U.S. patent application number 17/510958 was filed with the patent office on 2022-08-11 for compositions and methods for inhibiting expression of tmprss6 gene.
The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to Brian Bettencourt, David Bumcrot, Ivanka Toudjarska.
Application Number | 20220251570 17/510958 |
Document ID | / |
Family ID | 1000006284710 |
Filed Date | 2022-08-11 |
United States Patent
Application |
20220251570 |
Kind Code |
A1 |
Bumcrot; David ; et
al. |
August 11, 2022 |
COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF TMPRSS6
GENE
Abstract
The invention relates to double-stranded ribonucleic acid
(dsRNA) compositions targeting the TMPRSS6 gene, and methods of
using such dsRNA compositions to inhibit expression of TMPRSS6.
Inventors: |
Bumcrot; David; (Cambridge,
MA) ; Bettencourt; Brian; (Groton, MA) ;
Toudjarska; Ivanka; (Meford, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000006284710 |
Appl. No.: |
17/510958 |
Filed: |
October 26, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16121265 |
Sep 4, 2018 |
11198876 |
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17510958 |
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14866148 |
Sep 25, 2015 |
10100312 |
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16121265 |
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14007835 |
Feb 7, 2014 |
9175290 |
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PCT/US2012/030786 |
Mar 28, 2012 |
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14866148 |
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61568942 |
Dec 9, 2011 |
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61468830 |
Mar 29, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2320/30 20130101;
C12N 15/1137 20130101; C12N 2310/315 20130101; C12N 2310/321
20130101; C12Y 304/21 20130101; A61K 31/713 20130101; C12N 2310/344
20130101; C12N 2310/346 20130101; C12N 2310/351 20130101; C12N
2310/14 20130101; C12N 15/1138 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/713 20060101 A61K031/713 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting
expression of TMPRSS6, wherein said dsRNA comprises a sense strand
and an antisense strand, the antisense strand comprising a region
of complementarity to a TMPRSS6 transcript which comprises at least
15 contiguous nucleotides differing by no more than 3 nucleotides
from one of the antisense sequences listed in Tables 2, 3 or 4.
2. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
3. The dsRNA of claim 2, wherein at least one of said 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.
4. The dsRNA of claim 2, wherein said 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.
5. The dsRNA of claim 1, wherein the region of complementarity is
at least 17 nucleotides in length.
6. The dsRNA of claim 1, wherein the region of complementarity is
between 19 and 21 nucleotides in length.
7. (canceled)
8. The dsRNA of claim 1, wherein each strand is no more than 30
nucleotides in length.
9. The dsRNA of claim 1, wherein at least one strand comprises a 3'
overhang of at least 1 nucleotide or at least 2 nucleotides.
10. (canceled)
11. The dsRNA of claim 1, further comprising a ligand.
12. The dsRNA of claim 11, wherein the ligand is conjugated to the
3' end of the sense strand of the dsRNA.
13. The dsRNA of claim 1, wherein the region of complementarity
consists of one of the antisense sequences of Tables 2, 3 or 4.
14. The dsRNA of claim 1, wherein the dsRNA comprises a sense
strand consisting of a sense strand sequence selected from Tables
2, 3 or 4 and an antisense strand consisting of an antisense
sequence selected from Tables 2, 3 or 4.
15. A cell containing the dsRNA of claim 1.
16. A pharmaceutical composition for inhibiting expression of a
TMPRSS6 gene comprising the dsRNA of claim 1.
17. The pharmaceutical composition of claim 16, further comprising
a lipid formulation.
18. (canceled)
19. A method of inhibiting TMPRSS6 expression in a cell, the method
comprising: (a) introducing into the cell the dsRNA of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient
to obtain degradation of the mRNA transcript of a TMPRSS6 gene,
thereby inhibiting expression of the TMPRSS6 gene in the cell.
20. (canceled)
21. A method of treating a disorder mediated by TMPRSS6 expression
comprising administering to a human in need of such treatment a
therapeutically effective amount of the dsRNA of claim 1.
22.-26. (canceled)
27. 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 TMPRSS6, wherein said dsRNA is 30 base pairs or
less in length, and wherein said dsRNA targets said mRNA for
cleavage.
28.-31. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of Ser. No. 16/121,265,
filed on Sep. 4, 2018, which is a divisional of U.S. application
Ser. No. 14/866,148, filed Sep. 25, 2015, now U.S. Pat. No.
10,100,312, which is a divisional of U.S. application Ser. No.
14/007,835, filed Feb. 7, 2014, now U.S. Pat. No. 9,175,290, which
is a national phase application under 35 U.S.C. .sctn. 371 of PCT
International Application No. PCT/US2012/030786, filed Mar. 28,
2012, which claims the benefit of U.S. Provisional Application No.
61/568,942, filed Dec. 9, 2011, and U.S. Provisional Application
No. 61/468,830, filed Mar. 29, 2011. These prior applications are
incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the specific inhibition of the
expression of the TMPRSS6 gene.
BACKGROUND OF THE INVENTION
[0003] TMPRSS6 (Transmembrane Protease, Serine 6) encodes a type II
serine protease and is expressed mainly in the liver. TMPRSS6
influences iron levels in the liver by binding and proteolytically
degrading the hepcidin activator and BMP co-receptor HJV
(hemojuvelin), which causes down-regulation of hepcidin levels.
[0004] TMPRSS6 consists of a short N-terminal intracytoplasmic
tail, a type II transmembrane domain, a stem region composed of two
extracellular CUB (complement factor C1s/C1r, urchin embryonic
growth factor and BMP (bone morphogenetic protein)) domains, three
LDLR (low-density-lipoprotein receptor class A) domains, and a
C-terminal trypsin-like serine protease domain. There are also
consensus sites for N-glycosylation in the extracellular domain,
and a potential phosphorylation site in the intracytoplasmic tail
region.
SUMMARY OF THE INVENTION
[0005] Described herein are compositions and methods that effect
the RNA-induced silencing complex (RISC)-mediated cleavage of RNA
transcripts of the TMPRSS6 gene, such as in a cell or mammal. Also
described are compositions and methods for treating pathological
conditions and diseases caused by the expression of a TMPRSS6 gene,
such as a disorder characterized by iron overabundance (e.g.,
thalassemia, e.g., a .beta.-thalassemia intermedia or an
.alpha.-thalassemia). Also described are compositions and methods
for decreasing or preventing iron absorption or mobilization,
thereby ameliorating iron overload in certain pathological
conditions. The methods and compositions described herein are
generally useful for the treatment of hemochromatosis (iron
build-up in the body).
[0006] As used herein, the term "iRNA" refers to an agent that
contains RNA as that term is defined herein, and which mediates the
targeted cleavage of an RNA transcript via an RNA-induced silencing
complex (RISC) pathway. In one embodiment, an iRNA as described
herein inhibits the expression of TMPRSS6 in a cell or mammal.
Alternatively, in another embodiment, the iRNA up-regulates the
expression of TMPRSS6 in a cell or mammal.
[0007] The iRNAs included in the compositions featured herein
encompass a double-stranded RNA (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 a TMPRSS6
gene. In one embodiment, the dsRNA comprises a region of at least
15 contiguous nucleotides.
[0008] In one embodiment, an iRNA for inhibiting expression of a
TMPRSS6 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
TMPRSS6, and the region of complementarity is 30 nucleotides or
less, and at least 15 nucleotides in length. Generally, the iRNA is
19 to 24, e.g., 19 to 21 nucleotides in length. 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. The iRNA, upon contacting with a cell expressing
TMPRSS6, inhibits the expression of a TMPRSS6 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 TMPRSS6 iRNA is formulated in a stable
nucleic acid lipid particle (SNALP).
[0009] In one embodiment, an iRNA featured herein includes a first
sequence of a dsRNA that is selected from the group consisting of
the sense sequences of Tables 2, 3 or 4 and a second sequence that
is selected from the group consisting of the corresponding
antisense sequences of Tables 2, 3 or 4. 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. Generally, such a modified
sequence will be based on a first sequence of said iRNA selected
from the group consisting of the sense sequences of Tables 2, 3 or
4 and a second sequence selected from the group consisting of the
antisense sequences of Tables 2, 3 or 4.
[0010] In one embodiment, an iRNA featured herein includes a sense
strand of a TMPRSS6 dsRNA having a sequence selected from the group
consisting of SEQ ID NO:111, SEQ ID NO:455, SEQ ID NO:109, SEQ ID
NO:524, SEQ ID NO:89, SEQ ID NO:494, SEQ ID NO:445, SEQ ID NO:592,
SEQ ID NO:47, and SEQ ID NO:540; and an antisense strand consisting
of a sequence selected from the group consisting of SEQ ID NO:112,
SEQ ID NO:456, SEQ ID NO:110, SEQ ID NO:525, SEQ ID NO:90, SEQ ID
NO:495, SEQ ID NO:446, SEQ ID NO:593, SEQ ID NO:48 and SEQ ID
NO:541.
[0011] In another embodiment, a composition containing a dsRNA
targeting TMPRSS6 is administered to a subject who has elevated
iron levels, e.g., elevated levels of iron in the liver. A subject
who has elevated iron levels can be identified as a subject who has
elevated serum iron levels (e.g., over 350 .mu.g/dL, over 500
.mu.g/dL or over 1000 .mu.g/dL or more), elevated serum ferritin
levels, or a transferrin saturation level greater than 40%, greater
than 45%, greater than 50%, greater than 60% or more.
[0012] Mild-to-moderate iron overload is indicated by serum
ferritin levels of 300-2500 .mu.g/L, while levels >2500 .mu.g/L
are associated with an increased risk of cardiac disease. Serum
ferritin >1000 .mu.g/L has been shown to be associated with
adverse outcomes in both primary and secondary iron overload. Serum
ferritin levels higher than 200 .mu.g/L in premenopausal women, and
300 .mu.g/L in men and postmenopausal women indicate primary iron
overload due to hemochromatosis, and ferritin levels higher than
1000 .mu.g/L typically suggest liver damage due to iron overload. A
subject having a serum ferritin level higher than 300 .mu.g/L, 500
.mu.g/L, 1000 .mu.g/L, 1500 .mu.g/L, 2000 .mu.g/L, or 2500 .mu.g/L
or more is a candidate for treatment with a dsRNA targeting
TMPRSS6.
[0013] In another embodiment, a composition containing a dsRNA
targeting TMPRSS6 is administered to a subject who has elevated
transferrin levels, e.g., transferrin levels greater than 400
mg/dL, greater than 500 mg/dL, greater than 1000 mg/dL or more)
[0014] Iron levels can also be measured by a TIBC (Total Iron
Binding Capacity) test. The TIBC test measures the amount of iron
that the blood would carry if the transferrin were fully saturated.
Since transferrin is produced by the liver, the TIBC can be used to
monitor liver function and nutrition. A subject having TIBC values
greater than 400 .mu.g/dL, greater than 500 .mu.g/dL, or greater
than 1000 .mu.g/dL or more is a candidate for treatment with a
dsRNA targeting TMPRSS6.
[0015] In one embodiment, administration of the dsRNA lowers iron
levels, e.g., in the liver, or in serum, by at least 5%, e.g., by
at least 10%, by at least 15%, at least 20%, at least 25%, at least
30%, at least 40%, at least 50%, or at least 60%, or more. In some
embodiments, one or more of serum ferritin levels, serum
transferrin levels, transferrin saturation levels or TIBC values
are decreased by at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 40%, at least 50%, or at
least 60%, or more, as compared to pretreatment levels. In another
embodiment, the decrease in iron levels, decrease in serum ferritin
levels, decrease in transferrin or transferrin saturation levels,
or decrease in TIBC values is maintained for at least 5, 10, 20,
30, or 40 days or longer.
[0016] In one embodiment, the subject is selected, at least in
part, on the basis of needing (as opposed to merely selecting a
patient on the grounds of who happens to be in need of) lower iron
levels.
[0017] In one embodiment, an iRNA as described herein targets a
wildtype TMPRSS6 RNA transcript, and in another embodiment, the
iRNA targets a mutant transcript (e.g., a TMPRSS6 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 TMPRSS6. In another embodiment, the iRNA
targets both a wildtype and a mutant TMPRSS6 transcript. In yet
another embodiment, the iRNA targets a transcript variant of
TMPRSS6.
[0018] In one embodiment, an iRNA featured in the invention targets
a non-coding region of a TMPRSS6 RNA transcript, such as the 5' or
3' untranslated region.
[0019] In one embodiment, an iRNA featured in the invention is
delivered to the liver, e.g., hepatocytes of the liver or Kupffer
cells, e.g., hypertrophic Kupffer cells.
[0020] In one aspect, embodiments featured in the invention provide
a cell containing at least one of the iRNAs featured in the
invention. The cell is generally a mammalian cell, such as a human
cell.
[0021] In another aspect, embodiments featured in the invention
provide a pharmaceutical composition for inhibiting the expression
of a TMPRSS6 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
disorder that causes increased iron levels, e.g., hemochromatosis.
For example, the composition is useful for treating a thalassemia,
such as .beta.-thalassemia intermedia.
[0022] In another embodiment, the pharmaceutical composition is
formulated for administration of a dosage regimen described herein,
e.g., not more than once every two months, not more than once per
month, not more than twice per month, 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, 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 five years, or ten years, or longer,
including the remaining lifetime of a subject.
[0023] In another embodiment, a composition containing an iRNA
described herein, e.g., a dsRNA targeting TMPRSS6, is administered
with a non-iRNA therapeutic agent, such as an agent known to treat
hemochromatosis, or a disorder that causes hemochromatosis, such as
a thalassemia. For example, an iRNA featured in the invention can
be administered with an agent for treatment of a .beta.
thalassemia, e.g., .beta.-thalassemia intermedia, or another
disorder associated with increased iron levels.
[0024] In another embodiment, a TMPRSS6 iRNA is administered to a
patient, and then the non-iRNA agent is administered to the patient
(or vice versa). In another embodiment, a TMPRSS6 iRNA and the
non-iRNA therapeutic agent are administered at the same time. In
one embodiment, the agent is, for example, an agent that affects
iron levels, such as an iron chelator (e.g., desferrioxamine), or
folic acid.
[0025] In another aspect, provided herein is a method for
inhibiting the expression of a TMPRSS6 gene in a cell by performing
the following steps: [0026] (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 TMPRSS6, 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 TMPRSS6,
inhibits expression of a TMPRSS6 gene by at least 10%, preferably
at least 20%, at least 30%, at least 40% or more; and [0027] (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of the TMPRSS6 gene,
thereby inhibiting expression of a TMPRSS6 gene in the cell.
[0028] In another aspect, the invention provides methods and
compositions useful for activating expression of a TMPRSS6 gene in
a cell or mammal.
[0029] In another aspect, the invention provides a method for
modulating the expression of a TMPRSS6 gene in a cell by performing
the following steps: [0030] (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 TMPRSS6, 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 TMPRSS6,
modulates expression of a TMPRSS6 gene by at least 10%, preferably
at least 20%, at least 30%, at least 40% or more; and [0031] (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation or protection of the mRNA transcript of the
TMPRSS6 gene, thereby modulating expression of a TMPRSS6 gene in
the cell.
[0032] In one embodiment, the method is for inhibiting gene
expression in a liver cell, such as a hepatocyte, or a Kupffer
cell. In another embodiment, the method is for activating gene
expression in a liver cell.
[0033] In other aspects, the invention provides methods for
treating, preventing, reversing, or managing pathological processes
mediated by TMPRSS6 expression, such as a disorder associated with
hemochromatosis. In one embodiment, the method includes
administering to a patient in need of such treatment, prevention,
reversal, or management, a therapeutically or prophylactically
effective amount of one or more of the iRNAs featured in the
invention. In one embodiment the patient has a thalassemia, such as
.beta.-thalassemia intermedia. In another embodiment,
administration of the iRNA targeting TMPRSS6 alleviates or relieves
the severity of at least one symptom of a TMPRSS6-mediated disorder
in the patient, such as a symptom associated with iron overload,
e.g., joint pain, abdominal pain, or weakness.
[0034] In one aspect, the invention provides a vector for
inhibiting the expression of a TMPRSS6 gene in a cell. 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.
[0035] In another aspect, the invention provides a cell containing
a vector for inhibiting the expression of a TMPRSS6 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.
[0036] In yet another aspect, the invention provides a composition
containing a TMPRSS6 iRNA, in combination with a second iRNA
targeting a second gene involved in a pathological disease, and
useful for treating the disease, e.g., a .beta.-thalassemia. For
example, a second iRNA can target a negative regulator of hepcidin,
such as a hypoxia inducible factor, e.g., a HIF-1a or HIF-2a;
GDF15; or TWSG1. In one embodiment, the second iRNA targets a gene
involved in a second disorder that results from the
.beta.-thalassemia. For example, the second iRNA can target a gene
involved in diabetes mellitus, thrombosis or osteopenia.
[0037] 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
[0038] FIG. 1 is the sequence of human TMPRSS6 mRNA (Ref. Seq.
NM_153609.2, GI:56682967, Record dated Jan. 23, 2011, SEQ ID
NO:1).
[0039] FIGS. 2A and 2B depict the potency of two chemically
modified TMPRSS6 targeting siRNAs in the reduction of TMPRSS6 gene
expression in primary mouse hepatocytes.
[0040] FIGS. 3A and 3B depict the effect of LNP-TMPRSS6 siRNA-1
(AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286), on TMPRSS6 and HAMP1
gene expression, respectively, in WT C57BL/6 mice.
[0041] FIG. 4 depicts the duration of the TMPRSS6 siRNA mediated
effects on TMPRSS6 gene expression, HAMP1 gene expression, and
serum iron levels in WT C57BL/6 mice.
[0042] FIG. 5 depicts the level of TMPRSS6 siRNA mediated silencing
of TMPRSS6 necessary to maintain the TMPRSS6 siRNA mediated effects
on HAMP1 gene expression and serum iron levels in WT C57BL/6
mice.
[0043] FIGS. 6A and 6B depict the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on hematological parameters in WT C57BL/6
mice. FIG. 6A depicts the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on hemoglobin (HBG) in WT C57BL/6 mice 6
hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days post
administration. FIG. 6B depicts the effect of TMPRSS6 siRNA
mediated silencing of TMPRSS6 on hematocrit in WT C57BL/6 mice 6
hours, 24 hours, 48 hours, 72 hours, 7 days, and 14 days post
administration.
[0044] FIG. 7 depicts the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on serum iron parameters in thalassemic mice
(Th3/+), including serum iron levels, unsaturated iron binding
capacity (UIBC) levels, and transferrin saturation levels.
[0045] FIGS. 8A to 8C depict the effects of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on reticulocyte and erythrocyte parameters in
thalassemic mice (Th3/+). FIG. 8A depicts the effect on the number
of reticulocytes (%), FIG. 8B depicts the effect on the hemoglobin
content of reticulocytes (CHr), and FIG. 8C depicts the effect on
the number of mature red blood cells.
[0046] FIGS. 9A to 9D depicts the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on hematological parameters in thalassemic
mice (Th3/+). FIG. 9A depicts the effect on hematocrit (HCT)
levels, FIG. 9B depicts the effect on hemoglobin (HGB), FIG. 9C
depicts the effect on red blood cell (RBC) distribution width
(RDW), and FIG. 9D depicts the effect on mean corpuscle value
(MCV).
[0047] FIGS. 10A to 10C depict the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on spleen and liver iron content in
thalassemic mice (Th3/+). FIG. 10A depicts the effect on total
spleen iron content, FIG. 10B depicts the effect on spleen weight,
and FIG. 10C depicts the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on the concentration of iron in the liver.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Described herein are iRNAs and methods of using them for
inhibiting the expression of a TMPRSS6 gene in a cell or a mammal
where the iRNA targets a TMPRSS6 gene. Also provided are
compositions and methods for treating pathological conditions and
diseases caused by TMPRSS6 gene expression, such as conditions
associated with elevated levels of iron. iRNA directs the
sequence-specific degradation of mRNA through a process known as
RNA interference (RNAi). In an alternative embodiment, the iRNA
activates the expression of a TMPRSS6 gene in a cell or mammal,
where the iRNA targets a TMPRSS6 gene.
[0049] TMPRSS6 plays an important role in iron homeostasis as an
inhibitor of HAMP gene expression. The HAMP gene encodes for the
liver hormone hepcidin, which is a central regulator of iron
homeostasis. Hepcidin binds to the iron exporter protein
ferroportin (FPN1), which is localized mainly on absorptive
enterocytes, hepatocytes and macrophages. Hepcidin binding to the
extracellular domain of ferroportin leads to the internalization
and degradation of ferroportin, thus decreasing the absorption of
dietary iron from the intestine, and the release of iron from
macrophages and hepatocytes. HAMP gene expression can be stimulated
in response to iron through Bone Morphogenetic Protein (BMP)/Sons
of Mothers Against Decapentaplegic (SMAD)-dependent signal
transduction cascade mediated by the BMP-co-receptor hemojuvelin
(HJV). The key role of TMPRSS6 in HAMP regulation is in the
inhibition of BMP-mediated HAMP upregulation. TMPRSS6 inhibits
BMP-mediated HAMP upregulation by cleaving the BMP co-receptor HJV,
which is essential for BMP mediated HAMP upregulation; thus
preventing BMP signaling, SMAD translocation to the nucleus, and
HAMP transcriptional activation.
[0050] Several human and mouse studies have confirmed the role of
TMPRSS6 in HAMP regulation and iron homeostasis (Du et al. Science
2008, Vol. 320, pp 1088-1092; Folgueras et al. Blood 2008, Vol.
112, pp 2539-45). Studies have shown that loss of function
mutations in TMPRSS6 can lead to the upregulation of hepcidin
expression, causing an inherited iron deficiency anemia called iron
refractory iron deficiency anemia (IRIDA) (Finberg. Seminars in
Hematology 2009, Vol. 46, pp 378-86), which is characterized by
elevated hepcidin levels, hypochromic microcytic anemia, low mean
corpuscular volume (MCV), low transferrin saturation, poor
absorption of oral iron, and incomplete response to parenteral
iron. However, loss of function mutations in positive regulators of
HAMP (e.g., BMP1, BMP4, and HFE) have been shown to downregulate
hepcidin expression and cause iron overload disorders (Milet et al.
Am J Hum Gen 2007, Vol. 81, pp 799-80'7; Finberg et al. Blood 2011,
Vol. 117, pp 4590-9). In the primary iron overload disorders,
collectively called hereditary hemochromatosis (HH), in anemias
characterized by massive ineffective hematopoiesis, and in iron
overload (secondary hemochromatosis), such as .beta.-thalassemia
intermedia (TI), hepcidin levels are low despite elevated serum
iron concentrations and iron stores. A mouse model of
.beta.-thalassemia intermedia has demonstrated that the loss of
TMPRSS6 expression leads to elevated levels of hepcidin (Finberg
2010 Oral Presentation: "TMPRSS6, an inhibitor of Hepatic BMP/Smad
Signaling, is required for Hepcidin Suppression and Iron Loading in
a Mouse Model of .beta.-Thalassemia. American Society of Hematology
Annual Meeting 2010, Abstract No.:164).
[0051] The present invention describes methods and iRNA
compositions for modulating the expression of a TMPRSS6 gene. In
certain embodiments, expression of TMPRSS6 is reduced or inhibited
using a TMPRSS6-specific iRNA, thereby leading to increase HAMP
expression, and decreased serum iron levels. Thus, inhibition of
TMPRSS6 gene expression or activity using the iRNA compositions
featured in the invention can be a useful approach to therapies
aimed at reducing the iron levels in a subject. Such inhibition can
be useful for treating disorders associated with elevated iron
levels, such as hemochromatosis or thalassemia, e.g.,
.beta.-thalassemia.
[0052] The iRNAs of the compositions described 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 a TMPRSS6 gene. The use of these iRNAs enables the targeted
degradation of mRNAs of genes that are implicated in pathologies
associated with TMPRSS6 expression in mammals. Very low dosages of
TMPRSS6 iRNAs in particular can specifically and efficiently
mediate RNAi, resulting in significant inhibition of expression of
a TMPRSS6 gene. Using cell-based assays, the present inventors have
demonstrated that iRNAs targeting TMPRSS6 can specifically and
efficiently mediate RNAi, resulting in significant inhibition of
expression of a TMPRSS6 gene. Thus, methods and compositions
including these iRNAs are useful for treating pathological
processes that can be mediated by down regulating TMPRSS6, such as
in the treatment of a disorder that causes elevated iron levels,
e.g., a hemochromatosis, or a .beta.-thalassemia, e.g.,
.beta.-thalassemia intermedia. The following detailed description
discloses how to make and use compositions containing iRNAs to
inhibit the expression of a TMPRSS6 gene, as well as compositions
and methods for treating diseases and disorders caused by the
expression of this gene.
[0053] Embodiments of the pharmaceutical compositions featured
herein also 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 a TMPRSS6 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 a TMPRSS6 gene.
[0054] Accordingly, in some aspects, pharmaceutical compositions
containing a TMPRSS6 iRNA and a pharmaceutically acceptable
carrier, methods of using the compositions to inhibit expression of
a TMPRSS6 gene, and methods of using the pharmaceutical
compositions to treat diseases caused by expression of a TMPRSS6
gene are featured in the invention.
[0055] I. Definitions
[0056] 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.
[0057] "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 can be replaced in the nucleotide sequences of
dsRNA featured herein 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 described herein.
[0058] As used herein, "Transmembrane Protease, Serine 6"
("TMPSSR6") refers to a particular polypeptide expressed in a cell.
TMPRSS6 is also known as matriptase-2, IRIDA (iron refractory
iron-deficiency anemia), transmembrane protease serine 6, type II
transmembrane serine protease 6, and membrane-bound mosaic serine
proteinase matriptase-2. TMPRSS6 is a serine protease Type II
transmembrane protein of approximately 899 amino acids in length.
TMPRSS6 contains multiple domains, e.g., a short endo domain, a
transmembrane domain, a sea urchin sperm protein/enteropeptidase
domain/agrin (SEA) domain, two complement factor/urchin embryonic
growth factor/BMP domains (CUB), three LDL-R class a domains
(LDLa), and a trypsin-like serine protease domain with conserved
His-Asp-Ser triad (HDS). The sequence of a human TMPRSS6 mRNA
transcript can be found at NM_153609.2 (SEQ ID NO:1) (FIG. 1).
[0059] As used herein, the term "iRNA" refers to an agent that
contains RNA as that term is defined herein, and which mediates the
targeted cleavage of an RNA transcript via an RNA-induced silencing
complex (RISC) pathway. In one embodiment, an iRNA as described
herein effects inhibition of TMPRSS6 expression. Alternatively, in
another embodiment, an iRNA as described herein activates TMPRSS6
expression.
[0060] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of a TMPRSS6 gene, including messenger RNA
(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 there between. 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.
[0061] 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.
[0062] 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 can 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.
[0063] 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 can 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 (bp), 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.
[0064] "Complementary" sequences, as used herein, can 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 include,
but are not limited to, G:U Wobble or Hoogstein base pairing.
[0065] The terms "complementary," "fully complementary" and
"substantially complementary" herein can 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.
[0066] As used herein, a polynucleotide that is "substantially
complementary to at least part of" a messenger RNA (an mRNA) refers
to a polynucleotide that is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., an mRNA encoding
TMPRSS6). For example, a polynucleotide is complementary to at
least a part of a TMPRSS6 mRNA if the sequence is substantially
complementary to a non-interrupted portion of an mRNA encoding
TMPRSS6.
[0067] 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 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.
[0068] 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 nucleoside, 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 via
a RISC pathway.
[0069] 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."
[0070] 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 plants and
invertebrate 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] As used herein, in one embodiment, 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. Examples of "SNALP" formulations are
described elsewhere herein.
[0077] "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 can 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 are known in the art.
[0078] As used herein, the term "modulate the expression of,"
refers to at an least partial "inhibition" or partial "activation"
of TMPRSS6 gene expression in a cell treated with an iRNA
composition as described herein compared to the expression of
TMPRSS6 in an untreated cell.
[0079] The terms "activate," "enhance," "up-regulate the expression
of," "increase the expression of," and the like, in so far as they
refer to a TMPRSS6 gene, herein refer to the at least partial
activation of the expression of a TMPRSS6 gene, as manifested by an
increase in the amount of TMPRSS6 mRNA, which can be isolated from
or detected in a first cell or group of cells in which a TMPRSS6
gene is transcribed and which has or have been treated such that
the expression of a TMPRSS6 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).
[0080] In one embodiment, expression of a TMPRSS6 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,
a TMPRSS6 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 a TMPRSS6 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 TMPRSS6 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.
[0081] The terms "silence," "inhibit the expression of,"
"down-regulate the expression of," "suppress the expression of,"
and the like, in so far as they refer to a TMPRSS6 gene, herein
refer to the at least partial suppression of the expression of a
TMPRSS6 gene, as manifested by a reduction of the amount of TMPRSS6
mRNA which can be isolated from or detected in a first cell or
group of cells in which a TMPRSS6 gene is transcribed and which has
or have been treated such that the expression of a TMPRSS6 gene is
inhibited, 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). The degree
of inhibition is usually expressed in terms of
( mRNA .times. in .times. control .times. cells ) - ( mRNA .times.
in .times. treated .times. cells ) ( mRNA .times. in .times.
control .times. cells ) 100 .times. % ##EQU00001##
[0082] Alternatively, the degree of inhibition can be given in
terms of a reduction of a parameter that is functionally linked to
TMPRSS6 gene expression, e.g., the amount of protein encoded by a
TMPRSS6 gene, or the number of cells displaying a certain
phenotype, e.g., a decrease in iron levels, or in iron absorption.
In principle, TMPRSS6 gene silencing can be determined in any cell
expressing TMPRSS6, either constitutively or by genomic
engineering, and by any appropriate assay.
[0083] For example, in certain instances, expression of a TMPRSS6
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, a TMPRSS6 gene is suppressed by at
least about 60%, 70%, or 80% by administration of an iRNA described
herein. In some embodiments, a TMPRSS6 gene is suppressed by at
least about 85%, 90%, 95%, 98%, 99%, or more, by administration of
an iRNA as described herein.
[0084] As used herein in the context of TMPRSS6 expression, the
terms "treat," "treatment," and the like, refer to relief from or
alleviation of pathological processes mediated by TMPRSS6
expression. In the context of the present invention insofar as it
relates to any of the other conditions recited herein below (other
than pathological processes mediated by TMPRSS6 expression), the
terms "treat," "treatment," and the like mean to relieve or
alleviate at least one symptom associated with such condition, or
to slow or reverse the progression or anticipated progression of
such condition, such as slowing the progression of a
hemochromatosis, such as a .beta.-thalassemia.
[0085] By "lower" in the context of a disease marker or symptom is
meant a statistically 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 preferably down to a level
accepted as within the range of normal for an individual without
such disorder.
[0086] 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 mediated by TMPRSS6
expression or an overt symptom of pathological processes mediated
by TMPRSS6 expression. The specific amount that is therapeutically
effective can be readily determined by an ordinary medical
practitioner, and can vary depending on factors known in the art,
such as, for example, the type of pathological processes mediated
by TMPRSS6 expression, the patient's history and age, the stage of
pathological processes mediated by TMPRSS6 expression, and the
administration of other agents that inhibit pathological processes
mediated by TMPRSS6 expression.
[0087] 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, 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 TMPRSS6 can reduce TMPRSS6
protein levels by at least 10%. As used herein, the term
"thalassemia" refers to an inherited recessive blood disorder.
[0088] A loss-of-function mutation results in reduced rate of
synthesis or no synthesis of one of the globin chains that makes up
hemoglobin, and causes a deficiency in normal globin proteins.
Thalassemia patients produce a deficiency of either .alpha. globin
(called .alpha.-thalassemia), .beta. globin (called
.beta.-thalassemia) or, in rare cases, .delta. globin. In
.alpha.-thalassemia, an excess of .beta. chains form unstable
tetramers, which have abnormal oxygen dissociation curves.
.beta.-thalassemias can be minor, major or intermedia.
[0089] .beta. globin chains are encoded by a single gene called the
HBB (hemoglobin, .beta.) gene. .beta.-thalassemia minor occurs in
patients carrying one mutant .beta.-thalassemia allele, and one
wildtype allele. This condition has no effect on blood iron levels,
and patients do not require treatment. .beta.-thalassemia major
results when a patient carries two knock-out mutant
.beta.-thalassemia alleles. Excess iron accumulates in these
patients, and the excess iron is stored primarily in hypertrophic
Kupffer cells. Patients with .beta.-thalassemia major are typically
treated with chronic blood transfusion therapy, iron chelation,
splenectomy and allogeneic hematopoietic transplantation.
.beta.-thalassemia intermedia results when a patient carries one
knock-out allele of the .beta.-thalassemia gene and one partial
loss-of-function allele. Excess iron accumulates in these patients,
and the excess iron is stored primarily in hepatocytes. Patients
with thalassemia major and thalassemia intermedia have anemia
(hypoxia), which leads to an increase in EPO (erythropoietin) and
consequently, dramatic compensatory and ineffective erythropoiesis
(the production of red blood cells by stem cells in bone marrow).
Patients with thalassemia intermedia sometimes develop
hepatosplenomegaly, jaundice, osteopenia, thrombotic events, leg
ulcers, pulmonary hypotension, congestive heart failure, diabetes
mellitus, growth hormone deficiency, hypothyroidism,
hypoparathyroidism, hypogonadism, and facial deformities.
[0090] As used herein, the term "hemochromatosis" refers to a
disorder, which results in too much iron being absorbed from the
gastrointestinal tract. Hemochromatosis occurs in two forms:
primary and secondary. Primary hemochromatosis, the most common
genetic disorder in the United States (affecting an estimated 1 of
every 200 to 300 Americans), is usually caused by a specific
genetic problem that causes too much iron to be absorbed.
Secondary, or acquired, hemochromatosis, can be caused by diseases
such as thalassemia or sideroblastic anemia. Secondary
hemochromatosis sometimes develops in patients with hemolytic
anemia and chronic alcoholism. Symptoms of hemochromatosis include
abdominal pain, joint pain, fatigue, lack of energy, weakness,
darkening of the skin (often referred to as "bronzing"), and loss
of body hair.
[0091] 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.
[0092] As used herein, a "subject" is a mammal, e.g. a dog, horse,
cat, and other non-human primates. In a preferred embodiment, a
subject is a human.
[0093] As used herein, the term "LNPXX", wherein the "XX" are
numerals, is also referred to as "AFXX" herein. For example, LNP09
is also referred to AF09 and LNP12 is also known as or referred to
as AF12.
[0094] As used herein, the term "comprising" or "comprises" is used
in reference to compositions, methods, and respective component(s)
thereof, that are essential to the invention, yet open to the
inclusion of unspecified elements, whether essential or not.
[0095] As used herein, the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of elements that do not materially affect the basic
and novel or functional characteristic(s) of that embodiment
featured in the invention.
[0096] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0097] II. Double-Stranded Ribonucleic Acid (dsRNA)
[0098] Described herein are iRNA agents that modulate the
expression of the TMPRSS6 gene. In one embodiment, the iRNA agent
includes double-stranded ribonucleic acid (dsRNA) molecules for
inhibiting the expression of a TMPRSS6 gene in a cell or mammal,
e.g., in a human having elevated iron levels, such as in a patient
with a .beta.-thalassemia, or a hemachromatosis. 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 a TMPRSS6 gene. 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 TMPRSS6 gene, inhibits the expression of the TMPRSS6 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 a TMPRSS6 gene in a cell or mammal. Expression of a
TMPRSS6 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
TMPRSS6 mRNA levels, such as by bDNA or TaqMan.RTM. assay, or by
measuring protein levels, such as by immunofluorescence analysis,
using, for example, Western blotting or flow cytometric
techniques.
[0099] A dsRNA includes two RNA strands that are 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. The target sequence can be derived from the sequence of
an mRNA formed during the expression of a TMPRSS6 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,
preferably 15-30 nucleotides in length.
[0100] 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, 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 TMPRSS6
expression is not generated in the target cell by cleavage of a
larger dsRNA.
[0101] A dsRNA as described herein can 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, a TMPRSS6 gene is a human
TMPRSS6 gene. In another embodiment the TMPRSS6 gene is a mouse or
a rat TMPRSS6 gene. The sequence of mouse TMPRSS6 mRNA can be found
at GenBank Accession No. NM_027902 (GI:125656151, Record dated Dec.
28, 2010). The sequence of rat TMPRSS6 mRNA can be found at GenBank
Accession No. NM_001130556.1 (GI:194474097, Record dated Jan. 17,
2011). In specific embodiments, the first sequence is a sense
strand of a dsRNA that includes a sense sequence of one of Tables
2, 3 or 4 and the second sequence is an antisense strand of a dsRNA
that includes an antisense sequence of one of Tables 2, 3 or 4.
Alternative dsRNA agents that target elsewhere in the target
sequence provided in Tables 2, 3 or 4 can readily be determined
using the target sequence and the flanking TMPRSS6 sequence.
[0102] In one aspect, a dsRNA will include at least two nucleotide
sequences, a sense and an antisense sequence, whereby the sense
strand is selected from the groups of sequences provided in Tables
2, 3 or 4. In this aspect, 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 in the expression of a TMPRSS6 gene. As such, in
this aspect, a dsRNA will include two oligonucleotides, where one
oligonucleotide is described as the sense strand in Tables 2, 3 or
4 and the second oligonucleotide is described as the corresponding
antisense strand of the sense strand from Tables 2, 3 or 4. 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.
[0103] 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 or 4 dsRNAs described herein can include at least one strand
of a length of minimally 21 nt. It can be reasonably expected that
shorter duplexes having one of the sequences of Tables 2, 3 or 4
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 Tables 2,
3 or 4 and differing in their ability to inhibit the expression of
a TMPRSS6 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.
[0104] In addition, the RNAs provided in Tables 2, 3 or 4 identify
a site in a TMPRSS6 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 or 4 coupled to additional nucleotide sequences taken from the
region contiguous to the selected sequence in a TMPRSS6 gene.
[0105] 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 or 4 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.
[0106] Further, it is contemplated that for any sequence
identified, e.g., in Tables 2, 3 or 4 further optimization could be
achieved by systematically either adding or removing nucleotides to
generate longer or shorter sequences and testing those 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.
[0107] 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 a TMPRSS6 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 a TMPRSS6 gene. Consideration of the
efficacy of iRNAs with mismatches in inhibiting expression of a
TMPRSS6 gene is important, especially if the particular region of
complementarity in a TMPRSS6 gene is known to have polymorphic
sequence variation within the population.
[0108] 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. Such 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 the embodiments described herein 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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; 5,264,562; 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.
[0113] 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.
[0114] 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.
[0115] Modified RNAs can 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-dimethyl aminoethoxyethyl 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.
[0116] 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 can 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.
[0117] An iRNA can 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.
[0118] 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.
[0119] 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).
[0120] 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.
[0121] Another modification of the RNA of an iRNA featured in the
invention involves chemically linking to the RNA one or more
ligands, moieties or conjugates that enhance the activity, cellular
distribution, pharmacokinetic properties, 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).
[0122] In one embodiment, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g., molecule, cell or
cell type (e.g., a liver cell, such as a hepatocyte), 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.
Preferred ligands will not take part in duplex pairing in a
duplexed nucleic acid.
[0123] 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-glycolide) 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.
[0124] 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, vitamin A, biotin, or an RGD peptide or RGD peptide
mimetic.
[0125] 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]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.
[0126] 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.
[0127] 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.
[0128] In some embodiments, a ligand attached to an iRNA as
described herein acts as a PK modulator. As used herein, a "PK
modulator" refers to a pharmacokinetic 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 backbaone 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.
[0129] For macromolecular drugs and hydrophilic drug molecules,
which cannot easily cross bilayer membranes, entrapment in
endosomal/lysosomal compartments of the cell is thought to be the
biggest hurdle for effective delivery to their site of action. In
recent years, a number of approaches and strategies have been
devised to address this problem. For liposomal formulations, the
use of fusogenic lipids in the formulation have been the most
common approach (Singh, R. S., Goncalves, C. et al. (2004). On the
Gene Delivery Efficacies of pH-Sensitive Cationic Lipids via
Endosomal Protonation. A Chemical Biology Investigation. Chem.
Biol. 11, 713-723.). Other components, which exhibit pH-sensitive
endosomolytic activity through protonation and/or pH-induced
conformational changes, include charged polymers and peptides.
Examples may be found in Hoffman, A. S., Stayton, P. S. et al.
(2002). Design of "smart" polymers that can direct intracellular
drug delivery. Polymers Adv. Technol. 13, 992-999; Kakudo, Chaki,
T., S. et al. (2004). Transferrin-Modified Liposomes Equipped with
a pH-Sensitive Fusogenic Peptide: An Artificial Viral-like Delivery
System. Biochemistry 436, 5618-5628; Yessine, M. A. and Leroux, J.
C. (2004). Membrane-destabilizing polyanions: interaction with
lipid bilayers and endosomal escape of biomacromolecules. Adv. Drug
Deliv. Rev. 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007).
Fusogenic peptides enhance endosomal escape improving iRNA-induced
silencing of oncogenes. Int. J. Pharm. 331, 211-4. They have
generally been used in the context of drug delivery systems, such
as liposomes or lipoplexes. For folate receptor-mediated delivery
using liposomal formulations, for instance, a pH-sensitive
fusogenic peptide has been incorporated into the liposomes and
shown to enhance the activity through improving the unloading of
drug during the uptake process (Turk, M. J., Reddy, J. A. et al.
(2002). Characterization of a novel pH-sensitive peptide that
enhances drug release from folate-targeted liposomes at endosomal
pHs is described in Biochim. Biophys. Acta 1559, 56-68).
[0130] In certain embodiments, the endosomolytic components of the
present invention can be polyanionic peptides or peptidomimetics
which show pH-dependent membrane activity and/or fusogenicity. A
peptidomimetic can be a small protein-like chain designed to mimic
a peptide. A peptidomimetic can arise from modification of an
existing peptide in order to alter the molecule's properties, or
the synthesis of a peptide-like molecule using unnatural amino
acids or their analogs. In certain embodiments, they have improved
stability and/or biological activity when compared to a peptide. In
certain embodiments, the endosomolytic component assumes its active
conformation at endosomal pH (e.g., pH 5-6). The "active"
conformation is that conformation in which the endosomolytic
component promotes lysis of the endosome and/or transport of the
modular composition featured in the invention, or its any of its
components (e.g., a nucleic acid), from the endosome to the
cytoplasm of the cell.
[0131] Libraries of compounds can be screened for their
differential membrane activity at endosomal pH versus neutral pH
using a hemolysis assay. Promising candidates isolated by this
method may be used as components of the modular compositions
featured in the invention. A method for identifying an
endosomolytic component for use in the compositions and methods of
the present invention may comprise: providing a library of
compounds; contacting blood cells with the members of the library,
wherein the pH of the medium in which the contact occurs is
controlled; determining whether the compounds induce differential
lysis of blood cells at a low pH (e.g., about pH 5-6) versus
neutral pH (e.g., about pH 7-8).
[0132] Exemplary endosomolytic components include the GALA peptide
(Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA
peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586),
and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002,
1559: 56-68). In certain embodiments, the endosomolytic component
can contain a chemical group (e.g., an amino acid) which will
undergo a change in charge or protonation in response to a change
in pH. The endosomolytic component may be linear or branched.
Exemplary primary sequences of endosomolytic components include
H.sub.2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO:2);
H.sub.2N-(AALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO:3); and
H.sub.2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO:4).
[0133] In certain embodiments, more than one endosomolytic
component can be incorporated into the iRNA agent featured in the
invention. In some embodiments, this will entail incorporating more
than one of the same endosomolytic component into the iRNA agent.
In other embodiments, this will entail incorporating two or more
different endosomolytic components into iRNA agent.
[0134] These endosomolytic components can mediate endosomal escape
by, for example, changing conformation at endosomal pH. In certain
embodiments, the endosomolytic components can exist in a random
coil conformation at neutral pH and rearrange to an amphipathic
helix at endosomal pH. As a consequence of this conformational
transition, these peptides may insert into the lipid membrane of
the endosome, causing leakage of the endosomal contents into the
cytoplasm. Because the conformational transition is pH-dependent,
the endosomolytic components can display little or no fusogenic
activity while circulating in the blood (pH.about.7.4). "Fusogenic
activity," as used herein, is defined as that activity which
results in disruption of a lipid membrane by the endosomolytic
component. One example of fusogenic activity is the disruption of
the endosomal membrane by the endosomolytic component, leading to
endosomal lysis or leakage and transport of one or more components
of the modular composition featured in the invention (e.g., the
nucleic acid) from the endosome into the cytoplasm.
[0135] In addition to hemolysis assays, as described herein,
suitable endosomolytic components can be tested and identified by a
skilled artisan using other methods. For example, the ability of a
compound to respond to, e.g., change charge depending on, the pH
environment can be tested by routine methods, e.g., in a cellular
assay. In certain embodiments, a test compound is combined with or
contacted with a cell, and the cell is allowed to internalize the
test compound, e.g., by endocytosis. An endosome preparation can
then be made from the contacted cells and the endosome preparation
compared to an endosome preparation from control cells. A change,
e.g., a decrease, in the endosome fraction from the contacted cell
vs. the control cell indicates that the test compound can function
as a fusogenic agent. Alternatively, the contacted cell and control
cell can be evaluated, e.g., by microscopy, e.g., by light or
electron microscopy, to determine a difference in the endosome
population in the cells. The test compound and/or the endosomes can
labeled, e.g., to quantify endosomal leakage.
[0136] In another type of assay, an iRNA agent described herein is
constructed using one or more test or putative fusogenic agents.
The iRNA agent can be labeled for easy visualization. The ability
of the endosomolytic component to promote endosomal escape, once
the iRNA agent is taken up by the cell, can be evaluated, e.g., by
preparation of an endosome preparation, or by microscopy
techniques, which enable visualization of the labeled iRNA agent in
the cytoplasm of the cell. In certain other embodiments, the
inhibition of gene expression, or any other physiological
parameter, may be used as a surrogate marker for endosomal
escape.
[0137] In other embodiments, circular dichroism spectroscopy can be
used to identify compounds that exhibit a pH-dependent structural
transition.
[0138] A two-step assay can also be performed, wherein a first
assay evaluates the ability of a test compound alone to respond to
changes in pH, and a second assay evaluates the ability of a
modular composition that includes the test compound to respond to
changes in pH.
[0139] Lipid Conjugates
[0140] In one ligand, the ligand or conjugate is a lipid or
lipid-based molecule. Such a lipid or lipid-based molecule
preferably binds a serum protein, e.g., 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.
[0141] A lipid based ligand can be used to modulate, e.g., control
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.
[0142] In a preferred embodiment, the lipid based ligand binds HSA.
Preferably, it binds HSA with a sufficient affinity such that the
conjugate will be preferably distributed to a non-kidney tissue.
However, it is preferred that the affinity not be so strong that
the HSA-ligand binding cannot be reversed.
[0143] In another preferred embodiment, the lipid based ligand
binds HSA weakly or not at all, such that the conjugate will be
preferably distributed to the kidney. Other moieties that target to
kidney cells can also be used in place of or in addition to the
lipid based ligand.
[0144] 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. 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).
[0145] In another aspect, the ligand is a cell-permeation agent,
preferably a helical cell-permeation agent. Preferably, 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 preferably an .alpha.-helical agent, which preferably has
a lipophilic and a lipophobic phase.
[0146] Cell Permeation Peptides
[0147] Peptides suitable for use with the present invention can be
a natural peptide, e.g., tat or antennapedia peptide, a synthetic
peptide, or a peptidomimetic. Furthermore, the peptide can be a
modified peptide, for example peptide can comprise non-peptide or
pseudo-peptide linkages, and D-amino acids. 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.
[0148] 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:5). An
RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6))
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:7)) and the Drosophila
Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:8)) 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). Preferably, the peptide or peptidomimetic
tethered to the lipid 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.
[0149] An RGD peptide moiety can be used to target 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). Preferably, 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).
[0150] Peptides that target markers enriched in proliferating cells
can be used. E.g., RGD containing peptides and peptidomimetics can
target cancer cells, in particular cells that exhibit an
.alpha.v.beta.3 integrin. Thus, one could use RGD peptides, cyclic
peptides containing RGD, RGD peptides that include D-amino acids,
as well as synthetic RGD mimics. In addition to RGD, one can use
other moieties that target the .alpha.v.beta.3 integrin ligand.
Generally, such ligands can be used to control proliferating cells
and angiogeneis.
[0151] 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).
[0152] Carbohydrate Conjugates
[0153] In some embodiments, the iRNA oligonucleotides described
herein further comprise carbohydrate conjugates. The carbohydrate
conjugates 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 may
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 may 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
4-9 monosaccharide units), and polysaccharides such as starches,
glycogen, cellulose and polysaccharide gums. Specific
monosaccharides include C.sub.5 and above (preferably
C.sub.5-C.sub.8) sugars; di- and trisaccharides include sugars
having two or three monosaccharide units (preferably
C.sub.5-C.sub.8).
[0154] In one embodiment, the carbohydrate conjugate is selected
from the group consisting of:
##STR00001## ##STR00002## ##STR00003## ##STR00004##
i.e., Formula II-Formula XXII.
[0155] Another representative carbohydrate conjugate for use in the
embodiments described herein includes, but is not limited to,
##STR00005##
when one of X or Y is an oligonucleotide, the other is a
hydrogen.
[0156] In some embodiments, the carbohydrate conjugate further
comprises other ligand such as, but not limited to, PK modulator,
endosomolytic ligand, and cell permeation peptide.
[0157] Linkers
[0158] In some embodiments, the conjugates described herein can be
attached to the iRNA oligonucleotide with various linkers that can
be cleavable or non cleavable.
[0159] The term "linker" or "linking group" means an organic moiety
that connects two parts of a compound. Linkers typically comprise a
direct bond or an atom such as oxygen or sulfur, a unit such as
NR.sup.8, 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(R.sup.8), C(O), substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heterocyclic; where R.sup.8 is hydrogen, acyl,
aliphatic or substituted aliphatic. In one embodiment, the linker
is between 1-24 atoms, preferably 4-24 atoms, preferably 6-18
atoms, more preferably 8-18 atoms, and most preferably 8-16
atoms.
[0160] 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 10 times or more, preferably at least 100 times faster in the
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).
[0161] 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.
[0162] 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 the cationic lipid from the ligand inside the
cell, or into the desired compartment of the cell.
[0163] 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, liver targeting ligands can be linked to the
cationic lipids 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.
[0164] Linkers that contain peptide bonds can be used when
targeting cell types rich in peptidases, such as liver cells and
synoviocytes.
[0165] 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 may 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 2, 4, 10 or 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).
[0166] Redox Cleavable Linking Groups
[0167] One class of cleavable linking groups are redox cleavable
linking groups that are 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 a preferred embodiment, candidate compounds
are cleaved by at most 10% in the blood. In preferred embodiments,
useful candidate compounds are degraded at least 2, 4, 10 or 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.
[0168] Phosphate-Based Cleavable Linking Groups
[0169] Phosphate-based cleavable linking groups are 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.
[0170] Acid Cleavable Linking Groups
[0171] Acid cleavable linking groups are linking groups that are
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, 5.5, 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.
[0172] Ester-Based Linking Groups
[0173] Ester-based cleavable linking groups are 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.
[0174] Peptide-Based Cleaving Groups
[0175] Peptide-based cleavable linking groups are 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 --NHCHR.sup.AC(O)NHCHR.sup.BC(O)--, where
R.sup.A and R.sup.B are the R groups of the two adjacent amino
acids. These candidates can be evaluated using methods analogous to
those described above.
[0176] Representative carbohydrate conjugates with linkers include,
but are not limited to,
##STR00006## ##STR00007## ##STR00008##
when one of X or Y is an oligonucleotide, the other is a
hydrogen.
[0177] 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; each of
which is herein incorporated by reference.
[0178] 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 can 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.
"Chimeric" iRNA compounds or "chimeras," in the context of this
invention, are iRNA compounds, preferably dsRNAs, which 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.
[0179] 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 al., 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 RNA bearing an amino linker 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.
[0180] Delivery of iRNA
[0181] 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.
[0182] Direct Delivery of an iRNA Composition
[0183] 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. 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
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, a liposome, 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.
[0184] Vector Encoded iRNAs
[0185] In another aspect, iRNA targeting the TMPRSS6 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, 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).
[0186] 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, the strands of a dsRNA are expressed as inverted repeat
polynucleotides joined by a linker polynucleotide sequence such
that the dsRNA has a stem and loop structure.
[0187] iRNA expression vectors are generally DNA plasmids or viral
vectors. Expression vectors compatible with eukaryotic cells,
preferably those compatible 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 are provided containing 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.
[0188] iRNA expression plasmids can be transfected into target
cells as a complex with cationic lipid carriers (e.g.,
Oligofectamine) or non-cationic lipid-based carriers (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.
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] Another preferred 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.
[0196] 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.
[0197] 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
[0198] In one embodiment, provided herein are pharmaceutical
compositions containing an iRNA and a pharmaceutically acceptable
carrier. The pharmaceutical composition containing the iRNA is
useful for treating a disease or disorder associated with the
expression or activity of a TMPRSS6 gene, such as pathological
processes mediated by TMPRSS6 expression. Such pharmaceutical
compositions are formulated based on the mode of delivery. One
example is compositions that are formulated for systemic
administration via parenteral delivery, e.g., by intravenous (IV)
delivery.
[0199] The pharmaceutical compositions featured herein are
administered in dosages sufficient to inhibit expression of TMPRSS6
genes. 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 once
weekly, or once monthly, or once every other month. The composition
can alternatively be administered twice per week or twice per
month, or once every two, three or four weeks. In some embodiments,
the iRNA is 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 could be used with the agents of the present
invention. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0200] The effect of a single dose on TMPRSS6 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.
[0201] 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.
[0202] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
pathological processes mediated by TMPRSS6 expression. Such models
can be used for in vivo testing of iRNA, as well as for determining
a therapeutically effective dose. A suitable mouse model is, for
example, a mouse containing a transgene expressing human
TMPRSS6.
[0203] 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.
[0204] The iRNA can be delivered in a manner to target a particular
tissue, such as the liver (e.g., the hepatocytes of the liver).
[0205] 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, di stearolyphosphatidyl
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.
[0206] Liposomal Formulations
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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
[0214] 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. 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).
[0215] Liposomes which are pH-sensitive or negatively-charged,
entrap nucleic acids 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 nucleic acids 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).
[0216] 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.
[0217] 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).
[0218] 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 cyclosporine A into different layers
of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
[0219] 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).
[0220] 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).
[0221] 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.1215G, that contains a PEG moiety. Ilium 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.)
describes PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0222] 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.
[0223] 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.
[0224] 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).
[0225] 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.
[0226] 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.
[0227] 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.
[0228] 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.
[0229] 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).
[0230] Nucleic Acid Lipid Particles
[0231] In one embodiment, a TMPRSS6 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.
[0232] 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.
[0233] The cationic lipid can 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)tetrahydro-
-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 G1), 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] LNP01
[0240] 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 in its entirety),
Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar
Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e.,
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.
##STR00009##
[0241] LNP01 formulations are described, e.g., in International
Application Publication No. WO 2008/042973, which is hereby
incorporated by reference.
Additional exemplary lipid-dsRNA formulations are as follows:
TABLE-US-00001 cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid conjugate Cationic Lipid Lipid:siRNA
ratio SNALP- 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-
1 dimethylaminopropane (DLinDMA) cDMA (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]-dioxolane (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]dioxol-5-amine (ALN100) LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG
6,9,28,31-tetraen-19-yl 4- (MC3) 50/10/38.5/1.5
(dimethylamino)butanoate Lipid:siRNA 10:1 LNP12
1,1'-(2-(4-(24(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/GalNAc- 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)
[0242] SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane
(DLinDMA)) comprising formulations are described in International
Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby
incorporated by reference.
[0243] XTC comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S.
Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S.
Provisional Ser. No. 61/185,712, filed Jun. 10, 2009; U.S.
Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S.
Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and
International Application No. PCT/US2010/022614, filed Jan. 29,
2010, which are hereby incorporated by reference.
[0244] MC3 comprising formulations are described, e.g., in U.S.
Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S.
Provisional Ser. 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.
[0245] ALNY-100 comprising formulations are described, e.g.,
International patent application number PCT/US09/63933, filed on
Nov. 10, 2009, which is hereby incorporated by reference.
[0246] C12-200 comprising formulations are described in U.S.
Provisional Ser. 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.
[0247] As used herein, the term "LNPXX", wherein the "XX" are
numerals, is also referred to as "AFXX" herein. For example, LNP09
is also referred to AF09 and LNP12 is also known as or referred to
as AF12.
[0248] Synthesis of Cationic Lipids
[0249] Any of the compounds, e.g., cationic lipids and the like,
used in the nucleic acid-lipid particles featured in the invention
can 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.
[0250] "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.
Representative saturated cyclic alkyls include cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, and the like; while
unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl,
and the like.
[0251] "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.
[0252] "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.
[0253] "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.
[0254] "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 can 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.
[0255] 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.times.C(.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.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.
[0256] "Halogen" means fluoro, chloro, bromo and iodo.
[0257] In some embodiments, the methods featured in the invention
can 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.
[0258] Synthesis of Formula A
[0259] In some embodiments, nucleic acid-lipid particles featured
in the invention are formulated using a cationic lipid of formula
A:
##STR00010##
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.
##STR00011##
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.
##STR00012##
[0260] 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.
[0261] Synthesis of MC3
[0262] 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).
[0263] Synthesis of ALLAY-100
[0264] Synthesis of ketal 519 [ALNY-100] was performed using the
following scheme 3:
##STR00013##
[0265] Synthesis of 515
[0266] To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in
200 ml anhydrous THF 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).
[0267] Synthesis of 516
[0268] 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 (CDCl3, 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%).
[0269] Synthesis of 517A and 517E
[0270] 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
NaHCO3(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
[0271] 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.
[0272] Synthesis of 518
[0273] 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 (CDCl3, 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%.
[0274] General Procedure for the Synthesis of Compound 519
[0275] 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 .quadrature.=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.
[0276] 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.
[0277] 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.
[0278] 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.
[0279] 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. Particularly preferred are
formulations that target the liver when treating hepatic disorders
such as hepatic carcinoma.
[0280] The pharmaceutical formulations of 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.
[0281] The compositions of 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 of the present
invention 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.
[0282] Additional Formulations
[0283] Emulsions
[0284] The compositions of the present invention can 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.
[0285] 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).
[0286] 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).
[0287] 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.
[0288] 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).
[0289] 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.
[0290] 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.
[0291] 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.
[0292] 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, 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). 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).
[0293] 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.
[0294] 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.
[0295] 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.
[0296] 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.
[0297] Penetration Enhancers
[0298] 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.
[0299] 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.
[0300] 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).
[0301] 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).
[0302] 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).
[0303] 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).
[0304] 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 includes, 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).
[0305] 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.
[0306] 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.
[0307] Carriers
[0308] 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.
[0309] Excipients
[0310] 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.)
[0311] 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.
[0312] 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.
[0313] 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.
[0314] Other Components
[0315] 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.
[0316] Aqueous suspensions can contain substances that increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0317] In some embodiments, pharmaceutical compositions featured in
the invention include (a) one or more iRNA compounds and (b) one or
more anti-cytokine biologic agents which function by a non-RNAi
mechanism. Examples of such biologics include, biologics that
target IL1.beta. (e.g., anakinra), IL6 (e.g., tocilizumab), or TNF
(e.g., etanercept, infliximab, adlimumab, or certolizumab).
[0318] 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 preferred.
[0319] 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 herein lies generally
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage can 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 can 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.
[0320] In addition to their administration, as discussed above, the
iRNAs described herein can be administered in combination with
other known agents effective in treatment of pathological processes
mediated by TMPRSS6 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.
[0321] Methods for Treating Diseases Caused by Expression of a
TMPRSS6 Gene
[0322] The invention relates in particular to the use of an iRNA
targeting TMPRSS6 and compositions containing at least one such
iRNA for the treatment of a TMPRSS6-mediated disorder or disease.
For example, a composition containing an iRNA targeting a TMPRSS6
gene is used for treatment of a disorder associated with elevated
iron levels, such as a thalassemia, (e.g., .beta.-thalassemia
intermedia or .alpha.-thalassemia), primary hemochromatosis,
secondary hemochromatosis, severe juvenile hemochromatosis,
sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia,
or sickle-cell anemia. In one embodiment, a TMPRSS6 iRNA is used to
treat a hemoglobinopathy. The TMPRSS6 iRNAs featured in the
invention can also be used to treat elevated levels of iron due to
other conditions, such as, chronic alcoholism.
[0323] In thalassemias, the bone marrow synthesizes insufficient
amounts of a hemoglobin chain; this in turn reduces the production
of red blood cells and causes anemia. Either the a or the .beta.
chain may be affected, but .beta. thalassemias are more common;
newborn babies are healthy because their bodies still produce HbF,
which does not have .beta. chains; during the first few months of
life, the bone marrow switches to producing HbA, and symptoms start
to appear.
[0324] .beta.-thalassemias result from mutation with either
non-expressing (.beta..sup.0) or low expressing (.beta..sup.+)
alleles of the HBB gene. .beta.-thalassemias vary in severity
depending on the genotype, and include minor/trait
.beta.-thalassemia (.beta./.beta..sup.0 or .beta./.beta.+),
intermedia .beta.-thalassemia (.beta..sup.0/.beta.+), and major
.beta.-thalassemia (.beta..sup.0/.beta..sup.0 or
.beta..sup.+/.beta..sup.+).
[0325] Thalassemia intermedia (TI) typically presents with little
hemolysis, while major .beta.-thalassemia (TM) is typically
accompanied by abundant hemolysis which causes, e.g., anemia and
splenomegaly; and highly ineffective erythropoiesis, which causes
bone marrow drive (skeletal changes, oteopenia), increased
erythropoietin synthesis, hepato-splenomegaly, consumption of
haematinics (megablastic anemia), and high uric acid in blood. The
iRNAs featured in the invention, e.g., TMPRSS6 iRNAs, are better
suited for treating the iron overload that typically accompanies
thalassemia's that are more TI like (e.g., for treating individuals
having a .beta..sup.0/.beta.+, .beta./.beta..sup.0 or
.beta./.beta.+ genotype).
[0326] Symptoms of .beta.-thalassemias also include, e.g.,
complication due to therapy, e.g., iron overload, which causes
endocrinopathy, liver fibrosis and cardiac fibrosis. Administration
of an iRNA agent that targets TMPRSS6 can be effective to treat one
or more of these symptoms.
[0327] .alpha.-thalassemias result from mutation with either
non-expressing (.alpha..sup.0) or low expressing (.alpha..sup.+)
alleles of the HBA1 or HBA2 genes. .alpha.-thalassemias vary in
severity depending on the genotype, and include trait thalassemia
(-.alpha./.alpha..alpha.), Hb Bart and Hydrops fetalis
(.alpha..sup.0/.alpha..sup.0), .alpha.-Thalaseemia minor
(--/.alpha..alpha.), (-.alpha./-.alpha.), and HbH disease
(--/-.alpha.). Lower .alpha.-globin chains are produced, resulting
in an excess of .beta. chains in adults and excess y chains in
newborns. The excess .beta. chains form unstable tetramers (called
Hemoglobin H or HbH of 4 beta chains), which have abnormal oxygen
dissociation curves. Administration of an iRNA agent that targets
TMPRSS6 can be effective to treat iron overload in a subject who
has an .alpha.-thalassemias.
[0328] Symptoms of hemochromatosis include, e.g., abdominal pain,
joint pain, fatigue, lack of energy, weakness, darkening of the
skin (often referred to as "bronzing"), and loss of body hair.
Administration of an iRNA agent that targets TMPRSS6 can be
effective to treat one or more of these symptoms.
[0329] Other symptoms associated with iron overload include
increased risk for liver disease (cirrhosis, cancer), heart attack
or heart failure, diabetes mellitus, osteoarthritis, osteoporosis,
metabolic syndrome, hypothyroidism, hypogonadism, and in some cases
premature death. Iron mismanagement resulting in overload can also
accelerate such neurodegenerative diseases as Alzheimer's,
early-onset Parkinson's, Huntington's, epilepsy and multiple
sclerosis. Administration of an iRNA that targets TMPRSS6, e.g., an
iRNA described in Tables 2, 3 or 4 can treat one or more of these
symptoms, or prevent the development or progression of a disease or
disorder that is aggrevated by increased iron levels.
[0330] The invention further relates to the use of an iRNA or a
pharmaceutical composition thereof, e.g., for treating a disorder
associated with elevated iron levels, 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 these
disorders. For example, in certain embodiments, an iRNA targeting
TMPRSS6 is administered in combination with, e.g., iron chelators
(e.g., desferroxamine), folic acid, a blood transfusion, a
phlebotomy, agents to manage ulcers, agents to increase fetal
hemoglobin levels (e.g., hydroxyurea), agents to control infection
(e.g., antibiotics and antivirals), agents to treat thrombotic
state, or a stem cell or bone marrow transplant. A stem cell
transplant can utilize stem cells from an umbilical cord, such as
from a relative, e.g., a sibling. Exemplary iron chelators include
desferroxamine, Deferasirox (Exjade), deferoprone, vitamin E, wheat
germ oil, tocophersolan, and indicaxanthin.
[0331] The iRNA and an additional therapeutic agent can be
administered in the same composition, e.g., parenterally, or the
additional therapeutic agent can be administered as part of a
separate composition or by another method described herein.
Administration of the TMPRSS6 iRNA and the additional therapeutic
agent can be at the same time, or at different times and, in any
order.
[0332] The invention features a method of administering an iRNA
agent targeting TMPRSS6 to a patient having a disease or disorder
mediated by TMPRSS6 expression, such as a disorder associated with
elevated iron levels. Administration of the dsRNA can lower iron
levels, lower ferritin levels, and/or lower transferrin saturation
levels. For example, administration of the dsRNA can lower serum
iron levels and/or lower serum ferritin levels. Transferrin
saturation levels can be lowered by 5%, 10%, 15%, 20%, 25% or more.
Transferrin saturation levels can be lowered to below 50%, below
45%, below 40%, below 35%, below 35% or lower. Transferrin
saturation is a measure of the amount of iron bound to serum
transferrin, and corresponds to the ratio of serum iron and total
iron-binding capacity.
[0333] By "lower" in this context is meant a statistically
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 preferably down to a level accepted as within the
range of normal for an individual without such disorder.
[0334] Efficacy of treatment or prevention of disease can be
assessed, for example by measuring disease progression, disease
remission, symptom severity, reduction in pain, quality of life,
dose of a medication required to sustain a treatment effect, level
of a disease marker or any other measurable parameter appropriate
for a given disease being treated or targeted for prevention. 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. For example, the
levels of transferrin saturation or serum ferritin can be monitored
for efficacy of a given treatment regime.
[0335] Iron level tests are typically performed on a sample of a
pateint's blood. An iron level test measure the amount of iron in
the blood serum that is being carried by the proteins trasferrin. A
TIBC (Total iron-binding capacity) test measures the amount of iron
that the blood would carry if the transferrin were fully saturated.
Since transferrin is produced by the liver, the TIBC can be used to
monitor liver function and nutrition. The transferrin test is a
direct measure of transferrin (also called siderophilin) levels in
the blood. The saturation level of transferrin can be calculated by
dividing the serum iron level by the TIBC. The ferritin test
measures the level of a protein in the blood that stores iron for
later use by the body.
[0336] The iRNA treatments described herein can be used to treat
individuals having elevated iron levels, as may be indicated by
iron levels in serum e.g., iron levels measuring greater than 350
.mu.g/dL, greater than 500 .mu.g/dL, greater than 1000 .mu.g/dL, or
more. In an embodiment, elevated levels of iron in serum, e.g.,
greater than 15, 20, 25, or 30 mg/g dry weight.
[0337] The iRNA treatments described herein can be used to treat
individuals having elevated iron levels, as may be indicated by
elevated ferritin levels in serum, e.g., ferritin levels measuring
greater than 300 .mu.g/L, greater than 500 .mu.g/L, greater than
1000 .mu.g/L, greater than 1500 .mu.g/L, greater than 2000 .mu.g/L,
greater than 2500 .mu.g/L, or 3000 .mu.g/L, or more.
[0338] The iRNA treatments described herein can be used to treat
individuals having elevated iron levels, as may be indicated by
elevated transferrin levels in serum, e.g., transferrin levels
measuring greater than 400 mg/dL, greater than 500 mg/L, greater
than 1000 mg/dL, or more.
[0339] The iRNA treatments described herein can be used to treat
individuals having moderately elevated iron levels, as may be
indicated by moderately elevated transferrin saturation levels,
e.g., saturation levels of 40%, 45%, or 50% or more. In addition,
the treatment described herein may also be used to prevent elevated
iron levels in individuals with only minor elevations in
transferrin saturation. One of skill in the art can easily monitor
the transferrin saturation levels in subjects receiving treatment
with iRNA as described herein and assay for a reduction in
transferrin saturation levels of at least 5% or 10%.
[0340] The iRNA treatments described herein can be used to treat
individuals having elevated iron levels, as may be indicated by a
TIBC value greater than 400 .mu.g/dL, greater than 500 .mu.g/dL, or
greater than 1000 .mu.g/dL, or more.
[0341] In some embodiments, individuals in need of treatment with a
TMPRSS6 siRNA have decreased hematocrit levels, decreased
hemoglobin levels, increased red blood cell distribution width,
increased number of reticulocytes, decreased number of mature red
blood cells, increased unsaturated iron binding capacity, decreased
ineffective erythropoiesis, decreased extradedullary hematopoiesis,
and/or decreased HAMP1 expression levels.
[0342] A patient can be further monitored by assay of blood sugar
(glucose) level or a fetoprotein level, by echocardiogram (e.g., to
examine the heart's function), electrocardiogram (ECG) (e.g., to
look at the electrical activity of the heart), imaging tests (such
as CT scans, MRI and ultrasound), and liver function tests. Excess
iron staining or iron concentrations can be measured on liver
biopsy samples, or to confirm the extent of liver damage, e.g., the
stage of liver disease.
[0343] A treatment or preventive effect is evident when there is 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, and preferably 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
or symptom is observed.
[0344] Alternatively, the efficacy can be measured by a reduction
in the severity of disease as determined by one skilled in the art
of diagnosis based on a clinically accepted disease severity
grading scale.
[0345] Patients can be administered a therapeutic amount of iRNA,
such as 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg,
1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The iRNA can be
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. The administration is repeated, for example, on a regular
basis, such as 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. Administration of the iRNA can
reduce TMPRSS6 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.
[0346] Before administration of a full dose of the iRNA, patients
can be administered a smaller dose, such as a 5% infusion reaction,
and monitored for adverse effects, such as an allergic reaction or
a worsening of symptoms. In another example, the patient can be
monitored for unwanted immunostimulatory effects, such as increased
cytokine (e.g., TNF-.alpha. or INF-.alpha.) levels.
[0347] Many disorders associated with elevated iron levels are
hereditary. Therefore, a patient in need of a TMPRSS6 iRNA may be
identified by taking a family history. A healthcare provider, such
as a doctor, nurse, or family member, can take a family history
before prescribing or administering a TMPRSS6 dsRNA. A DNA test may
also be performed on the patient to identify a mutation in the
TMPRSS6 gene, before a TMPRSS6 dsRNA is administered to the
patient. For example, diagnosis of hereditary hemochromatosis can
be confirmed by identifying the two HFE (Hemochromatosis) gene
mutations C282Y and H63D, according to GenBank Accession No.
CAB07442.1 (GI:1890180, record dated Oct. 23, 2008).
[0348] Owing to the inhibitory effects on TMPRSS6 expression, a
composition according to the invention or a pharmaceutical
composition prepared therefrom can enhance the quality of life.
[0349] Methods for Modulating Expression of a TMPRSS6 Gene
[0350] In yet another aspect, the invention provides a method for
modulating (e.g., inhibiting or activating) the expression of a
TMPRSS6 gene in a mammal.
[0351] In one embodiment, the method includes administering a
composition featured in the invention to the mammal such that
expression of the target TMPRSS6 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.
The effect of the decreased target TMPRSS6 gene preferably results
in a decrease in iron absorption and/or mobilization in the body.
Decreased iron absorption or mobilization can be manifested by an
observed decrease in serum ferritin levels, serum or liver iron
levels, and/or serum transferrin saturation levels. In some
embodiments, one or more of serum ferritin levels, serum or liver
iron levels, or serum transferrin saturation levels are decreased
by at least 5%, at least 10%, at least 15%, at least 20%, at least
25%, at least 30%, at least 40%, at least 50%, or at least 60%, or
more, as compared to pretreatment levels. In some embodiments,
serum ferritin levels are decreased by at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 40%, at least
50%, or at least 60%, or more, as compared to pretreatment
levels.
[0352] In another embodiment, the method includes administering a
composition as described herein to a mammal such that expression of
the target TMPRSS6 gene is increased by e.g., at least 10% compared
to an untreated animal. In some embodiments, the activation of
TMPRSS6 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 TMPRSS6 expression by stabilizing the TMPRSS6 mRNA
transcript, interacting with a promoter in the genome, and/or
inhibiting an inhibitor of TMPRSS6 expression.
[0353] The iRNAs useful for the methods and compositions featured
in the invention specifically target RNAs (primary or processed) of
the target TMPRSS6 gene. Compositions and methods for inhibiting
the expression of these TMPRSS6 genes using iRNAs can be prepared
and performed as described elsewhere herein.
[0354] 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 TMPRSS6 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. In
certain embodiments, the compositions are administered by
intravenous infusion or injection.
[0355] 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. Interference RNA (iRNA) Synthesis
[0356] Source of Reagents
[0357] 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.
[0358] Oligonucleotide SYNTHESIS
[0359] Applicants have used several different methods to generate
the iRNA molecules described herein. This Example describes one
approach that has been used. The ordinarily skilled artisan can use
any method known in the art to prepare iRNAs as described
herein.
[0360] Oligonucleotides are synthesized on an AKTAoligopilot
synthesizer. Commercially available controlled pore glass solid
support (dT-CPG, 500 .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.
[0361] 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.
[0362] Deprotection I (Nucleobase Deprotection)
[0363] 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 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.
[0364] Deprotection II (Removal of 2'-TBDMS Group)
[0365] 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.
[0366] Analysis
[0367] 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.
[0368] HPLC Purification
[0369] 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.
[0370] iRNA Preparation
[0371] For the general preparation of iRNA, 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.
[0372] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 1.
TABLE-US-00002 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 C cytidine G guanosine T thymidine U uridine N any
nucleotide (G, A, C, T or U) a 2'-O-methyladenosine c
2'-O-methylcytidine g 2'-O-methylguanosine u 2'-O-methyluridine dT
2'-deoxythymidine s phosphorothioate linkage
Example 2. TMPRSS6 siRNA Design
[0373] Transcripts
[0374] siRNAs targeting TMPRSS6 were designed and synthesized. The
design used human transcript NM_153609.2 (SEQ ID NO:1, FIG. 1) from
the NCBI Refseq collection.
[0375] siRNA duplexes were designed with 100% identity to the
TMPRSS6 gene.
[0376] A total of 655 sense and 655 antisense human TMPRSS6 derived
siRNA oligos were designed. The oligos are presented in Table 2.
Additional sense and antisense human TMPRSS6 derived siRNA oligos
are presented in Table 3. Sense and antisense human TMPRSS6 derived
siRNA oligos with modifications are presented in Table 4.
TABLE-US-00003 TABLE 2 Sense and antisense strand sequences of
human TMPRSS6 dsRNAs position of 5' base on transcript
(NM_153609.2, Sense Sequence SEQ Antisense SEQ SEQ ID NO: 1) (5' to
3') ID NO: Sequence (5' to 3') ID NO: 36 CUCUGGUGCGAGCUGACCU 9
AGGUCAGCUCGCACCAGAG 10 46 AGCUGACCUGAGAUGCACU 11
AGUGCAUCUCAGGUCAGCU 12 72 UCUGUGAGCUGUCUCGGCA 13
UGCCGAGACAGCUCACAGA 14 78 AGCUGUCUCGGCACCCACU 15
AGUGGGUGCCGAGACAGCU 16 79 GCUGUCUCGGCACCCACUU 17
AAGUGGGUGCCGAGACAGC 18 100 AGUCACUGCCGCCUGAUGU 19
ACAUCAGGCGGCAGUGACU 20 104 ACUGCCGCCUGAUGUUGUU 21
AACAACAUCAGGCGGCAGU 22 105 CUGCCGCCUGAUGUUGUUA 23
UAACAACAUCAGGCGGCAG 24 107 GCCGCCUGAUGUUGUUACU 25
AGUAACAACAUCAGGCGGC 26 110 GCCUGAUGUUGUUACUCUU 27
AAGAGUAACAACAUCAGGC 28 124 CUCUUCCACUCCAAAAGGA 29
UCCUUUUGGAGUGGAAGAG 30 131 ACUCCAAAAGGAUGCCCGU 31
ACGGGCAUCCUUUUGGAGU 32 233 GUGAGGACUCCAAGAGAAA 33
UUUCUCUUGGAGUCCUCAC 34 311 CUUCGGCGGGGGUGCUACU 35
AGUAGCACCCCCGCCGAAG 36 313 UCGGCGGGGGUGCUACUCU 37
AGAGUAGCACCCCCGCCGA 38 316 GCGGGGGUGCUACUCUGGU 39
ACCAGAGUAGCACCCCCGC 40 318 GGGGGUGCUACUCUGGUAU 41
AUACCAGAGUAGCACCCCC 42 319 GGGGUGCUACUCUGGUAUU 43
AAUACCAGAGUAGCACCCC 44 329 UCUGGUAUUUCCUAGGGUA 45
UACCCUAGGAAAUACCAGA 46 331 UGGUAUUUCCUAGGGUACA 47
UGUACCCUAGGAAAUACCA 48 332 GGUAUUUCCUAGGGUACAA 49
UUGUACCCUAGGAAAUACC 50 363 GGUCAGCCAGGUGUACUCA 51
UGAGUACACCUGGCUGACC 52 367 AGCCAGGUGUACUCAGGCA 53
UGCCUGAGUACACCUGGCU 54 375 GUACUCAGGCAGUCUGCGU 55
ACGCAGACUGCCUGAGUAC 56 377 ACUCAGGCAGUCUGCGUGU 57
ACACGCAGACUGCCUGAGU 58 380 CAGGCAGUCUGCGUGUACU 59
AGUACACGCAGACUGCCUG 60 382 GGCAGUCUGCGUGUACUCA 61
UGAGUACACGCAGACUGCC 62 383 GCAGUCUGCGUGUACUCAA 63
UUGAGUACACGCAGACUGC 64 384 CAGUCUGCGUGUACUCAAU 65
AUUGAGUACACGCAGACUG 66 389 UGCGUGUACUCAAUCGCCA 67
UGGCGAUUGAGUACACGCA 68 391 CGUGUACUCAAUCGCCACU 69
AGUGGCGAUUGAGUACACG 70 392 GUGUACUCAAUCGCCACUU 71
AAGUGGCGAUUGAGUACAC 72 394 GUACUCAAUCGCCACUUCU 73
AGAAGUGGCGAUUGAGUAC 74 406 CACUUCUCCCAGGAUCUUA 75
UAAGAUCCUGGGAGAAGUG 76 418 GAUCUUACCCGCCGGGAAU 77
AUUCCCGGCGGGUAAGAUC 78 420 UCUUACCCGCCGGGAAUCU 79
AGAUUCCCGGCGGGUAAGA 80 421 CUUACCCGCCGGGAAUCUA 81
UAGAUUCCCGGCGGGUAAG 82 423 UACCCGCCGGGAAUCUAGU 83
ACUAGAUUCCCGGCGGGUA 84 427 CGCCGGGAAUCUAGUGCCU 85
AGGCACUAGAUUCCCGGCG 86 428 GCCGGGAAUCUAGUGCCUU 87
AAGGCACUAGAUUCCCGGC 88 446 UCCGCAGUGAAACCGCCAA 89
UUGGCGGUUUCACUGCGGA 90 447 CCGCAGUGAAACCGCCAAA 91
UUUGGCGGUUUCACUGCGG 92 502 CGCCUGGGAACUUACUACA 93
UGUAGUAAGUUCCCAGGCG 94 503 GCCUGGGAACUUACUACAA 95
UUGUAGUAAGUUCCCAGGC 96 505 CUGGGAACUUACUACAACU 97
AGUUGUAGUAAGUUCCCAG 98 517 UACAACUCCAGCUCCGUCU 99
AGACGGAGCUGGAGUUGUA 100 518 ACAACUCCAGCUCCGUCUA 101
UAGACGGAGCUGGAGUUGU 102 520 AACUCCAGCUCCGUCUAUU 103
AAUAGACGGAGCUGGAGUU 104 541 UUUGGGGAGGGACCCCUCA 105
UGAGGGGUCCCUCCCCAAA 106 550 GGACCCCUCACCUGCUUCU 107
AGAAGCAGGUGAGGGGUCC 108 563 GCUUCUUCUGGUUCAUUCU 109
AGAAUGAACCAGAAGAAGC 110 566 UCUUCUGGUUCAUUCUCCA 111
UGGAGAAUGAACCAGAAGA 112 593 AGCACCGCCGGCUGAUGCU 113
AGCAUCAGCCGGCGGUGCU 114 680 UCCCCUACAGGGCCGAGUA 115
UACUCGGCCCUGUAGGGGA 116 683 CCUACAGGGCCGAGUACGA 117
UCGUACUCGGCCCUGUAGG 118 686 ACAGGGCCGAGUACGAAGU 119
ACUUCGUACUCGGCCCUGU 120 689 GGGCCGAGUACGAAGUGGA 121
UCCACUUCGUACUCGGCCC 122 710 CCGAGGGCCUAGUGAUCCU 123
AGGAUCACUAGGCCCUCGG 124 735 CAGUGUGAAAGACAUAGCU 125
AGCUAUGUCUUUCACACUG 126 759 GAAUUCCACGCUGGGUUGU 127
ACAACCCAGCGUGGAAUUC 128 760 AAUUCCACGCUGGGUUGUU 129
AACAACCCAGCGUGGAAUU 130 766 ACGCUGGGUUGUUACCGCU 131
AGCGGUAACAACCCAGCGU 132 767 CGCUGGGUUGUUACCGCUA 133
UAGCGGUAACAACCCAGCG 134 769 CUGGGUUGUUACCGCUACA 135
UGUAGCGGUAACAACCCAG 136 772 GGUUGUUACCGCUACAGCU 137
AGCUGUAGCGGUAACAACC 138 776 GUUACCGCUACAGCUACGU 139
ACGUAGCUGUAGCGGUAAC 140 872 AGGACCUCAUGCUCAAACU 141
AGUUUGAGCAUGAGGUCCU 142 878 UCAUGCUCAAACUCCGGCU 143
AGCCGGAGUUUGAGCAUGA 144 970 AUCACCUCGGUGUACGGCU 145
AGCCGUACACCGAGGUGAU 146 973 ACCUCGGUGUACGGCUGCA 147
UGCAGCCGUACACCGAGGU 148 1033 AUCAUGGCGGUCGUCUGGA 149
UCCAGACGACCGCCAUGAU 150 1034 UCAUGGCGGUCGUCUGGAA 151
UUCCAGACGACCGCCAUGA 152 1067 GCUACUACGACCCCUUCGU 153
ACGAAGGGGUCGUAGUAGC 154 1091 CCGUGCAGCCGGUGGUCUU 155
AAGACCACCGGCUGCACGG 156 1106 UCUUCCAGGCCUGUGAAGU 157
ACUUCACAGGCCUGGAAGA 158 1114 GCCUGUGAAGUGAACCUGA 159
UCAGGUUCACUUCACAGGC 160 1118 GUGAAGUGAACCUGACGCU 161
AGCGUCAGGUUCACUUCAC 162 1133 CGCUGGACAACAGGCUCGA 163
UCGAGCCUGUUGUCCAGCG 164 1135 CUGGACAACAGGCUCGACU 165
AGUCGAGCCUGUUGUCCAG 166 1162 GUCCUCAGCACCCCGUACU 167
AGUACGGGGUGCUGAGGAC 168 1163 UCCUCAGCACCCCGUACUU 169
AAGUACGGGGUGCUGAGGA 170 1168 AGCACCCCGUACUUCCCCA 171
UGGGGAAGUACGGGGUGCU 172 1185 CAGCUACUACUCGCCCCAA 173
UUGGGGCGAGUAGUAGCUG 174 1186 AGCUACUACUCGCCCCAAA 175
UUUGGGGCGAGUAGUAGCU 176 1190 ACUACUCGCCCCAAACCCA 177
UGGGUUUGGGGCGAGUAGU 178 1195 UCGCCCCAAACCCACUGCU 179
AGCAGUGGGUUUGGGGCGA 180 1211 GCUCCUGGCACCUCACGGU 181
ACCGUGAGGUGCCAGGAGC 182 1231 CCCUCUCUGGACUACGGCU 183
AGCCGUAGUCCAGAGAGGG 184 1244 ACGGCUUGGCCCUCUGGUU 185
AACCAGAGGGCCAAGCCGU 186 1245 CGGCTTGGCCCTCTGGTTT 187
AAACCAGAGGGCCAAGCCG 188 1247 GCUUGGCCCUCUGGUUUGA 189
UCAAACCAGAGGGCCAAGC 190 1254 CCUCUGGUUUGAUGCCUAU 191
AUAGGCAUCAAACCAGAGG 192 1285 CAGAAGUAUGAUUUGCCGU 193
ACGGCAAAUCAUACUUCUG 194 1288 AAGUAUGAUUUGCCGUGCA 195
UGCACGGCAAAUCAUACUU 196 1292 AUGAUUUGCCGUGCACCCA 197
UGGGUGCACGGCAAAUCAU 198 1306 ACCCAGGGCCAGUGGACGA 199
UCGUCCACUGGCCCUGGGU 200 1310 AGGGCCAGUGGACGAUCCA 201
UGGAUCGUCCACUGGCCCU 202 1312 GGCCAGUGGACGAUCCAGA 203
UCUGGAUCGUCCACUGGCC 204 1313 GCCAGUGGACGAUCCAGAA 205
UUCUGGAUCGUCCACUGGC 206 1360 CAGCCCUACGCCGAGAGGA 207
UCCUCUCGGCGUAGGGCUG 208 1443 CGGUGUGCGGGUGCACUAU 209
AUAGUGCACCCGCACACCG 210 1447 GUGCGGGUGCACUAUGGCU 211
AGCCAUAGUGCACCCGCAC 212 1448 UGCGGGUGCACUAUGGCUU 213
AAGCCAUAGUGCACCCGCA 214 1451 GGGUGCACUAUGGCUUGUA 215
UACAAGCCAUAGUGCACCC 216 1454 UGCACUAUGGCUUGUACAA 217
UUGUACAAGCCAUAGUGCA 218 1486 UGCCCUGGAGAGUUCCUCU 219
AGAGGAACUCUCCAGGGCA 220 1565 UGGAUGAGAGAAACUGCGU 221
ACGCAGUUUCUCUCAUCCA 222 1611 GGACAGCACAUGCAUCUCA 223
UGAGAUGCAUGUGCUGUCC 224 1613 ACAGCACAUGCAUCUCACU 225
AGUGAGAUGCAUGUGCUGU 226 1634 CCAAGGUCUGUGAUGGGCA 227
UGCCCAUCACAGACCUUGG 228 1646 AUGGGCAGCCUGAUUGUCU 229
AGACAAUCAGGCUGCCCAU 230 1649 GGCAGCCUGAUUGUCUCAA 231
UUGAGACAAUCAGGCUGCC 232 1654 CCUGAUUGUCUCAACGGCA 233
UGCCGUUGAGACAAUCAGG 234 1662 UCUCAACGGCAGCGACGAA 235
UUCGUCGCUGCCGUUGAGA 236 1687 UGCCAGGAAGGGGUGCCAU 237
AUGGCACCCCUUCCUGGCA 238 1696 GGGGUGCCAUGUGGGACAU 239
AUGUCCCACAUGGCACCCC 240 1699 GUGCCAUGUGGGACAUUCA 241
UGAAUGUCCCACAUGGCAC 242 1703 CAUGUGGGACAUUCACCUU 243
AAGGUGAAUGUCCCACAUG 244 1723 CAGUGUGAGGACCGGAGCU 245
AGCUCCGGUCCUCACACUG 246 1745 UGAAGAAGCCCAACCCGCA 247
UGCGGGUUGGGCUUCUUCA 248 1749 GAAGCCCAACCCGCAGUGU 249
ACACUGCGGGUUGGGCUUC 250
1830 CCCCUCCAGCCGCAUUGUU 251 AACAAUGCGGCUGGAGGGG 252 1897
CAGGUUCGGGGUCGACACA 253 UGUGUCGACCCCGAACCUG 254 1898
AGGUUCGGGGUCGACACAU 255 AUGUGUCGACCCCGAACCU 256 1900
GUUCGGGGUCGACACAUCU 257 AGAUGUGUCGACCCCGAAC 258 1935
CGCUGACCGCUGGGUGAUA 259 UAUCACCCAGCGGUCAGCG 260 1936
GCUGACCGCUGGGUGAUAA 261 UUAUCACCCAGCGGUCAGC 262 1938
UGACCGCUGGGUGAUAACA 263 UGUUAUCACCCAGCGGUCA 264 1941
CCGCUGGGUGAUAACAGCU 265 AGCUGUUAUCACCCAGCGG 266 1997
UGCUGUGGACCGUGUUCCU 267 AGGAACACGGUCCACAGCA 268 2023
GUGUGGCAGAACUCGCGCU 269 AGCGCGAGUUCUGCCACAC 270 2078
UCCUGCACCCGUACCACGA 271 UCGUGGUACGGGUGCAGGA 272 2079
CCUGCACCCGUACCACGAA 273 UUCGUGGUACGGGUGCAGG 274 2081
UGCACCCGUACCACGAAGA 275 UCUUCGUGGUACGGGUGCA 276 2186
CGCGCUCCCACUUCUUCGA 277 UCGAAGAAGUGGGAGCGCG 278 2209
GGCCUGCACUGCUGGAUUA 279 UAAUCCAGCAGUGCAGGCC 280 2215
CACUGCUGGAUUACGGGCU 281 AGCCCGUAAUCCAGCAGUG 282 2283
GGAUGUGCAGUUGAUCCCA 283 UGGGAUCAACUGCACAUCC 284 2311
UGCAGCGAGGUCUAUCGCU 285 AGCGAUAGACCUCGCUGCA 286 2312
GCAGCGAGGUCUAUCGCUA 287 UAGCGAUAGACCUCGCUGC 288 2315
GCGAGGUCUAUCGCUACCA 289 UGGUAGCGAUAGACCUCGC 290 2320
GUCUAUCGCUACCAGGUGA 291 UCACCUGGUAGCGAUAGAC 292 2333
AGGUGACGCCACGCAUGCU 293 AGCAUGCGUGGCGUCACCU 294 2335
GUGACGCCACGCAUGCUGU 295 ACAGCAUGCGUGGCGUCAC 296 2337
GACGCCACGCAUGCUGUGU 297 ACACAGCAUGCGUGGCGUC 298 2470
GGCUGUGGCCGGCCUAACU 299 AGUUAGGCCGGCCACAGCC 300 2471
GCUGUGGCCGGCCUAACUA 301 UAGUUAGGCCGGCCACAGC 302 2473
UGUGGCCGGCCUAACUACU 303 AGUAGUUAGGCCGGCCACA 304 2480
GGCCUAACUACUUCGGCGU 305 ACGCCGAAGUAGUUAGGCC 306 2482
CCUAACUACUUCGGCGUCU 307 AGACGCCGAAGUAGUUAGG 308 2483
CUAACUACUUCGGCGUCUA 309 UAGACGCCGAAGUAGUUAG 310 2485
AACUACUUCGGCGUCUACA 311 UGUAGACGCCGAAGUAGUU 312 2501
ACACCCGCAUCACAGGUGU 313 ACACCUGUGAUGCGGGUGU 314 2506
CGCAUCACAGGUGUGAUCA 315 UGAUCACACCUGUGAUGCG 316 2525
GCUGGAUCCAGCAAGUGGU 317 ACCACUUGCUGGAUCCAGC 318 2551
GGAACUGCCCCCCUGCAAA 319 UUUGCAGGGGGGCAGUUCC 320 2671
AGGAGGUGGCAUCUUGUCU 321 AGACAAGAUGCCACCUCCU 322 2674
AGGUGGCAUCUUGUCUCGU 323 ACGAGACAAGAUGCCACCU 324 2678
GGCAUCUUGUCUCGUCCCU 325 AGGGACGAGACAAGAUGCC 326 2680
CAUCUUGUCUCGUCCCUGA 327 UCAGGGACGAGACAAGAUG 328 2681
AUCUUGUCUCGUCCCUGAU 329 AUCAGGGACGAGACAAGAU 330 2739
CAGCUGGGGGUCAAGACGU 331 ACGUCUUGACCCCCAGCUG 332 2744
GGGGGUCAAGACGUCCCCU 333 AGGGGACGUCUUGACCCCC 334 2746
GGGUCAAGACGUCCCCUGA 335 UCAGGGGACGUCUUGACCC 336 2825
CCACUGCUGCCUAAUGCAA 337 UUGCAUUAGGCAGCAGUGG 338 2829
UGCUGCCUAAUGCAAGGCA 339 UGCCUUGCAUUAGGCAGCA 340 2835
CUAAUGCAAGGCAGUGGCU 341 AGCCACUGCCUUGCAUUAG 342 2857
CAGCAAGAAUGCUGGUUCU 343 AGAACCAGCAUUCUUGCUG 344 2894
GAGGUGCGCCCCACUCUGU 345 ACAGAGUGGGGCGCACCUC 346 2958
CUUCGGAAGCCCCUGGUCU 347 AGACCAGGGGCUUCCGAAG 348 2960
UCGGAAGCCCCUGGUCUAA 349 UUAGACCAGGGGCUUCCGA 350 2962
GGAAGCCCCUGGUCUAACU 351 AGUUAGACCAGGGGCUUCC 352 2963
GAAGCCCCUGGUCUAACUU 353 AAGUUAGACCAGGGGCUUC 354 2968
CCCUGGUCUAACUUGGGAU 355 AUCCCAAGUUAGACCAGGG 356 2970
CUGGUCUAACUUGGGAUCU 357 AGAUCCCAAGUUAGACCAG 358 2975
CUAACUUGGGAUCUGGGAA 359 UUCCCAGAUCCCAAGUUAG 360 3006
CCAUCGGAGGGGACCCUCA 361 UGAGGGUCCCCUCCGAUGG 362 3045
UGGGCCUGCUGCCACUGUA 363 UACAGUGGCAGCAGGCCCA 364 3046
GGGCCUGCUGCCACUGUAA 365 UUACAGUGGCAGCAGGCCC 366 3052
GCUGCCACUGUAAGCCAAA 367 UUUGGCUUACAGUGGCAGC 368 3056
CCACUGUAAGCCAAAAGGU 369 ACCUUUUGGCUUACAGUGG 370 3071
AGGUGGGGAAGUCCUGACU 371 AGUCAGGACUUCCCCACCU 372 3174
GAAUAAAGCUGCCUGAUCA 373 UGAUCAGGCAGCUUUAUUC 374 3175
AAUAAAGCUGCCUGAUCAA 375 UUGAUCAGGCAGCUUUAUU 376 3180
AGCUGCCUGAUCAAAAAAA 377 UUUUUUUGAUCAGGCAGCU 378
TABLE-US-00004 TABLE 3 Unmodified sense and antisense strand
sequences of human TMPRSS6 dsRNAs SEQ SEQ ID ID NO.: NO.: Position
in Position in (anti- Duplex ID (sense) Sense Trans seq NM_153609.2
NM_153609.2 sense) Antisense Trans seq AD-46230.1 43
GGGGUGCUACUCUGGUAUU 319 319-337 44 AAUACCAGAGUAGCACCCC AD-46231.1
111 UCUUCUGGUUCAUUCUCCA 566 566-584 112 UGGAGAAUGAACCAGAAGA
AD-46232.1 131 ACGCUGGGUUGUUACCGCU 766 766-784 132
AGCGGUAACAACCCAGCGU AD-46233.1 193 CAGAAGUAUGAUUUGCCGU 1285
1285-1303 194 ACGGCAAAUCAUACUUCUG AD-46234.1 259
CGCUGACCGCUGGGUGAUA 1935 1935-1953 260 UAUCACCCAGCGGUCAGCG
AD-46235.1 45 UCUGGUAUUUCCUAGGGUA 329 329-347 46
UACCCUAGGAAAUACCAGA AD-46236.1 117 CCUACAGGGCCGAGUACGA 683 683-701
118 UCGUACUCGGCCCUGUAGG AD-46237.1 133 CGCUGGGUUGUUACCGCUA 767
767-785 134 UAGCGGUAACAACCCAGCG AD-46238.1 203 GGCCAGUGGACGAUCCAGA
1312 1312-1330 204 UCUGGAUCGUCCACUGGCC AD-46239.1 263
UGACCGCUGGGUGAUAACA 1938 1938-1956 264 UGUUAUCACCCAGCGGUCA
AD-46240.1 51 GGUCAGCCAGGUGUACUCA 363 363-381 52
UGAGUACACCUGGCUGACC AD-46241.1 379 CUACAGGGCCGAGUACGAA 684 684-702
380 UUCGUACUCGGCCCUGUAG AD-46242.1 135 CUGGGUUGUUACCGCUACA 769
769-787 136 UGUAGCGGUAACAACCCAG AD-46243.1 217 UGCACUAUGGCUUGUACAA
1454 1454-1472 218 UUGUACAAGCCAUAGUGCA AD-46244.1 604
CCUGGAGAGGUGUCCUUCA 2044 2044-2062 605 UGAAGGACACCUCUCCAGG
AD-46244.2 604 CCUGGAGAGGUGUCCUUCA 2044 2044-2062 605
UGAAGGACACCUCUCCAGG AD-46245.1 53 AGCCAGGUGUACUCAGGCA 367 367-385
54 UGCCUGAGUACACCUGGCU AD-46246.1 119 ACAGGGCCGAGUACGAAGU 686
686-704 120 ACUUCGUACUCGGCCCUGU AD-46247.1 137 GGUUGUUACCGCUACAGCU
772 772-790 138 AGCUGUAGCGGUAACAACC AD-46248.1 381
UGUGAUGGGGUCAAGGACU 1534 1534-1552 382 AGUCCUUGACCCCAUCACA
AD-46249.1 383 CUGGAGAGGUGUCCUUCAA 2045 2045-2063 384
UUGAAGGACACCUCUCCAG AD-46250.1 89 UCCGCAGUGAAACCGCCAA 446 446-464
90 UUGGCGGUUUCACUGCGGA AD-46251.1 121 GGGCCGAGUACGAAGUGGA 689
689-707 122 UCCACUUCGUACUCGGCCC AD-46252.1 385 GGACCGACUGGCCAUGUAU
921 921-939 386 AUACAUGGCCAGUCGGUCC AD-46253.1 606
CAACGGCCUGGAUGAGAGA 1557 1557-1575 607 UCUCUCAUCCAGGCCGUUG
AD-46253.2 606 CAACGGCCUGGAUGAGAGA 1557 1557-1575 607
UCUCUCAUCCAGGCCGUUG AD-46254.1 387 AGUUGAUCCCACAGGACCU 2291
2291-2309 388 AGGUCCUGUGGGAUCAACU AD-46255.1 91 CCGCAGUGAAACCGCCAAA
447 447-465 92 UUUGGCGGUUUCACUGCGG AD-46256.1 123
CCGAGGGCCUAGUGAUCCU 710 710-728 124 AGGAUCACUAGGCCCUCGG AD-46257.1
169 UCCUCAGCACCCCGUACUU 1163 1163-1181 170 AAGUACGGGGUGCUGAGGA
AD-46258.1 253 CAGGUUCGGGGUCGACACA 1897 1897-1915 254
UGUGUCGACCCCGAACCUG AD-46259.1 293 AGGUGACGCCACGCAUGCU 2333
2333-2351 294 AGCAUGCGUGGCGUCACCU AD-46260.1 389
AAACCGCCAAAGCCCAGAA 455 455-473 390 UUCUGGGCUUUGGCGGUUU AD-46261.1
125 CAGUGUGAAAGACAUAGCU 735 735-753 126 AGCUAUGUCUUUCACACUG
AD-46262.1 183 CCCUCUCUGGACUACGGCU 1231 1231-1249 184
AGCCGUAGUCCAGAGAGGG AD-46263.1 257 GUUCGGGGUCGACACAUCU 1900
1900-1918 258 AGAUGUGUCGACCCCGAAC AD-46264.1 391
UGUGUGCCGGCUACCGCAA 2351 2351-2369 392 UUGCGGUAGCCGGCACACA
AD-46265.1 109 GCUUCUUCUGGUUCAUUCU 563 563-581 110
AGAAUGAACCAGAAGAAGC AD-46266.1 393 AUUCCACGCUGGGUUGUUA 761 761-779
394 UAACAACCCAGCGUGGAAU AD-46267.1 185 ACGGCUUGGCCCUCUGGUU 1244
1244-1262 186 AACCAGAGGGCCAAGCCGU AD-46268.1 395
UCGCUGACCGCUGGGUGAU 1934 1934-1952 396 AUCACCCAGCGGUCAGCGA
AD-46269.1 608 AGUGGUGACCUGAGGAACU 2538 2538-2556 609
AGUUCCUCAGGUCACCACU AD-46269.2 608 AGUGGUGACCUGAGGAACU 2538
2538-2556 609 AGUUCCUCAGGUCACCACU AD-46270.1 397
CAAGCAGGGGGACAAGUAU 2612 2612-2630 398 AUACUUGUCCCCCUGCUUG
AD-46271.1 399 UGAUGUCUGCUCCAGUGAU 2696 2696-2714 400
AUCACUGGAGCAGACAUCA AD-46272.1 359 CUAACUUGGGAUCUGGGAA 2975
2975-2993 360 UUCCCAGAUCCCAAGUUAG AD-46273.1 47 UGGUAUUUCCUAGGGUACA
331 331-349 48 UGUACCCUAGGAAAUACCA AD-46273.2 47
UGGUAUUUCCUAGGGUACA 331 331-349 48 UGUACCCUAGGAAAUACCA AD-46273.3
47 UGGUAUUUCCUAGGGUACA 331 331-349 48 UGUACCCUAGGAAAUACCA
AD-46274.1 401 GAGGUGUCCUUCAAGGUGA 2050 2050-2068 402
UCACCUUGAAGGACACCUC AD-46276.1 403 AAGCAGGGGGACAAGUAUU 2613
2613-2631 404 AAUACUUGUCCCCCUGCUU AD-46277.1 331
CAGCUGGGGGUCAAGACGU 2739 2739-2757 332 ACGUCUUGACCCCCAGCUG
AD-46278.1 405 CUUGGGAUCUGGGAAUGGA 2979 2979-2997 406
UCCAUUCCCAGAUCCCAAG AD-46279.1 407 GGUAUUUCCUAGGGUACAA 332 332-350
408 UUGUACCCUAGGAAAUACC AD-46280.1 409 GGCUACCGCAAGGGCAAGA 2359
2359-2377 410 UCUUGCCCUUGCGGUAGCC AD-46282.1 411
GCAGGGGGACAAGUAUUCU 2615 2615-2633 412 AGAAUACUUGUCCCCCUGC
AD-46283.1 413 GCUCAGCAGCAAGAAUGCU 2851 2851-2869 414
AGCAUUCUUGCUGCUGAGC AD-46284.1 415 UUGGGAUCUGGGAAUGGAA 2980
2980-2998 416 UUCCAUUCCCAGAUCCCAA AD-46285.1 417
CCAAAGCCCAGAAGAUGCU 461 461-479 418 AGCAUCUUCUGGGCUUUGG AD-46286.1
419 GCUACCGCAAGGGCAAGAA 2360 2360-2378 420 UUCUUGCCCUUGCGGUAGC
AD-46286.2 419 GCUACCGCAAGGGCAAGAA 2360 2360-2378 420
UUCUUGCCCUUGCGGUAGC AD-46288.1 423 UGGUGGCAGGAGGUGGCAU 2664
2664-2682 424 AUGCCACCUCCUGCCACCA AD-46289.1 425
CCCACUCUGUACAGAGGCU 2903 2903-2921 426 AGCCUCUGUACAGAGUGGG
AD-46290.1 427 CUCACAGCCCAGACCCUCA 3128 3128-3146 428
UGAGGGUCUGGGCUGUGAG AD-46291.1 429 CCUCUCUGGACUACGGCUU 1232
1232-1250 430 AAGCCGUAGUCCAGAGAGG AD-46293.1 431
GUGGCAGGAGGUGGCAUCU 2666 2666-2684 432 AGAUGCCACCUCCUGCCAC
AD-46294.1 433 UUCGGAAGCCCCUGGUCUA 2959 2959-2977 434
UAGACCAGGGGCUUCCGAA AD-46295.1 435 AGCUCAGCUGCCCUUUGGA 3157
3157-3175 436 UCCAAAGGGCAGCUGAGCU AD-46296.1 437
GGCCUGGAUGAGAGAAACU 1561 1561-1579 438 AGUUUCUCUCAUCCAGGCC
AD-46297.1 439 UGGCAGGAGGUGGCAUCUU 2667 2667-2685 440
AAGAUGCCACCUCCUGCCA AD-46298.1 349 UCGGAAGCCCCUGGUCUAA 2960
2960-2978 350 UUAGACCAGGGGCUUCCGA AD-46299.1 421
GCUCAGCUGCCCUUUGGAA 3158 3158-3176 422 UUCCAAAGGGCAGCUGAGC
AD-46299.2 421 GCUCAGCUGCCCUUUGGAA 3158 3158-3176 422
UUCCAAAGGGCAGCUGAGC AD-46300.1 441 ACUGUGACUGUGGCCUCCA 1808
1808-1826 442 UGGAGGCCACAGUCACAGU AD-46301.1 321
AGGAGGUGGCAUCUUGUCU 2671 2671-2689 322 AGACAAGAUGCCACCUCCU
AD-46302.1 443 CCCCUGGUCUAACUUGGGA 2967 2967-2985 444
UCCCAAGUUAGACCAGGGG AD-46303.1 445 UCAGCUGCCCUUUGGAAUA 3160
3160-3178 446 UAUUCCAAAGGGCAGCUGA AD-46304.1 447
UCGGGGUCGACACAUCUGU 1902 1902-1920 448 ACAGAUGUGUCGACCCCGA
AD-46305.1 449 GUCCCUGAUGUCUGCUCCA 2691 2691-2709 450
UGGAGCAGACAUCAGGGAC AD-46306.1 355 CCCUGGUCUAACUUGGGAU 2968
2968-2986 356 AUCCCAAGUUAGACCAGGG AD-46307.1 610
CAGCUGCCCUUUGGAAUAA 3161 3161-3179 611 UUAUUCCAAAGGGCAGCUG
AD-46307.2 610 CAGCUGCCCUUUGGAAUAA 3161 3161-3179 611
UUAUUCCAAAGGGCAGCUG AD-46308.1 451 UCAUCGCUGACCGCUGGGU 1931
1931-1949 452 ACCCAGCGGUCAGCGAUGA
TABLE-US-00005 TABLE 4 Modified sense and antisense strand
sequences of human TMPRSS6 dsRNAs SEQ SEQ ID ID NO.: NO.: Position
in Position in (anti- Duplex ID (sense) Sense sequence NM_153609.2
NM_153609.2 sense) Antisense sequence AD-46230.1 453
GGGGuGcuAcucuGGuAuudTsdT 319 319-337 454 AAuACcAGAGuAGcACCCCdTsdT
AD-46231.1 455 ucuucuGGuucAuucuccAdTsdT 566 566-584 456
UGGAGAAUGAACcAGAAGAdTsdT AD-46232.1 457 AcGcuGGGuuGuuAccGcudTsdT
766 766-784 458 AGCGGuAAcAACCcAGCGUdTsdT AD-46233.1 459
cAGAAGuAuGAuuuGccGudTsdT 1285 1285-1303 460
ACGGcAAAUcAuACUUCUGdTsdT AD-46234.1 461 cGcuGAccGcuGGGuGAuAdTsdT
1935 1935-1953 462 uAUcACCcAGCGGUcAGCGdTsdT AD-46235.1 463
ucuGGuAuuuccuAGGGuAdTsdT 329 329-347 464 uACCCuAGGAAAuACcAGAdTsdT
AD-46236.1 465 ccuAcAGGGccGAGuAcGAdTsdT 683 683-701 466
UCGuACUCGGCCCUGuAGGdTsdT AD-46237.1 467 cGcuGGGuuGuuAccGcuAdTsdT
767 767-785 468 uAGCGGuAAcAACCcAGCGdTsdT AD-46238.1 469
GGccAGuGGAcGAuccAGAdTsdT 1312 1312-1330 470
UCUGGAUCGUCcACUGGCCdTsdT AD-46239.1 471 uGAccGcuGGGuGAuAAcAdTsdT
1938 1938-1956 472 UGUuAUcACCcAGCGGUcAdTsdT AD-46240.1 473
GGucAGccAGGuGuAcucAdTsdT 363 363-381 474 UGAGuAcACCUGGCUGACCdTsdT
AD-46241.1 475 cuAcAGGGccGAGuAcGAAdTsdT 684 684-702 476
UUCGuACUCGGCCCUGuAGdTsdT AD-46242.1 478 cuGGGuuGuuAccGcuAcAdTsdT
769 769-787 479 UGuAGCGGuAAcAACCcAGdTsdT AD-46243.1 480
uGcAcuAuGGcuuGuAcAAdTsdT 1454 1454-1472 481
UUGuAcAAGCcAuAGUGcAdTsdT AD-46244.1 482 ccuGGAGAGGuGuccuucAdTsdT
2044 2044-2062 483 UGAAGGAcACCUCUCcAGGdTsdT AD-46244.2 482
ccuGGAGAGGuGuccuucAdTsdT 2044 2044-2062 483
UGAAGGAcACCUCUCcAGGdTsdT AD-46245.1 484 AGccAGGuGuAcucAGGcAdTsdT
367 367-385 485 UGCCUGAGuAcACCUGGCUdTsdT AD-46246.1 486
AcAGGGccGAGuAcGAAGudTsdT 686 686-704 487 ACUUCGuACUCGGCCCUGUdTsdT
AD-46247.1 488 GGuuGuuAccGcuAcAGcudTsdT 772 772-790 489
AGCUGuAGCGGuAAcAACCdTsdT AD-46248.1 490 uGuGAuGGGGucAAGGAcudTsdT
1534 1534-1552 491 AGUCCUUGACCCcAUcAcAdTsdT AD-46249.1 492
cuGGAGAGGuGuccuucAAdTsdT 2045 2045-2063 493
UUGAAGGAcACCUCUCcAGdTsdT AD-46250.1 494 uccGcAGuGAAAccGccAAdTsdT
446 446-464 495 UUGGCGGUUUcACUGCGGAdTsdT AD-46251.1 496
GGGccGAGuAcGAAGuGGAdTsdT 689 689-707 497 UCcACUUCGuACUCGGCCCdTsdT
AD-46252.1 498 GGAccGAcuGGccAuGuAudTsdT 921 921-939 499
AuAcAUGGCcAGUCGGUCCdTsdT AD-46253.1 500 cAAcGGccuGGAuGAGAGAdTsdT
1557 1557-1575 501 UCUCUcAUCcAGGCCGUUGdTsdT AD-46253.2 500
cAAcGGccuGGAuGAGAGAdTsdT 1557 1557-1575 501
UCUCUcAUCcAGGCCGUUGdTsdT AD-46254.1 502 AGuuGAucccAcAGGAccudTsdT
2291 2291-2309 503 AGGUCCUGUGGGAUcAACUdTsdT AD-46255.1 504
ccGcAGuGAAAccGccAAAdTsdT 447 447-465 505 UUUGGCGGUUUcACUGCGGdTsdT
AD-46256.1 506 ccGAGGGccuAGuGAuccudTsdT 710 710-728 507
AGGAUcACuAGGCCCUCGGdTsdT AD-46257.1 508 uccucAGcAccccGuAcuudTsdT
1163 1163-1181 509 AAGuACGGGGUGCUGAGGAdTsdT AD-46258.1 510
cAGGuucGGGGucGAcAcAdTsdT 1897 1897-1915 511
UGUGUCGACCCCGAACCUGdTsdT AD-46259.1 512 AGGuGAcGccAcGcAuGcudTsdT
2333 2333-2351 513 AGcAUGCGUGGCGUcACCUdTsdT AD-46260.1 514
AAAccGccAAAGcccAGAAdTsdT 455 455-473 515 UUCUGGGCUUUGGCGGUUUdTsdT
AD-46261.1 516 cAGuGuGAAAGAcAuAGcudTsdT 735 735-753 517
AGCuAUGUCUUUcAcACUGdTsdT AD-46262.1 518 cccucucuGGAcuAcGGcudTsdT
1231 1231-1249 519 AGCCGuAGUCcAGAGAGGGdTsdT AD-46263.1 520
GuucGGGGucGAcAcAucudTsdT 1900 1900-1918 521
AGAUGUGUCGACCCCGAACdTsdT AD-46264.1 522 uGuGuGccGGcuAccGcAAdTsdT
2351 2351-2369 523 UUGCGGuAGCCGGcAcAcAdTsdT AD-46265.1 524
GcuucuucuGGuucAuucudTsdT 563 563-581 525 AGAAUGAACcAGAAGAAGCdTsdT
AD-46266.1 526 AuuccAcGcuGGGuuGuuAdTsdT 761 761-779 527
uAAcAACCcAGCGUGGAAUdTsdT AD-46267.1 528 AcGGcuuGGcccucuGGuudTsdT
1244 1244-1262 529 AACcAGAGGGCcAAGCCGUdTsdT AD-46268.1 530
ucGcuGAccGcuGGGuGAudTsdT 1934 1934-1952 531
AUcACCcAGCGGUcAGCGAdTsdT AD-46269.1 532 AGuGGuGAccuGAGGAAcudTsdT
2538 2538-2556 533 AGUUCCUcAGGUcACcACUdTsdT AD-46269.2 532
AGuGGuGAccuGAGGAAcudTsdT 2538 2538-2556 533
AGUUCCUcAGGUcACcACUdTsdT AD-46270.1 534 cAAGcAGGGGGAcAAGuAudTsdT
2612 2612-2630 535 AuACUUGUCCCCCUGCUUGdTsdT AD-46271.1 536
uGAuGucuGcuccAGuGAudTsdT 2696 2696-2714 537
AUcACUGGAGcAGAcAUcAdTsdT AD-46272.1 538 cuAAcuuGGGAucuGGGAAdTsdT
2975 2975-2993 539 UUCCcAGAUCCcAAGUuAGdTsdT AD-46273.1 540
uGGuAuuuccuAGGGuAcAdTsdT 331 331-349 541 UGuACCCuAGGAAAuACcAdTsdT
AD-46273.2 540 uGGuAuuuccuAGGGuAcAdTsdT 331 331-349 541
UGuACCCuAGGAAAuACcAdTsdT AD-46273.3 540 uGGuAuuuccuAGGGuAcAdTsdT
331 331-349 541 UGuACCCuAGGAAAuACcAdTsdT AD-46274.1 542
GAGGuGuccuucAAGGuGAdTsdT 2050 2050-2068 543
UcACCUUGAAGGAcACCUCdTsdT AD-46276.1 544 AAGcAGGGGGAcAAGuAuudTsdT
2613 2613-2631 545 AAuACUUGUCCCCCUGCUUdTsdT AD-46277.1 546
cAGcuGGGGGucAAGAcGudTsdT 2739 2739-2757 547
ACGUCUUGACCCCcAGCUGdTsdT AD-46278.1 548 cuuGGGAucuGGGAAuGGAdTsdT
2979 2979-2997 549 UCcAUUCCcAGAUCCcAAGdTsdT AD-46279.1 550
GGuAuuuccuAGGGuAcAAdTsdT 332 332-350 551 UUGuACCCuAGGAAAuACCdTsdT
AD-46280.1 552 GGcuAccGcAAGGGcAAGAdTsdT 2359 2359-2377 553
UCUUGCCCUUGCGGuAGCCdTsdT AD-46282.1 554 GcAGGGGGAcAAGuAuucudTsdT
2615 2615-2633 555 AGAAuACUUGUCCCCCUGCdTsdT AD-46283.1 556
GcucAGcAGcAAGAAuGcudTsdT 2851 2851-2869 557
AGcAUUCUUGCUGCUGAGCdTsdT AD-46284.1 558 uuGGGAucuGGGAAuGGAAdTsdT
2980 2980-2998 559 UUCcAUUCCcAGAUCCcAAdTsdT AD-46285.1 560
ccAAAGcccAGAAGAuGcudTsdT 461 461-479 561 AGcAUCUUCUGGGCUUUGGdTsdT
AD-46286.1 562 GcuAccGcAAGGGcAAGAAdTsdT 2360 2360-2378 563
UUCUUGCCCUUGCGGuAGCdTsdT AD-46286.2 562 GcuAccGcAAGGGcAAGAAdTsdT
2360 2360-2378 563 UUCUUGCCCUUGCGGuAGCdTsdT AD-46288.1 564
uGGuGGcAGGAGGuGGcAudTsdT 2664 2664-2682 565
AUGCcACCUCCUGCcACcAdTsdT AD-46289.1 566 cccAcucuGuAcAGAGGcudTsdT
2903 2903-2921 567 AGCCUCUGuAcAGAGUGGGdTsdT AD-46290.1 568
cucAcAGcccAGAcccucAdTsdT 3128 3128-3146 569
UGAGGGUCUGGGCUGUGAGdTsdT AD-46291.1 570 ccucucuGGAcuAcGGcuudTsdT
1232 1232-1250 571 AAGCCGuAGUCcAGAGAGGdTsdT AD-46293.1 572
GuGGcAGGAGGuGGcAucudTsdT 2666 2666-2684 573
AGAUGCcACCUCCUGCcACdTsdT AD-46294.1 574 uucGGAAGccccuGGucuAdTsdT
2959 2959-2977 575 uAGACcAGGGGCUUCCGAAdTsdT AD-46295.1 576
AGcucAGcuGcccuuuGGAdTsdT 3157 3157-3175 577
UCcAAAGGGcAGCUGAGCUdTsdT AD-46296.1 578 GGccuGGAuGAGAGAAAcudTsdT
1561 1561-1579 579 AGUUUCUCUcAUCcAGGCCdTsdT AD-46297.1 580
uGGcAGGAGGuGGcAucuudTsdT 2667 2667-2685 581
AAGAUGCcACCUCCUGCcAdTsdT AD-46298.1 582 ucGGAAGccccuGGucuAAdTsdT
2960 2960-2978 583 UuAGACcAGGGGCUUCCGAdTsdT AD-46299.1 584
GcucAGcuGcccuuuGGAAdTsdT 3158 3158-3176 585
UUCcAAAGGGcAGCUGAGCdTsdT AD-46299.2 584 GcucAGcuGcccuuuGGAAdTsdT
3158 3158-3176 585 UUCcAAAGGGcAGCUGAGCdTsdT AD-46300.1 586
AcuGuGAcuGuGGccuccAdTsdT 1808 1808-1826 587
UGGAGGCcAcAGUcAcAGUdTsdT AD-46301.1 588 AGGAGGuGGcAucuuGucudTsdT
2671 2671-2689 589 AGAcAAGAUGCcACCUCCUdTsdT AD-46302.1 590
ccccuGGucuAAcuuGGGAdTsdT 2967 2967-2985 591
UCCcAAGUuAGACcAGGGGdTsdT AD-46303.1 592 ucAGcuGcccuuuGGAAuAdTsdT
3160 3160-3178 593 uAUUCcAAAGGGcAGCUGAdTsdT AD-46304.1 594
ucGGGGucGAcAcAucuGudTsdT 1902 1902-1920 595
AcAGAUGUGUCGACCCCGAdTsdT AD-46305.1 596 GucccuGAuGucuGcuccAdTsdT
2691 2691-2709 597 UGGAGcAGAcAUcAGGGACdTsdT AD-46306.1 598
cccuGGucuAAcuuGGGAudTsdT 2968 2968-2986 599
AUCCcAAGUuAGACcAGGGdTsdT AD-46307.1 600 cAGcuGcccuuuGGAAuAAdTsdT
3161 3161-3179 601
UuAUUCcAAAGGGcAGCUGdTsdT AD-46307.2 600 cAGcuGcccuuuGGAAuAAdTsdT
3161 3161-3179 201 UuAUUCcAAAGGGcAGCUGdTsdT AD-46308.1 602
ucAucGcuGAccGcuGGGudTsdT 1931 1931-1949 603
ACCcAGCGGUcAGCGAUGAdTsdT
[0377] Synthesis of TMPRSS6 Sequences
[0378] TMPRSS6 iRNA sequences can be synthesized on a MerMade 192
synthesizer at 1 .mu.mol scale.
[0379] Endolight chemistry can be applied as detailed below. [0380]
All pyrimidines (cytosine and uridine) in the sense strand
contained 2'-O-Methyl bases (2' O-Methyl C and 2'-O-Methyl U)
[0381] In the antisense strand, pyrimidines adjacent to (towards 5'
position) ribo A nucleoside can be replaced with their
corresponding 2-O-Methyl nucleosides [0382] A two base dTsdT
extension at 3' end of both sense and anti sense sequences can be
introduced [0383] The sequence file can be converted to a text file
to make it compatible for loading in the MerMade 192 synthesis
software
[0384] Synthesis, Cleavage and deprotection
[0385] The synthesis of TMPRSS6 sequences use solid supported
oligonucleotide synthesis using phosphoramidite chemistry.
[0386] The synthesis of the above sequences can be performed at 1
.mu.m scale in 96 well plates. The amidite solutions can be
prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in
Acetonitrile) can be used as activator.
[0387] The synthesized sequences can be cleaved and deprotected in
96 well plates, using methylamine in the first step and fluoride
reagent in the second step. The crude sequences can be precipitated
using acetone:ethanol (80:20) mix and the pellets re-suspended in
0.02M sodium acetate buffer. Samples from each sequence can be
analyzed by LC-MS to confirm the identity, and by UV for
quantification. A selected set of samples can also be analyzed by
IEX chromatography to determine purity.
[0388] Purification and Desalting
[0389] All sequences can be purified on AKTA explorer purification
system using Source 15Q column. Sample injection and collection can
be performed in 96 well (1.8 mL-deep well) plates. A single peak
corresponding to the full length sequence can be collected in the
eluent. The purified sequences can be desalted on a Sephadex G25
column using AKTA purifier. The desalted TMPRSS6 sequences can be
analyzed for concentration (by UV measurement at A260) and purity
(by ion exchange HPLC). The single strands can then be submitted
for annealing.
Example 3. In Vitro Screening of TMPRSS6 siRNA Duplexes for TMPRSS6
Knockdown Activity
[0390] TMPRSS6 siRNA duplexes were screened for the ability to
knockdown TMPRSS6 expression in vitro. Single dose screening, dose
response screening, and viability of host cells were evaluated.
[0391] In Vitro Screening:
[0392] Cell Culture and Transfections for Single Dose and Dose
Response Studies:
[0393] HeLa or Hep3B cells (ATCC, Manassas, Va.) were grown to near
confluence at 37.degree. C. in an atmosphere of 5% CO.sub.2 in X
(ATCC) supplemented with 10% FBS, streptomycin, and glutamine
(ATCC) before being released from the plate by trypsinization.
Transfection was carried out by adding 14.8 .mu.l of Opti-MEM plus
0.41 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 without antibiotic containing
.about.2.times.10.sup.4 HeLa or 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, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001,
0.0005, 0.0001, 0.00005, 0.00001 nM final duplex concentration.
[0394] Total RNA Isolation Using DYNABEADS.RTM. mRNA Isolation Kit
(Invitrogen, Part #: 610-12):
[0395] Cells were harvested and lysed in 150 .mu.l of Lysis/Binding
Buffer then mixed for 5 minutes at 850 rpm using an Eppendorf.RTM.
Thermomixer (the mixing speed was the same throughout the process).
Ten microliters of magnetic beads and 80 .mu.l of 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, the magnetic beads were washed twice with 150 .mu.l of
Wash Buffer A and mixed for one 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. Beads were next washed with 150 .mu.l Elution Buffer,
captured and supernatant removed. The beads were then allowed to
dry for two minutes. After drying, 50 .mu.l of Elution Buffer was
added and mixed for five minutes at 70.degree. C. The beads were
captured on a magnet for five minutes. 40 .mu.l of supernatant was
removed and added to another 96 well plate.
[0396] cDNA Synthesis Using ABI High Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Foster City, Calif., Cat
#4368813):
[0397] A master mix of 2 .mu.l of 10.times. Buffer, 0.8 .mu.l of
25.times.dNTPs, 2 .mu.l of Random primers, 1 .mu.l of Reverse
Transcriptase, 1 .mu.l of RNase inhibitor and 3.2 .mu.l of H2O 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.
[0398] Real Time PCR:
[0399] 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 TMPRSS6 TaqMan probe (Applied Biosystems cat #Hs00542184_m1)
and 5 .mu.l Lightcycler 480 probe master mix (Roche Cat
#04887301001) per well in a 384 well 50 plates (Roche cat
#04887301001). Real time PCR was done in an ABI 7900HT Real Time
PCR system (Applied Biosystems) using the .DELTA..DELTA.Ct(RQ)
assay. Each duplex was tested in two independent transfections and
each transfection was assayed in duplicate, unless otherwise noted
in the summary tables.
[0400] 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
over the same dose range, or to its own lowest dose.
[0401] Viability screens. HeLa or Hep3B cells (ATCC, Manassas, Va.)
were grown to near confluence at 37.degree. C. in an atmosphere of
5% CO.sub.2 in X (ATCC) supplemented with 10% FBS, streptomycin,
and glutamine (ATCC) before being released from the plate by
trypsinization. Cell viability was measured on days 3 and 5 in HeLa
and Hep3B cells following transfection with 100, 10, 1, 0.1, 0.01
and 0.0001 nM siRNA. Cells were plated at a density of
2.5.times.10.sup.3-5.times.10.sup.3 cells per well in 96 well
plates. Each siRNA was assayed in triplicate and the data averaged.
siRNAs targeting PLK1 and AD-19200 were included as positive
controls for loss of viability and AD-1955 as a negative control.
PLK1 and AD-19200 result in a dose dependant loss of viability. To
measure viability, 20 ul of CellTiter Blue (Promega) was added to
each well of the 96 well plates after 3 and 5 days and incubated at
37.degree. C. for 2 hours. Plates were then read in a
Spectrophotometer (Molecular Devices) at 560.sub.Ex/590.sub.Em.
Viability was expressed as the average value of light units from
three replicate transfections+/-standard deviation.
In Vitro Knockdown of TMPRSS6 Expression by TMPRSS6 siRNA
Duplexes.
[0402] Table 5 presents data indicating the knockdown of TMPRSS6 in
Hep3B cells transfected with siRNAs targeting TMPRSS6. The data is
expressed as a fraction of TMPRSS6 message remaining in cells
transfected with siRNAs targeting TMPRSS6, relative to cells
transfected with a negative control siRNA, AD-1955. Cells that were
not treated ("naive" cells) served as a second negative control.
All siRNAs were tested at least twice, and qPCR reactions were also
performed in duplicate. Single dose experiments were performed at
10 nM and 0.1 nM final siRNA duplex concentration.
TABLE-US-00006 TABLE 5 TMPRSS6 expression in single dose screen in
vitro. 10 nM 0.1 nM 10 nM 0.1 nM Duplex ID Ave Ave SD SD AD-46230.1
0.89 1.14 0.036 0.145 AD-46230.1 0.85 1.22 0.039 0.063 AD-46231.1
0.11 0.29 0.017 0.007 AD-46232.1 0.78 0.87 0.03 0.023 AD-46233.1
0.6 0.98 0.033 0.046 AD-46234.1 0.79 1.06 0.082 0.068 AD-46235.1
0.18 0.87 0.009 0.066 AD-46235.1 0.18 0.96 0.009 0.132 AD-46236.1
0.15 1.06 0.007 0.036 AD-46237.1 0.81 0.98 0.043 0.027 AD-46238.1
0.71 0.99 0.069 0.031 AD-46239.1 0.83 1.3 0.035 0.073 AD-46240.1
0.89 0.99 0.027 0.079 AD-46240.1 0.88 1 0.009 0.034 AD-46241.1 0.6
0.9 0.029 0.029 AD-46242.1 0.81 0.91 0.016 0.049 AD-46243.1 0.82
0.87 0.029 0.066 AD-46244.1 0.19 0.43 0.018 0.028 AD-46245.1 0.48
0.79 0.148 0.016 AD-46245.1 0.51 0.82 0.147 0.028 AD-46246.1 0.39
0.89 0.012 0.043 AD-46247.1 0.84 0.9 0.047 0.019 AD-46248.1 0.68
0.95 0.059 0.075 AD-46249.1 0.17 0.29 0.005 0.152 AD-46250.1 0.19
0.53 0.017 0.011 AD-46251.1 0.16 0.47 0.007 0.005 AD-46252.1 1.04
1.08 0.031 0.038 AD-46253.1 0.27 0.45 0.02 0.031 AD-46254.1 1.03
1.08 0.221 0.021 AD-46255.1 0.52 0.84 0.029 0.036 AD-46256.1 0.81
1.02 0.025 0.015 AD-46257.1 0.64 0.97 0.016 0.076 AD-46258.1 0.91
0.98 0.054 0.059 AD-46259.1 0.77 1.03 0.052 0.067 AD-46260.1 1.24 1
0.634 0.031 AD-46261.1 0.12 0.19 0.007 0.006 AD-46262.1 0.58 1.27
0.016 0.024 AD-46263.1 0.79 0.95 0.03 0.021 AD-46264.1 0.93 1.16
0.052 0.095 AD-46265.1 0.09 0.47 0.007 0.017 AD-46266.1 0.25 0.8
0.024 0.018 AD-46267.1 0.65 0.84 0.058 0.02 AD-46268.1 0.92 1 0.008
0.048 AD-46269.1 0.37 0.52 0.037 0.024 AD-46270.1 0.26 0.55 0.01
0.03 AD-46271.1 0.35 0.8 0.044 0.029 AD-46272.1 0.62 0.91 0.015
0.061 AD-46273.1 0.18 0.3 0.02 0.012 AD-46274.1 0.88 0.85 0.04
0.016 AD-46276.1 0.33 0.64 0.024 0.024 AD-46277.1 0.85 0.89 0.12
0.026 AD-46278.1 0.24 0.7 0.019 0.059 AD-46279.1 0.55 0.79 0.008
0.025 AD-46280.1 0.96 0.84 0.059 0.042 AD-46282.1 0.21 0.47 0.017
0.004 AD-46283.1 0.62 1.01 0.05 0.03 AD-46284.1 0.42 0.78 0.016
0.019 AD-46285.1 0.37 0.86 0.014 0.042 AD-46286.1 0.19 0.49 0.019
0.027 AD-46288.1 0.65 0.88 0.052 0.032 AD-46289.1 0.89 0.92 0.062
0.032 AD-46290.1 0.83 0.9 0.035 0.029 AD-46291.1 0.65 0.87 0.014
0.014 AD-46293.1 0.31 0.68 0.012 0.054 AD-46294.1 0.25 0.7 0.015
0.031 AD-46295.1 0.2 0.42 0.004 0.029 AD-46296.1 0.43 0.83 0.012
0.043 AD-46297.1 0.3 0.6 0.009 0.017 AD-46298.1 0.91 0.91 0.08
0.008 AD-46299.1 0.26 0.57 0.018 0.052 AD-46300.1 0.98 0.91 0.037
0.024 AD-46301.1 0.65 0.87 0.018 0.051 AD-46302.1 0.92 1.01 0.021
0.048 AD-46303.1 0.13 0.43 0.008 0.019 AD-46304.1 1.11 1.01 0.016
0.056 AD-46305.1 0.21 0.73 0.029 0.011 AD-46306.1 0.84 0.96 0.114
0.092 AD-46307.1 0.27 0.49 0.007 0.019 AD-46308.1 0.69 0.83 0.02
0.024 Naive 1.04 1.06 0.021 0.018 Naive 1.07 1.29 0.065 0.059
AD-1955 0.85 0.85 0.055 0.071 AD-1955 1.1 0.97 0.034 0.04 AD-1955 1
0.98 0.036 0.058 AD-1955 1.04 0.98 0.053 0.049 AD-1955 1.04 1.08
0.021 0.039 AD-1955 0.98 1.19 0.049 0.058
IC.sub.50 of Select TMPRSS6 siRNA Duplexes in In Vitro Dose
Response Screen.
[0403] Table 6 presents the IC.sub.50 values of select TMPRSS6
siRNA duplexes determined from in vitro dose response screens.
TMPRSS6 siRNA duplexes that were efficacious in the 10 nM and 0.1
nM single dose screen (Table 5), were tested for TMPRSS6 knockdown
activity in a dose response at 1 and 5 days following transfection
in Hep3B cells. Dose response experiments were conducted at 10, 1,
0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005,
0.00001 nM final siRNA duplex concentration. For normalization,
knockdown of TMPRSS6 was measured relative to the non-targeting
control, AD-1955, or the value obtained at the lowest siRNA
concentration for each duplex tested.
TABLE-US-00007 TABLE 6 IC.sub.50 of select TMPRSS6 siRNA duplexes
in in vitro dose response screen. Normalized to low dose Normalized
to AD-1955 Day 1 Day 5 Day 1 Day 5 Duplex ID (nM) (nM) (nM) (nM)
AD-46250.1 0.57 0.08 0.22 0.04 AD-46265.1 0.14 0.07 0.2 0.03
AD-46231.1 0.07 0.04 0.06 0.02 AD-46251.1 0.27 0.1 0.37 0.07
AD-46261.1 0.04 0.09 0.08 0.05 AD-46253.1 0.78 0.07 0.35 0.13
AD-46244.1 0.14 0.13 0.2 0.32 AD-46269.1 0.06 0.57 0.07 1.16
AD-46270.1 0.94 No IC50 0 No IC50 AD-46282.1 1.16 No IC50 0.02 No
IC50 AD-46297.1 0.05 No IC50 0.08 No IC50 AD-46299.1 0.01 3.89 0.03
0.69 AD-46303.1 0.01 2.47 0.03 0.04 AD-46307.1 1.02 0.02 2.68 0.15
AD-46273.1 0.23 0.03 0.72 0.1 AD-46286.1 0.22 0.46 0.53 0.46
AD-46249.1 0.27 1.96 0.31 5.87 AD-46295.1 0.76 0.31 0.24 0.1
In Vitro Viability Screening of HeLa and HEP3B Cell Lines
Transfected with TMPRSS6 siRNA Duplexes.
[0404] Table 7 presents viability data of HeLa and HEP3B cell lines
transfected with TMPRSS6 siRNA duplexes. Viability data are
expressed as average raw fluorescence units, where smaller values
represent lower viability. Error is expressed as standard deviation
from three replicate transfections.
TABLE-US-00008 TABLE 7 Viability of HeLa and HEP3B cell lines
transfected with TMPRSS6 siRNA duplexes. HeLa HeLa HeLa HeLa HeLa
HeLa HeLa HeLa HeLa HeLa HeLa HeLa Day 3 Day 3 Day 3 Day 3 Day 3
Day 3 Day 3 Day 3 Day 3 Day 3 Day 3 Day 3 10 1 0.1 0.01 0.001
0.0001 10 1 0.1 0.01 0.001 0.0001 nM nM nM nM nM nM nM SD nM SD nM
SD nM SD nM SD nM SD AD-46250.1 5260 13504 29520 30542 30924 30956
150 62 272 220 799 751 AD-46265.1 12234 29940 32497 33323 32124
32882 968 884 1071 946 707 595 AD-46231.1 25177 28407 32021 32650
33375 32704 710 420 127 1697 356 667 AD-46251.1 29528 30151 30215
32163 31743 31726 416 102 31 1588 518 1091 AD-46261.1 16677 26331
30594 31681 32847 31544 390 277 431 1375 681 583 AD-46253.1 21580
28887 30953 31684 32457 31491 1158 437 524 944 229 455 AD-46244.1
13230 16369 26545 31359 32753 32280 197 165 255 357 589 1318
AD-46269.1 9978 19514 29290 30839 31529 31173 597 360 1406 400 743
626 AD-46270.1 17543 17834 31180 31087 32793 31314 370 1026 771 552
391 1293 AD-46282.1 29055 32421 31840 31006 34287 32185 446 618 430
855 323 133 AD-46297.1 8126 16696 28128 33928 33955 32322 193 598
733 895 1266 392 AD-46299.1 31922 30196 30880 30447 31900 32608
1459 617 58 194 773 964 AD-46303.1 27309 28325 27975 29319 30310
30935 1363 572 421 295 306 95 AD-46307.1 33156 33240 32059 33072
32135 33307 667 258 775 1164 102 286 AD-46273.1 24465 29130 30417
33043 34639 31876 142 768 271 261 853 800 AD-46286.1 3640 9590
29713 33138 32877 30814 34 631 371 1185 1641 599 AD-46249.1 17315
25591 30443 31599 32719 29855 981 258 578 482 1412 886 AD-46295.1
30565 31730 30772 31777 32874 30916 403 261 1223 1880 981 441
AD-19200 9727 15752 31352 32521 30110 30650 648 699 763 1543 55 9
PLK 1166 1626 27849 29902 30512 30273 23 44 91 299 362 563 AD-1955
26502 30164 30267 31906 33309 30906 5669 134 353 645 233 696 Naive
32821 32311 30805 31683 33238 31470 1455 631 555 557 288 164 Naive
33594 32373 32005 34024 35629 33401 554 253 754 899 55 649 Naive
30695 30651 29956 31377 32734 32527 304 299 807 874 646 225 HeLa
HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa HeLa Day 5 Day 5
Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 10 1
0.1 0.01 0.001 0.0001 10 1 0.1 0.01 0.001 0.0001 nM nM nM nM nM nM
nM SD nM SD nM SD nM SD nM SD nM SD AD-46250.1 2344 25502 46627
44986 46479 46070 44 1916 157 913 598 2016 AD-46265.1 10411 46611
48725 47425 47238 47942 300 327 602 1479 2145 1690 AD-46231.1 41079
46963 48575 48060 47467 48500 1645 319 243 998 1821 1203 AD-46251.1
42551 47044 49088 48269 47755 48719 1597 420 162 1105 1434 1232
AD-46261.1 37500 46441 48702 47953 47776 48878 689 441 451 1447
1614 1159 AD-46253.1 31772 45899 48606 47801 47693 49237 1310 65
648 1550 1365 789 AD-46244.1 11597 28046 46020 47413 47670 49430
967 527 395 1336 937 869 AD-46269.1 10704 37735 47496 47629 47496
49194 317 161 198 1359 1502 986 AD-46270.1 16356 26284 48520 48011
48016 49358 382 663 497 1121 1024 681 AD-46282.1 22372 42327 47297
47478 47450 49349 656 715 343 1513 2057 883 AD-46297.1 4228 26993
47037 47269 46961 48993 41 657 593 1847 1574 639 AD-46299.1 45283
45485 46334 43966 42922 46772 1088 908 382 2057 3580 1131
AD-46303.1 42669 46358 46240 45624 46920 46764 849 183 791 890 90
539 AD-46307.1 47710 47466 47974 47671 47911 48505 273 539 680 399
238 309 AD-46273.1 36834 45018 47522 47912 48316 48804 680 432 104
619 308 248 AD-46286.1 2970 31215 47504 47883 48062 48999 515 1262
1093 826 87 541 AD-46249.1 20356 44959 48534 47988 48053 49145 884
1033 1238 1045 619 530 AD-46295.1 46448 48014 49195 48654 48432
49355 685 1021 746 1183 645 407 AD-19200 26444 35772 39724 48377
48373 49509 725 1009 1246 540 762 408 PLK 1105 1804 37258 47955
47893 49416 44 95 1110 781 474 515 AD-1955 44857 47272 48354 48050
48668 49721 322 388 756 880 585 490 Naive 48734 48454 48549 47246
48004 49067 850 303 743 1166 349 102 Naive 48318 47839 45252 47098
47128 48914 969 527 223 797 548 526 Naive 45189 45096 45508 44334
45177 47004 1327 938 579 1342 930 350 Hep3B Hep3B Hep3B Hep3B Hep3B
Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Day 3 Day 3 Day 3 Day 3
Day 3 Day 3 Day 3 Day 3 Day 3 Day 3 Day 3 Day 3 10 1 0.1 0.01 0.001
0.0001 10 1 0.1 0.01 0.001 0.0001 nM nM nM nM nM nM nM SD nM SD nM
SD nM SD nM SD nM SD AD-46250.1 4495 4905 6786 7022 6122 6033 225
105 56 85 151 49 AD-46265.1 6453 6990 6917 6685 6165 5974 187 79
103 70 121 21 AD-46231.1 6478 7042 6808 6444 6173 5987 97 19 35 66
131 69 AD-46251.1 5663 5990 6084 6241 5869 6298 445 38 73 69 63 88
AD-46261.1 5380 6025 5824 6325 5801 6076 376 14 29 67 81 65
AD-46253.1 5417 6078 5840 6113 5568 6503 549 29 103 81 20 72
AD-46244.1 4743 5479 5884 6078 6170 6593 29 43 51 168 60 70
AD-46269.1 2788 2958 5479 5878 5899 5739 64 14 97 215 80 22
AD-46270.1 4378 4720 5579 6127 6066 6522 235 94 167 17 43 260
AD-46282.1 5096 5932 6258 5988 6068 6724 101 34 32 107 59 20
AD-46297.1 1134 1325 4477 6051 6199 6626 40 64 80 101 134 55
AD-46299.1 5875 5836 6251 5872 6016 6726 47 64 39 54 81 104
AD-46303.1 6879 7060 6801 6793 6306 6827 43 32 59 60 126 65
AD-46307.1 6951 6826 6613 6511 6119 7093 97 148 46 82 97 91
AD-46273.1 6628 6749 6711 6839 6237 6958 122 24 59 59 48 109
AD-46286.1 5384 5405 5755 6469 6299 6207 81 5 45 33 95 58
AD-46249.1 3955 4239 5214 6549 6171 6537 141 70 134 37 35 27
AD-46295.1 6186 6535 5776 6500 6247 6252 96 34 141 35 41 35
AD-19200 2304 3860 5592 6634 6063 6111 95 24 43 41 67 74 PLK 1484
1668 3385 6283 5714 6015 36 52 130 94 112 143 AD-1955 5718 5826
5633 6356 6369 6460 27 60 16 80 108 122 Naive 5799 6503 6350 6351
6002 6449 69 98 44 40 72 66 Naive 5623 6550 5950 6103 5574 6489 23
49 37 82 59 93 Naive 5895 6021 5550 5908 5573 6769 72 27 55 90 64
42 Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B Hep3B
Hep3B Hep3B Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5 Day 5
Day 5 Day 5 Day 5 10 1 0.1 0.01 0.001 0.0001 10 1 0.1 0.01 0.001
0.0001 nM nM nM nM nM nM nM SD nM SD nM SD nM SD nM SD nM SD
AD-46250.1 4758 5572 9636 12294 11079 10674 311 285 180 901 575 403
AD-46265.1 9937 12543 10822 13430 12967 12089 323 1714 1094 107
1407 704 AD-46231.1 12650 13786 11763 13765 14003 13857 1002 422
1551 177 213 320 AD-46251.1 8543 9397 10581 13642 12990 13568 518
1054 707 289 1247 475 AD-46261.1 10459 11700 11735 13764 13738
13210 148 1308 459 277 712 210 AD-46253.1 11125 12124 11533 14213
13967 11946 473 1531 772 262 679 1015 AD-46244.1 7330 7939 10428
12695 13584 11852 451 416 1104 61 358 1473 AD-46269.1 2316 2442
11476 13621 12821 11090 507 623 574 299 831 298 AD-46270.1 6643
5235 11774 12788 13487 11895 179 386 709 1032 635 760 AD-46282.1
7767 10214 12650 12859 12980 11175 214 1116 569 1282 925 169
AD-46297.1 1012 1124 9438 12403 12063 11599 47 96 162 990 1118 83
AD-46299.1 13643 13396 12404 12113 12782 12913 1585 2086 202 896
1040 1209 AD-46303.1 10567 12918 10617 11203 11189 11260 456 1263
106 309 310 153 AD-46307.1 13787 14089 11830 13512 13489 12773 208
467 900 60 504 203 AD-46273.1 13801 13484 12719 14212 14305 12499
386 219 1250 382 128 176 AD-46286.1 5783 6472 10990 14352 14424
12234 93 78 472 632 103 649 AD-46249.1 3763 5086 10729 14293 14283
12608 269 124 453 570 443 633 AD-46295.1 14870 15096 11289 14697
14336 12000 539 224 453 698 689 903 AD-19200 1546 6337 10310 14261
13551 11486 132 379 456 250 646 754 PLK 1337 1636 6996 14661 13860
12555 31 79 759 740 423 296 AD-1955 11717 12560 12164 14504 13008
11077 1146 1210 1289 392 1405 56 Naive 13989 14873 11512 14022
13458 11399 404 316 267 412 635 114 Naive 14167 14550 11269 14247
13793 11771 197 426 230 640 664 888 Naive 13857 14632 10432 13485
14164 12808 231 1150 474 546 177 1028
Example 4. TMPRSS6 siRNA Duplex Lead Selection
[0405] To select specific TMPRSS6 siRNAs for use in further in vivo
experimentation, chemically modified siRNAs were screened by
transfection in HEP3B human hepatoma cells for TMPRSS6 gene
silencing activity. Two highly potent siRNAs with minimal predicted
off-target potential and with multi-species reactivity, including
human cynomolgus monkey, rat, and mouse, were selected for
evaluation in vivo. Potency of the two selected TMPRSS6 siRNAs was
also confirmed in primary mouse hepatocytes, wherein both TMPRSS6
siRNA-1 (AD-46273) and TMPRSS6 siRNA-2 (AD-46286) demonstrated
strong TMPRSS6 gene silencing activity, with TMPRSS6 siRNA-1
(AD-46273) demonstrating an IC.sub.50 of 70 pM (FIG. 2A) and
TMPRSS6 siRNA-2 (AD-46286) demonstrating an IC.sub.50 of 140 pM
(FIG. 2B).
Example 5. The Effect of TMPRSS6 siRNA Mediated Silencing of
TMPRSS6 in WT C57BL/6 Mice
[0406] The Effect of TMPRSS6 siRNA on TMPRSS6 and HAMP1 mRNA
Expression in WT C57BL/6 Mice.
[0407] In order to evaluate the effect of LNP-TMPRSS6 siRNA-1
(AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286) in vivo, eight week
old female WT C57BL/6 mice were dosed via tail vein IV injection
with 1 mg/kg LNP-TMPRSS6 siRNA-1 (AD-46273) or LNP-TMPRSS6 siRNA-2
(AD-46286) or LNP-AD-19551 (siRNA targeting the non-mammalian gene
LUCIFERASE). The TMPRSS6 siRNAs were formulated with LNP11 (MC3).
The mice were sacrificed 24 hours post dosing, and livers removed,
flash frozen, and ground into powder. A small amount (.about.20 mg)
of liver powder was disrupted in lysis buffer and used for mRNA
analysis by TaqMan.RTM.. A total of five mice were used per group.
The data are expressed as a percent of LNP-Luc control ratios of
target TMPRSS6 mRNA relative to B-actin mRNA. As shown in FIG. 3A,
there was a specific and potent dose dependent inhibition of liver
TMPRSS6 mRNA expression by LNP-TMPRSS6 siRNA-1 (AD-46273) and
LNP-TMPRSS6 siRNA-2 (AD-46286) (data represent mean+/-standard
deviation), with an ED50 of 0.035 mg/kg, and an ED50 of 0.18 mg/kg,
respectively. As shown in FIG. 3B, there was also a dose dependent
inhibition of liver HAMP1 mRNA expression by LNP-TMPRSS6 siRNA-1
(AD-46273) and LNP-TMPRSS6 siRNA-2 (AD-46286).
[0408] The Duration of TMPRSS6 siRNA Mediated Silencing of TMPRSS6
and HAMP1 Gene Expression in WT C57BL/6 Mice.
[0409] In order to evaluate the duration of the TMPRSS6 siRNA
mediated knockdown of TMPRSS6 and HAMP1 gene expression, eight week
old WT C57BL/6 mice were administered a single 1 mg/kg dose via
tail vein IV injection with LNP-TMPRSS6 siRNA-1 (AD-46273), or
LNP-Luc control (LNP-AD-1955), or PBS; all siRNA agents were
delivered as LNP11 formulations. The mice were sacrificed at 6
hours, 24 hours, 48 hours, 3 days, 7 days, and 14 days. The mRNA
expression level of TMPRSS6 and HAMP1 in the liver was analyzed
using TaqMan.RTM. assay and normalized to B-actin. Five mice were
used per group, and the data is represented in FIG. 4 as
mean+/-standard deviation. As shown in FIG. 4, 1 mg/kg single dose
of LNP-TMPRSS6 siRNA-1 (AD-46273) knocked down TMPRSS6 mRNA
expression as early as six hours post dosing, and reduced TMPRSS6
mRNA expression to approximately 90% of LNP-Luc control or PBS
control for the duration of the two week time period. HAMP1 gene
expression was increased starting 24 hours post dosing and was
maintained for the duration of the two week time period, with a
maximum increase of 200% of control on day 14 post dosing (FIG. 4).
In addition, serum iron levels were assayed as the percentage of
transferrin (Tf) saturation using an Olympus AU 400. The level of
transferrin saturation was calculated as the ratio of serum iron to
total iron binding capacity (TIBC) and is expressed as a percent of
transferrin saturation. The percent of transferrin saturation was
reduced by approximately 50% starting 24 hours post dosing and
maintained over the two week time period, indicating that the
circulating iron levels in the serum were decreased (FIG. 4). Level
of TMPRSS6 siRNA mediated silencing of TMPRSS6 necessary to
maintain the TMPRSS6 siRNA mediated effects on HAMP1 gene
expression and serum iron levels in WT C57BL/6 Mice.
[0410] In order to evaluate the level of TMPRSS6 siRNA mediated
silencing of TMPRSS6 necessary to maintain the TMPRSS6 siRNA
mediated effects on HAMP1 gene expression and serum iron levels in
WT C57BL/6 mice; C57BL/6 mice were dosed with 0.3 mg/kg LNP-TMPRSS6
siRNA-1 (AD-46273), or LNP-Luc control, or PBS; all siRNA agents
were delivered as LNP11 formulations. The mice were sacrificed at 5
hours, 24 hours, 48 hours, 3 days, 7 days, 14 days, 21, days, and
28 days post dosing. The mRNA expression level of TMPRSS6 and HAMP1
was analyzed using TaqMan.RTM. assay and normalized to B-actin.
Five mice were used per group, and the data is represented in FIG.
5 as mean+/-standard deviation. As shown in FIG. 5, the maximal
reduction of TMPRSS6 gene expression of 90% was achieved 24 hours
post dosing and maintained up until day three post dosing. At day
seven post dosing, TMPRSS6 gene expression was reduced by
approximately 85%; HAMP1 gene expression was induced to
approximately 250% of control; and transferrin saturation (%) was
reduced by approximately 50% (FIG. 5). At day 21 post dosing,
TMPRSS6 gene expression was reduced by approximately 40%; HAMP1
gene expression had normalized; and serum iron levels, as measured
by transferrin saturation (%), began to return to normal levels
(FIG. 5). In summary, maximal knockdown of TMPRSS6 mRNA expression
was achieved at 24 hours post dosing and returned to approximately
50% of normal expression levels by 3 weeks post dosing; hepcidin
mRNA levels were increased as early as 24 hours and maintained up
to seven days post dosing; hepcidin levels returned to control
levels on day fourteen post dosing; and transferrin saturation, as
an indicator of circulating iron levels, was reduced by 50% of
control levels as early as 24 hours post dosing, and was normalized
towards week four. Thus the data presented in FIG. 5 illustrates
that more than 50% TMPRSS6 silencing is required to maintain the
LNP-TMPRSS6 siRNA-1 (AD-46273) mediated effects on HAMP1 gene
expression and serum iron levels.
[0411] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Hematological Parameters in WT C57BL/6 Mice.
[0412] In order to evaluate the effect of TMPRSS6 siRNA mediated
silencing of TMPRSS6 on hematological parameters, including
hemoglobin (HGB) and hematocrit; WT C57BL/6 mice were dosed with 1
mg/kg single dose of TMPRSS6 siRNA-1 (AD-46273) or LNP-Luc control,
or PBS; and subsequently sacrificed at different time points up to
two weeks post dosing. Hematological parameters including,
hemoglobin (HGB), hematocrit, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), and the reticulocyte hemoglobin content (Chr)
were assayed using Advia 120 analyzer. As shown in FIGS. 6A and 6B,
silencing of TMPRSS6 in Th3/+ mice led to a decrease HGB (FIG. 6A),
and a decrease in hematocrit (FIG. 6B) in WT C57BL/6 mice. There
was a similar effect on mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin
concentration (MCHC), and the reticulocyte hemoglobin content
(Chr).
Example 6. The Effect of TMPRSS6 siRNA Mediated Silencing of
TMPRSS6 in Thalassemic Mice (Th3/+)
[0413] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Serum Iron Parameters in Thalassemic Mice (Th3/+).
[0414] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on serum iron parameters, including iron levels,
unsaturated iron-binding capacity (UIBC), and Tf saturation in
thalassemic mice (Th3/+), six week old Th3/+mice were dosed via
tail vein injection with 1 mg/kg LNP-TMPRSS6 siRNA-1 (AD-46273), or
LNP-Luc control, or PBS, and the mice were sacrificed two weeks
post dosing. Five mice were used per group, and data represented in
FIG. 7 as mean+/-standard deviation, with ** denoting a
P-value<0.01 and *** denoting a P-value<0.001. As shown in
FIG. 7, silencing of TMPRSS6 in Th3/+mice led to a significant
reduction of serum iron, UIBC, and Tf saturation compared to the
control PBS group.
[0415] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Reticulocyte and Erthyrocyte Parameters in Thalassemic Mice
(Th3/+).
[0416] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on reticulocyte and erythrocyte parameters, including
reticulocyte number, reticulocyte hemoglobin content (CHr), and
erythrocyte number (RBC), in thalassemic mice (Th3/+); six week old
Th3/+ mice were dosed via tail vein injection, with 1 mg/kg
LNP-TMPRSS6 siRNA-1 (AD-46273), or LNP-Luc control, or PBS, and the
mice were sacrificed two weeks post dosing. Reticulocyte and
erythrocyte parameters, including reticulocyte number, reticulocyte
hemoglobin content (CHr), and erythrocyte number (RBC) were assayed
using Advia 120 analyzer. Five mice were used per group, and the
data is represented in FIGS. 8A-8C as mean+/-standard deviation,
with ** denoting a P-value<0.01 and *** denoting a
P-value<0.001. As shown in FIGS. 8A and 8B, respectively,
silencing of TMPRSS6 in Th3/+ mice led to a significant reduction
in the number of reticulocytes as well as the hemoglobin content of
reticulocytes (Chr). In addition, silencing of TMPRSS6 in Th3/+
mice led to a significant increase in the number of mature
erythrocytes (RBC) (FIG. 8C), demonstrating a significant
improvement in ineffective erythropoiesis, extramedullary
hematopoiesis, and red blood cell production.
[0417] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Hematological Parameters in Thalassemic Mice (Th3/+).
[0418] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on hematological parameters, including hematocrit (HCT),
hemoglobin (HGB), red blood cell distribution width (RDW), and mean
corpuscle value (MCV) in thalassemic mice (Th3/+); six week old
Th3/+ mice were dosed via tail vein injection, with 1 mg/kg
LNP-TMPRSS6 siRNA-1 (AD-46273), or LNP-Luc control, or PBS, and the
mice were sacrificed two weeks post dosing. Hematological paramters
including hematocrit (HCT), hemoglobin (HGB), red blood cell
distribution width (RDW), and mean corpuscle value (MCV); were
assayed using Advia 120 analyzer. Five mice were used per group,
and the data is represented in FIG. 9 as mean+/-standard deviation,
with ** denoting a P-value<0.01 and *** denoting a
P-value<0.001. Silencing of TMPRSS6 in Th3/+ mice led to a
significant increase in HCT (FIG. 9A), a significant increase in
HGB (FIG. 9B), a significant decrease in RDW (FIG. 9C), and a
significant decrease in MCV (FIG. 9D). The data presented in FIG. 9
illustrates a normalization of the .beta.-thalassemia phenotype in
these hematological parameters post administration of the
LNP-TMPRSS6 siRNA-1 (AD-46273).
[0419] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Peripheral Blood Morphology in Thalassemic Mice (Th3/+).
[0420] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on peripheral blood morphology in thalassemic mice
(Th3/+); six week old Th3/+ mice were dosed via tail vein injection
with, 1 mg/kg LNP-TMPRSS6 siRNA-1 (AD-46273) or LNP-Luc control,
and the mice were sacrificed two weeks post dosing.
May-Grunwald/Gimsa stain at 10.times. magnification showed a marked
decrease in polychromasia in the Th3/+ mice treated with the
TMPRSS6 siRNA compared to control, representative of the decreased
reticulocyte number as well as an overall trend toward
normalization of the mature red blood cell morphology.
May-Grunwald/Gimsa stain at 10.times. magnification also showed
slight anisocytosis was induced by the WT TMPRSS6 siRNA animal when
compared to WT control animal.
[0421] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Splenic Architecture in Thalassemic Mice (Th3/+).
[0422] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on splenic architecture in thalassemic mice (Th3/+); six
week old Th3/+ mice were dosed via tail vein injection, with 1
mg/kg LNP-TMPRSS6 siRNA-1 (AD-46273), or LNP-Luc control, or PBS,
and mice were sacrificed two weeks post dosing. Hematoxylin and
eosin (H&E) stain at 10.times. magnification showed Th3/+ mice
treated with the TMPRSS6 siRNA compared to control had a
normalization of splenic architecture, including a reduction in
sinusoidal extramedullary erythropoiesis and the reappearance of
white pulp nodules.
[0423] The Effect of TMPRSS6 siRNA Mediated Silencing of TMPRSS6 on
Spleen and Liver Iron Content in Thalassemic Mice (Th3/+).
[0424] To evaluate the effect of TMPRSS6 siRNA mediated silencing
of TMPRSS6 on spleen and liver iron content in thalassemic mice
(Th3/+); six week old Th3/+ mice were dosed via tail vein
injection, with 1 mg/kg LNP-TMPRSS6 siRNA-1 (AD-46273), or LNP-Luc
control, or PBS, and the mice were sacrificed two weeks post
dosing. Five mice were used per group, and the data is represented
in FIGS. 10A-10C as mean+/-standard deviation, with ** denoting a
P-value<0.01 and *** denoting a P-value<0.001. Silencing of
TMPRSS6 in Th3/+ mice led to a significant reduction of spleen iron
content and spleen weight (FIG. 10A and FIG. 10B, respectively),
indicating a normalization of extramedullary hematopoiesis. A trend
towards a reduction liver iron content was also observed, but was
not statistically significant (FIG. 10C).
[0425] The above results demonstrate that silencing of TMPRSS6 by
systemic administration of formulated siRNAs increases HAMP
expression to levels sufficient to ameliorate the phenotype in a
mouse model of .beta.-thalassemia intermedia. Therefore,
LNP-TMPRSS6-siRNAs are being developed for congenital iron overload
disorders characterized by abnormally low hepcidin levels, (e.g.,
.beta.-thalassemia intermedia and hereditary hemochromatosis).
EQUIVALENTS
[0426] 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 1
1
61113212DNAHomo sapiens 1cttgagccag acccagtcca gctctggtgc
ctgccctctg gtgcgagctg acctgagatg 60cacttccctc ctctgtgagc tgtctcggca
cccacttgca gtcactgccg cctgatgttg 120ttactcttcc actccaaaag
gatgcccgtg gccgaggccc cccaggtggc tggcgggcag 180ggggacggag
gtgatggcga ggaagcggag ccggagggga tgttcaaggc ctgtgaggac
240tccaagagaa aagcccgggg ctacctccgc ctggtgcccc tgtttgtgct
gctggccctg 300ctcgtgctgg cttcggcggg ggtgctactc tggtatttcc
tagggtacaa ggcggaggtg 360atggtcagcc aggtgtactc aggcagtctg
cgtgtactca atcgccactt ctcccaggat 420cttacccgcc gggaatctag
tgccttccgc agtgaaaccg ccaaagccca gaagatgctc 480aaggagctca
tcaccagcac ccgcctggga acttactaca actccagctc cgtctattcc
540tttggggagg gacccctcac ctgcttcttc tggttcattc tccaaatccc
cgagcaccgc 600cggctgatgc tgagccccga ggtggtgcag gcactgctgg
tggaggagct gctgtccaca 660gtcaacagct cggctgccgt cccctacagg
gccgagtacg aagtggaccc cgagggccta 720gtgatcctgg aagccagtgt
gaaagacata gctgcattga attccacgct gggttgttac 780cgctacagct
acgtgggcca gggccaggtc ctccggctga aggggcctga ccacctggcc
840tccagctgcc tgtggcacct gcagggcccc aaggacctca tgctcaaact
ccggctggag 900tggacgctgg cagagtgccg ggaccgactg gccatgtatg
acgtggccgg gcccctggag 960aagaggctca tcacctcggt gtacggctgc
agccgccagg agcccgtggt ggaggttctg 1020gcgtcggggg ccatcatggc
ggtcgtctgg aagaagggcc tgcacagcta ctacgacccc 1080ttcgtgctct
ccgtgcagcc ggtggtcttc caggcctgtg aagtgaacct gacgctggac
1140aacaggctcg actcccaggg cgtcctcagc accccgtact tccccagcta
ctactcgccc 1200caaacccact gctcctggca cctcacggtg ccctctctgg
actacggctt ggccctctgg 1260tttgatgcct atgcactgag gaggcagaag
tatgatttgc cgtgcaccca gggccagtgg 1320acgatccaga acaggaggct
gtgtggcttg cgcatcctgc agccctacgc cgagaggatc 1380cccgtggtgg
ccacggccgg gatcaccatc aacttcacct cccagatctc cctcaccggg
1440cccggtgtgc gggtgcacta tggcttgtac aaccagtcgg acccctgccc
tggagagttc 1500ctctgttctg tgaatggact ctgtgtccct gcctgtgatg
gggtcaagga ctgccccaac 1560ggcctggatg agagaaactg cgtttgcaga
gccacattcc agtgcaaaga ggacagcaca 1620tgcatctcac tgcccaaggt
ctgtgatggg cagcctgatt gtctcaacgg cagcgacgaa 1680gagcagtgcc
aggaaggggt gccatgtggg acattcacct tccagtgtga ggaccggagc
1740tgcgtgaaga agcccaaccc gcagtgtgat gggcggcccg actgcaggga
cggctcggat 1800gaggagcact gtgactgtgg cctccagggc ccctccagcc
gcattgttgg tggagctgtg 1860tcctccgagg gtgagtggcc atggcaggcc
agcctccagg ttcggggtcg acacatctgt 1920gggggggccc tcatcgctga
ccgctgggtg ataacagctg cccactgctt ccaggaggac 1980agcatggcct
ccacggtgct gtggaccgtg ttcctgggca aggtgtggca gaactcgcgc
2040tggcctggag aggtgtcctt caaggtgagc cgcctgctcc tgcacccgta
ccacgaagag 2100gacagccatg actacgacgt ggcgctgctg cagctcgacc
acccggtggt gcgctcggcc 2160gccgtgcgcc ccgtctgcct gcccgcgcgc
tcccacttct tcgagcccgg cctgcactgc 2220tggattacgg gctggggcgc
cttgcgcgag ggcggcccca tcagcaacgc tctgcagaaa 2280gtggatgtgc
agttgatccc acaggacctg tgcagcgagg tctatcgcta ccaggtgacg
2340ccacgcatgc tgtgtgccgg ctaccgcaag ggcaagaagg atgcctgtca
gggtgactca 2400ggtggtccgc tggtgtgcaa ggcactcagt ggccgctggt
tcctggcggg gctggtcagc 2460tggggcctgg gctgtggccg gcctaactac
ttcggcgtct acacccgcat cacaggtgtg 2520atcagctgga tccagcaagt
ggtgacctga ggaactgccc ccctgcaaag cagggcccac 2580ctcctggact
cagagagccc agggcaactg ccaagcaggg ggacaagtat tctggcgggg
2640ggtgggggag agagcaggcc ctgtggtggc aggaggtggc atcttgtctc
gtccctgatg 2700tctgctccag tgatggcagg aggatggaga agtgccagca
gctgggggtc aagacgtccc 2760ctgaggaccc aggcccacac ccagcccttc
tgcctcccaa ttctctctcc tccgtcccct 2820tcctccactg ctgcctaatg
caaggcagtg gctcagcagc aagaatgctg gttctacatc 2880ccgaggagtg
tctgaggtgc gccccactct gtacagaggc tgtttgggca gccttgcctc
2940cagagagcag attccagctt cggaagcccc tggtctaact tgggatctgg
gaatggaagg 3000tgctcccatc ggaggggacc ctcagagccc tggagactgc
caggtgggcc tgctgccact 3060gtaagccaaa aggtggggaa gtcctgactc
cagggtcctt gccccacccc tgcctgccac 3120ctgggccctc acagcccaga
ccctcactgg gaggtgagct cagctgccct ttggaataaa 3180gctgcctgat
caaaaaaaaa aaaaaaaaaa aa 3212229PRTUnknownDescription of Unknown
Exemplary primary peptide of endosomolytic components 2Ala Ala Leu
Glu Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10 15Leu Glu
Ala Leu Ala Glu Ala Ala Ala Ala Gly Gly Cys 20
25330PRTUnknownDescription of Unknown Exemplary primary peptide of
endosomolytic components 3Ala Ala Leu Ala Glu Ala Leu Ala Glu Ala
Leu Ala Glu Ala Leu Ala1 5 10 15Glu Ala Leu Ala Glu Ala Leu Ala Ala
Ala Ala Gly Gly Cys 20 25 30415PRTUnknownDescription of Unknown
Exemplary primary peptide of endosomolytic components 4Ala Leu Glu
Ala Leu Ala Glu Ala Leu Glu Ala Leu Ala Glu Ala1 5 10
15516PRTUnknownDescription of Unknown Hydrophobic membrane
translocation peptide 5Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu
Ala Leu Leu Ala Pro1 5 10 15611PRTUnknownDescription of Unknown
Hydrophobic membrane translocation peptide 6Ala Ala Leu Leu Pro Val
Leu Leu Ala Ala Pro1 5 10713PRTHuman immunodeficiency virus 7Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln1 5
10816PRTDrosophila sp. 8Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg
Met Lys Trp Lys Lys1 5 10 15919RNAHomo sapiens 9cucuggugcg
agcugaccu 191019RNAHomo sapiens 10aggucagcuc gcaccagag
191119RNAHomo sapiens 11agcugaccug agaugcacu 191219RNAHomo sapiens
12agugcaucuc aggucagcu 191319RNAHomo sapiens 13ucugugagcu gucucggca
191419RNAHomo sapiens 14ugccgagaca gcucacaga 191519RNAHomo sapiens
15agcugucucg gcacccacu 191619RNAHomo sapiens 16agugggugcc gagacagcu
191719RNAHomo sapiens 17gcugucucgg cacccacuu 191819RNAHomo sapiens
18aagugggugc cgagacagc 191919RNAHomo sapiens 19agucacugcc gccugaugu
192019RNAHomo sapiens 20acaucaggcg gcagugacu 192119RNAHomo sapiens
21acugccgccu gauguuguu 192219RNAHomo sapiens 22aacaacauca ggcggcagu
192319RNAHomo sapiens 23cugccgccug auguuguua 192419RNAHomo sapiens
24uaacaacauc aggcggcag 192519RNAHomo sapiens 25gccgccugau guuguuacu
192619RNAHomo sapiens 26aguaacaaca ucaggcggc 192719RNAHomo sapiens
27gccugauguu guuacucuu 192819RNAHomo sapiens 28aagaguaaca acaucaggc
192919RNAHomo sapiens 29cucuuccacu ccaaaagga 193019RNAHomo sapiens
30uccuuuugga guggaagag 193119RNAHomo sapiens 31acuccaaaag gaugcccgu
193219RNAHomo sapiens 32acgggcaucc uuuuggagu 193319RNAHomo sapiens
33gugaggacuc caagagaaa 193419RNAHomo sapiens 34uuucucuugg aguccucac
193519RNAHomo sapiens 35cuucggcggg ggugcuacu 193619RNAHomo sapiens
36aguagcaccc ccgccgaag 193719RNAHomo sapiens 37ucggcggggg ugcuacucu
193819RNAHomo sapiens 38agaguagcac ccccgccga 193919RNAHomo sapiens
39gcgggggugc uacucuggu 194019RNAHomo sapiens 40accagaguag cacccccgc
194119RNAHomo sapiens 41gggggugcua cucugguau 194219RNAHomo sapiens
42auaccagagu agcaccccc 194319RNAHomo sapiens 43ggggugcuac ucugguauu
194419RNAHomo sapiens 44aauaccagag uagcacccc 194519RNAHomo sapiens
45ucugguauuu ccuagggua 194619RNAHomo sapiens 46uacccuagga aauaccaga
194719RNAHomo sapiens 47ugguauuucc uaggguaca 194819RNAHomo sapiens
48uguacccuag gaaauacca 194919RNAHomo sapiens 49gguauuuccu aggguacaa
195019RNAHomo sapiens 50uuguacccua ggaaauacc 195119RNAHomo sapiens
51ggucagccag guguacuca 195219RNAHomo sapiens 52ugaguacacc uggcugacc
195319RNAHomo sapiens 53agccaggugu acucaggca 195419RNAHomo sapiens
54ugccugagua caccuggcu 195519RNAHomo sapiens 55guacucaggc agucugcgu
195619RNAHomo sapiens 56acgcagacug ccugaguac 195719RNAHomo sapiens
57acucaggcag ucugcgugu 195819RNAHomo sapiens 58acacgcagac ugccugagu
195919RNAHomo sapiens 59caggcagucu gcguguacu 196019RNAHomo sapiens
60aguacacgca gacugccug 196119RNAHomo sapiens 61ggcagucugc guguacuca
196219RNAHomo sapiens 62ugaguacacg cagacugcc 196319RNAHomo sapiens
63gcagucugcg uguacucaa 196419RNAHomo sapiens 64uugaguacac gcagacugc
196519RNAHomo sapiens 65cagucugcgu guacucaau 196619RNAHomo sapiens
66auugaguaca cgcagacug 196719RNAHomo sapiens 67ugcguguacu caaucgcca
196819RNAHomo sapiens 68uggcgauuga guacacgca 196919RNAHomo sapiens
69cguguacuca aucgccacu 197019RNAHomo sapiens 70aguggcgauu gaguacacg
197119RNAHomo sapiens 71guguacucaa ucgccacuu 197219RNAHomo sapiens
72aaguggcgau ugaguacac 197319RNAHomo sapiens 73guacucaauc gccacuucu
197419RNAHomo sapiens 74agaaguggcg auugaguac 197519RNAHomo sapiens
75cacuucuccc aggaucuua 197619RNAHomo sapiens 76uaagauccug ggagaagug
197719RNAHomo sapiens 77gaucuuaccc gccgggaau 197819RNAHomo sapiens
78auucccggcg gguaagauc 197919RNAHomo sapiens 79ucuuacccgc cgggaaucu
198019RNAHomo sapiens 80agauucccgg cggguaaga 198119RNAHomo sapiens
81cuuacccgcc gggaaucua 198219RNAHomo sapiens 82uagauucccg gcggguaag
198319RNAHomo sapiens 83uacccgccgg gaaucuagu 198419RNAHomo sapiens
84acuagauucc cggcgggua 198519RNAHomo sapiens 85cgccgggaau cuagugccu
198619RNAHomo sapiens 86aggcacuaga uucccggcg 198719RNAHomo sapiens
87gccgggaauc uagugccuu 198819RNAHomo sapiens 88aaggcacuag auucccggc
198919RNAHomo sapiens 89uccgcaguga aaccgccaa 199019RNAHomo sapiens
90uuggcgguuu cacugcgga 199119RNAHomo sapiens 91ccgcagugaa accgccaaa
199219RNAHomo sapiens 92uuuggcgguu ucacugcgg 199319RNAHomo sapiens
93cgccugggaa cuuacuaca 199419RNAHomo sapiens 94uguaguaagu ucccaggcg
199519RNAHomo sapiens 95gccugggaac uuacuacaa 199619RNAHomo sapiens
96uuguaguaag uucccaggc 199719RNAHomo sapiens 97cugggaacuu acuacaacu
199819RNAHomo sapiens 98aguuguagua aguucccag 199919RNAHomo sapiens
99uacaacucca gcuccgucu 1910019RNAHomo sapiens 100agacggagcu
ggaguugua 1910119RNAHomo sapiens 101acaacuccag cuccgucua
1910219RNAHomo sapiens 102uagacggagc uggaguugu 1910319RNAHomo
sapiens 103aacuccagcu ccgucuauu 1910419RNAHomo sapiens
104aauagacgga gcuggaguu 1910519RNAHomo sapiens 105uuuggggagg
gaccccuca 1910619RNAHomo sapiens 106ugaggggucc cuccccaaa
1910719RNAHomo sapiens 107ggaccccuca ccugcuucu 1910819RNAHomo
sapiens 108agaagcaggu gaggggucc 1910919RNAHomo sapiens
109gcuucuucug guucauucu 1911019RNAHomo sapiens 110agaaugaacc
agaagaagc 1911119RNAHomo sapiens 111ucuucugguu cauucucca
1911219RNAHomo sapiens 112uggagaauga accagaaga 1911319RNAHomo
sapiens 113agcaccgccg gcugaugcu 1911419RNAHomo sapiens
114agcaucagcc ggcggugcu 1911519RNAHomo sapiens 115uccccuacag
ggccgagua 1911619RNAHomo sapiens 116uacucggccc uguagggga
1911719RNAHomo sapiens 117ccuacagggc cgaguacga 1911819RNAHomo
sapiens 118ucguacucgg cccuguagg 1911919RNAHomo sapiens
119acagggccga guacgaagu 1912019RNAHomo sapiens 120acuucguacu
cggcccugu 1912119RNAHomo sapiens 121gggccgagua cgaagugga
1912219RNAHomo sapiens 122uccacuucgu acucggccc 1912319RNAHomo
sapiens 123ccgagggccu agugauccu 1912419RNAHomo sapiens
124aggaucacua ggcccucgg 1912519RNAHomo sapiens 125cagugugaaa
gacauagcu 1912619RNAHomo sapiens 126agcuaugucu uucacacug
1912719RNAHomo sapiens 127gaauuccacg cuggguugu 1912819RNAHomo
sapiens 128acaacccagc guggaauuc 1912919RNAHomo sapiens
129aauuccacgc uggguuguu 1913019RNAHomo sapiens 130aacaacccag
cguggaauu 1913119RNAHomo sapiens 131acgcuggguu guuaccgcu
1913219RNAHomo sapiens 132agcgguaaca acccagcgu 1913319RNAHomo
sapiens 133cgcuggguug uuaccgcua 1913419RNAHomo sapiens
134uagcgguaac aacccagcg 1913519RNAHomo sapiens 135cuggguuguu
accgcuaca 1913619RNAHomo sapiens 136uguagcggua acaacccag
1913719RNAHomo sapiens 137gguuguuacc gcuacagcu 1913819RNAHomo
sapiens 138agcuguagcg guaacaacc 1913919RNAHomo sapiens
139guuaccgcua cagcuacgu 1914019RNAHomo sapiens 140acguagcugu
agcgguaac 1914119RNAHomo sapiens
141aggaccucau gcucaaacu 1914219RNAHomo sapiens 142aguuugagca
ugagguccu 1914319RNAHomo sapiens 143ucaugcucaa acuccggcu
1914419RNAHomo sapiens 144agccggaguu ugagcauga 1914519RNAHomo
sapiens 145aucaccucgg uguacggcu 1914619RNAHomo sapiens
146agccguacac cgaggugau 1914719RNAHomo sapiens 147accucggugu
acggcugca 1914819RNAHomo sapiens 148ugcagccgua caccgaggu
1914919RNAHomo sapiens 149aucauggcgg ucgucugga 1915019RNAHomo
sapiens 150uccagacgac cgccaugau 1915119RNAHomo sapiens
151ucauggcggu cgucuggaa 1915219RNAHomo sapiens 152uuccagacga
ccgccauga 1915319RNAHomo sapiens 153gcuacuacga ccccuucgu
1915419RNAHomo sapiens 154acgaaggggu cguaguagc 1915519RNAHomo
sapiens 155ccgugcagcc gguggucuu 1915619RNAHomo sapiens
156aagaccaccg gcugcacgg 1915719RNAHomo sapiens 157ucuuccaggc
cugugaagu 1915819RNAHomo sapiens 158acuucacagg ccuggaaga
1915919RNAHomo sapiens 159gccugugaag ugaaccuga 1916019RNAHomo
sapiens 160ucagguucac uucacaggc 1916119RNAHomo sapiens
161gugaagugaa ccugacgcu 1916219RNAHomo sapiens 162agcgucaggu
ucacuucac 1916319RNAHomo sapiens 163cgcuggacaa caggcucga
1916419RNAHomo sapiens 164ucgagccugu uguccagcg 1916519RNAHomo
sapiens 165cuggacaaca ggcucgacu 1916619RNAHomo sapiens
166agucgagccu guuguccag 1916719RNAHomo sapiens 167guccucagca
ccccguacu 1916819RNAHomo sapiens 168aguacggggu gcugaggac
1916919RNAHomo sapiens 169uccucagcac cccguacuu 1917019RNAHomo
sapiens 170aaguacgggg ugcugagga 1917119RNAHomo sapiens
171agcaccccgu acuucccca 1917219RNAHomo sapiens 172uggggaagua
cggggugcu 1917319RNAHomo sapiens 173cagcuacuac ucgccccaa
1917419RNAHomo sapiens 174uuggggcgag uaguagcug 1917519RNAHomo
sapiens 175agcuacuacu cgccccaaa 1917619RNAHomo sapiens
176uuuggggcga guaguagcu 1917719RNAHomo sapiens 177acuacucgcc
ccaaaccca 1917819RNAHomo sapiens 178uggguuuggg gcgaguagu
1917919RNAHomo sapiens 179ucgccccaaa cccacugcu 1918019RNAHomo
sapiens 180agcagugggu uuggggcga 1918119RNAHomo sapiens
181gcuccuggca ccucacggu 1918219RNAHomo sapiens 182accgugaggu
gccaggagc 1918319RNAHomo sapiens 183cccucucugg acuacggcu
1918419RNAHomo sapiens 184agccguaguc cagagaggg 1918519RNAHomo
sapiens 185acggcuuggc ccucugguu 1918619RNAHomo sapiens
186aaccagaggg ccaagccgu 1918719DNAHomo sapiens 187cggcttggcc
ctctggttt 1918819RNAHomo sapiens 188aaaccagagg gccaagccg
1918919RNAHomo sapiens 189gcuuggcccu cugguuuga 1919019RNAHomo
sapiens 190ucaaaccaga gggccaagc 1919119RNAHomo sapiens
191ccucugguuu gaugccuau 1919219RNAHomo sapiens 192auaggcauca
aaccagagg 1919319RNAHomo sapiens 193cagaaguaug auuugccgu
1919419RNAHomo sapiens 194acggcaaauc auacuucug 1919519RNAHomo
sapiens 195aaguaugauu ugccgugca 1919619RNAHomo sapiens
196ugcacggcaa aucauacuu 1919719RNAHomo sapiens 197augauuugcc
gugcaccca 1919819RNAHomo sapiens 198ugggugcacg gcaaaucau
1919919RNAHomo sapiens 199acccagggcc aguggacga 1920019RNAHomo
sapiens 200ucguccacug gcccugggu 1920119RNAHomo sapiens
201agggccagug gacgaucca 1920219RNAHomo sapiens 202uggaucgucc
acuggcccu 1920319RNAHomo sapiens 203ggccagugga cgauccaga
1920419RNAHomo sapiens 204ucuggaucgu ccacuggcc 1920519RNAHomo
sapiens 205gccaguggac gauccagaa 1920619RNAHomo sapiens
206uucuggaucg uccacuggc 1920719RNAHomo sapiens 207cagcccuacg
ccgagagga 1920819RNAHomo sapiens 208uccucucggc guagggcug
1920919RNAHomo sapiens 209cggugugcgg gugcacuau 1921019RNAHomo
sapiens 210auagugcacc cgcacaccg 1921119RNAHomo sapiens
211gugcgggugc acuauggcu 1921219RNAHomo sapiens 212agccauagug
cacccgcac 1921319RNAHomo sapiens 213ugcgggugca cuauggcuu
1921419RNAHomo sapiens 214aagccauagu gcacccgca 1921519RNAHomo
sapiens 215gggugcacua uggcuugua 1921619RNAHomo sapiens
216uacaagccau agugcaccc 1921719RNAHomo sapiens 217ugcacuaugg
cuuguacaa 1921819RNAHomo sapiens 218uuguacaagc cauagugca
1921919RNAHomo sapiens 219ugcccuggag aguuccucu 1922019RNAHomo
sapiens 220agaggaacuc uccagggca 1922119RNAHomo sapiens
221uggaugagag aaacugcgu 1922219RNAHomo sapiens 222acgcaguuuc
ucucaucca 1922319RNAHomo sapiens 223ggacagcaca ugcaucuca
1922419RNAHomo sapiens 224ugagaugcau gugcugucc 1922519RNAHomo
sapiens 225acagcacaug caucucacu 1922619RNAHomo sapiens
226agugagaugc augugcugu 1922719RNAHomo sapiens 227ccaaggucug
ugaugggca 1922819RNAHomo sapiens 228ugcccaucac agaccuugg
1922919RNAHomo sapiens 229augggcagcc ugauugucu 1923019RNAHomo
sapiens 230agacaaucag gcugcccau 1923119RNAHomo sapiens
231ggcagccuga uugucucaa 1923219RNAHomo sapiens 232uugagacaau
caggcugcc 1923319RNAHomo sapiens 233ccugauuguc ucaacggca
1923419RNAHomo sapiens 234ugccguugag acaaucagg 1923519RNAHomo
sapiens 235ucucaacggc agcgacgaa 1923619RNAHomo sapiens
236uucgucgcug ccguugaga 1923719RNAHomo sapiens 237ugccaggaag
gggugccau 1923819RNAHomo sapiens 238auggcacccc uuccuggca
1923919RNAHomo sapiens 239ggggugccau gugggacau 1924019RNAHomo
sapiens 240augucccaca uggcacccc 1924119RNAHomo sapiens
241gugccaugug ggacauuca 1924219RNAHomo sapiens 242ugaauguccc
acauggcac 1924319RNAHomo sapiens 243caugugggac auucaccuu
1924419RNAHomo sapiens 244aaggugaaug ucccacaug 1924519RNAHomo
sapiens 245cagugugagg accggagcu 1924619RNAHomo sapiens
246agcuccgguc cucacacug 1924719RNAHomo sapiens 247ugaagaagcc
caacccgca 1924819RNAHomo sapiens 248ugcggguugg gcuucuuca
1924919RNAHomo sapiens 249gaagcccaac ccgcagugu 1925019RNAHomo
sapiens 250acacugcggg uugggcuuc 1925119RNAHomo sapiens
251ccccuccagc cgcauuguu 1925219RNAHomo sapiens 252aacaaugcgg
cuggagggg 1925319RNAHomo sapiens 253cagguucggg gucgacaca
1925419RNAHomo sapiens 254ugugucgacc ccgaaccug 1925519RNAHomo
sapiens 255agguucgggg ucgacacau 1925619RNAHomo sapiens
256augugucgac cccgaaccu 1925719RNAHomo sapiens 257guucgggguc
gacacaucu 1925819RNAHomo sapiens 258agaugugucg accccgaac
1925919RNAHomo sapiens 259cgcugaccgc ugggugaua 1926019RNAHomo
sapiens 260uaucacccag cggucagcg 1926119RNAHomo sapiens
261gcugaccgcu gggugauaa 1926219RNAHomo sapiens 262uuaucaccca
gcggucagc 1926319RNAHomo sapiens 263ugaccgcugg gugauaaca
1926419RNAHomo sapiens 264uguuaucacc cagcgguca 1926519RNAHomo
sapiens 265ccgcugggug auaacagcu 1926619RNAHomo sapiens
266agcuguuauc acccagcgg 1926719RNAHomo sapiens 267ugcuguggac
cguguuccu 1926819RNAHomo sapiens 268aggaacacgg uccacagca
1926919RNAHomo sapiens 269guguggcaga acucgcgcu 1927019RNAHomo
sapiens 270agcgcgaguu cugccacac 1927119RNAHomo sapiens
271uccugcaccc guaccacga 1927219RNAHomo sapiens 272ucgugguacg
ggugcagga 1927319RNAHomo sapiens 273ccugcacccg uaccacgaa
1927419RNAHomo sapiens 274uucgugguac gggugcagg 1927519RNAHomo
sapiens 275ugcacccgua ccacgaaga 1927619RNAHomo sapiens
276ucuucguggu acgggugca 1927719RNAHomo sapiens 277cgcgcuccca
cuucuucga 1927819RNAHomo sapiens 278ucgaagaagu gggagcgcg
1927919RNAHomo sapiens 279ggccugcacu gcuggauua 1928019RNAHomo
sapiens 280uaauccagca gugcaggcc 1928119RNAHomo sapiens
281cacugcugga uuacgggcu 1928219RNAHomo sapiens 282agcccguaau
ccagcagug 1928319RNAHomo sapiens 283ggaugugcag uugauccca
1928419RNAHomo sapiens 284ugggaucaac ugcacaucc 1928519RNAHomo
sapiens 285ugcagcgagg ucuaucgcu 1928619RNAHomo sapiens
286agcgauagac cucgcugca 1928719RNAHomo sapiens 287gcagcgaggu
cuaucgcua 1928819RNAHomo sapiens 288uagcgauaga ccucgcugc
1928919RNAHomo sapiens 289gcgaggucua ucgcuacca 1929019RNAHomo
sapiens 290ugguagcgau agaccucgc 1929119RNAHomo sapiens
291gucuaucgcu accagguga 1929219RNAHomo sapiens 292ucaccuggua
gcgauagac 1929319RNAHomo sapiens 293aggugacgcc acgcaugcu
1929419RNAHomo sapiens 294agcaugcgug gcgucaccu 1929519RNAHomo
sapiens 295gugacgccac gcaugcugu 1929619RNAHomo sapiens
296acagcaugcg uggcgucac 1929719RNAHomo sapiens 297gacgccacgc
augcugugu 1929819RNAHomo sapiens 298acacagcaug cguggcguc
1929919RNAHomo sapiens 299ggcuguggcc ggccuaacu 1930019RNAHomo
sapiens 300aguuaggccg gccacagcc 1930119RNAHomo sapiens
301gcuguggccg gccuaacua 1930219RNAHomo sapiens 302uaguuaggcc
ggccacagc 1930319RNAHomo sapiens 303uguggccggc cuaacuacu
1930419RNAHomo sapiens 304aguaguuagg ccggccaca 1930519RNAHomo
sapiens 305ggccuaacua cuucggcgu 1930619RNAHomo sapiens
306acgccgaagu aguuaggcc 1930719RNAHomo sapiens 307ccuaacuacu
ucggcgucu 1930819RNAHomo sapiens 308agacgccgaa guaguuagg
1930919RNAHomo sapiens 309cuaacuacuu cggcgucua 1931019RNAHomo
sapiens 310uagacgccga aguaguuag 1931119RNAHomo sapiens
311aacuacuucg gcgucuaca 1931219RNAHomo sapiens 312uguagacgcc
gaaguaguu 1931319RNAHomo sapiens 313acacccgcau cacaggugu
1931419RNAHomo sapiens 314acaccuguga ugcgggugu 1931519RNAHomo
sapiens 315cgcaucacag gugugauca 1931619RNAHomo sapiens
316ugaucacacc ugugaugcg 1931719RNAHomo sapiens 317gcuggaucca
gcaaguggu 1931819RNAHomo sapiens 318accacuugcu ggauccagc
1931919RNAHomo sapiens 319ggaacugccc cccugcaaa 1932019RNAHomo
sapiens 320uuugcagggg ggcaguucc 1932119RNAHomo sapiens
321aggagguggc aucuugucu 1932219RNAHomo sapiens 322agacaagaug
ccaccuccu 1932319RNAHomo sapiens 323agguggcauc uugucucgu
1932419RNAHomo sapiens 324acgagacaag augccaccu 1932519RNAHomo
sapiens 325ggcaucuugu cucgucccu 1932619RNAHomo sapiens
326agggacgaga caagaugcc 1932719RNAHomo sapiens 327caucuugucu
cgucccuga 1932819RNAHomo sapiens 328ucagggacga gacaagaug
1932919RNAHomo sapiens 329aucuugucuc gucccugau
1933019RNAHomo sapiens 330aucagggacg agacaagau 1933119RNAHomo
sapiens 331cagcuggggg ucaagacgu 1933219RNAHomo sapiens
332acgucuugac ccccagcug 1933319RNAHomo sapiens 333gggggucaag
acguccccu 1933419RNAHomo sapiens 334aggggacguc uugaccccc
1933519RNAHomo sapiens 335gggucaagac guccccuga 1933619RNAHomo
sapiens 336ucaggggacg ucuugaccc 1933719RNAHomo sapiens
337ccacugcugc cuaaugcaa 1933819RNAHomo sapiens 338uugcauuagg
cagcagugg 1933919RNAHomo sapiens 339ugcugccuaa ugcaaggca
1934019RNAHomo sapiens 340ugccuugcau uaggcagca 1934119RNAHomo
sapiens 341cuaaugcaag gcaguggcu 1934219RNAHomo sapiens
342agccacugcc uugcauuag 1934319RNAHomo sapiens 343cagcaagaau
gcugguucu 1934419RNAHomo sapiens 344agaaccagca uucuugcug
1934519RNAHomo sapiens 345gaggugcgcc ccacucugu 1934619RNAHomo
sapiens 346acagaguggg gcgcaccuc 1934719RNAHomo sapiens
347cuucggaagc cccuggucu 1934819RNAHomo sapiens 348agaccagggg
cuuccgaag 1934919RNAHomo sapiens 349ucggaagccc cuggucuaa
1935019RNAHomo sapiens 350uuagaccagg ggcuuccga 1935119RNAHomo
sapiens 351ggaagccccu ggucuaacu 1935219RNAHomo sapiens
352aguuagacca ggggcuucc 1935319RNAHomo sapiens 353gaagccccug
gucuaacuu 1935419RNAHomo sapiens 354aaguuagacc aggggcuuc
1935519RNAHomo sapiens 355cccuggucua acuugggau 1935619RNAHomo
sapiens 356aucccaaguu agaccaggg 1935719RNAHomo sapiens
357cuggucuaac uugggaucu 1935819RNAHomo sapiens 358agaucccaag
uuagaccag 1935919RNAHomo sapiens 359cuaacuuggg aucugggaa
1936019RNAHomo sapiens 360uucccagauc ccaaguuag 1936119RNAHomo
sapiens 361ccaucggagg ggacccuca 1936219RNAHomo sapiens
362ugaggguccc cuccgaugg 1936319RNAHomo sapiens 363ugggccugcu
gccacugua 1936419RNAHomo sapiens 364uacaguggca gcaggccca
1936519RNAHomo sapiens 365gggccugcug ccacuguaa 1936619RNAHomo
sapiens 366uuacaguggc agcaggccc 1936719RNAHomo sapiens
367gcugccacug uaagccaaa 1936819RNAHomo sapiens 368uuuggcuuac
aguggcagc 1936919RNAHomo sapiens 369ccacuguaag ccaaaaggu
1937019RNAHomo sapiens 370accuuuuggc uuacagugg 1937119RNAHomo
sapiens 371agguggggaa guccugacu 1937219RNAHomo sapiens
372agucaggacu uccccaccu 1937319RNAHomo sapiens 373gaauaaagcu
gccugauca 1937419RNAHomo sapiens 374ugaucaggca gcuuuauuc
1937519RNAHomo sapiens 375aauaaagcug ccugaucaa 1937619RNAHomo
sapiens 376uugaucaggc agcuuuauu 1937719RNAHomo sapiens
377agcugccuga ucaaaaaaa 1937819RNAHomo sapiens 378uuuuuuugau
caggcagcu 1937919RNAHomo sapiens 379cuacagggcc gaguacgaa
1938019RNAHomo sapiens 380uucguacucg gcccuguag 1938119RNAHomo
sapiens 381ugugaugggg ucaaggacu 1938219RNAHomo sapiens
382aguccuugac cccaucaca 1938319RNAHomo sapiens 383cuggagaggu
guccuucaa 1938419RNAHomo sapiens 384uugaaggaca ccucuccag
1938519RNAHomo sapiens 385ggaccgacug gccauguau 1938619RNAHomo
sapiens 386auacauggcc agucggucc 1938719RNAHomo sapiens
387aguugauccc acaggaccu 1938819RNAHomo sapiens 388agguccugug
ggaucaacu 1938919RNAHomo sapiens 389aaaccgccaa agcccagaa
1939019RNAHomo sapiens 390uucugggcuu uggcgguuu 1939119RNAHomo
sapiens 391ugugugccgg cuaccgcaa 1939219RNAHomo sapiens
392uugcgguagc cggcacaca 1939319RNAHomo sapiens 393auuccacgcu
ggguuguua 1939419RNAHomo sapiens 394uaacaaccca gcguggaau
1939519RNAHomo sapiens 395ucgcugaccg cugggugau 1939619RNAHomo
sapiens 396aucacccagc ggucagcga 1939719RNAHomo sapiens
397caagcagggg gacaaguau 1939819RNAHomo sapiens 398auacuugucc
cccugcuug 1939919RNAHomo sapiens 399ugaugucugc uccagugau
1940019RNAHomo sapiens 400aucacuggag cagacauca 1940119RNAHomo
sapiens 401gagguguccu ucaagguga 1940219RNAHomo sapiens
402ucaccuugaa ggacaccuc 1940319RNAHomo sapiens 403aagcaggggg
acaaguauu 1940419RNAHomo sapiens 404aauacuuguc ccccugcuu
1940519RNAHomo sapiens 405cuugggaucu gggaaugga 1940619RNAHomo
sapiens 406uccauuccca gaucccaag 1940719RNAHomo sapiens
407gguauuuccu aggguacaa 1940819RNAHomo sapiens 408uuguacccua
ggaaauacc 1940919RNAHomo sapiens 409ggcuaccgca agggcaaga
1941019RNAHomo sapiens 410ucuugcccuu gcgguagcc 1941119RNAHomo
sapiens 411gcagggggac aaguauucu 1941219RNAHomo sapiens
412agaauacuug ucccccugc 1941319RNAHomo sapiens 413gcucagcagc
aagaaugcu 1941419RNAHomo sapiens 414agcauucuug cugcugagc
1941519RNAHomo sapiens 415uugggaucug ggaauggaa 1941619RNAHomo
sapiens 416uuccauuccc agaucccaa 1941719RNAHomo sapiens
417ccaaagccca gaagaugcu 1941819RNAHomo sapiens 418agcaucuucu
gggcuuugg 1941919RNAHomo sapiens 419gcuaccgcaa gggcaagaa
1942019RNAHomo sapiens 420uucuugcccu ugcgguagc 1942119RNAHomo
sapiens 421gcucagcugc ccuuuggaa 1942219RNAHomo sapiens
422uuccaaaggg cagcugagc 1942319RNAHomo sapiens 423ugguggcagg
agguggcau 1942419RNAHomo sapiens 424augccaccuc cugccacca
1942519RNAHomo sapiens 425cccacucugu acagaggcu 1942619RNAHomo
sapiens 426agccucugua cagaguggg 1942719RNAHomo sapiens
427cucacagccc agacccuca 1942819RNAHomo sapiens 428ugagggucug
ggcugugag 1942919RNAHomo sapiens 429ccucucugga cuacggcuu
1943019RNAHomo sapiens 430aagccguagu ccagagagg 1943119RNAHomo
sapiens 431guggcaggag guggcaucu 1943219RNAHomo sapiens
432agaugccacc uccugccac 1943319RNAHomo sapiens 433uucggaagcc
ccuggucua 1943419RNAHomo sapiens 434uagaccaggg gcuuccgaa
1943519RNAHomo sapiens 435agcucagcug cccuuugga 1943619RNAHomo
sapiens 436uccaaagggc agcugagcu 1943719RNAHomo sapiens
437ggccuggaug agagaaacu 1943819RNAHomo sapiens 438aguuucucuc
auccaggcc 1943919RNAHomo sapiens 439uggcaggagg uggcaucuu
1944019RNAHomo sapiens 440aagaugccac cuccugcca 1944119RNAHomo
sapiens 441acugugacug uggccucca 1944219RNAHomo sapiens
442uggaggccac agucacagu 1944319RNAHomo sapiens 443ccccuggucu
aacuuggga 1944419RNAHomo sapiens 444ucccaaguua gaccagggg
1944519RNAHomo sapiens 445ucagcugccc uuuggaaua 1944619RNAHomo
sapiens 446uauuccaaag ggcagcuga 1944719RNAHomo sapiens
447ucggggucga cacaucugu 1944819RNAHomo sapiens 448acagaugugu
cgaccccga 1944919RNAHomo sapiens 449gucccugaug ucugcucca
1945019RNAHomo sapiens 450uggagcagac aucagggac 1945119RNAHomo
sapiens 451ucaucgcuga ccgcugggu 1945219RNAHomo sapiens
452acccagcggu cagcgauga 1945321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotideDescription of
Combined DNA/RNA Molecule Synthetic oligonucleotide 453ggggugcuac
ucugguauut t 2145421DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotideDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 454aauaccagag uagcacccct t
2145521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 455ucuucugguu cauucuccat t
2145621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 456uggagaauga accagaagat t
2145721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 457acgcuggguu guuaccgcut t
2145821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 458agcgguaaca acccagcgut t
2145921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 459cagaaguaug auuugccgut t
2146021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 460acggcaaauc auacuucugt t
2146121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 461cgcugaccgc ugggugauat t
2146221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 462uaucacccag cggucagcgt t
2146321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 463ucugguauuu ccuaggguat t
2146421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 464uacccuagga aauaccagat t
2146521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 465ccuacagggc cgaguacgat t
2146621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 466ucguacucgg cccuguaggt t
2146721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 467cgcuggguug uuaccgcuat t
2146821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 468uagcgguaac aacccagcgt t
2146921DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 469ggccagugga cgauccagat t
2147021DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 470ucuggaucgu ccacuggcct t
2147121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 471ugaccgcugg gugauaacat t
2147221DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 472uguuaucacc cagcggucat t
2147321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 473ggucagccag guguacucat t
2147421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 474ugaguacacc uggcugacct t
2147521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 475cuacagggcc gaguacgaat t
2147621DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 476uucguacucg gcccuguagt t
2147721DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotideDescription of Combined DNA/RNA Molecule
Synthetic
oligonucleotide 477uuauuccaaa gggcagcugt t 2147821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 478cuggguuguu accgcuacat t 2147921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 479uguagcggua acaacccagt t 2148021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 480ugcacuaugg cuuguacaat t 2148121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 481uuguacaagc cauagugcat t 2148221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 482ccuggagagg uguccuucat t 2148321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 483ugaaggacac cucuccaggt t 2148421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 484agccaggugu acucaggcat t 2148521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 485ugccugagua caccuggcut t 2148621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 486acagggccga guacgaagut t 2148721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 487acuucguacu cggcccugut t 2148821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 488gguuguuacc gcuacagcut t 2148921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 489agcuguagcg guaacaacct t 2149021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 490ugugaugggg ucaaggacut t 2149121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 491aguccuugac cccaucacat t 2149221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 492cuggagaggu guccuucaat t 2149321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 493uugaaggaca ccucuccagt t 2149421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 494uccgcaguga aaccgccaat t 2149521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 495uuggcgguuu cacugcggat t 2149621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 496gggccgagua cgaaguggat t 2149721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 497uccacuucgu acucggccct t 2149821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 498ggaccgacug gccauguaut t 2149921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 499auacauggcc agucggucct t 2150021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 500caacggccug gaugagagat t 2150121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 501ucucucaucc aggccguugt t 2150221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 502aguugauccc acaggaccut t 2150321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 503agguccugug ggaucaacut t 2150421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 504ccgcagugaa accgccaaat t 2150521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 505uuuggcgguu ucacugcggt t 2150621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 506ccgagggccu agugauccut t 2150721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 507aggaucacua ggcccucggt t 2150821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 508uccucagcac cccguacuut t 2150921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 509aaguacgggg ugcugaggat t 2151021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 510cagguucggg gucgacacat t 2151121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 511ugugucgacc ccgaaccugt t 2151221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 512aggugacgcc acgcaugcut t 2151321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 513agcaugcgug gcgucaccut t 2151421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 514aaaccgccaa agcccagaat t 2151521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 515uucugggcuu uggcgguuut t 2151621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 516cagugugaaa gacauagcut t 2151721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 517agcuaugucu uucacacugt t 2151821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 518cccucucugg acuacggcut t 2151921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 519agccguaguc cagagagggt t 2152021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 520guucgggguc gacacaucut t 2152121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 521agaugugucg accccgaact t 2152221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 522ugugugccgg cuaccgcaat t 2152321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 523uugcgguagc cggcacacat t 2152421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 524gcuucuucug guucauucut t 2152521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 525agaaugaacc agaagaagct t 2152621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 526auuccacgcu ggguuguuat t 2152721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 527uaacaaccca gcguggaaut t 2152821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 528acggcuuggc ccucugguut t 2152921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 529aaccagaggg ccaagccgut t 2153021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 530ucgcugaccg cugggugaut t 2153121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 531aucacccagc ggucagcgat t 2153221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 532aguggugacc ugaggaacut t 2153321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 533aguuccucag gucaccacut t 2153421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 534caagcagggg gacaaguaut t 2153521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 535auacuugucc cccugcuugt t 2153621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 536ugaugucugc uccagugaut t 2153721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 537aucacuggag cagacaucat t 2153821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 538cuaacuuggg aucugggaat t 2153921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 539uucccagauc ccaaguuagt t 2154021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 540ugguauuucc uaggguacat t 2154121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 541uguacccuag gaaauaccat t 2154221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 542gagguguccu ucaaggugat t 2154321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 543ucaccuugaa ggacaccuct t 2154421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 544aagcaggggg acaaguauut t 2154521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 545aauacuuguc ccccugcuut t 2154621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 546cagcuggggg ucaagacgut t 2154721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 547acgucuugac ccccagcugt t 2154821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 548cuugggaucu gggaauggat t 2154921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 549uccauuccca gaucccaagt t 2155021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 550gguauuuccu aggguacaat t 2155121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 551uuguacccua ggaaauacct t 2155221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 552ggcuaccgca agggcaagat t 2155321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 553ucuugcccuu gcgguagcct t 2155421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 554gcagggggac aaguauucut t 2155521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 555agaauacuug ucccccugct t 2155621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 556gcucagcagc aagaaugcut t 2155721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 557agcauucuug cugcugagct t 2155821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 558uugggaucug ggaauggaat t 2155921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 559uuccauuccc agaucccaat t 2156021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 560ccaaagccca gaagaugcut t 2156121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 561agcaucuucu gggcuuuggt t 2156221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 562gcuaccgcaa gggcaagaat t 2156321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 563uucuugcccu ugcgguagct t 2156421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 564ugguggcagg agguggcaut t 2156521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 565augccaccuc cugccaccat t 2156621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 566cccacucugu acagaggcut t 2156721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 567agccucugua cagagugggt t 2156821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 568cucacagccc agacccucat t 2156921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 569ugagggucug ggcugugagt t 2157021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 570ccucucugga cuacggcuut t 2157121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 571aagccguagu ccagagaggt t 2157221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 572guggcaggag guggcaucut t 2157321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 573agaugccacc uccugccact t 2157421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 574uucggaagcc ccuggucuat t 2157521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 575uagaccaggg gcuuccgaat t 2157621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 576agcucagcug cccuuuggat t 2157721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 577uccaaagggc agcugagcut t 2157821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 578ggccuggaug agagaaacut t 2157921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 579aguuucucuc auccaggcct t 2158021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 580uggcaggagg uggcaucuut t 2158121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 581aagaugccac cuccugccat t 2158221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 582ucggaagccc cuggucuaat t 2158321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 583uuagaccagg ggcuuccgat t 2158421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 584gcucagcugc ccuuuggaat t 2158521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 585uuccaaaggg cagcugagct t 2158621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 586acugugacug uggccuccat t 2158721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 587uggaggccac agucacagut t 2158821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 588aggagguggc aucuugucut t 2158921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 589agacaagaug ccaccuccut t 2159021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 590ccccuggucu aacuugggat t 2159121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 591ucccaaguua gaccaggggt t 2159221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 592ucagcugccc uuuggaauat t 2159321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 593uauuccaaag ggcagcugat t 2159421DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 594ucggggucga cacaucugut t 2159521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 595acagaugugu cgaccccgat t 2159621DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 596gucccugaug ucugcuccat t 2159721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 597uggagcagac aucagggact t 2159821DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 598cccuggucua acuugggaut t 2159921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 599aucccaaguu agaccagggt t 2160021DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 600cagcugcccu uuggaauaat t 2160121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 601uuauuccaaa gggcagcugt t 2160221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 602ucaucgcuga ccgcugggut t 2160321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotideDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 603acccagcggu cagcgaugat t 2160419RNAHomo sapiens
604ccuggagagg uguccuuca 1960519RNAHomo sapiens 605ugaaggacac
cucuccagg 1960619RNAHomo sapiens 606caacggccug gaugagaga
1960719RNAHomo sapiens 607ucucucaucc aggccguug 1960819RNAHomo
sapiens 608aguggugacc ugaggaacu 1960919RNAHomo sapiens
609aguuccucag gucaccacu 1961019RNAHomo sapiens 610cagcugcccu
uuggaauaa 1961119RNAHomo sapiens 611uuauuccaaa gggcagcug 19
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