGCCGCCAGCAC GUGCUGGCGGCGAAGCACC 2707 2708
1832-1850 GUGCUUCGCCGCCAGCACC GGUGCUGGCGGCGAAGCAC 2709 2710
1833-1851 UGCUUCGCCGCCAGCACCA UGGUGCUGGCGGCGAAGCA 2711 2712
1834-1852 GCUUCGCCGCCAGCACCAG CUGGUGCUGGCGGCGAAGC 2713 2714
1835-1853 CUUCGCCGCCAGCACCAGC GCUGGUGCUGGCGGCGAAG 2715 2716
1836-1854 UUCGCCGCCAGCACCAGCG CGCUGGUGCUGGCGGCGAA 2717 2718
1837-1855 UCGCCGCCAGCACCAGCGC GCGCUGGUGCUGGCGGCGA 2719 2720
1838-1856 CGCCGCCAGCACCAGCGCA UGCGCUGGUGCUGGCGGCG 2721 2722
1839-1857 GCCGCCAGCACCAGCGCAA UUGCGCUGGUGCUGGCGGC 2723 2724
1840-1858 CCGCCAGCACCAGCGCAAC GUUGCGCUGGUGCUGGCGG 2725 2726
1841-1859 CGCCAGCACCAGCGCAACG CGUUGCGCUGGUGCUGGCG 2727 2728
1842-1860 GCCAGCACCAGCGCAACGU ACGUUGCGCUGGUGCUGGC 2729 2730
1865-1883 CUCAUGAGACAGAUGCUAA UUAGCAUCUGUCUCAUGAG 2731 2732
1866-1884 UCAUGAGACAGAUGCUAAU AUUAGCAUCUGUCUCAUGA 2733 2734
1867-1885 CAUGAGACAGAUGCUAAUG CAUUAGCAUCUGUCUCAUG 2735 2736
1868-1886 AUGAGACAGAUGCUAAUGG CCAUUAGCAUCUGUCUCAU 2737 2738
1869-1887 UGAGACAGAUGCUAAUGGA UCCAUUAGCAUCUGUCUCA 2739 2740
1871-1889 AGACAGAUGCUAAUGGAUG CAUCCAUUAGCAUCUGUCU 2741 2742
1872-1890 GACAGAUGCUAAUGGAUGC GCAUCCAUUAGCAUCUGUC 2743 2744
1873-1891 ACAGAUGCUAAUGGAUGCC GGCAUCCAUUAGCAUCUGU 2745 2746
1874-1892 CAGAUGCUAAUGGAUGCCG CGGCAUCCAUUAGCAUCUG 2747 2748
1875-1893 AGAUGCUAAUGGAUGCCGG CCGGCAUCCAUUAGCAUCU 2749 2750
1876-1894 GAUGCUAAUGGAUGCCGGC GCCGGCAUCCAUUAGCAUC 2751 2752
1877-1895 AUGCUAAUGGAUGCCGGCC GGCCGGCAUCCAUUAGCAU 2753 2754
1878-1896 UGCUAAUGGAUGCCGGCCU AGGCCGGCAUCCAUUAGCA 2755 2756
1879-1897 GCUAAUGGAUGCCGGCCUC GAGGCCGGCAUCCAUUAGC 2757 2758
1880-1898 CUAAUGGAUGCCGGCCUCC GGAGGCCGGCAUCCAUUAG 2759 2760
1881-1899 UAAUGGAUGCCGGCCUCCC GGGAGGCCGGCAUCCAUUA 2761 2762
1882-1900 AAUGGAUGCCGGCCUCCCU AGGGAGGCCGGCAUCCAUU 2763 2764
1883-1901 AUGGAUGCCGGCCUCCCUG CAGGGAGGCCGGCAUCCAU 2765 2766
1884-1902 UGGAUGCCGGCCUCCCUGU ACAGGGAGGCCGGCAUCCA 2767 2768
1885-1903 GGAUGCCGGCCUCCCUGUU AACAGGGAGGCCGGCAUCC 2769 2770
1886-1904 GAUGCCGGCCUCCCUGUUG CAACAGGGAGGCCGGCAUC 2771 2772
1887-1905 AUGCCGGCCUCCCUGUUGU ACAACAGGGAGGCCGGCAU 2773 2774
1888-1906 UGCCGGCCUCCCUGUUGUC GACAACAGGGAGGCCGGCA 2775 2776
1889-1907 GCCGGCCUCCCUGUUGUCC GGACAACAGGGAGGCCGGC 2777 2778
1890-1908 CCGGCCUCCCUGUUGUCCA UGGACAACAGGGAGGCCGG 2779 2780
1891-1909 CGGCCUCCCUGUUGUCCAC GUGGACAACAGGGAGGCCG 2781 2782
1892-1910 GGCCUCCCUGUUGUCCACU AGUGGACAACAGGGAGGCC 2783 2784
1893-1911 GCCUCCCUGUUGUCCACUG CAGUGGACAACAGGGAGGC 2785 2786
1894-1912 CCUCCCUGUUGUCCACUGC GCAGUGGACAACAGGGAGG 2787 2788
1895-1913 CUCCCUGUUGUCCACUGCC GGCAGUGGACAACAGGGAG 2789 2790
1896-1914 UCCCUGUUGUCCACUGCCC GGGCAGUGGACAACAGGGA 2791 2792
1897-1915 CCCUGUUGUCCACUGCCCC GGGGCAGUGGACAACAGGG 2793 2794
1898-1916 CCUGUUGUCCACUGCCCCA UGGGGCAGUGGACAACAGG 2795 2796
1899-1917 CUGUUGUCCACUGCCCCAG CUGGGGCAGUGGACAACAG 2797 2798
1900-1918 UGUUGUCCACUGCCCCAGC GCUGGGGCAGUGGACAACA 2799 2800
1901-1919 GUUGUCCACUGCCCCAGCC GGCUGGGGCAGUGGACAAC 2801 2802
1902-1920 UUGUCCACUGCCCCAGCCA UGGCUGGGGCAGUGGACAA
2803 2804 1903-1921 UGUCCACUGCCCCAGCCAC GUGGCUGGGGCAGUGGACA 2805
2806 1904-1922 GUCCACUGCCCCAGCCACA UGUGGCUGGGGCAGUGGAC 2807 2808
1905-1923 UCCACUGCCCCAGCCACAU AUGUGGCUGGGGCAGUGGA 2809 2810
1906-1924 CCACUGCCCCAGCCACAUC GAUGUGGCUGGGGCAGUGG 2811 2812
1907-1925 CACUGCCCCAGCCACAUCA UGAUGUGGCUGGGGCAGUG 2813 2814
1908-1926 ACUGCCCCAGCCACAUCAU AUGAUGUGGCUGGGGCAGU 2815 2816
1909-1927 CUGCCCCAGCCACAUCAUC GAUGAUGUGGCUGGGGCAG 2817 2818
1910-1928 UGCCCCAGCCACAUCAUCC GGAUGAUGUGGCUGGGGCA 2819 2820
1911-1929 GCCCCAGCCACAUCAUCCC GGGAUGAUGUGGCUGGGGC 2821 2822
1912-1930 CCCCAGCCACAUCAUCCCU AGGGAUGAUGUGGCUGGGG 2823 2824
1913-1931 CCCAGCCACAUCAUCCCUG CAGGGAUGAUGUGGCUGGG 2825 2826
1914-1932 CCAGCCACAUCAUCCCUGU ACAGGGAUGAUGUGGCUGG 2827 2828
1915-1933 CAGCCACAUCAUCCCUGUG CACAGGGAUGAUGUGGCUG 2829 2830
1916-1934 AGCCACAUCAUCCCUGUGC GCACAGGGAUGAUGUGGCU 2831 2832
1917-1935 GCCACAUCAUCCCUGUGCG CGCACAGGGAUGAUGUGGC 2833 2834
1918-1936 CCACAUCAUCCCUGUGCGG CCGCACAGGGAUGAUGUGG 2835 2836
1919-1937 CACAUCAUCCCUGUGCGGG CCCGCACAGGGAUGAUGUG 2837 2838
1920-1938 ACAUCAUCCCUGUGCGGGU ACCCGCACAGGGAUGAUGU 2839 2840
1922-1940 AUCAUCCCUGUGCGGGUUG CAACCCGCACAGGGAUGAU 2841 2842
1923-1941 UCAUCCCUGUGCGGGUUGC GCAACCCGCACAGGGAUGA 2843 2844
1924-1942 CAUCCCUGUGCGGGUUGCA UGCAACCCGCACAGGGAUG 2845 2846
1925-1943 AUCCCUGUGCGGGUUGCAG CUGCAACCCGCACAGGGAU 2847 2848
1926-1944 UCCCUGUGCGGGUUGCAGA UCUGCAACCCGCACAGGGA 2849 2850
1928-1946 CCUGUGCGGGUUGCAGAUG CAUCUGCAACCCGCACAGG 2851 2852
1929-1947 CUGUGCGGGUUGCAGAUGC GCAUCUGCAACCCGCACAG 2853 2854
1930-1948 UGUGCGGGUUGCAGAUGCU AGCAUCUGCAACCCGCACA 2855 2856
1931-1949 GUGCGGGUUGCAGAUGCUG CAGCAUCUGCAACCCGCAC 2857 2858
1932-1950 UGCGGGUUGCAGAUGCUGC GCAGCAUCUGCAACCCGCA 2859 2860
1933-1951 GCGGGUUGCAGAUGCUGCU AGCAGCAUCUGCAACCCGC 2861 2862
1934-1952 CGGGUUGCAGAUGCUGCUA UAGCAGCAUCUGCAACCCG 2863 2864
1935-1953 GGGUUGCAGAUGCUGCUAA UUAGCAGCAUCUGCAACCC 2865 2866
1936-1954 GGUUGCAGAUGCUGCUAAA UUUAGCAGCAUCUGCAACC 2867 2868
1937-1955 GUUGCAGAUGCUGCUAAAA UUUUAGCAGCAUCUGCAAC 2869 2870
1938-1956 UUGCAGAUGCUGCUAAAAA UUUUUAGCAGCAUCUGCAA 2871 2872
1939-1957 UGCAGAUGCUGCUAAAAAC GUUUUUAGCAGCAUCUGCA 2873 2874
1940-1958 GCAGAUGCUGCUAAAAACA UGUUUUUAGCAGCAUCUGC 2875 2876
1941-1959 CAGAUGCUGCUAAAAACAC GUGUUUUUAGCAGCAUCUG 2877 2878
1961-1979 GAAGUCUGUGAUGAACUAA UUAGUUCAUCACAGACUUC 2879 2880
1963-1981 AGUCUGUGAUGAACUAAUG CAUUAGUUCAUCACAGACU 2881 2882
1965-1983 UCUGUGAUGAACUAAUGAG CUCAUUAGUUCAUCACAGA 2883 2884
1966-1984 CUGUGAUGAACUAAUGAGC GCUCAUUAGUUCAUCACAG 2885 2886
1968-1986 GUGAUGAACUAAUGAGCAG CUGCUCAUUAGUUCAUCAC 2887 2888
1969-1987 UGAUGAACUAAUGAGCAGA UCUGCUCAUUAGUUCAUCA 2889 2890
1970-1988 GAUGAACUAAUGAGCAGAC GUCUGCUCAUUAGUUCAUC 2891 2892
1971-1989 AUGAACUAAUGAGCAGACA UGUCUGCUCAUUAGUUCAU 2893 2894
1972-1990 UGAACUAAUGAGCAGACAU AUGUCUGCUCAUUAGUUCA 2895 2896
1973-1991 GAACUAAUGAGCAGACAUA UAUGUCUGCUCAUUAGUUC 2897 2898
1974-1992 AACUAAUGAGCAGACAUAA UUAUGUCUGCUCAUUAGUU 2899 2900
1975-1993 ACUAAUGAGCAGACAUAAC GUUAUGUCUGCUCAUUAGU 2901 2902
1978-1996 AAUGAGCAGACAUAACAUC GAUGUUAUGUCUGCUCAUU 2903 2904
1979-1997 AUGAGCAGACAUAACAUCU AGAUGUUAUGUCUGCUCAU 2905 2906
1980-1998 UGAGCAGACAUAACAUCUA UAGAUGUUAUGUCUGCUCA 2907 2908
2000-2018 GUGCAAGCAAUCAAUUACC GGUAAUUGAUUGCUUGCAC 2909 2910
2001-2019 UGCAAGCAAUCAAUUACCC GGGUAAUUGAUUGCUUGCA 2911 2912
2002-2020 GCAAGCAAUCAAUUACCCU AGGGUAAUUGAUUGCUUGC 2913 2914
2004-2022 AAGCAAUCAAUUACCCUAC GUAGGGUAAUUGAUUGCUU 2915 2916
2024-2042 GUGCCCCGGGGAGAAGAGC GCUCUUCUCCCCGGGGCAC 2917 2918
2025-2043 UGCCCCGGGGAGAAGAGCU AGCUCUUCUCCCCGGGGCA 2919 2920
2026-2044 GCCCCGGGGAGAAGAGCUC GAGCUCUUCUCCCCGGGGC 2921 2922
2027-2045 CCCCGGGGAGAAGAGCUCC GGAGCUCUUCUCCCCGGGG 2923 2924
2028-2046 CCCGGGGAGAAGAGCUCCU AGGAGCUCUUCUCCCCGGG 2925 2926
2029-2047 CCGGGGAGAAGAGCUCCUA UAGGAGCUCUUCUCCCCGG 2927 2928
2030-2048 CGGGGAGAAGAGCUCCUAC GUAGGAGCUCUUCUCCCCG 2929 2930
2031-2049 GGGGAGAAGAGCUCCUACG CGUAGGAGCUCUUCUCCCC 2931 2932
2032-2050 GGGAGAAGAGCUCCUACGG CCGUAGGAGCUCUUCUCCC 2933 2934
2033-2051 GGAGAAGAGCUCCUACGGA UCCGUAGGAGCUCUUCUCC 2935 2936
2034-2052 GAGAAGAGCUCCUACGGAU AUCCGUAGGAGCUCUUCUC 2937 2938
2060-2078 ACCCCUCACCACACACCCC GGGGUGUGUGGUGAGGGGU 2939 2940
2061-2079 CCCCUCACCACACACCCCA UGGGGUGUGUGGUGAGGGG 2941 2942
2062-2080 CCCUCACCACACACCCCAG CUGGGGUGUGUGGUGAGGG 2943 2944
2063-2081 CCUCACCACACACCCCAGA UCUGGGGUGUGUGGUGAGG 2945 2946
2064-2082 CUCACCACACACCCCAGAU AUCUGGGGUGUGUGGUGAG 2947 2948
2065-2083 UCACCACACACCCCAGAUG CAUCUGGGGUGUGUGGUGA 2949 2950
2066-2084 CACCACACACCCCAGAUGA UCAUCUGGGGUGUGUGGUG 2951 2952
2067-2085 ACCACACACCCCAGAUGAU AUCAUCUGGGGUGUGUGGU 2953 2954
2068-2086 CCACACACCCCAGAUGAUG CAUCAUCUGGGGUGUGUGG 2955 2956
2069-2087 CACACACCCCAGAUGAUGA UCAUCAUCUGGGGUGUGUG 2957 2958
2070-2088 ACACACCCCAGAUGAUGAA UUCAUCAUCUGGGGUGUGU 2959 2960
2071-2089 CACACCCCAGAUGAUGAAC GUUCAUCAUCUGGGGUGUG 2961 2962
2072-2090 ACACCCCAGAUGAUGAACU AGUUCAUCAUCUGGGGUGU 2963 2964
2073-2091 CACCCCAGAUGAUGAACUA UAGUUCAUCAUCUGGGGUG 2965 2966
2074-2092 ACCCCAGAUGAUGAACUAC GUAGUUCAUCAUCUGGGGU 2967 2968
2076-2094 CCCAGAUGAUGAACUACUU AAGUAGUUCAUCAUCUGGG 2969 2970
2077-2095 CCAGAUGAUGAACUACUUC GAAGUAGUUCAUCAUCUGG 2971 2972
2078-2096 CAGAUGAUGAACUACUUCC GGAAGUAGUUCAUCAUCUG 2973 2974
2079-2097 AGAUGAUGAACUACUUCCU AGGAAGUAGUUCAUCAUCU 2975 2976
2080-2098 GAUGAUGAACUACUUCCUU AAGGAAGUAGUUCAUCAUC 2977 2978
2081-2099 AUGAUGAACUACUUCCUUG CAAGGAAGUAGUUCAUCAU 2979 2980
2082-2100 UGAUGAACUACUUCCUUGA UCAAGGAAGUAGUUCAUCA 2981 2982
2083-2101 GAUGAACUACUUCCUUGAG CUCAAGGAAGUAGUUCAUC 2983 2984
2084-2102 AUGAACUACUUCCUUGAGA UCUCAAGGAAGUAGUUCAU 2985 2986
2085-2103 UGAACUACUUCCUUGAGAA UUCUCAAGGAAGUAGUUCA 2987 2988
2086-2104 GAACUACUUCCUUGAGAAU AUUCUCAAGGAAGUAGUUC 2989 2990
2087-2105 AACUACUUCCUUGAGAAUC GAUUCUCAAGGAAGUAGUU 2991 2992
2088-2106 ACUACUUCCUUGAGAAUCU AGAUUCUCAAGGAAGUAGU 2993 2994
2089-2107 CUACUUCCUUGAGAAUCUG CAGAUUCUCAAGGAAGUAG 2995 2996
2090-2108 UACUUCCUUGAGAAUCUGC GCAGAUUCUCAAGGAAGUA 2997 2998
2091-2109 ACUUCCUUGAGAAUCUGCU AGCAGAUUCUCAAGGAAGU 2999 3000
2117-2135 UGGAAGCAAGUGGGGCUGG CCAGCCCCACUUGCUUCCA 3001 3002
2118-2136 GGAAGCAAGUGGGGCUGGA UCCAGCCCCACUUGCUUCC 3003 3004
2119-2137 GAAGCAAGUGGGGCUGGAA UUCCAGCCCCACUUGCUUC 3005 3006
2120-2138 AAGCAAGUGGGGCUGGAAC GUUCCAGCCCCACUUGCUU 3007 3008
2121-2139 AGCAAGUGGGGCUGGAACU AGUUCCAGCCCCACUUGCU 3009 3010
2122-2140 GCAAGUGGGGCUGGAACUG CAGUUCCAGCCCCACUUGC 3011 3012
2123-2141 CAAGUGGGGCUGGAACUGA UCAGUUCCAGCCCCACUUG 3013 3014
2124-2142 AAGUGGGGCUGGAACUGAA UUCAGUUCCAGCCCCACUU 3015 3016
2125-2143 AGUGGGGCUGGAACUGAAG CUUCAGUUCCAGCCCCACU 3017 3018
2126-2144 GUGGGGCUGGAACUGAAGC GCUUCAGUUCCAGCCCCAC 3019 3020
2127-2145 UGGGGCUGGAACUGAAGCC GGCUUCAGUUCCAGCCCCA 3021 3022
2147-2165 CAUUCCUCAGCUGAGUGCA UGCACUCAGCUGAGGAAUG 3023 3024
2148-2166 AUUCCUCAGCUGAGUGCAA UUGCACUCAGCUGAGGAAU 3025 3026
2149-2167 UUCCUCAGCUGAGUGCAAC GUUGCACUCAGCUGAGGAA 3027 3028
2150-2168 UCCUCAGCUGAGUGCAACU AGUUGCACUCAGCUGAGGA 3029 3030
2151-2169 CCUCAGCUGAGUGCAACUU AAGUUGCACUCAGCUGAGG 3031 3032
2152-2170 CUCAGCUGAGUGCAACUUC GAAGUUGCACUCAGCUGAG 3033 3034
2153-2171 UCAGCUGAGUGCAACUUCU AGAAGUUGCACUCAGCUGA 3035 3036
2154-2172 CAGCUGAGUGCAACUUCUG CAGAAGUUGCACUCAGCUG 3037 3038
2155-2173 AGCUGAGUGCAACUUCUGC GCAGAAGUUGCACUCAGCU 3039 3040
2156-2174 GCUGAGUGCAACUUCUGCA UGCAGAAGUUGCACUCAGC 3041 3042
2157-2175 CUGAGUGCAACUUCUGCAG CUGCAGAAGUUGCACUCAG 3043 3044
2158-2176 UGAGUGCAACUUCUGCAGG CCUGCAGAAGUUGCACUCA 3045 3046
2159-2177 GAGUGCAACUUCUGCAGGA UCCUGCAGAAGUUGCACUC 3047 3048
2160-2178 AGUGCAACUUCUGCAGGAG CUCCUGCAGAAGUUGCACU 3049 3050
2161-2179 GUGCAACUUCUGCAGGAGG CCUCCUGCAGAAGUUGCAC 3051 3052
2162-2180 UGCAACUUCUGCAGGAGGC GCCUCCUGCAGAAGUUGCA
3053 3054 2163-2181 GCAACUUCUGCAGGAGGCC GGCCUCCUGCAGAAGUUGC 3055
3056 2164-2182 CAACUUCUGCAGGAGGCCA UGGCCUCCUGCAGAAGUUG 3057 3058
2165-2183 AACUUCUGCAGGAGGCCAC GUGGCCUCCUGCAGAAGUU 3059 3060
2166-2184 ACUUCUGCAGGAGGCCACU AGUGGCCUCCUGCAGAAGU 3061 3062
2167-2185 CUUCUGCAGGAGGCCACUG CAGUGGCCUCCUGCAGAAG 3063 3064
2168-2186 UUCUGCAGGAGGCCACUGC GCAGUGGCCUCCUGCAGAA 3065 3066
2169-2187 UCUGCAGGAGGCCACUGCA UGCAGUGGCCUCCUGCAGA 3067 3068
2170-2188 CUGCAGGAGGCCACUGCAU AUGCAGUGGCCUCCUGCAG 3069 3070
2171-2189 UGCAGGAGGCCACUGCAUU AAUGCAGUGGCCUCCUGCA 3071 3072
2172-2190 GCAGGAGGCCACUGCAUUU AAAUGCAGUGGCCUCCUGC 3073 3074
2173-2191 CAGGAGGCCACUGCAUUUU AAAAUGCAGUGGCCUCCUG 3075 3076
2174-2192 AGGAGGCCACUGCAUUUUG CAAAAUGCAGUGGCCUCCU 3077 3078
2175-2193 GGAGGCCACUGCAUUUUGA UCAAAAUGCAGUGGCCUCC 3079 3080
2176-2194 GAGGCCACUGCAUUUUGAA UUCAAAAUGCAGUGGCCUC 3081 3082
2177-2195 AGGCCACUGCAUUUUGAAG CUUCAAAAUGCAGUGGCCU 3083 3084
2178-2196 GGCCACUGCAUUUUGAAGU ACUUCAAAAUGCAGUGGCC 3085 3086
2179-2197 GCCACUGCAUUUUGAAGUG CACUUCAAAAUGCAGUGGC 3087 3088
2180-2198 CCACUGCAUUUUGAAGUGA UCACUUCAAAAUGCAGUGG 3089 3090
2181-2199 CACUGCAUUUUGAAGUGAU AUCACUUCAAAAUGCAGUG 3091 3092
2182-2200 ACUGCAUUUUGAAGUGAUG CAUCACUUCAAAAUGCAGU 3093 3094
2183-2201 CUGCAUUUUGAAGUGAUGA UCAUCACUUCAAAAUGCAG 3095 3096
2184-2202 UGCAUUUUGAAGUGAUGAG CUCAUCACUUCAAAAUGCA 3097 3098
2185-2203 GCAUUUUGAAGUGAUGAGU ACUCAUCACUUCAAAAUGC 3099 3100
2186-2204 CAUUUUGAAGUGAUGAGUG CACUCAUCACUUCAAAAUG 3101 3102
2187-2205 AUUUUGAAGUGAUGAGUGA UCACUCAUCACUUCAAAAU 3103 3104
2188-2206 UUUUGAAGUGAUGAGUGAA UUCACUCAUCACUUCAAAA 3105 3106
2190-2208 UUGAAGUGAUGAGUGAAAG CUUUCACUCAUCACUUCAA 3107 3108
2191-2209 UGAAGUGAUGAGUGAAAGA UCUUUCACUCAUCACUUCA 3109 3110
2192-2210 GAAGUGAUGAGUGAAAGAG CUCUUUCACUCAUCACUUC 3111 3112
2193-2211 AAGUGAUGAGUGAAAGAGA UCUCUUUCACUCAUCACUU 3113 3114
2194-2212 AGUGAUGAGUGAAAGAGAG CUCUCUUUCACUCAUCACU 3115 3116
2195-2213 GUGAUGAGUGAAAGAGAGA UCUCUCUUUCACUCAUCAC 3117 3118
2196-2214 UGAUGAGUGAAAGAGAGAA UUCUCUCUUUCACUCAUCA 3119 3120
2197-2215 GAUGAGUGAAAGAGAGAAG CUUCUCUCUUUCACUCAUC 3121 3122
2198-2216 AUGAGUGAAAGAGAGAAGU ACUUCUCUCUUUCACUCAU 3123 3124
2199-2217 UGAGUGAAAGAGAGAAGUC GACUUCUCUCUUUCACUCA 3125 3126
2200-2218 GAGUGAAAGAGAGAAGUCC GGACUUCUCUCUUUCACUC 3127 3128
2201-2219 AGUGAAAGAGAGAAGUCCU AGGACUUCUCUCUUUCACU 3129 3130
2202-2220 GUGAAAGAGAGAAGUCCUA UAGGACUUCUCUCUUUCAC 3131 3132
2203-2221 UGAAAGAGAGAAGUCCUAU AUAGGACUUCUCUCUUUCA 3133 3134
2204-2222 GAAAGAGAGAAGUCCUAUU AAUAGGACUUCUCUCUUUC 3135 3136
2205-2223 AAAGAGAGAAGUCCUAUUU AAAUAGGACUUCUCUCUUU 3137 3138
2206-2224 AAGAGAGAAGUCCUAUUUC GAAAUAGGACUUCUCUCUU 3139 3140
2207-2225 AGAGAGAAGUCCUAUUUCU AGAAAUAGGACUUCUCUCU 3141 3142
2208-2226 GAGAGAAGUCCUAUUUCUC GAGAAAUAGGACUUCUCUC 3143 3144
2209-2227 AGAGAAGUCCUAUUUCUCA UGAGAAAUAGGACUUCUCU 3145 3146
2210-2228 GAGAAGUCCUAUUUCUCAG CUGAGAAAUAGGACUUCUC 3147 3148
2211-2229 AGAAGUCCUAUUUCUCAGG CCUGAGAAAUAGGACUUCU 3149 3150
2212-2230 GAAGUCCUAUUUCUCAGGC GCCUGAGAAAUAGGACUUC 3151 3152
2213-2231 AAGUCCUAUUUCUCAGGCU AGCCUGAGAAAUAGGACUU 3153 3154
2214-2232 AGUCCUAUUUCUCAGGCUU AAGCCUGAGAAAUAGGACU 3155 3156
2215-2233 GUCCUAUUUCUCAGGCUUG CAAGCCUGAGAAAUAGGAC 3157 3158
2216-2234 UCCUAUUUCUCAGGCUUGA UCAAGCCUGAGAAAUAGGA 3159 3160
2217-2235 CCUAUUUCUCAGGCUUGAG CUCAAGCCUGAGAAAUAGG 3161 3162
2218-2236 CUAUUUCUCAGGCUUGAGC GCUCAAGCCUGAGAAAUAG 3163 3164
2219-2237 UAUUUCUCAGGCUUGAGCA UGCUCAAGCCUGAGAAAUA 3165 3166
2220-2238 AUUUCUCAGGCUUGAGCAA UUGCUCAAGCCUGAGAAAU 3167 3168
2221-2239 UUUCUCAGGCUUGAGCAAG CUUGCUCAAGCCUGAGAAA 3169 3170
2222-2240 UUCUCAGGCUUGAGCAAGU ACUUGCUCAAGCCUGAGAA 3171 3172
2223-2241 UCUCAGGCUUGAGCAAGUU AACUUGCUCAAGCCUGAGA 3173 3174
2224-2242 CUCAGGCUUGAGCAAGUUG CAACUUGCUCAAGCCUGAG 3175 3176
2225-2243 UCAGGCUUGAGCAAGUUGG CCAACUUGCUCAAGCCUGA 3177 3178
2226-2244 CAGGCUUGAGCAAGUUGGU ACCAACUUGCUCAAGCCUG 3179 3180
2229-2247 GCUUGAGCAAGUUGGUAUC GAUACCAACUUGCUCAAGC 3181 3182
2231-2249 UUGAGCAAGUUGGUAUCUG CAGAUACCAACUUGCUCAA 3183 3184
2232-2250 UGAGCAAGUUGGUAUCUGC GCAGAUACCAACUUGCUCA 3185 3186
2233-2251 GAGCAAGUUGGUAUCUGCU AGCAGAUACCAACUUGCUC 3187 3188
2234-2252 AGCAAGUUGGUAUCUGCUC GAGCAGAUACCAACUUGCU 3189 3190
2235-2253 GCAAGUUGGUAUCUGCUCA UGAGCAGAUACCAACUUGC 3191 3192
2236-2254 CAAGUUGGUAUCUGCUCAG CUGAGCAGAUACCAACUUG 3193 3194
2237-2255 AAGUUGGUAUCUGCUCAGG CCUGAGCAGAUACCAACUU 3195 3196
2238-2256 AGUUGGUAUCUGCUCAGGC GCCUGAGCAGAUACCAACU 3197 3198
2239-2257 GUUGGUAUCUGCUCAGGCC GGCCUGAGCAGAUACCAAC 3199 3200
2240-2258 UUGGUAUCUGCUCAGGCCU AGGCCUGAGCAGAUACCAA 3201 3202
2241-2259 UGGUAUCUGCUCAGGCCUG CAGGCCUGAGCAGAUACCA 3203 3204
2242-2260 GGUAUCUGCUCAGGCCUGA UCAGGCCUGAGCAGAUACC 3205 3206
2243-2261 GUAUCUGCUCAGGCCUGAG CUCAGGCCUGAGCAGAUAC 3207 3208
2244-2262 UAUCUGCUCAGGCCUGAGC GCUCAGGCCUGAGCAGAUA 3209 3210
2245-2263 AUCUGCUCAGGCCUGAGCA UGCUCAGGCCUGAGCAGAU 3211 3212
2246-2264 UCUGCUCAGGCCUGAGCAU AUGCUCAGGCCUGAGCAGA 3213 3214
2247-2265 CUGCUCAGGCCUGAGCAUG CAUGCUCAGGCCUGAGCAG 3215 3216
2248-2266 UGCUCAGGCCUGAGCAUGA UCAUGCUCAGGCCUGAGCA 3217 3218
2249-2267 GCUCAGGCCUGAGCAUGAC GUCAUGCUCAGGCCUGAGC 3219 3220
2250-2268 CUCAGGCCUGAGCAUGACC GGUCAUGCUCAGGCCUGAG 3221 3222
2251-2269 UCAGGCCUGAGCAUGACCU AGGUCAUGCUCAGGCCUGA 3223 3224
2252-2270 CAGGCCUGAGCAUGACCUC GAGGUCAUGCUCAGGCCUG 3225 3226
2253-2271 AGGCCUGAGCAUGACCUCA UGAGGUCAUGCUCAGGCCU 3227 3228
2279-2297 CACUUAACCCCAGGCCAUU AAUGGCCUGGGGUUAAGUG 3229 3230
2280-2298 ACUUAACCCCAGGCCAUUA UAAUGGCCUGGGGUUAAGU 3231 3232
2281-2299 CUUAACCCCAGGCCAUUAU AUAAUGGCCUGGGGUUAAG 3233 3234
2282-2300 UUAACCCCAGGCCAUUAUC GAUAAUGGCCUGGGGUUAA 3235 3236
2283-2301 UAACCCCAGGCCAUUAUCA UGAUAAUGGCCUGGGGUUA 3237 3238
2284-2302 AACCCCAGGCCAUUAUCAU AUGAUAAUGGCCUGGGGUU 3239 3240
2285-2303 ACCCCAGGCCAUUAUCAUA UAUGAUAAUGGCCUGGGGU 3241 3242
2287-2305 CCCAGGCCAUUAUCAUAUC GAUAUGAUAAUGGCCUGGG 3243 3244
2288-2306 CCAGGCCAUUAUCAUAUCC GGAUAUGAUAAUGGCCUGG 3245 3246
2289-2307 CAGGCCAUUAUCAUAUCCA UGGAUAUGAUAAUGGCCUG 3247 3248
2290-2308 AGGCCAUUAUCAUAUCCAG CUGGAUAUGAUAAUGGCCU 3249 3250
2291-2309 GGCCAUUAUCAUAUCCAGA UCUGGAUAUGAUAAUGGCC 3251 3252
2292-2310 GCCAUUAUCAUAUCCAGAU AUCUGGAUAUGAUAAUGGC 3253 3254
2314-2332 CUUCAGAGUUGUCUUUAUA UAUAAAGACAACUCUGAAG 3255 3256
2315-2333 UUCAGAGUUGUCUUUAUAU AUAUAAAGACAACUCUGAA 3257 3258
2316-2334 UCAGAGUUGUCUUUAUAUG CAUAUAAAGACAACUCUGA 3259 3260
2318-2336 AGAGUUGUCUUUAUAUGUG CACAUAUAAAGACAACUCU 3261 3262
2322-2340 UUGUCUUUAUAUGUGAAUU AAUUCACAUAUAAAGACAA 3263 3264
2323-2341 UGUCUUUAUAUGUGAAUUA UAAUUCACAUAUAAAGACA 3265 3266
2324-2342 GUCUUUAUAUGUGAAUUAA UUAAUUCACAUAUAAAGAC 3267 3268
2325-2343 UCUUUAUAUGUGAAUUAAG CUUAAUUCACAUAUAAAGA 3269 3270
2326-2344 CUUUAUAUGUGAAUUAAGU ACUUAAUUCACAUAUAAAG 3271 3272
2327-2345 UUUAUAUGUGAAUUAAGUU AACUUAAUUCACAUAUAAA 3273 3274
2328-2346 UUAUAUGUGAAUUAAGUUA UAACUUAAUUCACAUAUAA 3275 3276
2329-2347 UAUAUGUGAAUUAAGUUAU AUAACUUAAUUCACAUAUA 3277 3278
2330-2348 AUAUGUGAAUUAAGUUAUA UAUAACUUAAUUCACAUAU 3279 3280
2331-2349 UAUGUGAAUUAAGUUAUAU AUAUAACUUAAUUCACAUA 3281 3282
2332-2350 AUGUGAAUUAAGUUAUAUU AAUAUAACUUAAUUCACAU 3283 3284
2333-2351 UGUGAAUUAAGUUAUAUUA UAAUAUAACUUAAUUCACA 3285 3286
2334-2352 GUGAAUUAAGUUAUAUUAA UUAAUAUAACUUAAUUCAC 3287 3288
2335-2353 UGAAUUAAGUUAUAUUAAA UUUAAUAUAACUUAAUUCA 3289 3290
2336-2354 GAAUUAAGUUAUAUUAAAU AUUUAAUAUAACUUAAUUC 3291 3292
2337-2355 AAUUAAGUUAUAUUAAAUU AAUUUAAUAUAACUUAAUU 3293 3294
2338-2356 AUUAAGUUAUAUUAAAUUU AAAUUUAAUAUAACUUAAU 3295 3296
2339-2357 UUAAGUUAUAUUAAAUUUU AAAAUUUAAUAUAACUUAA 3297 3298
2340-2358 UAAGUUAUAUUAAAUUUUA UAAAAUUUAAUAUAACUUA 3299 3300
2341-2359 AAGUUAUAUUAAAUUUUAA UUAAAAUUUAAUAUAACUU 3301 3302
2342-2360 AGUUAUAUUAAAUUUUAAU AUUAAAAUUUAAUAUAACU 3303 3304
2343-2361 GUUAUAUUAAAUUUUAAUC GAUUAAAAUUUAAUAUAAC
3305 3306 2345-2363 UAUAUUAAAUUUUAAUCUA UAGAUUAAAAUUUAAUAUA 3307
3308 2346-2364 AUAUUAAAUUUUAAUCUAU AUAGAUUAAAAUUUAAUAU 3309 3310
2347-2365 UAUUAAAUUUUAAUCUAUA UAUAGAUUAAAAUUUAAUA 3311 3312
2348-2366 AUUAAAUUUUAAUCUAUAG CUAUAGAUUAAAAUUUAAU 3313 3314
2349-2367 UUAAAUUUUAAUCUAUAGU ACUAUAGAUUAAAAUUUAA 3315 3316
2350-2368 UAAAUUUUAAUCUAUAGUA UACUAUAGAUUAAAAUUUA 3317 3318
2351-2369 AAAUUUUAAUCUAUAGUAA UUACUAUAGAUUAAAAUUU 3319 3320
2354-2372 UUUUAAUCUAUAGUAAAAA UUUUUACUAUAGAUUAAAA 3321 3322
2355-2373 UUUAAUCUAUAGUAAAAAC GUUUUUACUAUAGAUUAAA 3323 3324
2356-2374 UUAAUCUAUAGUAAAAACA UGUUUUUACUAUAGAUUAA 3325 3326
2357-2375 UAAUCUAUAGUAAAAACAU AUGUUUUUACUAUAGAUUA 3327 3328
2358-2376 AAUCUAUAGUAAAAACAUA UAUGUUUUUACUAUAGAUU 3329 3330
2359-2377 AUCUAUAGUAAAAACAUAG CUAUGUUUUUACUAUAGAU 3331 3332
2360-2378 UCUAUAGUAAAAACAUAGU ACUAUGUUUUUACUAUAGA 3333 3334
2361-2379 CUAUAGUAAAAACAUAGUC GACUAUGUUUUUACUAUAG 3335 3336
2362-2380 UAUAGUAAAAACAUAGUCC GGACUAUGUUUUUACUAUA 3337 3338
2363-2381 AUAGUAAAAACAUAGUCCU AGGACUAUGUUUUUACUAU 3339 3340
2364-2382 UAGUAAAAACAUAGUCCUG CAGGACUAUGUUUUUACUA 3341 3342
2365-2383 AGUAAAAACAUAGUCCUGG CCAGGACUAUGUUUUUACU 3343 3344
2366-2384 GUAAAAACAUAGUCCUGGA UCCAGGACUAUGUUUUUAC 3345 3346
2367-2385 UAAAAACAUAGUCCUGGAA UUCCAGGACUAUGUUUUUA 3347 3348
2368-2386 AAAAACAUAGUCCUGGAAA UUUCCAGGACUAUGUUUUU 3349 3350
2369-2387 AAAACAUAGUCCUGGAAAU AUUUCCAGGACUAUGUUUU 3351 3352
2370-2388 AAACAUAGUCCUGGAAAUA UAUUUCCAGGACUAUGUUU 3353 3354
2371-2389 AACAUAGUCCUGGAAAUAA UUAUUUCCAGGACUAUGUU 3355 3356
2372-2390 ACAUAGUCCUGGAAAUAAA UUUAUUUCCAGGACUAUGU 3357 3358
2373-2391 CAUAGUCCUGGAAAUAAAU AUUUAUUUCCAGGACUAUG 3359 3360
2374-2392 AUAGUCCUGGAAAUAAAUU AAUUUAUUUCCAGGACUAU 3361 3362
2375-2393 UAGUCCUGGAAAUAAAUUC GAAUUUAUUUCCAGGACUA 3363 3364
2377-2395 GUCCUGGAAAUAAAUUCUU AAGAAUUUAUUUCCAGGAC 3365 3366
2378-2396 UCCUGGAAAUAAAUUCUUG CAAGAAUUUAUUUCCAGGA
Example 9. Suppression of Porphyrin Precursors Using ALAS1 siRNA in
an Acute Treatment Paradigm
[0730] The AIP mouse model (see Example 5) was used to investigate
whether ALAS1 siRNA would work an an acute treatment paradigm to
lower already elevated levels of ALA and PBG, as would be present,
for example, when a human porphyria patient suffers from an acute
attack. Administration of the AD-53558 LNP11 formulation siRNA at a
1 mg/kg dose 12 hours after the last dose of phenobarbitol rapidly
decreased the levels of both ALA and PBG in mouse plasma, whereas
in Luc control treated animals the levels continued to rise (FIG.
14). These results indicate that ALAS siRNA is effective for
treating an acute attack. The ALAS1 siRNA was effective to lower
and prevent further increases in ALA and PBG levels.
Example 10. siRNAs that Target ALAS1
[0731] Further unmodified and modified siRNA sequences that target
ALAS1 siRNA were designed and produced as described in Example 2.
The in vitro activity of the modified duplexes was tested as
described below.
Methods
Lipid Mediated Transfection
[0732] For Hep3B, PMH, and primary Cynomolgus hepatocytes,
transfection was carried out by adding 14.8 .mu.l of Opti-MEM plus
0.2 .mu.l of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad
Calif. catalog number 13778-150) to 5 .mu.l of each siRNA duplex to
an individual well in a 96-well plate. The mixture was then
incubated at room temperature for 20 minutes. Eighty .mu.l of
complete growth media without antibiotic containing the appropriate
cell number were then added to the siRNA mixture. Cells were
incubated for 24 hours prior to RNA purification.
[0733] Single dose experiments were performed at 1 uM, 500 nM, 20
nM, 10 nM and 0.2 nM final duplex concentration for GalNAc
modified.
[0734] Free Uptake Transfection
[0735] Cryopreserved Primary Cynomolgus Hepatocytes (Celsius In
Vitro Technologies, MO03055-P) were thawed at 37.degree. C. water
bath immediately prior to usage and re-suspended at
0.26.times.10.sup.6 cells/ml in InVitroGRO CP (plating) medium
(Celsius In Vitro Technologies, catalog number Z99029). During
transfections, cells were plated onto a BD BioCoat 96 well collagen
plate (BD, 356407) at 25,000 cells per well and incubated at
37.degree. C. in an atmosphere of 5% CO.sub.2. Free Uptake
experiments were performed by adding 10 .mu.l of siRNA duplexes in
PBS per well into a 96 well (96w) plate. Ninety .mu.l of complete
growth media containing appropriate cell number for the cell type
was then added to the siRNA. Cells were incubated for 24 hours
prior to RNA purification. Single dose experiments were performed
at 1 uM, 500 nM, 20 nM and 10 nM final duplex.
[0736] Total RNA Isolation Using DYNABEADS mRNA Isolation Kit
(Invitrogen, Part #: 610-12)
[0737] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding
Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf
Thermomixer (the mixing speed was the same throughout the process).
Ten microliters of magnetic beads and 80 .mu.l Lysis/Binding Buffer
mixture were added to a round bottom plate and mixed for 1 minute.
Magnetic beads were captured using a magnetic stand and the
supernatant was removed without disturbing the beads. After
removing the supernatant, the lysed cells were added to the
remaining beads and mixed for 5 minutes. After removing the
supernatant, magnetic beads were washed 2 times with 150 .mu.l Wash
Buffer A and mixed for 1 minute. The beads were captured again and
the supernatant was removed. The beads were then washed with 150
.mu.l Wash Buffer B, captured and the supernatant was removed. The
beads were next washed with 150 .mu.l Elution Buffer, captured and
the supernatant removed. Finally, the beads were allowed to dry for
2 minutes. After drying, 50 .mu.l of Elution Buffer was added and
mixed for 5 minutes at 70.degree. C. The beads were captured on
magnet for 5 minutes. Forty-five .mu.l of supernatant was removed
and added to another 96 well plate.
[0738] cDNA Synthesis Using ABI High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, Calif., Cat
#4368813)
[0739] A master mix of 2 .mu.l 10.times. Buffer, 0.8 .mu.l
25.times.dNTPs, 2 .mu.l Random primers, 1 .mu.l Reverse
Transcriptase, 1 .mu.l RNase inhibitor and 3.2 .mu.l of H2O per
reaction as prepared. Equal volumes master mix and RNA were mixed
for a final volume of 12 .mu.l for in vitro screened or 20 .mu.l
for in vivo screened samples. cDNA was generated using a Bio-Rad
C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the
following steps: 25.degree. C. for 10 minutes, 37.degree. C. for
120 minutes, 85.degree. C. for 5 seconds, and 4.degree. C.
hold.
[0740] Real time PCR
[0741] Two .mu.l of cDNA were added to a master mix containing 2
.mu.l of H.sub.2O, 0.5 .mu.l GAPDH TaqMan Probe (Life Technologies
catalog number 4326317E for Hep3B cells, catalog number 352339E for
primary mouse hepatocytes or custom probe for cynomolgus primary
hepatocytes), 0.5 .mu.l C5 TaqMan probe (Life Technologies catalog
number Hs00167441_m1 for Hep3B cells or Mm00457879_m1 for Primary
Mouse Hepatoctyes or custom probe for cynomolgus primary
hepatocytes) and 5 .mu.l Lightcycler 480 probe master mix (Roche
catalog number 04887301001) per well in a 384 well (384 w) plates
(Roche catalog number 04887301001). Real time PCR was performed in
an Roche LC480 Real Time PCR system (Roche) using the
.DELTA..DELTA.Ct(RQ) assay. For in vitro screening, each duplex was
tested with two biological replicates unless otherwise noted and
each Real Time PCR was performed in duplicate technical replicates.
For in vivo screening, each duplex was tested in one or more
experiments (3 mice per group) and each Real Time PCR was run in
duplicate technical replicates.
[0742] To calculate relative fold change in ALAS1 mRNA levels, real
time data were analyzed using the .DELTA..DELTA.Ct method and
normalized to assays performed with cells transfected with 10 nM
AD-1955, or mock transfected cells. IC.sub.50s were calculated
using a 4 parameter fit model using XLFit and normalized to cells
transfected with AD-1955 over the same dose range, or to its own
lowest dose.
[0743] The sense and antisense sequences of AD-1955 are:
TABLE-US-00023 SENSE: (SEQ ID NO: 3682) cuuAcGcuGAGuAcuucGAdTsdT
ANTISENSE: (SEQ ID NO: 3683) UCGAAGuACUcAGCGuAAGdTsdT.
[0744] The single strand and duplex sequences of the modified and
unmodified siRNAs are provided in Table 14 and Table 15,
respectively.
TABLE-US-00024 TABLE 14 Human ALAS1 Modified Single Strands and
Duplex Sequences SEQ Target ID sites of SEQ ID NO: antisense NO:
(anti- Duplex sequence on (sense) sense) Name Sense Sequence
(5'-3') Antisense Sequence (5'-3') NM_000688.4 3371 3372 AD-58848
CfsasUfgCfcAfaAfAfAfuGfgAfcA asUfsgAfuGfuCfcAfuuuUfuGfgCfaU
1635-1657 fuCfaUfL96 fgsAfsc 3373 3374 AD-58849
AfsusUfuUfgAfaGfUfGfaUfgAfgU usUfsuCfaCfuCfaUfcacUfuCfaAfaA
2189-2211 fgAfaAfL96 fusGfsc 3375 3376 AD-58850
AfsgsUfuAfuAfuUfAfAfaUfuUfuA asGfsaUfuAfaAfaUfuuaAfuAfuAfaC
2344-2366 faUfcUfL96 fusUfsa 3377 3378 AD-58851
GfscsAfuUfuUfgAfAfGfuGfaUfgA usCfsaCfuCfaUfcAfcuuCfaAfaAfuG
2187-2209 fgUfgAfL96 fcsAfsg 3379 3380 AD-58852
GfsasAfcUfaAfuGfAfGfcAfgAfcA gsUfsuAfuGfuCfuGfcucAfuUfaGfuU
1975-1997 fuAfaCfL96 fcsAfsu 3381 3382 AD-58853
AfsasUfgAfcCfaCfAfCfcUfaUfcG asAfscUfcGfaUfaGfgugUfgGfuCfaU 973-995
faGfuUfL96 fusCfsu 3383 3384 AD-58854 UfsasAfaUfuUfuAfAfUfcUfaUfaG
usUfsuAfcUfaUfaGfauuAfaAfaUfuU 2352-2374 fuAfaAfL96 fasAfsu 3385
3386 AD-58855 UfsusCfaGfuAfuGfAfUfcGfuUfuC
csAfsaAfgAfaAfcGfaucAfuAfcUfgA 929-951 fuUfuGfL96 fasAfsa 3387 3388
AD-58856 CfsasCfuUfuUfcAfGfUfaUfgAfuC
asAfsaCfgAfuCfaUfacuGfaAfaAfgU 924-946 fgUfuUfL96 fgsGfsa 3389 3390
AD-58857 AfsasAfuCfuGfuUfUfCfcAfcUfuU
csUfsgAfaAfaGfuGfgaaAfcAfgAfuU 913-935 fuCfaGfL96 fusUfsg 3391 3392
AD-58858 CfsasUfuUfgAfaAfCfUfgUfcCfaU
usUfsgAfaUfgGfaCfaguUfuCfaAfaU 1478-1500 fuCfaAfL96 fgsCfsc 3393
3394 AD-58859 CfscsUfaUfcGfaGfUfUfuUfuAfaA
csAfsgUfuUfuAfaAfaacUfcGfaUfaG 983-1005 faCfuGfL96 fgsUfsg 3395
3396 AD-58861 GfsasCfcAfgAfaAfGfAfgUfgUfcU
gsAfsuGfaGfaCfaCfucuUfuCfuGfgU 872-894 fcAfuCfL96 fcsUfsu 3397 3398
AD-58862 AfscsCfaGfaAfaGfAfGfuGfuCfuC
asGfsaUfgAfgAfcAfcucUfuUfcUfgG 873-895 faUfcUfL96 fusCfsu 3399 3400
AD-58863 AfscsUfaAfuGfaGfCfAfgAfcAfuA
asUfsgUfuAfuGfuCfugcUfcAfuUfaG 1977-1999 faCfaUfL96 fusUfsc 3401
3402 AD-58864 UfsasGfuAfaAfaAfCfAfuAfgUfcC
usCfscAfgGfaCfuAfuguUfuUfuAfcU 2366-2388 fuGfgAfL96 fasUfsa 3403
3404 AD-58865 UfsasUfuUfcUfgGfAfAfcUfaGfuA
asAfsuUfuAfcUfaGfuucCfaGfaAfaU 1185-1207 faAfuUfL96 fasUfsu 3405
3406 AD-58867 UfsusCfuGfcAfaAfGfCfcAfgUfcU
csUfscAfaGfaCfuGfgcuUfuGfcAfgA 706-728 fuGfaGfL96 fasGfsa 3407 3408
AD-58868 GfsasGfgAfaAfgAfGfGfuUfgCfuG
gsUfsuUfcAfgCfaAfccuCfuUfuCfcU 759-781 faAfaCfL96 fcsAfsc 3409 3410
AD-58869 GfsgsUfaCfuAfgAfAfAfuAfuUfuC
usCfscAfgAfaAfuAfuuuCfuAfgUfaC 1174-1196 fuGfgAfL96 fcsAfsc 3411
3412 AD-58870 GfsasCfaUfcAfuGfCfAfaAfaGfcA
usCfsuUfuGfcUfuUfugcAfuGfaUfgU 853-875 faAfgAfL96 fcsCfsu 3413 3414
AD-58871 AfsasAfuUfuUfaAfUfCfuAfuAfgU
usUfsuUfaCfuAfuAfgauUfaAfaAfuU 2353-2375 faAfaAfL96 fusAfsa 3415
3416 AD-58873 CfsasUfgAfuCfcAfAfGfgGfaUfuC
usUfsuCfgAfaUfcCfcuuGfgAfuCfaU 1362-1384 fgAfaAfL96 fgsGfsa 3417
3418 AD-58874 AfsgsAfcCfaGfaAfAfGfaGfuGfuC
asUfsgAfgAfcAfcUfcuuUfcUfgGfuC 871-893 fuCfaUfL96 fusUfsu 3419 3420
AD-58875 AfsusCfcUfgAfaGfAfGfcGfcUfgA
usCfscCfuCfaGfcGfcucUfuCfaGfgA 1810-1832 fgGfgAfL96 fusCfsc 3421
3422 AD-58876 GfsusCfuGfuGfaUfGfAfaCfuAfaU
gsCfsuCfaUfuAfgUfucaUfcAfcAfgA 1966-1988 fgAfgCfL96 fcsUfsu 3423
3424 AD-58877 CfsasGfaAfaGfaGfUfGfuCfuCfaU
gsAfsaGfaUfgAfgAfcacUfcUfuUfcU 875-897 fcUfuCfL96 fgsGfsu 3425 3426
AD-58878 AfscsUfuUfuCfaGfUfAfuGfaUfcG
gsAfsaAfcGfaUfcAfuacUfgAfaAfaG 925-947 fuUfuCfL96 fusGfsg 3427 3428
AD-58879 UfscsAfuGfcCfaAfAfAfaUfgGfaC
usGfsaUfgUfcCfaUfuuuUfgGfcAfuG 1634-1656 faUfcAfL96 fasCfsu 3429
3430 AD-58880 AfsasUfaUfuUfcUfGfGfaAfcUfaG
usUfsuAfcUfaGfuUfccaGfaAfaUfaU 1183-1205 fuAfaAfL96 fusUfsc 3431
3432 AD-58881 CfsusUfcUfuCfaAfGfAfuAfaCfuU
usGfsgCfaAfgUfuAfucuUfgAfaGfaA 892-914 fgCfcAfL96 fgsAfsu 3433 3434
AD-58882 UfsusUfcAfgUfaUfGfAfuCfgUfuU
asAfsaGfaAfaCfgAfucaUfaCfuGfaA 928-950 fcUfuUfL96 fasAfsg 3435 3436
AD-58883 CfscsCfaGfuGfuGfGfUfuAfgUfgU
usUfsuCfaCfaCfuAfaccAfcAfcUfgG 790-812 fgAfaAfL96 fgsGfsc 3437 3438
AD-58884 GfscsUfgUfgAfgAfUfUfuAfcUfcU
asAfsuCfaGfaGfuAfaauCfuCfaCfaG 1325-1347 fgAfuUfL96 fcsCfsu 3439
3440 AD-58885 AfsgsGfcUfuGfaGfCfAfaGfuUfgG
gsAfsuAfcCfaAfcUfugcUfcAfaGfcC 2229-2251 fuAfuCfL96 fusGfsa 3441
3442 AD-58886 GfsasAfaGfaGfuGfUfCfuCfaUfcU
asAfsgAfaGfaUfgAfgacAfcUfcUfuU 877-899 fuCfuUfL96 fcsUfsg 3443 3444
AD-58887 AfsusUfuCfuGfgAfAfCfuAfgUfaA
gsAfsaUfuUfaCfuAfguuCfcAfgAfaA 1186-1208 faUfuCfL96 fusAfsu 3445
3446 AD-58888 UfsgsUfgAfuGfuGfGfCfcCfaUfgA
asAfsaCfuCfaUfgGfgccAfcAfuCfaC 1531-1553 fgUfuUfL96 fasCfsa 3447
3448 AD-58889 AfsasGfaGfaGfaAfGfUfcCfuAfuU
gsAfsgAfaAfuAfgGfacuUfcUfcUfcU 2208-2230 fuCfuCfL96 fusUfsc 3449
3450 AD-58890 UfsgsGfcAfgCfaCfAfGfaUfgAfaU
usCfsuGfaUfuCfaUfcugUfgCfuGfcC 671-693 fcAfgAfL96 fasGfsg 3451 3452
AD-58891 AfsusGfaUfcGfuUfUfCfuUfuGfaG
usUfsuUfcUfcAfaAfgaaAfcGfaUfcA 935-957 faAfaAfL96 fusAfsc 3453 3454
AD-58892 UfscsUfgGfaAfcUfAfGfuAfaAfuU
asUfsgGfaAfuUfuAfcuaGfuUfcCfaG 1189-1211 fcCfaUfL96 fasAfsa 3455
3456 AD-59095 GfscsCfcAfuUfcUfUfAfuCfcCfgA
asCfsuCfgGfgAfuAfagaAfuGfgsgsc 360-382 fgUfL96 3457 3458 AD-59096
GfsgsAfaCfcAfuGfCfCfuCfcAfuG asUfscAfuGfgAfgGfcauGfgUfuscsc
1347-1369 faUfL96 3459 3460 AD-59097 UfsgsGfaGfuCfuGfUfGfcGfgAfuC
asGfsgAfuCfcGfcAfcagAfcUfcscsa 1794-1816 fcUfL96 3461 3462 AD-59098
CfsasCfcCfaCfgGfGfUfgUfgUfgG usCfscCfaCfaCfaCfccgUfgGfgsusg
1112-1134 fgAfL96 3463 3464 AD-59099 GfsgsAfgUfcUfgUfGfCfgGfaUfcC
usAfsgGfaUfcCfgCfacaGfaCfuscsc 1795-1817 fuAfL96 3465 3466 AD-59100
CfsasAfaAfcUfgCfCfCfcAfaGfaU usCfsaUfcUfuGfgGfgcaGfuUfususg 428-450
fgAfL96 3467 3468 AD-59101 GfscsCfuCfcAfuGfAfUfcCfaAfgG
usCfscCfuUfgGfaUfcauGfgAfgsgsc 1355-1377 fgAfL96 3469 3470 AD-59102
CfsasUfcAfuCfcCfUfGfuGfcGfgG asAfscCfcGfcAfcAfgggAfuGfasusg
1921-1943 fuUfL96 3471 3472 AD-59103 AfscsCfcAfcGfgGfUfGfuGfuGfgG
usCfscCfcAfcAfcAfcccGfuGfgsgsu 1113-1135 fgAfL96 3473 3474 AD-59104
CfsasCfaUfcAfuCfCfCfuGfuGfcG usCfscGfcAfcAfgGfgauGfaUfgsusg
1919-1941 fgAfL96 3475 3476 AD-59105 CfsasGfaAfaGfaGfUfGfuCfuCfaU
asGfsaUfgAfgAfcAfcucUfuUfcsusg 873-895 fcUfL96 3477 3478 AD-59106
CfscsUfcCfaUfgAfUfCfcAfaGfgG asUfscCfcUfuGfgAfucaUfgGfasgsg
1356-1378 faUfL96 3479 3480 AD-59107 UfsgsCfcCfaUfuCfUfUfaUfcCfcG
usUfscGfgGfaUfaAfgaaUfgGfgscsa 359-381 faAfL96 3481 3482 AD-59108
CfsusUfcAfcCfcUfGfGfcUfaAfgA usAfsuCfuUfaGfcCfaggGfuGfasasg
1297-1319 fuAfL96 3483 3484 AD-59109 AfsusCfaUfcCfcUfGfUfgCfgGfgU
usAfsaCfcCfgCfaCfaggGfaUfgsasu 1922-1944 fuAfL96 3485 3486 AD-59110
AfsgsAfaAfgAfgUfGfUfcUfcAfuC asAfsgAfuGfaGfaCfacuCfuUfuscsu 874-896
fuUfL96 3487 3488 AD-59111 CfsusCfcAfuGfaUfCfCfaAfgGfgA
asAfsuCfcCfuUfgGfaucAfuGfgsasg 1357-1379 fuUfL96 3489 3490 AD-59112
CfscsAfuUfcUfuAfUfCfcCfgAfgU usGfsaCfuCfgGfgAfuaaGfaAfusgsg 362-384
fcAfL96
3491 3492 AD-59113 CfsasCfcCfuGfgCfUfAfaGfaUfgA
usAfsuCfaUfcUfuAfgccAfgGfgsusg 1300-1322 fuAfL96 3493 3494 AD-59114
UfscsAfuCfcCfuGfUfGfcGfgGfuU usCfsaAfcCfcGfcAfcagGfgAfusgsa
1923-1945 fgAfL96 3495 3496 AD-59115 AfsasGfaGfuGfuCfUfCfaUfcUfuC
asAfsgAfaGfaUfgAfgacAfcUfcsusu 877-899 fuUfL96 3497 3498 AD-59116
GfsusCfaUfgCfcAfAfAfaAfuGfgA usGfsuCfcAfuUfuUfuggCfaUfgsasc
1631-1653 fcAfL96 3499 3500 AD-59117 CfsasUfuCfuUfaUfCfCfcGfaGfuC
usGfsgAfcUfcGfgGfauaAfgAfasusg 363-385 fcAfL96 3501 3502 AD-59118
AfscsCfcUfgGfcUfAfAfgAfuGfaU usCfsaUfcAfuCfuUfagcCfaGfgsgsu
1301-1323 fgAfL96 3503 3504 AD-59119 CfsusCfuUfcAfcCfCfUfgGfcUfaA
usCfsuUfaGfcCfaGfgguGfaAfgsasg 1295-1317 fgAfL96 3505 3506 AD-59120
AfsusGfcCfaAfaAfAfUfgGfaCfaU usGfsaUfgUfcCfaUfuuuUfgGfcsasu
1634-1656 fcAfL96 3507 3508 AD-59121 UfsgsCfcCfcAfaGfAfUfgAfuGfgA
asUfsuCfcAfuCfaUfcuuGfgGfgscsa 434-456 faUfL96 3509 3510 AD-59122
GfsasAfcCfaUfgCfCfUfcCfaUfgA usAfsuCfaUfgGfaGfgcaUfgGfususc
1348-1370 fuAfL96 3511 3512 AD-59123 UfscsUfuCfaCfcCfUfGfgCfuAfaG
asUfscUfuAfgCfcAfgggUfgAfasgsa 1296-1318 faUfL96 3513 3514 AD-59124
UfsgsCfcAfaAfaAfUfGfgAfcAfuC asUfsgAfuGfuCfcAfuuuUfuGfgscsa
1635-1657 faUfL96 3515 3516 AD-59125 CfscsAfgAfaAfgAfGfUfgUfcUfcA
usAfsuGfaGfaCfaCfucuUfuCfusgsg 872-894 fuAfL96 3517 3518 AD-59126
GfsasAfaCfuGfuCfCfAfuUfcAfaU usCfsaUfuGfaAfuGfgacAfgUfususc
1481-1503 fgAfL96 3519 3520 AD-59127 UfscsAfcCfcUfgGfCfUfaAfgAfuG
asUfscAfuCfuUfaGfccaGfgGfusgsa 1299-1321 faUfL96 3521 3522 AD-59128
CfscsCfuGfgAfgUfCfUfgUfgCfgG asUfscCfgCfaCfaGfacuCfcAfgsgsg
1791-1813 faUfL96 3523 3524 AD-59129 GfsasAfaGfaGfuGfUfCfuCfaUfcU
usAfsaGfaUfgAfgAfcacUfcUfususc 875-897 fuAfL96 3525 3526 AD-59130
UfsgsGfaGfcCfcUfGfGfaGfuCfuG usAfscAfgAfcUfcCfaggGfcUfcscsa
1786-1808 fuAfL96
TABLE-US-00025 TABLE 15 Human ALASl Unmodified Single Strands and
Duplex Sequences Target SEQ sites of SEQ ID ID NO: antisense NO:
(anti- Duplex Sense Sequence Antisense Sequence sequence on (sense)
sense) Name (5'-3') (5'-3') NM_000688.4 3684 3527 AD-58848
CAUGCCAAAAAUGGACAUCAU AUGAUGUCCAUUUUUGGCAUGAC 1635-1657 3528 3529
AD-58849 AUUUUGAAGUGAUGAGUGAAA UUUCACUCAUCACUUCAAAAUGC 2189-2211
3530 3531 AD-58850 AGUUAUAUUAAAUUUUAAUCU AGAUUAAAAUUUAAUAUAACUUA
2344-2366 3532 3533 AD-58851 GCAUUUUGAAGUGAUGAGUGA
UCACUCAUCACUUCAAAAUGCAG 2187-2209 3534 3535 AD-58852
GAACUAAUGAGCAGACAUAAC GUUAUGUCUGCUCAUUAGUUCAU 1975-1997 3536 3537
AD-58853 AAUGACCACACCUAUCGAGUU AACUCGAUAGGUGUGGUCAUUCU 973-995 3538
3539 AD-58854 UAAAUUUUAAUCUAUAGUAAA UUUACUAUAGAUUAAAAUUUAAU
2352-2374 3540 3541 AD-58855 UUCAGUAUGAUCGUUUCUUUG
CAAAGAAACGAUCAUACUGAAAA 929-951 3542 3543 AD-58856
CACUUUUCAGUAUGAUCGUUU AAACGAUCAUACUGAAAAGUGGA 924-946 3544 3545
AD-58857 AAAUCUGUUUCCACUUUUCAG CUGAAAAGUGGAAACAGAUUUUG 913-935 3546
3547 AD-58858 CAUUUGAAACUGUCCAUUCAA UUGAAUGGACAGUUUCAAAUGCC
1478-1500 3548 3549 AD-58859 CCUAUCGAGUUUUUAAAACUG
CAGUUUUAAAAACUCGAUAGGUG 983-1005 3550 3551 AD-58861
GACCAGAAAGAGUGUCUCAUC GAUGAGACACUCUUUCUGGUCUU 872-894 3552 3553
AD-58862 ACCAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUGGUCU 873-895 3554
3555 AD-58863 ACUAAUGAGCAGACAUAACAU AUGUUAUGUCUGCUCAUUAGUUC
1977-1999 3556 3557 AD-58864 UAGUAAAAACAUAGUCCUGGA
UCCAGGACUAUGUUUUUACUAUA 2366-2388 3558 3559 AD-58865
UAUUUCUGGAACUAGUAAAUU AAUUUACUAGUUCCAGAAAUAUU 1185-1207 3560 3561
AD-58867 UUCUGCAAAGCCAGUCUUGAG CUCAAGACUGGCUUUGCAGAAGA 706-728 3562
3563 AD-58868 GAGGAAAGAGGUUGCUGAAAC GUUUCAGCAACCUCUUUCCUCAC 759-781
3564 3565 AD-58869 GGUACUAGAAAUAUUUCUGGA UCCAGAAAUAUUUCUAGUACCAC
1174-1196 3566 3567 AD-58870 GACAUCAUGCAAAAGCAAAGA
UCUUUGCUUUUGCAUGAUGUCCU 853-875 3568 3569 AD-58871
AAAUUUUAAUCUAUAGUAAAA UUUUACUAUAGAUUAAAAUUUAA 2353-2375 3570 3571
AD-58873 CAUGAUCCAAGGGAUUCGAAA UUUCGAAUCCCUUGGAUCAUGGA 1362-1384
3572 3573 AD-58874 AGACCAGAAAGAGUGUCUCAU AUGAGACACUCUUUCUGGUCUUU
871-893 3574 3575 AD-58875 AUCCUGAAGAGCGCUGAGGGA
UCCCUCAGCGCUCUUCAGGAUCC 1810-1832 3576 3577 AD-58876
GUCUGUGAUGAACUAAUGAGC GCUCAUUAGUUCAUCACAGACUU 1966-1988 3578 3579
AD-58877 CAGAAAGAGUGUCUCAUCUUC GAAGAUGAGACACUCUUUCUGGU 875-897 3580
3581 AD-58878 ACUUUUCAGUAUGAUCGUUUC GAAACGAUCAUACUGAAAAGUGG 925-947
3582 3583 AD-58879 UCAUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAUGACU
1634-1656 3584 3585 AD-58880 AAUAUUUCUGGAACUAGUAAA
UUUACUAGUUCCAGAAAUAUUUC 1183-1205 3586 3587 AD-58881
CUUCUUCAAGAUAACUUGCCA UGGCAAGUUAUCUUGAAGAAGAU 892-914 3588 3589
AD-58882 UUUCAGUAUGAUCGUUUCUUU AAAGAAACGAUCAUACUGAAAAG 928-950 3590
3591 AD-58883 CCCAGUGUGGUUAGUGUGAAA UUUCACACUAACCACACUGGGGC 790-812
3592 3593 AD-58884 GCUGUGAGAUUUACUCUGAUU AAUCAGAGUAAAUCUCACAGCCU
1325-1347 3594 3595 AD-58885 AGGCUUGAGCAAGUUGGUAUC
GAUACCAACUUGCUCAAGCCUGA 2229-2251 3596 3597 AD-58886
GAAAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUUUCUG 877-899 3598 3599
AD-58887 AUUUCUGGAACUAGUAAAUUC GAAUUUACUAGUUCCAGAAAUAU 1186-1208
3600 3601 AD-58888 UGUGAUGUGGCCCAUGAGUUU AAACUCAUGGGCCACAUCACACA
1531-1553 3602 3603 AD-58889 AAGAGAGAAGUCCUAUUUCUC
GAGAAAUAGGACUUCUCUCUUUC 2208-2230 3604 3605 AD-58890
UGGCAGCACAGAUGAAUCAGA UCUGAUUCAUCUGUGCUGCCAGG 671-693 3606 3607
AD-58891 AUGAUCGUUUCUUUGAGAAAA UUUUCUCAAAGAAACGAUCAUAC 935-957 3608
3609 AD-58892 UCUGGAACUAGUAAAUUCCAU AUGGAAUUUACUAGUUCCAGAAA
1189-1211 3610 3611 AD-59095 GCCCAUUCUUAUCCCGAGU
ACUCGGGAUAAGAAUGGGC 360-382 3612 3613 AD-59096 GGAACCAUGCCUCCAUGAU
AUCAUGGAGGCAUGGUUCC 1347-1369 3614 3615 AD-59097
UGGAGUCUGUGCGGAUCCU AGGAUCCGCACAGACUCCA 1794-1816 3616 3617
AD-59098 CACCCACGGGUGUGUGGGA UCCCACACACCCGUGGGUG 1112-1134 3618
3619 AD-59099 GGAGUCUGUGCGGAUCCUA UAGGAUCCGCACAGACUCC 1795-1817
3620 3621 AD-59100 CAAAACUGCCCCAAGAUGA UCAUCUUGGGGCAGUUUUG 428-450
3622 3623 AD-59101 GCCUCCAUGAUCCAAGGGA UCCCUUGGAUCAUGGAGGC
1355-1377 3624 3625 AD-59102 CAUCAUCCCUGUGCGGGUU
AACCCGCACAGGGAUGAUG 1921-1943 3626 3627 AD-59103
ACCCACGGGUGUGUGGGGA UCCCCACACACCCGUGGGU 1113-1135 3628 3629
AD-59104 CACAUCAUCCCUGUGCGGA UCCGCACAGGGAUGAUGUG 1919-1941 3630
3631 AD-59105 CAGAAAGAGUGUCUCAUCU AGAUGAGACACUCUUUCUG 873-895 3632
3633 AD-59106 CCUCCAUGAUCCAAGGGAU AUCCCUUGGAUCAUGGAGG 1356-1378
3634 3635 AD-59107 UGCCCAUUCUUAUCCCGAA UUCGGGAUAAGAAUGGGCA 359-381
3636 3637 AD-59108 CUUCACCCUGGCUAAGAUA UAUCUUAGCCAGGGUGAAG
1297-1319 3638 3639 AD-59109 AUCAUCCCUGUGCGGGUUA
UAACCCGCACAGGGAUGAU 1922-1944 3640 3641 AD-59110
AGAAAGAGUGUCUCAUCUU AAGAUGAGACACUCUUUCU 874-896 3642 3643 AD-59111
CUCCAUGAUCCAAGGGAUU AAUCCCUUGGAUCAUGGAG 1357-1379 3644 3645
AD-59112 CCAUUCUUAUCCCGAGUCA UGACUCGGGAUAAGAAUGG 362-384 3646 3647
AD-59113 CACCCUGGCUAAGAUGAUA UAUCAUCUUAGCCAGGGUG 1300-1322 3648
3649 AD-59114 UCAUCCCUGUGCGGGUUGA UCAACCCGCACAGGGAUGA 1923-1945
3650 3651 AD-59115 AAGAGUGUCUCAUCUUCUU AAGAAGAUGAGACACUCUU 877-899
3652 3653 AD-59116 GUCAUGCCAAAAAUGGACA UGUCCAUUUUUGGCAUGAC
1631-1653 3654 3655 AD-59117 CAUUCUUAUCCCGAGUCCA
UGGACUCGGGAUAAGAAUG 363-385 3656 3657 AD-59118 ACCCUGGCUAAGAUGAUGA
UCAUCAUCUUAGCCAGGGU 1301-1323 3658 3659 AD-59119
CUCUUCACCCUGGCUAAGA UCUUAGCCAGGGUGAAGAG 1295-1317 3660 3661
AD-59120 AUGCCAAAAAUGGACAUCA UGAUGUCCAUUUUUGGCAU 1634-1656 3662
3663 AD-59121 UGCCCCAAGAUGAUGGAAU AUUCCAUCAUCUUGGGGCA 434-456 3664
3665 AD-59122 GAACCAUGCCUCCAUGAUA UAUCAUGGAGGCAUGGUUC 1348-1370
3666 3667 AD-59123 UCUUCACCCUGGCUAAGAU AUCUUAGCCAGGGUGAAGA
1296-1318 3668 3669 AD-59124 UGCCAAAAAUGGACAUCAU
AUGAUGUCCAUUUUUGGCA 1635-1657 3670 3671 AD-59125
CCAGAAAGAGUGUCUCAUA UAUGAGACACUCUUUCUGG 872-894 3672 3673 AD-59126
GAAACUGUCCAUUCAAUGA UCAUUGAAUGGACAGUUUC 1481-1503 3674 3675
AD-59127 UCACCCUGGCUAAGAUGAU AUCAUCUUAGCCAGGGUGA 1299-1321 3676
3677 AD-59128 CCCUGGAGUCUGUGCGGAU AUCCGCACAGACUCCAGGG 1791-1813
3678 3679 AD-59129 GAAAGAGUGUCUCAUCUUA UAAGAUGAGACACUCUUUC 875-897
3680 3681 AD-59130 UGGAGCCCUGGAGUCUGUA UACAGACUCCAGGGCUCCA
1786-1808
[0745] The results of the in vitro assays are provided in Table 16.
Table 16 also notes the target species of each of the siRNAs.
TABLE-US-00026 TABLE 16 Results of Functional Assays Cyno Free
Uptake Cyno Transfection Hep3b Transfection Target 1 uM 20 nM 20 nM
0.2 nM 10 nM 0.1 nM Duplex ID Species Type Avg 500 nM Avg 10 nM Avg
Avg Avg Avg AD-58848 M/R/Rh/H 21/23 131.6 176.0 104.4 128.0 43.5
44.8 25.3 76.8 AD-58849 H/Rh 21/23 91.9 88.1 92.2 105.0 29.4 35.4
11.5 47.1 AD-58850 H/Rh 21/23 79.4 103.4 80.0 111.2 NA 62.2 31.3
72.0 AD-58851 H/Rh 21/23 99.7 74.7 94.8 104.7 NA 40.7 8.6 81.3
AD-58852 H/Rh 21/23 108.1 91.8 103.3 111.9 101.1 128.8 43.4 129.0
AD-58853 H/Rh 21/23 74.8 67.7 84.2 93.5 24.7 52.9 14.1 61.2
AD-58854 H/Rh 21/23 145.9 124.1 106.6 115.3 119.0 83.9 85.0 84.0
AD-58855 H/Rh 21/23 81.5 97.9 92.7 101.8 39.5 40.3 15.3 67.6
AD-58856 H/Rh 21/23 74.1 90.6 84.6 82.6 22.4 30.7 8.7 33.3 AD-58857
H/Rh 21/23 64.7 91.4 62.3 87.1 22.0 31.6 9.8 106.3 AD-58858 H/Rh
21/23 67.4 91.7 68.6 98.3 27.9 40.3 17.4 44.8 AD-58859 H/Rh 21/23
71.2 77.2 92.4 90.1 19.1 34.3 13.1 39.7 AD-58861 H/Rh 21/23 104.6
107.2 102.0 100.6 25.9 35.1 18.0 69.8 AD-58862 H/Rh 21/23 66.8 77.0
68.7 88.5 20.3 31.1 24.2 49.9 AD-58863 H/Rh 21/23 70.8 66.8 76.8
98.5 21.5 29.7 8.7 54.9 AD-58864 H/Rh 21/23 76.2 85.6 83.7 100.8
60.4 61.0 56.4 87.3 AD-58865 H/Rh 21/23 67.9 77.9 95.9 98.4 21.3
38.6 15.5 81.4 AD-58867 H/Rh 21/23 95.9 93.3 107.0 97.5 32.3 42.7
16.6 79.8 AD-58868 H/Rh 21/23 95.2 92.1 116.2 94.7 54.6 69.2 61.5
105.9 AD-58869 H/Rh 21/23 65.0 78.2 75.8 88.2 17.4 25.0 13.0 63.9
AD-58870 H/Rh 21/23 69.4 92.3 81.0 88.1 29.2 43.8 33.7 79.1
AD-58871 H/Rh 21/23 61.2 77.3 88.2 77.0 71.2 73.2 36.7 110.3
AD-58873 H/Rh 21/23 95.2 100.9 83.3 94.6 54.2 52.8 36.6 73.3
AD-58874 H/Rh 21/23 75.8 76.8 63.8 85.3 22.3 31.2 15.0 38.2
AD-58875 H/Rh 21/23 80.7 88.7 78.6 97.9 48.6 73.6 61.2 90.6
AD-58876 H/Rh 21/23 90.8 93.1 82.5 100.2 41.1 56.9 21.2 58.7
AD-58877 H/Rh 21/23 68.3 85.1 51.2 78.7 18.5 46.6 11.9 27.4
AD-58878 H/Rh 21/23 78.3 68.3 81.2 91.2 24.1 23.4 6.2 37.1 AD-58879
H/Rh 21/23 87.9 94.1 79.7 95.4 32.0 47.8 15.7 82.5 AD-58880 H/Rh
21/23 74.9 72.2 88.9 88.1 20.1 27.5 14.0 60.7 AD-58881 H/Rh 21/23
85.9 76.8 78.8 118.0 22.2 36.7 27.6 71.6 AD-58882 H/Rh 21/23 54.1
53.4 60.3 85.8 14.6 27.2 8.2 23.8 AD-58883 H/Rh 21/23 80.4 69.9
75.7 80.3 31.8 25.8 12.3 63.0 AD-58884 H/Rh 21/23 57.7 55.3 64.8
78.2 20.0 30.0 11.8 68.9 AD-58885 H/Rh 21/23 101.8 91.8 104.1 101.5
85.9 71.9 61.8 71.2 AD-58886 M/R/Rh/H 21/23 47.1 58.0 36.3 93.3
16.0 26.6 9.2 32.0 AD-58887 H/Rh 21/23 73.6 98.7 82.6 95.2 28.5
33.5 12.8 65.2 AD-58888 H/Rh 21/23 90.2 69.9 69.4 85.6 46.9 45.0
16.6 72.0 AD-58889 H/Rh 21/23 83.6 98.6 82.4 92.2 36.5 40.3 31.6
99.4 AD-58890 H/Rh 21/23 69.5 95.4 84.2 88.2 50.8 45.6 21.7 92.9
AD-58891 H/Rh 21/23 62.8 75.7 75.4 109.2 23.6 34.3 15.6 55.8
AD-58892 H/Rh 21/23 60.2 92.9 89.8 92.9 22.8 43.3 20.2 75.6
AD-59095 M/R/Rh/H 19mer 88.9 NA 132.8 NA 48.3 97.4 54.3 99.0
AD-59096 M/R/Rh/H 19mer 95.5 NA 90.5 NA 105.7 138.6 131.4 120.7
AD-59097 M/R/Rh/H 19mer 92.5 NA 84.2 NA 75.0 NA 94.7 108.5 AD-59098
M/R/Rh/H 19mer 84.0 NA 87.7 NA 109.3 NA 130.0 87.3 AD-59099
M/R/Rh/H 19mer 89.7 NA 90.0 NA 77.8 85.4 46.8 74.9 AD-59100
M/R/Rh/H 19mer 84.8 NA 144.3 NA 70.6 108.1 91.5 117.6 AD-59101
M/R/Rh/H 19mer 79.0 NA 103.8 NA 89.8 102.9 124.2 107.0 AD-59102
M/R/Rh/H 19mer 85.9 NA 100.6 NA 72.2 68.5 87.9 95.1 AD-59103
M/R/Rh/H 19mer 86.0 NA 91.1 NA 93.0 81.3 130.0 96.0 AD-59104
M/R/Rh/H 19mer 92.6 NA 96.9 NA 94.9 91.4 124.4 83.1 AD-59105
M/R/Rh/H 19mer 48.9 NA 101.7 NA 18.4 48.9 17.0 34.7 AD-59106
M/R/Rh/H 19mer 63.2 NA 76.7 NA 28.5 40.7 28.6 46.4 AD-59107
M/R/Rh/H 19mer 71.4 NA 68.7 NA 37.1 45.3 26.8 63.6 AD-59108
M/R/Rh/H 19mer 70.7 NA 85.1 NA 89.9 84.8 139.2 101.7 AD-59109
M/R/Rh/H 19mer 86.1 NA 83.4 NA 84.9 96.2 131.7 86.7 AD-59110
M/R/Rh/H 19mer 70.8 NA 119.7 NA 38.5 60.4 67.4 80.3 AD-59111
M/R/Rh/H 19mer 66.1 NA 76.5 NA 52.2 61.0 69.7 87.6 AD-59112
M/R/Rh/H 19mer 71.2 NA 80.2 NA 91.2 83.4 127.4 89.0 AD-59113
M/R/Rh/H 19mer 67.0 NA 77.8 NA 49.1 59.0 66.8 91.4 AD-59114
M/R/Rh/H 19mer 81.7 NA 79.3 NA 96.3 88.0 129.6 72.4 AD-59115
M/R/Rh/H 19mer 40.4 NA 69.6 NA 19.6 35.7 9.3 16.9 AD-59116 M/R/Rh/H
19mer 72.2 NA 78.3 NA 53.5 77.8 70.1 107.8 AD-59117 M/R/Rh/H 19mer
70.7 NA 75.6 NA 75.8 74.9 129.0 103.5 AD-59118 M/R/Rh/H 19mer 68.8
NA 75.9 NA 81.4 82.1 114.1 89.7 AD-59119 M/R/Rh/H 19mer 64.9 NA
86.5 NA 85.1 125.1 122.8 124.8 AD-59120 M/R/Rh/H 19mer 63.5 NA 75.1
NA 29.9 52.0 16.1 54.1 AD-59121 M/R/Rh/H 19mer 67.6 NA 72.0 NA 88.8
77.4 108.0 103.1 AD-59122 M/R/Rh/H 19mer 60.2 NA 62.3 NA 25.1 45.3
16.2 54.8 AD-59123 M/R/Rh/H 19mer 68.6 NA 108.2 NA 59.2 84.6 80.0
97.7 AD-59124 M/R/Rh/H 19mer 47.5 NA 56.5 NA 23.9 40.0 9.8 18.9
AD-59125 M/R/Rh/H 19mer 45.4 NA 47.2 NA 15.2 40.7 14.7 15.1
AD-59126 M/R/Rh/H 19mer 64.3 NA 74.6 NA 51.6 57.1 35.5 54.4
AD-59127 M/R/Rh/H 19mer 103.4 NA 105.8 NA 94.0 156.4 135.9 113.7
AD-59128 M/R/Rh/H 19mer 102.4 NA 81.4 NA 66.3 89.3 60.2 74.9
AD-59129 M/R/Rh/H 19mer 41.3 NA 38.8 NA 17.9 41.4 8.6 12.6 AD-59130
M/R/Rh/H 19mer 58.3 NA 80.8 NA 94.9 78.3 106.7 88.0
[0746] Table 17 illustrates the IC.sub.50s of select ALAS1 siRNA
duplexes. The IC50 were determined from the knockdown of
endogenously expressed ALAS1 in the Hep3B cell line, at 24 hours
following transfection of each ALAS1 modified siRNA duplex (see
Table 14). At least seven duplexes, including AD-58882, AD-58878,
AD-58886, AD-58877, AD-59115, AD-58856, and AD-59129, consistently
demonstrated IC.sub.50s of less than 0.1 nm, indicating that these
duplexes were particularly effective in suppressing ALAS1
expression.
TABLE-US-00027 TABLE 17 IC.sub.50S of select ALAS1 siRNA duplexes
Duplex ID 384w IC50 (nM) 96w IC50 (nM) AD-58882 0.008 0.014
AD-58878 0.040 0.031 AD-58886 0.037 0.033 AD-58877 0.031 0.034
AD-59115 0.093 0.052 AD-58856 0.061 0.066 AD-59129 0.085 0.071
AD-59124 0.572 0.078 AD-58874 0.140 0.102 AD-59125 0.118 0.115
AD-59105 0.511 0.144 AD-59120 180.592 0.498 AD-59122 36.646 0.646
AD-59106 7.906 0.847 AD-59126 n/a 1.014 AD-59107 n/a 1.971
EQUIVALENTS
[0747] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210087558A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210087558A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References