U.S. patent application number 14/794042 was filed with the patent office on 2016-03-10 for rnai modulation of scap and therapeutic uses thereof.
The applicant listed for this patent is Alnylam Pharmaceuticals, Inc., The Board of Regents of The University of Texas System. Invention is credited to Michael S. BROWN, Joseph L. GOLDSTEIN, Jay D. HORTON, Young-Ah MOON, Juergen SOUTSCHEK, Pamela TAN.
Application Number | 20160068840 14/794042 |
Document ID | / |
Family ID | 39201196 |
Filed Date | 2016-03-10 |
United States Patent
Application |
20160068840 |
Kind Code |
A1 |
SOUTSCHEK; Juergen ; et
al. |
March 10, 2016 |
RNAI MODULATION OF SCAP AND THERAPEUTIC USES THEREOF
Abstract
The invention relates to a double-stranded ribonucleic acid
(dsRNA) for inhibiting the expression of a SCAP gene (Human SCAP
gene), comprising an antisense strand having a nucleotide sequence
which is less that 30 nucleotides in length, generally 19-25
nucleotides in length, and which is substantially complementary to
at least a part of a SCAP gene. The invention also relates to a
pharmaceutical composition comprising the dsRNA together with a
pharmaceutically acceptable carrier; methods for treating diseases
caused by Human SCAP expression and the expression of a SCAP gene
using the pharmaceutical composition; and methods for inhibiting
the expression of a SCAP gene in a cell.
Inventors: |
SOUTSCHEK; Juergen;
(Innsbruck, AT) ; TAN; Pamela; (Ebenhausen,
DE) ; HORTON; Jay D.; (Plano, TX) ; BROWN;
Michael S.; (Dallas, TX) ; GOLDSTEIN; Joseph L.;
(Dallas, TX) ; MOON; Young-Ah; (Irving,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc.
The Board of Regents of The University of Texas System |
Cambridge
Austin |
MA
TX |
US
US |
|
|
Family ID: |
39201196 |
Appl. No.: |
14/794042 |
Filed: |
July 8, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13743713 |
Jan 17, 2013 |
9102940 |
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14794042 |
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13080334 |
Apr 5, 2011 |
8383805 |
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13743713 |
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12749159 |
Mar 29, 2010 |
7919613 |
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13080334 |
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11857120 |
Sep 18, 2007 |
7737266 |
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12749159 |
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60845289 |
Sep 18, 2006 |
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Current U.S.
Class: |
514/44A ;
435/320.1; 435/352; 435/375; 536/24.5 |
Current CPC
Class: |
A61P 3/10 20180101; C12N
2310/321 20130101; C12N 2310/314 20130101; C12N 15/113 20130101;
C12N 2310/346 20130101; A61P 3/06 20180101; A61P 9/10 20180101;
C12N 2310/3521 20130101; A61P 1/16 20180101; A61P 3/00 20180101;
A61P 1/00 20180101; C12N 2310/315 20130101; C12N 2310/321 20130101;
A61P 3/04 20180101; C12N 2310/3515 20130101; A61P 9/00 20180101;
C12N 2310/14 20130101; C12N 2310/322 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the
expression of a SCAP gene in a cell, wherein said dsRNA comprises
at least two sequences that are complementary to each other and
wherein a sense strand comprises a first sequence and an antisense
strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding a SCAP gene, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said SCAP, inhibits
expression of said SCAP gene by at least 20%.
2. The dsRNA of claim 1, wherein said SCAP gene is a human SCAP
gene, and preferably a Homo sapiens SCAP gene.
3. The dsRNA of claim 1, wherein said at least 20% inhibition of
expression of a SCAP gene is effected in primary hamster
hepatocytes.
4. The dsRNA of claim 1, wherein said first sequence and said
second sequence are selected from even and uneven numbers of the
group consisting of SEQ ID NO:1-48 respectively.
5. The dsRNA of claim 1, wherein the dsRNA is chosen from the group
of AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493, AD-9501,
AD-9497, AD-9509 and AD-9513.
6. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
7. The dsRNA of claim 6, wherein said modified nucleotide is chosen
from the group 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.
8. The dsRNA of claim 6, wherein said modified nucleotide is 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.
9. The dsRNA of claim 6, wherein said first sequence and said
second sequence are selected from even and uneven numbers of the
group consisting of SEQ ID NO:1-48 respectively.
10. A cell comprising the dsRNA of claim 1.
11. A pharmaceutical composition for inhibiting the expression of a
SCAP gene in an organism, comprising a dsRNA and a pharmaceutically
acceptable carrier, wherein the dsRNA comprises at least two
sequences that are complementary to each other and wherein a sense
strand comprises a first sequence and an antisense strand comprises
a second sequence comprising a region of complementarity which is
substantially complementary to at least a part of a mRNA encoding
SCAP, and wherein said region of complementarity is less than 30
nucleotides in length and wherein said dsRNA, upon contact with a
cell expressing said Human SCAP, inhibits expression of said Homo
sapiens SCAP gene by at least 20%.
12. The pharmaceutical composition of claim 11, wherein said SCAP
gene is a human SCAP gene, and preferably a Homo sapiens SCAP
gene.
13. The pharmaceutical composition of claim 11, wherein said at
least 20% inhibition of expression of a SCAP gene is effected in
primary hamster hepatocytes.
14. The pharmaceutical composition of claim 11, wherein said first
sequence and said second sequence are selected from even and uneven
numbers of the group consisting of SEQ NO:1-48, respectively.
15. The pharmaceutical composition of claim 11, wherein the dsRNA
is chosen from the group of AD-9505, AD-9498, AD-9512, AD-9490,
AD-9495, AD-9503, AD-9494, AD-9500, AD-9492, AD-9499, AD-9496,
AD-9510, AD-9511, AD-9491, AD-9506, AD-9508, AD-9502, AD-9504,
AD-9507, AD-9493, AD-9501, AD-9497, AD-9509 and AD-9513.
16. A method for inhibiting the expression of a SCAP gene in a
cell, the method comprising: (a) introducing into the cell a
double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding SCAP, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
said dsRNA, upon contact with a cell expressing said SCAP gene,
inhibits expression of said SCAP gene by at least 20%; and (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of a SCAP gene, thereby
inhibiting expression of a SCAP gene in the cell.
17. The method of claim 16, wherein the gene is a human SCAP gene,
and preferably a Homo sapiens SCAP gene.
18. A method of treating, preventing or managing pathological
processes mediated by SCAP expression comprising administering to a
patient in need of such treatment, prevention or management a
therapeutically or prophylactically effective amount of a dsRNA,
wherein the dsRNA comprises at least two sequences that are
complementary to each other and wherein a sense strand comprises a
first sequence and an antisense strand comprises a second sequence
comprising a region of complementarity which is substantially
complementary to at least a part of an mRNA encoding SCAP, and
wherein said region of complementarity is less than 30 nucleotides
in length and wherein said dsRNA, upon contact with a cell
expressing said SCAP gene, inhibits expression of said SCAP gene by
at least 20%.
19. The method of claim 18, wherein the patient suffers from
non-alcoholic liver disease, fatty liver, hyperlipemia,
hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia and/or
hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulin
dependent diabetes mellitus (NIDDM), coronary heart disease,
obesity, metabolic syndrome, peripheral arterial disease, and
cerebrovascular disease
20. A vector for inhibiting the expression of a SCAP gene in a
cell, said vector comprising a regulatory sequence operably linked
to a nucleotide sequence that encodes at least one strand of a
dsRNA, wherein one of the strands of said dsRNA is substantially
complementary to at least a part of a mRNA encoding SCAP and
wherein said dsRNA is less than 30 base pairs in length and wherein
said dsRNA, upon contact with a cell expressing said SCAP gene,
inhibits the expression of said SCAP gene by at least 20%.
21. A cell comprising the vector of claim 20.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Application Ser.
No. 60/845,289, filed Sep. 18, 2006, which is incorporated herein
by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods of treatment using
modulators of the gene SREPB cleavage activating protein (SCAP).
More specifically, the invention concerns methods of treating
disorders associated with undesired SCAP activity, by administering
short interfering RNA that down-regulate the expression of SCAP,
and agents useful therein.
BACKGROUND OF THE INVENTION
[0003] Lipid homeostasis is essential to all living beings that
rely on lipid membranes to separate their cell's vital functions
from the environment, including all animals, and humans.
Furthermore, lipids are used as energy reservoirs by many
organisms. A vast array of different lipidic substances, including,
for example, phospholipids, triglycerides, fatty acids, and
sterols, perform a wide variety of essential functions in cells.
Altogether, lipid homeostasis is a tightly regulated,
multi-branched, intricate web of interdependent processes in
essentially all higher organisms.
[0004] Naturally, the more complex a system, the more can go awry.
A large number of diseases and conditions, e.g. in humans, are
known to be, in whole or in part, consequences of lipid homeostasis
dysfunctions. These include both inherited diseases, where one or a
number of the many genes involved in lipid homeostasis completely
or partially loses its function, or is mis-regulated, as well as
acquired diseases, where gene function or gene regulation in the
body is altered after single or repeated contact with one or a
combination of substances.
[0005] In many a species including humans, the body's needs for
lipids are filled partially by dietary intake as well as by the
synthesis of lipids from precursors. The liver stands out as the
single organ responsible for the collection of dietary lipid
intake, lipid synthesis, and the control of lipid release to and
re-uptake from the bloodstream. Consequentially, it is involved in
many, if not all, lipid metabolism disorders.
[0006] Many such disorders are caused by, or accompanied with, an
overabundance of certain lipids in all or parts of the body, be it
from excessive intake, faulty degradation or transport, or
excessive de novo synthesis.
[0007] For example, Non-Alcoholic Fatty Liver Disease (NAFLD) is a
condition where excess triglycerides accumulate in the liver, and
is associated with various drugs, nutritional factors, multiple
genetic defects in energy metabolism, and, most prominently,
insulin resistance (Browning J D and Horton J D, J. Clin.
Investigation 2004, 114:147). Conversely, a hallmark of
atherosclerosis is the appearance of so-called foam cells,
macrophages filled with excess cholesterol and cholesterol esters
(Kruth H S, Front Biosci 2001, 6:D429). Other non-limiting examples
of disorders associated with excessive levels of lipids in the body
are: non-alcoholic liver disease, fatty liver, hyperlipemia,
hyperlipidemia, hyperlipoproteinemia, hypercholesterolemia and/or
hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulin
dependent diabetes mellitus (NIDDM), coronary heart disease,
obesity, metabolic syndrome, peripheral arterial disease, and
cerebrovascular disease. The treatment of disorders of this type
could potentially be aided by attenuating the body's own synthesis
of lipids.
[0008] A central element in the regulation of lipid biosynthesis in
the human liver is a group of transcription factors termed Sterol
Regulatory Element Binding Proteins (SREBPs). There are three SREBP
isoforms called SREBP-1a, SREBP-1c and SREBP-2. They are located in
the endoplasmatic reticulum (ER) in a precursor form (Yokoyama C.
et al., Cell 1993, 75:187; Hua X. et al., Proc. Natl. Acad. Sci.
1993, 90:11603) which, in the presence of cholesterol, is bound to
cholesterol and two other proteins: SCAP (SREBP-cleavage activating
protein) and Insig1 (Insulin-induced gene 1). When cholesterol
levels fall, Insig-1 dissociates from the SREBP-SCAP complex,
allowing the complex to migrate to the Golgi apparatus, where SREBP
is cleaved by S1P and S2P (site 1/2 protease; Sakai J et al, Mol.
Cell 1998, 2:505; Rawson R. B. et al, Mol. Cell 1997, 1:47), two
enzymes that are activated by SCAP. The cleaved SREBP then migrates
to the nucleus and acts as a transcription factor by binding to the
SRE (sterol regulatory element) of a number of genes and
stimulating their transcription (Briggs M. R. et al., J. Biol.
Chem. 1993, 268:14490). Among the genes transcribed are the
LDL-Receptor, up-regulation of which leads to increased in-flux of
cholesterol from the bloodstream, HMG-CoA reductase, the rate
limiting enzyme in de-novo cholesterol synthesis (Anderson et al,
Trends Cell Biol 2003, 13:534), as well as a number of genes
involved in fatty acid synthesis.
[0009] In an attempt to lower the body's own production of lipids,
one attractive option therefore would seem to be the blocking of
SREBP activation. Since SCAP-binding is a prerequisite for the
transport and activation of all three SREBP isoforms, an inhibition
of SCAP's activity could lead to a general down-regulation of
cellular lipid synthesis and uptake. For example, SCAP activity
could be inhibited by agents binding to the sterol sensing domain
(SSD) of SCAP with higher affinity than cholesterol, and preferably
in an irreversible manner, thereby prohibiting SREBP transport and
activation. Alternatively, inhibiting the translation and/or
transcription of the gene encoding SCAP could lead to lower levels
of SCAP present in the ER membrane and available for SREBP-binding
and activation.
[0010] Recently, double-stranded RNA molecules (dsRNA) have been
shown to block gene expression in a highly conserved regulatory
mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et
al.) discloses the use of a dsRNA of at least 25 nucleotides in
length to inhibit the expression of genes in C. elegans. dsRNA has
also been shown to degrade target RNA in other organisms, including
plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631,
Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr.
Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer;
and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has
now become the focus for the development of a new class of
pharmaceutical agents for treating disorders that are caused by the
aberrant or unwanted regulation of a gene.
[0011] Despite significant advances in the field of RNAi and
advances in the treatment of pathological processes mediated by
excessive levels of lipids, there remains a need for an agent that
can selectively and efficiently attenuate the body's own lipid
biosynthesis, e.g by inhibiting SCAP, and thereby SREBP, activity,
using the cell's own RNAi machinery. Such agent shall possess both
high biological activity and in vivo stability, and shall
effectively inhibit expression of a target SCAP gene, such as human
SCAP, for use in treating pathological processes mediated directly
or indirectly by SCAP expression.
SUMMARY OF THE INVENTION
[0012] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of a SCAP gene in a cell or mammal using such dsRNA. The
invention also provides compositions and methods for treating
pathological conditions and diseases mediated by the expression of
a SCAP gene, such as in conditions and diseases associated with
excessive levels of lipids and/or unwanted lipid biosynthesis. The
dsRNA of the invention comprises an RNA strand (the antisense
strand) having a region which is less than 30 nucleotides in
length, generally 19-24 nucleotides in length, and is substantially
complementary to at least part of an mRNA transcript of a SCAP
gene. The SCAP gene is preferably a human SCAP gene, and more
preferably a Homo sapiens SCAP gene.
[0013] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
a SCAP gene. The dsRNA comprises at least two sequences that are
complementary to each other. The dsRNA comprises a sense strand
comprising a first sequence and an antisense strand comprising a
second sequence. The antisense strand comprises a nucleotide
sequence which is substantially complementary to at least part of
an mRNA encoding a SCAP gene, and the region of complementarity is
less than 30 nucleotides in length, generally 19-24 nucleotides in
length. The dsRNA, upon contacting with a cell expressing the SCAP
gene, inhibits the expression of the SCAP gene by at least 20%, or
at least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90%
or 95%, e.g. in primary hamster hepatocytes.
[0014] For example, the dsRNA molecules of the invention can be
comprised of a first sequence of the dsRNA that is selected from
the group consisting of the sense strand sequences of the RNAi
agents AD-9490-AD-9513 (uneven numbers of the group of SEQ ID NO:
1-48, Table 1), and the second sequence is selected from the group
consisting of the antisense strand sequences of AD-9490-AD-9513
(even numbers of the group of SEQ ID NO: 1-48, Table 1). The dsRNA
molecules of the invention can be comprised of naturally occurring
nucleotides or can be comprised of at least one modified
nucleotide, such as a 2'-O-methyl modified nucleotide, a nucleotide
comprising 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 T-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 modified sequence will be based on a first sequence
of said dsRNA selected from the group consisting of the sense
sequences of AD-9490-AD-9513 (Table 1) and a second sequence
selected from the group consisting of the antisense sequences of
AD-9490-AD-9513 (Table 1).
TABLE-US-00001 TABLE 1 RNAi agents selected for the down-regulation
of homo sapiens (NM_012235.2), mus musculus (NM_001001144.1) and
Cricetus cricetus (U67060) SCAP, and minimal off-target
interactions in humans Duplex Sense SEQ ID Antisense SEQ ID
identifier strand sequence.sup.1 NO: strand sequence.sup.1 NO:
AD-9490 gauuggcauccugguauacTT 1 guauaccaggaugccaaucTT 2 AD-9491
agcgccucaucauggcuggTT 3 ccagccaugaugaggcgcuTT 4 AD-9492
ggccuucuacaaccaugggTT 5 cccaugguuguagaaggccTT 6 AD-9493
gaggugugggacgccauugTT 7 caauggcgucccacaccucTT 8 AD-9494
uggauuggcauccugguauTT 9 auaccaggaugccaauccaTT 10 AD-9495
gccauugucugcaacuuugTT 11 caaaguugcagacaauggcTT 12 AD-9496
ccaucacccuggucuuccaTT 13 uggaagaccagggugauggTT 14 AD-9497
uguccuuccgccacuggccTT 15 ggccaguggcggaaggacaTT 16 AD-9498
ccuucuacaaccaugggcuTT 17 agcccaugguuguagaaggTT 18 AD-9499
gaccgcagcacaggcaucaTT 19 ugaugccugugcugeggucTT 20 AD-9500
ggauuggcauccugguauaTT 21 uauaccaggaugccaauccTT 22 AD-9501
aucugggaccgcagcacagTT 23 cugugcugcggucccagauTT 24 AD-9502
ucugcaucuuagccugcugTT 25 cagcaggcuaagaugcagaTT 26 AD-9503
agaucgacauggucaagucTT 27 gacuugaccaugucgaucuTT 28 AD-9504
caucacccuggucuuccagTT 29 cuggaagaccagggugaugTT 30 AD-9505
caucuuagccugcugcuacTT 31 guagcagcaggcuaagaugTT 32 AD-9506
ugcaucuuagccugcugcuTT 33 agcagcaggcuaagaugcaTT 34 AD-9507
aagaucgacauggucaaguTT 35 acuugaccaugucgaucuuTT 36 AD-9508
aggugugggacgccauugaTT 37 ucaauggcgucccacaccuTT 38 AD-9509
cagcgccucaucauggcugTT 39 cagccaugaugaggcgcugTT 40 AD-9510
ggaccgcagcacaggcaucTT 41 gaugccugugcugegguccTT 42 AD-9511
cugccauugucugcaacuuTT 43 aaguugcagacaauggcagTT 44 AD-9512
cugcaucuuagccugcugcTT 45 gcagcaggcuaagaugcagTT 46 AD-9513
ucuuagccugcugcuacccTT 47 ggguagcagcaggcuaagaTT 48 .sup.1Capital
letters = desoxyribonucleotides; small letters = ribonucleotides;
underlined: nucleoside thiophosphate
[0015] In a preferred embodiment, the dsRNA is chosen from the
group of AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503,
AD-9494, AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511,
AD-9491, AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493,
AD-9501, AD-9497, AD-9509 and AD-9513, and inhibits the expression
of a SCAP gene in a cell, e.g. a primary hamster hepatocyte, by at
least 20%.
[0016] More preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493, AD-9501,
AD-9497, and AD-9509, and inhibits the expression of a SCAP gene in
a cell, e.g. a primary hamster hepatocyte, by at least 30%.
[0017] Yet more preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, and inhibits the
expression of a SCAP gene in a cell, e.g. a primary hamster
hepatocyte, by at least 40%.
[0018] Yet more preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, and inhibits the expression of a SCAP
gene in a cell, e.g. a primary hamster hepatocyte, by at least
50%.
[0019] Yet more preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, and inhibits
the expression of a SCAP gene in a cell, e.g. a primary hamster
hepatocyte, by at least 60%.
[0020] Yet more preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, and inhibits the expression of a SCAP gene in a cell, e.g.
a primary hamster hepatocyte, by at least 70%.
[0021] Most preferably, the dsRNA is chosen from the group of
AD-9505, AD-9498, AD-9512, and inhibits the expression of a SCAP
gene in a cell, e.g. a primary hamster hepatocyte, by at least
75%.
[0022] In another embodiment, the invention provides a cell
comprising one of the dsRNAs of the invention. The cell is
generally a mammalian cell, such as a human cell.
[0023] In another embodiment, the invention provides a
pharmaceutical composition for inhibiting the expression of a SCAP
gene, e.g. a human or Homo sapiens SCAP gene, in an organism,
generally a human subject, comprising one or more of the dsRNA of
the invention and a pharmaceutically acceptable carrier or delivery
vehicle. Therein, the dsRNA is preferably chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493, AD-9501,
AD-9497, AD-9509, and AD-9513, and inhibits the expression of a
SCAP gene in a cell, e.g. a primary hamster hepatocyte, by at least
20%. For further preferred embodiments of the pharmaceutical
composition, the dsRNA is chosen from the groups given above.
[0024] In another embodiment, the invention provides a method for
inhibiting the expression of a SCAP gene, e.g. a human SCAP gene,
and preferably a Homo sapiens SCAP gene, in a cell, comprising the
following steps: [0025] (a) introducing into the cell a
double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
comprises at least two sequences that are complementary to each
other. The dsRNA comprises a sense strand comprising a first
sequence and an antisense strand comprising a second sequence. The
antisense strand comprises a region of complementarity which is
substantially complementary to at least a part of a mRNA encoding a
SCAP gene, and wherein the region of complementarity is less than
30 nucleotides in length, generally 19-24 nucleotides in length,
and wherein the dsRNA, upon contact with a cell expressing the SCAP
gene inhibits expression of the SCAP gene by at least 20%, or at
least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90% or
95%, e.g. in primary hamster hepatocytes; and [0026] (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of the SCAP gene, thereby
inhibiting expression of the SCAP gene in the cell.
[0027] Therein, the dsRNA is preferably chosen from the group of
AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503, AD-9494,
AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511, AD-9491,
AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493, AD-9501,
AD-9497, AD-9509, and AD-9513, and inhibits the expression of a
SCAP gene in a cell, e.g. a primary hamster hepatocyte, by at least
20%. For further preferred embodiments of the above method, the
dsRNA is chosen from the groups given above.
[0028] In another embodiment, the invention provides methods for
treating, preventing or managing pathological processes mediated by
SCAP expression, e.g. disorders of lipid metabolism, lipid
homeostasis, and/or lipid distribution, e.g. non-alcoholic liver
disease, fatty liver, hyperlipemia, hyperlipidemia,
hyperlipoproteinemia, hypercholesterolemia and/or
hypertriglyceridemia, atherosclerosis, pancreatitis, non-insulin
dependent diabetes mellitus (NIDDM), coronary heart disease,
obesity, metabolic syndrome, peripheral arterial disease, and
cerebrovascular disease, comprising administering to a patient in
need of such treatment, prevention or management a therapeutically
or prophylactically effective amount of one or more of the dsRNAs
of the invention. Therein, the dsRNA is preferably chosen from the
group of AD-9505, AD-9498, AD-9512, AD-9490, AD-9495, AD-9503,
AD-9494, AD-9500, AD-9492, AD-9499, AD-9496, AD-9510, AD-9511,
AD-9491, AD-9506, AD-9508, AD-9502, AD-9504, AD-9507, AD-9493,
AD-9501, AD-9497, AD-9509, and AD-9513, and inhibits the expression
of a SCAP gene in a cell, e.g. a primary hamster hepatocyte, by at
least 20%. For further preferred embodiments of the pharmaceutical
composition, the dsRNA is chosen from the groups given above.
[0029] In another embodiment, the invention provides vectors for
inhibiting the expression of a SCAP gene in a cell, comprising a
regulatory sequence operably linked to a nucleotide sequence that
encodes at least one strand of one of the dsRNA of the
invention.
[0030] In another embodiment, the invention provides a cell
comprising a vector for inhibiting the expression of a SCAP gene in
a cell. The vector comprises a regulatory sequence operably linked
to a nucleotide sequence that encodes at least one strand of one of
the dsRNA of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0031] No Figures are presented.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of a SCAP gene in a cell or mammal using the dsRNA. The
invention also provides compositions and methods for treating
pathological conditions and diseases in a mammal caused by the
expression of a SCAP gene using dsRNA. dsRNA directs the
sequence-specific degradation of mRNA through a process known as
RNA interference (RNAi).
[0033] The dsRNA of the invention comprises an RNA strand (the
antisense strand) having a region which is less than 30 nucleotides
in length, generally 19-24 nucleotides in length, and is
substantially complementary to at least part of an mRNA transcript
of a SCAP gene. The use of these dsRNAs enables the targeted
degradation of mRNAs of genes that are implicated in diseases
involving faulty regulation of disorders of lipid metabolism, lipid
homeostasis, and/or lipid distribution, e.g. non-alcoholic liver
disease, fatty liver, or various forms of dyslipidemia. Using
cell-based and animal assays, the present inventors have
demonstrated that very low dosages of these dsRNA can specifically
and efficiently mediate RNAi, resulting in significant inhibition
of expression of a SCAP gene. Thus, the methods and compositions of
the invention comprising these dsRNAs are useful for treating
pathological processes mediated by SCAP expression, e.g. disorders
of lipid metabolism, lipid homeostasis, and/or lipid distribution,
e.g. non-alcoholic liver disease, fatty liver, or various forms of
dyslipidemia, by targeting a gene involved in the regulation of
lipid metabolism, lipid homeostasis, and/or lipid distribution.
[0034] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
expression of a SCAP gene, as well as compositions and methods for
treating diseases and disorders caused by the expression of a SCAP
gene, such as non-alcoholic liver disease, fatty liver, or various
forms of dyslipidemia. The pharmaceutical compositions of the
invention comprise a dsRNA having an antisense strand comprising a
region of complementarity which is less than 30 nucleotides in
length, generally 19-24 nucleotides in length, and is substantially
complementary to at least part of an RNA transcript of a SCAP gene,
together with a pharmaceutically acceptable carrier.
[0035] Accordingly, certain aspects of the invention provide
pharmaceutical compositions comprising the dsRNA of the invention
together with a pharmaceutically acceptable carrier, methods of
using the compositions to inhibit expression of a SCAP gene,
methods of using the pharmaceutical compositions to treat diseases
caused by expression of a SCAP gene, vectors encoding dsRNAs of the
invention, and cells comprising such dsRNAs or vectors of the
invention.
I. DEFINITIONS
[0036] 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.
[0037] "G," "C," "A", "T" and "U" (irrespective of whether written
in capital or small letters) each generally stand for a nucleotide
that contains guanine, cytosine, adenine, thymine, 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, thymine, and uracil may be replaced by other moieties
without substantially altering the base pairing properties of an
oligonucleotide comprising a nucleotide bearing such replacement
moiety. For example, without limitation, a nucleotide comprising
inosine as its base may base pair with nucleotides containing
adenine, cytosine, or uracil. Hence, nucleotides containing uracil,
guanine, or adenine may be replaced in the nucleotide sequences of
the invention by a nucleotide containing, for example, inosine.
Sequences comprising such replacement moieties are embodiments of
the invention.
[0038] As used herein, "SCAP" or "SCAP gene" refers to genes
encoding SREBP activating proteins, non-exhaustive examples of
which are found under GenBank accession numbers NM.sub.--012235.2
(Homo sapiens), NM.sub.--001001144.1 (Mus musculus) and U67060
(Cricetus cricetus).
[0039] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of a SCAP gene, including mRNA that is a
product of RNA processing of a primary transcription product. The
target sequence of any given RNAi agent of the invention means an
mRNA-sequence of X nucleotides that is targeted by the RNAi agent
by virtue of the complementarity of the antisense strand of the
RNAi agent to such sequence and to which the antisense strand may
hybridize when brought into contact with the mRNA, wherein X is the
number of nucleotides in the antisense strand plus the number of
nucleotides in a single-stranded overhang of the sense strand, if
any.
[0040] 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.
[0041] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0042] This includes base-pairing of the oligonucleotide or
polynucleotide comprising the first nucleotide sequence to the
oligonucleotide or polynucleotide comprising the second nucleotide
sequence over the entire length of the first and second nucleotide
sequence. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but generally not more
than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. 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 of the
invention.
[0043] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled.
[0044] The terms "complementary", "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0045] As used herein, a polynucleotide which is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide which is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., encoding a SCAP
gene). For example, a polynucleotide is complementary to at least a
part of a SCAP gene mRNA if the sequence is substantially
complementary to a non-interrupted portion of an mRNA encoding the
SCAP gene.
[0046] The term "double-stranded RNA" or "dsRNA", as used herein,
refers to a complex of ribonucleic acid molecules, having a duplex
structure comprising two anti-parallel and substantially
complementary, as defined above, nucleic acid strands. The two
strands forming the duplex structure may be different portions of
one larger RNA molecule, or they may be separate RNA molecules.
Where the two strands are part of one larger molecule, and
therefore are connected by an uninterrupted chain of nucleotides
between the 3'-end of one strand and the 5' end of the respective
other strand forming the duplex structure, the connecting RNA chain
is referred to as a "hairpin loop". Where the two strands are
connected covalently by means other than an uninterrupted chain of
nucleotides between the 3'-end of one strand and the 5' end of the
respective other strand forming the duplex structure, the
connecting structure is referred to as a "linker". The RNA strands
may have the same or a different number of nucleotides. The maximum
number of base pairs is the number of nucleotides in the shortest
strand of the dsRNA minus any overhangs that are present in the
duplex. In addition to the duplex structure, a dsRNA may comprise
one or more nucleotide overhangs.
[0047] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of a dsRNA when a 3'-end of one strand of the dsRNA
extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt
ended" dsRNA is a dsRNA that has no nucleotide overhang at either
end of the molecule.
[0048] The term "antisense strand" refers to the strand of 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 are
most tolerated in the terminal regions and, if present, are
generally in a terminal region or regions, e.g., within 6, 5, 4, 3,
or 2 nucleotides of the 5' and/or 3' terminus. Most preferably, the
mismatches are located within 6, 5, 4, 3, or 2 nucleotides of the
5' terminus of the antisense strand and/or the 3' terminus of the
sense strand.
[0049] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0050] "Introducing into a cell", when referring to a dsRNA, means
facilitating uptake or absorption into the cell, as is understood
by those skilled in the art. Absorption or uptake of dsRNA 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; a dsRNA may also be "introduced into a
cell", wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to
the organism. For example, for in vivo delivery, dsRNA can be
injected into a tissue site or administered systemically. In vitro
introduction into a cell includes methods known in the art such as
electroporation and lipofection.
[0051] The terms "silence" and "inhibit the expression of", in as
far as they refer to a SCAP gene, e.g. a human SCAP gene, herein
refer to the at least partial suppression of the expression of a
SCAP gene, e.g. a human SCAP gene, as manifested by a reduction of
the amount of mRNA transcribed from a SCAP gene which may be
isolated from a first cell or group of cells in which a SCAP gene
is transcribed and which has or have been treated such that the
expression of a SCAP 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). Preferably, the cells are primary hamster hepatocytes. The
degree of inhibition is usually expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0052] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
SCAP gene transcription, e.g. the amount of protein encoded by a
SCAP gene which is secreted by a cell, or found in solution after
lysis of such cells, or the number of cells displaying a certain
phenotype, e.g. surface expression of LDL receptor, or lipid
synthesis. In principle, SCAP gene silencing may be determined in
any cell expressing the target, either constitutively or by genomic
engineering, and by any appropriate assay. However, when a
reference is needed in order to determine whether a given dsRNA
inhibits the expression of a SCAP gene by a certain degree and
therefore is encompassed by the instant invention, the assays
provided in the Examples below shall serve as such reference.
[0053] Generally, the expression of a SCAP gene shall be considered
to be at least partially suppressed, if the probability of the
difference in the results of measurements of SCAP mRNA content, or
other functional parameter, obtained from treated cells and control
cells essentially resulting from random effects is less than 5%. In
other words, expression of a SCAP gene shall be considered to be at
least partially suppressed, if said difference is statistically
significant.
[0054] For example, in certain instances, expression of a SCAP
gene, e.g. a human SCAP gene, is suppressed by at least 20%, 25%,
35%, 40%, 45%, or 50% by administration of the double-stranded
oligonucleotide of the invention. In some embodiment, a SCAP gene,
e.g. a human SCAP gene, is suppressed by at least 55%, 60%, 65%,
70%, 75%, or 80% by administration of the double-stranded
oligonucleotide of the invention. In some embodiments, a SCAP gene,
e.g. a human SCAP gene, is suppressed by at least 85%, 90%, or 95%
by administration of the double-stranded oligonucleotide of the
invention. Table 5 provides values for inhibition of SCAP
expression using various dsRNA molecules of the invention.
[0055] As used herein in the context of SCAP expression, e.g.
expression of a human SCAP, the terms "treat", "treatment", and the
like, refer to relief from or alleviation of pathological processes
mediated by SCAP 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
SCAP 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 of such
condition.
[0056] 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 SCAP expression
or an overt symptom of pathological processes mediated by SCAP
expression. The specific amount that is therapeutically effective
can be readily determined by ordinary medical practitioner, and may
vary depending on factors known in the art, such as, e.g. the type
of pathological processes mediated by SCAP expression, the
patient's history and age, the stage of pathological processes
mediated by SCAP expression, and the administration of other
anti-SCAP expression agents.
[0057] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA 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 25% 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 25% reduction in that parameter.
[0058] 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.
[0059] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (DSRNA)
[0060] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
a SCAP gene, e.g. a human SCAP gene, in a cell or mammal, wherein
the dsRNA comprises an antisense strand comprising a region of
complementarity which is complementary to at least a part of an
mRNA formed in the expression of a SCAP gene, e.g. a human SCAP
gene, and wherein the region of complementarity is less than 30
nucleotides in length, generally 19-24 nucleotides in length, and
wherein said dsRNA, upon contact with a cell expressing said SCAP
gene, inhibits the expression of said SCAP gene by at least 20%.
The dsRNA comprises two RNA strands that are sufficiently
complementary to hybridize to form a duplex structure. One strand
of the dsRNA (the antisense strand) comprises a region of
complementarity that is substantially complementary, and generally
fully complementary, to a target sequence, derived from the
sequence of an mRNA formed during the expression of a SCAP gene,
the other strand (the sense strand) comprises a region which 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,
more generally between 18 and 25, yet more generally between 19 and
24, and most generally between 19 and 21 base pairs in length.
Similarly, the region of complementarity to the target sequence is
between 15 and 30, more generally between 18 and 25, yet more
generally between 19 and 24, and most generally between 19 and 21
nucleotides in length. The dsRNA of the invention may further
comprise one or more single-stranded nucleotide overhang(s).
[0061] 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 a preferred
embodiment, a SCAP gene is the human SCAP gene. In specific
embodiments, the first strand of the dsRNA comprises the sense
strand sequences of the RNAi agents AD-9490-AD-9513 (uneven numbers
of the group of SEQ ID NO: 1-48, Table 1), and the second sequence
is selected from the group consisting of the antisense strand
sequences of AD-9490-AD-9513 (even numbers of the group of SEQ ID
NO: 1-48, Table 1).
[0062] In further embodiments, the dsRNA comprises at least one
nucleotide sequence selected from the groups of sequences provided
above for the RNAi agents AD-9490-AD-9513 (Table 1). In other
embodiments, the dsRNA comprises at least two sequences selected
from this group, wherein one of the at least two sequences is
complementary to another of the at least two sequences, and one of
the at least two sequences is substantially complementary to a
sequence of an mRNA generated in the expression of a SCAP gene,
e.g. a human SCAP gene. Generally, the dsRNA comprises two
oligonucleotides, wherein one oligonucleotide is described as the
sense strand in one of the RNAi agents AD-9490-AD-9513 (Table 1),
and the second oligonucleotide is described as the antisense strand
in one of the RNAi agents AD-9490-AD-9513 (Table 1).
[0063] The skilled person is well aware that dsRNAs comprising 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 dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided for the RNAi agents
AD-9490-AD-9513 (Table 1), the dsRNAs of the invention can comprise
at least one strand of a length of minimally 21 nt. It can be
reasonably expected that shorter dsRNAs comprising one of the
sequences provided herein for the RNAi agents AD-9490-AD-9513
(Table 1), minus only a few nucleotides on one or both ends may be
similarly effective as compared to the dsRNAs described above.
Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17,
18, 19, 20, or more contiguous nucleotides from one of the
sequences of the RNAi agents AD-9490-AD-9513 (Table 1), and
differing in their ability to inhibit the expression of a SCAP
gene, e.g. a human SCAP gene, in an assay as described herein below
by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA
comprising the full sequence, are contemplated by the invention.
Further dsRNAs that cleave within the target sequence of the RNAi
agents AD-9490-AD-9513 (Table 1), can readily be made using a SCAP
mRNA sequence, e.g. a human SCAP mRNA sequence, e.g. GeneBank
accession no. NM.sub.--012235.2, and the respective target
sequence.
[0064] In addition, the RNAi agents provided in Table 1 identify a
site in the SCAP mRNA that is susceptible to RNAi-effected
cleavage. As such the present invention further includes RNAi
agents that target within the sequence targeted by one of the
agents of the present invention. As used herein a second RNAi agent
is said to target within the sequence of a first RNAi agent if the
second RNAi agent cleaves the message anywhere within the mRNA that
is complementary to the antisense strand of the first RNAi agent.
Such a second agent will generally consist of at least 15
contiguous nucleotides from one of the sequences provided in Table
1 coupled to additional nucleotide sequences taken from the region
contiguous to the selected sequence in a SCAP gene. For example,
the 3'-most 15 nucleotides of the target sequence of AD-9509
combined with the next 6 nucleotides from the target SCAP gene
produces a single strand agent of 21 nucleotides that is based on
one of the sequences provided in Table 1.
[0065] The dsRNA of the invention can contain one or more
mismatches to the target sequence. In a preferred embodiment, the
dsRNA of the invention contains no more than 3 mismatches. If the
antisense strand of the dsRNA 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 dsRNA contains mismatches to the target sequence, it
is preferable that the mismatch be restricted to 5 nucleotides from
either end, for example 5, 4, 3, 2, or 1 nucleotide from either the
5' or 3' end of the region of complementarity, and preferably from
the 5'-end. For example, for a 23 nucleotide dsRNA strand which is
complementary to a region of a SCAP gene, the dsRNA generally does
not contain any mismatch within the central 13 nucleotides. In
another embodiment, the antisense strand of the dsRNA does not
contain any mismatch in the region from positions 1, or 2, to
positions 9, 10, 11, or 12, of the antisense strand (counting
5'-3'). These positions are generally considered as the seed region
(positions 1, or 2, to 9, or 10) and the site of mRNA cleavage
(positions 11 and 12), respectively, and seem most sensitive to
mismatches. The methods described within the invention can be used
to determine whether a dsRNA containing a mismatch to a target
sequence is effective in inhibiting the expression of a SCAP gene.
Consideration of the efficacy of dsRNAs with mismatches in
inhibiting expression of a SCAP gene is important, especially if
the particular region of complementarity in a SCAP gene is known to
have polymorphic sequence variation within the population.
[0066] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, generally 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties than their blunt-ended
counterparts. Moreover, the present inventors have discovered that
the presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Generally, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, generally
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Generally, the antisense strand of the
dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is
blunt. In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
[0067] In yet another embodiment, the dsRNA is chemically modified
to enhance stability. The nucleic acids of 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. Specific examples of preferred dsRNA compounds useful in
this invention include dsRNAs containing modified backbones or no
natural internucleoside linkages. As defined in this specification,
dsRNAs having modified backbones include those that retain a
phosphorus atom in the backbone and 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
dsRNAs that do not have a phosphorus atom in their internucleoside
backbone can also be considered to be oligonucleosides.
[0068] Preferred modified dsRNA 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.
[0069] 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; and 5,625,050, each of which is
herein incorporated by reference
[0070] Preferred modified dsRNA 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.
[0071] 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.
[0072] In other preferred dsRNA mimetics, 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 dsRNA 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
a dsRNA 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 in Nielsen et al., Science, 1991,
254, 1497-1500.
[0073] Most preferred embodiments of the invention are dsRNAs 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.2N(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. Also
preferred are dsRNAs having morpholino backbone structures of the
above-referenced U.S. Pat. No. 5,034,506.
[0074] Modified dsRNAs may also contain one or more substituted
sugar moieties. Preferred dsRNAs comprise 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. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.n--OCH.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. Other preferred dsRNAs comprise 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 dsRNA, or a group for improving
the pharmacodynamic properties of an dsRNA, and other substituents
having similar properties. A preferred modification includes
2'-methoxyethoxy (T-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. A further
preferred modification includes T-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 hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0075] Other preferred modifications include 2'-methoxy
(2'-OCH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and 2'-fluoro (2'-F).
Similar modifications may also be made at other positions on the
dsRNA, 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. DsRNAs 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 in its entirety.
[0076] DsRNAs may also include nucleobase (often referred to in the
art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl
anal other 8-substituted adenines and guanines, 5-halo,
particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylguanine and 7-methyladenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine
and 3-deazaguanine and 3-deazaadenine. Further nucleobases include
those disclosed in U.S. Pat. No. 3,687,808, those disclosed in 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 of 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 presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0077] 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; and 5,681,941, each of which is
herein incorporated by reference, and U.S. Pat. No. 5,750,692, also
herein incorporated by reference.
[0078] Another modification of the dsRNAs of the invention involves
chemically linking to the dsRNA one or more moieties or conjugates
which enhance the activity, cellular distribution or cellular
uptake of the dsRNA. Such moieties include but are not limited to
lipid moieties such as a cholesterol moiety (Letsinger et al.,
Proc. Natl. Acid. Sci. USA, 199, 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-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0079] Representative U.S. patents that teach the preparation of
such dsRNA 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, each of
which is herein incorporated by reference.
[0080] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an dsRNA. The
present invention also includes dsRNA compounds which are chimeric
compounds. "Chimeric" dsRNA compounds or "chimeras," in the context
of this invention, are dsRNA compounds, particularly 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 dsRNAs typically contain at least one region
wherein the dsRNA is modified so as to confer upon the dsRNA
increased resistance to nuclease degradation, increased cellular
uptake, and/or increased binding affinity for the target nucleic
acid. An additional region of the dsRNA may serve as a substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
Cleavage of the RNA target can be routinely detected by gel
electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0081] In certain instances, the dsRNA may be modified by a
non-ligand group. A number of non-ligand molecules have been
conjugated to dsRNAs in order to enhance the activity, cellular
distribution or cellular uptake of the dsRNA, and procedures for
performing such conjugations are available in the scientific
literature. Such non-ligand moieties have included lipid moieties,
such as cholesterol (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 dsRNA conjugates have
been listed above. Typical conjugation protocols involve the
synthesis of dsRNAs bearing an aminolinker at one or more positions
of the sequence. The amino group is then reacted with the molecule
being conjugated using appropriate coupling or activating reagents.
The conjugation reaction may be performed either with the dsRNA
still bound to the solid support or following cleavage of the dsRNA
in solution phase. Purification of the dsRNA conjugate by HPLC
typically affords the pure conjugate.
[0082] Vector Encoded RNAi Agents
[0083] The dsRNA of the invention can also be expressed from
recombinant viral vectors intracellularly in vivo. The recombinant
viral vectors of the invention comprise sequences encoding the
dsRNA of the invention and any suitable promoter for expressing the
dsRNA sequences. Suitable promoters include, for example, the U6 or
H1 RNA pol III promoter sequences and the cytomegalovirus promoter.
Selection of other suitable promoters is within the skill in the
art. The recombinant viral vectors of the invention can also
comprise inducible or regulatable promoters for expression of the
dsRNA in a particular tissue or in a particular intracellular
environment. The use of recombinant viral vectors to deliver dsRNA
of the invention to cells in vivo is discussed in more detail
below.
[0084] dsRNA of the invention can be expressed from a recombinant
viral vector either as two separate, complementary RNA molecules,
or as a single RNA molecule with two complementary regions.
[0085] Any viral vector capable of accepting the coding sequences
for the dsRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. 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.
[0086] For example, lentiviral vectors of the invention can be
pseudotyped with surface proteins from vesicular stomatitis virus
(VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the
invention can be made to target different cells by engineering the
vectors to express different capsid protein serotypes. For example,
an AAV vector expressing a serotype 2 capsid on a serotype 2 genome
is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2
vector can be replaced by a serotype 5 capsid gene to produce an
AAV 2/5 vector. Techniques for constructing AAV vectors which
express different capsid protein serotypes are within the skill in
the art; see, e.g., Rabinowitz J E et al. (2002), J Virol
76:791-801, the entire disclosure of which is herein incorporated
by reference.
[0087] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the dsRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310;
Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990),
Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30;
and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire
disclosures of which are herein incorporated by reference.
[0088] Preferred viral vectors are those derived from AV and AAV.
In a particularly preferred embodiment, the dsRNA of the invention
is expressed as two separate, complementary single-stranded RNA
molecules from a recombinant AAV vector comprising, for example,
either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV)
promoter.
[0089] A suitable AV vector for expressing the dsRNA of 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.
[0090] Suitable AAV vectors for expressing the dsRNA of 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. No. 5,252,479; U.S. Pat. No.
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.
III. PHARMACEUTICAL COMPOSITIONS COMPRISING DSRNA
[0091] In one embodiment, the invention provides pharmaceutical
compositions comprising a dsRNA, as described herein, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
comprising the dsRNA is useful for treating a disease or disorder
associated with the expression or activity of a SCAP gene, such as
pathological processes mediated by human SCAP expression, or
diseases or disorders which can be treated by downregulation of
SCAP 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.
[0092] The pharmaceutical compositions of the invention are
administered in dosages sufficient to inhibit expression of a SCAP
gene. The present inventors have found that, because of their
improved efficiency, compositions comprising the dsRNA of the
invention can be administered at surprisingly low dosages. A
maximum dosage of 5 mg dsRNA per kilogram body weight of recipient
per day is sufficient to inhibit or completely suppress expression
of a SCAP gene.
[0093] In general, a suitable dose of dsRNA will be in the range of
0.01 microgram to 5.0 milligrams per kilogram body weight of the
recipient per day, generally in the range of 1 microgram to 1 mg
per kilogram body weight per day. The pharmaceutical composition
may be administered once daily, or the dsRNA may be administered as
two, three, or more sub-doses at appropriate intervals throughout
the day or even using continuous infusion or delivery through a
controlled release formulation. In that case, the dsRNA 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 dsRNA over a several day period. Sustained release
formulations are well known in the art and are particularly useful
for vaginal delivery of agents, 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.
[0094] 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
dsRNAs 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.
[0095] Advances in genetics have generated a number of laboratory
animal models for the study of various human diseases, such as
pathological processes mediated by SCAP expression. Such models are
used for in vivo testing of dsRNA, as well as for determining a
therapeutically effective dose.
[0096] The present invention also includes pharmaceutical
compositions and formulations which include the dsRNA compounds of
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, 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; or intracranial, e.g., intrathecal or
intraventricular, administration.
[0097] 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. Preferred
topical formulations include those in which the dsRNAs of the
invention are in admixture with a topical delivery agent such as
lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl ethanolamine=DOPE,
dimyristoylphosphatidyl choline=DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol=DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl=DOTAP and
dioleoylphosphatidyl ethanolamine=DOTMA). DsRNAs of the invention
may be encapsulated within liposomes or may form complexes thereto,
in particular to cationic liposomes. Alternatively, dsRNAs may be
complexed to lipids, in particular to cationic lipids. Preferred
fatty acids and esters include but are not limited 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.1-10 alkyl ester (e.g.
isopropylmyristate IPM), monoglyceride, diglyceride or
pharmaceutically acceptable salt thereof. Topical formulations are
described in detail in U.S. patent application Ser. No. 09/315,298
filed on May 20, 1999 which is incorporated herein by reference in
its entirety.
[0098] 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. Preferred oral formulations are those in which
dsRNAs of the invention are administered in conjunction with one or
more penetration enhancers, surfactants, and chelators. Preferred
surfactants include fatty acids and/or esters or salts thereof,
bile acids and/or salts thereof. Preferred 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. Preferred 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). Also preferred are combinations of
penetration enhancers, for example, fatty acids/salts in
combination with bile acids/salts. A particularly preferred
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 of 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. Particularly preferred 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. application. Ser. No.
08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1,
1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No.
09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May
20, 1999), each of which is incorporated herein by reference in
their entirety.
[0099] Compositions and formulations for parenteral, intrathecal or
intraventricular 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
[0104] Emulsions
[0105] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter (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.
[0106] 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 (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0107] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (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 (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0108] 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.
[0109] 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).
[0110] 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.
[0111] 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.
[0112] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (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 (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.
[0113] In one embodiment of the present invention, the compositions
of dsRNAs 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 (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).
[0114] 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
(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.
[0115] 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 (C.sub.8-C.sub.12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0116] 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 (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 (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 dsRNAs. 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
dsRNAs and nucleic acids from the gastrointestinal tract, as well
as improve the local cellular uptake of dsRNAs and nucleic acids
within the gastrointestinal tract, vagina, buccal cavity and other
areas of administration.
[0117] 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
dsRNAs 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.
[0118] Liposomes
[0119] 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.
[0120] 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.
[0121] In order to cross 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.
[0122] 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.
[0123] 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.
[0124] 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.
[0125] 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
[0126] 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).
[0127] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0128] 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.
[0129] 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).
[0130] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
[0131] 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).
[0132] 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-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499
(Lim et al).
[0133] 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.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0134] A limited 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 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.
[0135] 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.
[0136] 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).
[0137] 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.
[0138] 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.
[0139] 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.
[0140] 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.
[0141] 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).
[0142] Penetration Enhancers
[0143] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly dsRNAs, 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.
[0144] 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 (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.
[0145] 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 dsRNAs 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) (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).
[0146] 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-C.sub.10 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.)
(Lee et al., Critical Reviews in Therapeutic Drug Carryier 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).
[0147] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (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. The bile salts of the invention 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) (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).
[0148] 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 dsRNAs 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). Chelating agents of the invention 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)(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).
[0149] 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
dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of
penetration enhancers include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0150] Agents that enhance uptake of dsRNAs 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.
[0151] 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.
[0152] Carriers
[0153] 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., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183.
[0154] Excipients:
[0155] 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).
[0156] Pharmaceutically acceptable organic or inorganic excipient
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.
[0157] 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.
[0158] 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.
[0159] Pharmaceutical compositions for the delivery to the
respiratory tract
[0160] Another aspect of the invention provides for the delivery of
IRNA agents to the respiratory tract. The respiratory tract
includes the upper airways, including the oropharynx and larynx,
followed by the lower airways, which include the trachea followed
by bifurcations into the bronchi and bronchioli. The upper and
lower airways are called the conductive airways. The terminal
bronchioli then divide into respiratory bronchioli which then lead
to the ultimate respiratory zone, the alveoli, or deep lung. The
deep lung, or alveoli, are the primary target of inhaled
therapeutic aerosols for systemic delivery of iRNA agents.
[0161] Pulmonary delivery compositions can be delivered by
inhalation by the patient of a dispersion so that the composition,
preferably the iRNA agent, within the dispersion can reach the lung
where it can, for example, be readily absorbed through the alveolar
region directly into blood circulation. Pulmonary delivery can be
effective both for systemic delivery and for localized delivery to
treat diseases of the lungs.
[0162] Pulmonary delivery can be achieved by different approaches,
including the use of nebulized, aerosolized, micellular and dry
powder-based formulations; administration by inhalation may be oral
and/or nasal. Delivery can be achieved with liquid nebulizers,
aerosol-based inhalers, and dry powder dispersion devices.
Metered-dose devices are preferred. One of the benefits of using an
atomizer or inhaler is that the potential for contamination is
minimized because the devices are self contained. Dry powder
dispersion devices, for example, deliver drugs that may be readily
formulated as dry powders. An iRNA composition may be stably stored
as lyophilized or spray-dried powders by itself or in combination
with suitable powder carriers. The delivery of a composition for
inhalation can be mediated by a dosing timing element which can
include a timer, a dose counter, time measuring device, or a time
indicator which when incorporated into the device enables dose
tracking, compliance monitoring, and/or dose triggering to a
patient during administration of the aerosol medicament.
[0163] Examples of pharmaceutical devices for aerosol delivery
include metered dose inhalers (MDIs), dry powder inhalers (DPIs),
and air-jet nebulizers. Exemplary delivery systems by inhalation
which can be readily adapted for delivery of the subject iRNA
agents are described in, for example, U.S. Pat. Nos. 5,756,353;
5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359;
WO01/54664; WO02/060412. Other aerosol formulations that may be
used for delivering the iRNA agents are described in U.S. Pat. Nos.
6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078;
WO02/053190; WO01/60420; WO00/66206. Further, methods for
delivering iRNA agents can be adapted from those used in delivering
other oligonucleotides (e.g., an antisense oligonucleotide) by
inhalation, such as described in Templin et al., Antisense Nucleic
Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al., Expert Opin
Biol Ther, 2001, 1:979-83; Sandrasagra et al., Antisense Nucleic
Acid Drug Dev, 2002, 12:177-81.
[0164] The delivery of the inventive agents may also involve the
administration of so called "pro-drugs", i.e. formulations or
chemical modifications of a therapeutic substance that require some
form of processing or transport by systems innate to the subject
organism to release the therapeutic substance, preferably at the
site where its action is desired; this latter embodiment may be
used in conjunction with delivery of the respiratory tract, but
also together with other embodiments of the present invention. For
example, the human lungs can remove or rapidly degrade
hydrolytically cleavable deposited aerosols over periods ranging
from minutes to hours. In the upper airways, ciliated epithelia
contribute to the "mucociliary excalator" by which particles are
swept from the airways toward the mouth. Pavia, D., "Lung
Mucociliary Clearance," in Aerosols and the Lung: Clinical and
Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,
Butterworths, London, 1984. In the deep lungs, alveolar macrophages
are capable of phagocytosing particles soon after their deposition.
Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and
Brain, J. D., "Physiology and Pathophysiology of Pulmonary
Macrophages," in The Reticuloendothelial System, S. M. Reichard and
J. Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.
[0165] In preferred embodiments, particularly where systemic dosing
with the iRNA agent is desired, the aerosoled iRNA agents are
formulated as microparticles. Microparticles having a diameter of
between 0.5 and ten microns can penetrate the lungs, passing
through most of the natural barriers. A diameter of less than ten
microns is required to bypass the throat; a diameter of 0.5 microns
or greater is required to avoid being exhaled.
[0166] Other Components
[0167] 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.
[0168] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0169] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more dsRNA agents and (b) one or
more other chemotherapeutic agents which function by a non-RNA
interference mechanism. Examples of such chemotherapeutic agents
include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU
and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide
for a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-dsRNA chemotherapeutic agents are also within the scope
of this invention. Two or more combined compounds may be used
together or sequentially.
[0170] 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 which exhibit
high therapeutic indices are preferred.
[0171] 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 of the invention lies generally
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
of the compound or, when appropriate, of the polypeptide product of
a target sequence (e.g., achieving a decreased concentration of the
polypeptide) that includes the IC50 (i.e., the concentration of the
test compound which achieves a half-maximal inhibition of symptoms)
as determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high performance liquid
chromatography.
[0172] In addition to their administration individually or as a
plurality, as discussed above, the dsRNAs of the invention can be
administered in combination with other known agents effective in
treatment of pathological processes mediated by SCAP expression. In
any event, the administering physician can adjust the amount and
timing of dsRNA administration on the basis of results observed
using standard measures of efficacy known in the art or described
herein.
[0173] Methods for Treating Diseases Caused by Expression of a SCAP
Gene
[0174] The invention relates in particular to the use of a dsRNA or
a pharmaceutical composition prepared therefrom for the treatment
of disorders of lipid metabolism, lipid homeostasis, and/or lipid
distribution, e.g non-alcoholic liver disease, fatty liver,
hyperlipemia, hyperlipidemia, hyperlipoproteinemia,
hypercholesterolemia and/or hypertriglyceridemia, atherosclerosis,
pancreatitis, non-insulin dependent diabetes mellitus (NIDDM),
coronary heart disease, obesity, metabolic syndrome, peripheral
arterial disease, and cerebrovascular disease. Owing to the
inhibitory effect on SCAP expression, a dsRNA according to the
invention or a pharmaceutical composition prepared therefrom can
enhance the quality of life of patients with such diseases or
disorders.
[0175] The invention furthermore relates to the use of an dsRNA or
a pharmaceutical composition thereof 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 diseases
or disorders of lipid metabolism, lipid homeostasis, and/or lipid
distribution, e.g. non-alcoholic liver disease, fatty liver,
hyperlipemia, hyperlipidemia, hyperlipoproteinemia,
hypercholesterolemia and/or hypertriglyceridemia, atherosclerosis,
pancreatitis, non-insulin dependent diabetes mellitus (NIDDM),
coronary heart disease, obesity, metabolic syndrome, peripheral
arterial disease, and cerebrovascular disease. Where the
pharmaceutical composition aims for the treatment of diseases or
disorders of lipid metabolism, lipid homeostasis, and/or lipid
distribution, preference is given to a combination with lipid
lowering drugs, e.g. statins, insulin for diabetes, and medication
for liver disease.
[0176] Methods for Inhibiting Expression of a SCAP Gene
[0177] In yet another aspect, the invention provides a method for
inhibiting the expression of a SCAP gene in a mammal. The method
comprises administering a composition of the invention to the
mammal such that expression of the target SCAP gene, e.g. human
SCAP, is silenced. Because of their high specificity, the dsRNAs of
the invention specifically target RNAs (primary or processed) of
the target SCAP gene. Compositions and methods for inhibiting the
expression of these SCAP genes using dsRNAs can be performed as
described elsewhere herein.
[0178] In one embodiment, the method comprises administering a
composition comprising a dsRNA, wherein the dsRNA comprises a
nucleotide sequence which is complementary to at least a part of an
RNA transcript of a SCAP gene, e.g. human SCAP, 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 or parenteral routes,
including intravenous, intramuscular, subcutaneous, transdermal,
airway (aerosol), nasal, rectal, vaginal and topical (including
buccal and sublingual) administration. In preferred embodiments,
the compositions are administered by intravenous infusion or
injection.
[0179] 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 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
Design of siRNAs
[0180] siRNA design was carried out to identify siRNAs targeting
hamster, mouse, and/or human SCAP. First, the mRNA sequences of Mus
musculus (NM.sub.--001001144.1) and Cricetus cricetus (U67060; Hua
et al., Cell. 1996, 87:415) SCAP were examined by computer analysis
to identify homologous sequences of 19 nucleotides that yield RNAi
agents cross-reactive between these two species.
[0181] In identifying agents cross-reactive between mouse and
hamster, the selection was limited to 19mer sequences having at
least 3 mismatches to any other sequence in the mouse genome by
using a software tool provided by the Whitehead Institute at
http://jura.wi.mit.edu/bioc/siRNAext/ in the version of May 18,
2005. The sequences thus identified formed the basis for the
synthesis of the iRNA agents given in Table 2, which contain
modified nucleotides. Therein, all pyrimidine-base bearing
nucleotides in the sense strand, and all cytidines occurring in a
sequence context 5'-ca-3' and all uridines occurring in a sequence
context 5'-ua-3' in the antisense strand, are 2'-O-methyl-modified
nucleotides, and the 3'-terminal deoxythymidines
TABLE-US-00002 TABLE 2 RNAi agents selected for the down-regulation
of mus musculus (NM_001001144.1) and Cricetus cricetus (U67060)
SCAP, and minimal off-target interactions in mice Duplex Sense SEQ
ID Antisense SEQ ID identifier strand sequence.sup.1 NO: strand
sequence.sup.1 NO: AL-DP-6054 ggcmgacmaumumacmcmumumgumacmaTT 49
ugumacmaaggumaaugucgccTT 50 AL-DP-6055
gumcmcmumgumcmgaumcmgacmaumumcmTT 51 gaaugucgaucgacmaggacTT 52
AL-DP-6056 cmacmumcmaaumggcmggumgagaumTT 53 aucucmaccgccmauugagugTT
54 AL-DP-6057 umcmcmumgumcmgaumcmgacmaumumcmgTT 55
cgaaugucgaucgacmaggaTT 56 AL-DP-6058 gagumgumcmumggcmumagcmgaumgTT
57 cmaucgcumagccmagacmacucTT 58 AL-DP-6059
cmumcmacmcmumgcmumumaaumcmgacmaTT 59 ugucgauumaagcmaggugagTT 60
AL-DP-6060 ggaumumgumagcmumgcmumcmggcmumTT 61
agccgagcmagcumacmaauccTT 62 AL-DP-6061
umumgumagcmumgcmumcmggcmumumaaTT 63 uumaagccgagcmagcumacmaaTT 64
AL-DP-6062 gcmumumaaumggumumcmcmcmumumgaumTT 65
aucmaagggaaccmauumaagcTT 66 AL-DP-6063 acmacmumcmaaumggcmggumgagaTT
67 ucucmaccgccmauugaguguTT 68 AL-DP-6064
acmcmumcmacmcmumgcmumumaaumcmgaTT 69 ucgauumaagcmaggugagguTT 70
AL-DP-6065 gaggumgaagcmumumcmggaumumgTT 71 cmaauccgaagcuucmaccucTT
72 .sup.1Capital letters = desoxyribonucleotides; small letters =
ribonucleotides; underlined: nucleoside thiophosphates; cm =
2'-O-methyl-cytidine; um = 2'-O-methyl-uridine
[0182] In order to furthermore identify agents useful for
inhibiting the expression of human SCAP, the sequences of Homo
sapiens (NM.sub.--012235.2), Mus musculus (NM.sub.--001001144.1)
and Cricetus cricetus (U67060) SCAP were compared in a second step,
and homologous sequences of 19 nucleotides that yield RNAi agents
cross-reactive between these three species were identified. To
minimize non-specific effects in humans, the selection was further
narrowed by fastA comparison to the gene sequences in the human
RefSeq database (available from
http://www.ncbi.nlm.nih.gov/RefSeq/), Rev. 17. The 24 19mer
sequences of the RNAi agents in Table 1 were identified which had
at least 2 mismatches to any other gene in the human RefSeq
database for both the sense and the antisense strand, wherein, for
the closest matching gene, at least one of these mismatches came to
lie in the "seed region" (Pos. 2 to 10, counting 5' to 3'), which
is particularly sensitive to mismatches, and which had at least two
mismatches, or at least one seed region mismatch, to any other gene
in the mouse RefSeq database.
[0183] dsRNA Synthesis
[0184] Source of Reagents
[0185] 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.
[0186] siRNA Synthesis
[0187] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 .mu.mole using an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
and controlled pore glass (CPG, 500 .ANG., Proligo Biochemie GmbH,
Hamburg, Germany) as solid support. RNA and RNA containing
2'-O-methyl nucleotides were generated by solid phase synthesis
employing the corresponding phosphoramidites and 2'-O-methyl
phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,
Germany). These building blocks were incorporated at selected sites
within the sequence of the oligoribonucleotide chain using standard
nucleoside phosphoramidite chemistry such as described in Current
protocols in nucleic acid chemistry, Beaucage, S. L. et al.
(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA.
Phosphorothioate linkages were introduced by replacement of the
iodine oxidizer solution with a solution of the Beaucage reagent
(Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further
ancillary reagents were obtained from Mallinckrodt Baker
(Griesheim, Germany).
[0188] Deprotection and purification of the crude
oligoribonucleotides by anion exchange HPLC were carried out
according to established procedures. Yields and concentrations were
determined by UV absorption of a solution of the respective RNA at
a wavelength of 260 nm using a spectral photometer (DU 640B,
Beckman Coulter GmbH, Unterschlei.beta.heim, Germany). Double
stranded RNA was generated by mixing an equimolar solution of
complementary strands in annealing buffer (20 mM sodium phosphate,
pH 6.8; 100 mM sodium chloride), heated in a water bath at
85-90.degree. C. for 3 minutes and cooled to room temperature over
a period of 3-4 hours. The annealed RNA solution was stored at
-20.degree. C. until use.
[0189] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as -Chol-3'), an appropriately modified solid
support was used for RNA synthesis. The modified solid support was
prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00001##
[0191] A 4.7 M aqueous solution of sodium hydroxide (50 mL) was
added into a stirred, ice-cooled solution of ethyl glycinate
hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl
acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred
at room temperature until completion of the reaction was
ascertained by TLC. After 19 h the solution was partitioned with
dichloromethane (3.times.100 mL). The organic layer was dried with
anhydrous sodium sulfate, filtered and evaporated. The residue was
distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl-
]-amino}-propionic acid ethyl ester AB
##STR00002##
[0193] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was
dissolved in dichloromethane (50 mL) and cooled with ice.
Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to
the solution at 0.degree. C. It was then followed by the addition
of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and
dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was
brought to room temperature and stirred further for 6 h. Completion
of the reaction was ascertained by TLC. The reaction mixture was
concentrated under vacuum and ethyl acetate was added to
precipitate diisopropyl urea. The suspension was filtered. The
filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium
bicarbonate and water. The combined organic layer was dried over
sodium sulfate and concentrated to give the crude product which was
purified by column chromatography (50% EtOAC/Hexanes) to yield
11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC
##STR00003##
[0195]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-he-
xanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol)
was dissolved in 20% piperidine in dimethylformamide at 0.degree.
C. The solution was continued stirring for 1 h. The reaction
mixture was concentrated under vacuum, water was added to the
residue, and the product was extracted with ethyl acetate. The
crude product was purified by conversion into its hydrochloride
salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h-
exanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester
AD
##STR00004##
[0197] The hydrochloride salt of
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane.
The suspension was cooled to 0.degree. C. on ice. To the suspension
diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the
resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol)
was added. The reaction mixture was stirred overnight. The reaction
mixture was diluted with dichloromethane and washed with 10%
hydrochloric acid. The product was purified by flash chromatography
(10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15-
,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-he-
xanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE
##STR00005##
[0199] Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL
of dry toluene. The mixture was cooled to 0.degree. C. on ice and 5
g (6.6 mmol) of diester AD was added slowly with stirring within 20
mins. The temperature was kept below 5.degree. C. during the
addition. The stirring was continued for 30 mins at 0.degree. C.
and 1 mL of glacial acetic acid was added, immediately followed by
4 g of NaH.sub.2PO.sub.4.H.sub.2O in 40 mL of water The resultant
mixture was extracted twice with 100 mL of dichloromethane each and
the combined organic extracts were washed twice with 10 mL of
phosphate buffer each, dried, and evaporated to dryness. The
residue was dissolved in 60 mL of toluene, cooled to 0.degree. C.
and extracted with three 50 mL portions of cold pH 9.5 carbonate
buffer. The aqueous extracts were adjusted to pH 3 with phosphoric
acid, and extracted with five 40 mL portions of chloroform which
were combined, dried and evaporated to dryness. The residue was
purified by column chromatography using 25% ethylacetate/hexane to
afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic
acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
##STR00006##
[0201] Methanol (2 mL) was added dropwise over a period of 1 h to a
refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium
borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring
was continued at reflux temperature for 1 h. After cooling to room
temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted
with ethylacetate (3.times.40 mL). The combined ethylacetate layer
was dried over anhydrous sodium sulfate and concentrated under
vacuum to yield the product which was purified by column
chromatography (10% MeOH/CHCl.sub.3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-
-yl}-6-oxo-hexyl)-carbamic acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
##STR00007##
[0203] Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with
pyridine (2.times.5 mL) in vacuo. Anhydrous pyridine (10 mL) and
4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with
stirring. The reaction was carried out at room temperature
overnight. The reaction was quenched by the addition of methanol.
The reaction mixture was concentrated under vacuum and to the
residue dichloromethane (50 mL) was added. The organic layer was
washed with 1M aqueous sodium bicarbonate. The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated. The
residual pyridine was removed by evaporating with toluene. The
crude product was purified by column chromatography (2%
MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl.sub.3) (1.75 g, 95%).
[0204] Succinic acid
mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{641741,5-dimethy-
l-hexyl)-10,13-dimethyl
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H
cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)
ester AH
##STR00008##
[0205] Compound AG (1.0 g, 1.05 mmol) was mixed with succinic
anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and
dried in a vacuum at 40.degree. C. overnight. The mixture was
dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318
g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at
room temperature under argon atmosphere for 16 h. It was then
diluted with dichloromethane (40 mL) and washed with ice cold
aqueous citric acid (5 wt %, 30 mL) and water (2.times.20 mL). The
organic phase was dried over anhydrous sodium sulfate and
concentrated to dryness. The residue was used as such for the next
step.
[0206] Cholesterol Derivatised CPG AI
##STR00009##
[0207] Succinate AH (0.254 g, 0.242 mmol) was dissolved in a
mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that
solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL),
2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively.
To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol)
in acetonitrile (0.6 ml) was added. The reaction mixture turned
bright orange in color. The solution was agitated briefly using a
wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG)
(1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The
CPG was filtered through a sintered funnel and washed with
acetonitrile, dichloromethane and ether successively. Unreacted
amino groups were masked using acetic anhydride/pyridine. The
achieved loading of the CPG was measured by taking UV measurement
(37 mM/g).
[0208] The synthesis of siRNAs bearing a 5'-12-dodecanoic acid
bisdecylamide group (herein referred to as "5'-C32-") or a
5'-cholesteryl derivative group (herein referred to as "5'-Chol-")
was performed as described in WO 2004/065601, except that, for the
cholesteryl derivative, the oxidation step was performed using the
Beaucage reagent in order to introduce a phosphorothioate linkage
at the 5'-end of the nucleic acid oligomer.
[0209] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 2.
TABLE-US-00003 TABLE 3 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.sup.a
Nucleotide(s) A, a 2'-deoxy-adenosine-5'-phosphate,
adenosine-5'-phosphate C, c 2'-deoxy-cytidine-5'-phosphate,
cytidine-5'-phosphate G, g 2'-deoxy-guanosine-5'-phosphate,
guanosine-5'-phosphate T, t 2'-deoxy-thymidine-5'-phosphate,
thymidine-5'-phosphate U, u 2'-deoxy-uridine-5'-phosphate,
uridine-5'-phosphate N, n any 2'-deoxy-nucleotide/nucleotide (G, A,
C, or T, g, a, c or u) Am 2'-O-methyladenosine-5'-phosphate Cm
2'-O-methylcytidine-5'-phosphate Gm
2'-O-methylguanosine-5'-phosphate Tm
2'-O-methyl-thymidine-5'-phosphate Um
2'-O-methyluridine-5'-phosphate Af
2'-fluoro-2'-deoxy-adenosine-5'-phosphate Cf
2'-fluoro-2'-deoxy-cytidine-5'-phosphate Gf
2'-fluoro-2'-deoxy-guanosine-5'-phosphate Tf
2'-fluoro-2'-deoxy-thymidine-5'-phosphate Uf
T-fluoro-2'-deoxy-uridine-5'-phosphate A, C, G, T, underlined:
nucleoside-5'-phosphorothioate U, a, c, g, t, u am, cm, gm,
underlined: 2-O-methyl-nucleoside-5'-phosphorothioate tm, um
.sup.acapital letters represent 2'-deoxyribonucleotides (DNA),
lower case letters represent ribonucleotides (RNA)
[0210] dsRNA Expression Vectors
[0211] In another aspect of the invention, Human SCAP specific
dsRNA molecules that modulate Human SCAP gene expression activity
are expressed from transcription units inserted into DNA or RNA
vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10;
Skillern, A., et al., International PCT Publication No. WO
00/22113, Conrad, International PCT Publication No. WO 00/22114,
and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be
introduced as a linear construct, a circular plasmid, or a viral
vector, which can be incorporated and inherited as a transgene
integrated into the host genome. 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).
[0212] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and co-transfected
into a target cell. Alternatively each individual strand of the
dsRNA can be transcribed by promoters both of which are located on
the same expression plasmid. In a preferred embodiment, a dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0213] The recombinant dsRNA expression vectors are generally DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al.,
BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science
252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or
alphavirus as well as others known in the art. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, in vitro and/or in vivo (see,
e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and
Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et
al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991,
Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad.
Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy
3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). Recombinant retroviral vectors
capable of transducing and expressing genes inserted into the
genome of a cell can be produced by transfecting the recombinant
retroviral genome into suitable packaging cell lines such as PA317
and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone
et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant
adenoviral vectors can be used to infect a wide variety of cells
and tissues in susceptible hosts (e.g., rat, hamster, dog, and
chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and
also have the advantage of not requiring mitotically active cells
for infection.
[0214] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector of the invention may be a eukaryotic RNA
polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g.
CMV early promoter or actin promoter or U1 snRNA promoter) or
generally RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter,
provided the expression plasmid also encodes T7 RNA polymerase
required for transcription from a T7 promoter. The promoter can
also direct transgene expression to the pancreas (see, e.g. the
insulin regulatory sequence for pancreas (Bucchini et al., 1986,
Proc. Natl. Acad. Sci. USA 83:2511-2515)).
[0215] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a 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 transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the dsRNA transgene.
[0216] Generally, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA 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.
[0217] dsRNA expression DNA plasmids are typically 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 dsRNA-mediated
knockdowns targeting different regions of a single Human SCAP gene
or multiple Human SCAP genes over a period of a week or more are
also contemplated by the invention. Successful introduction of the
vectors of the invention 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 ex vivo
cells 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.
[0218] The Human SCAP specific dsRNA molecules can also be inserted
into vectors and used as gene therapy vectors for human patients.
Gene therapy vectors can be delivered to a subject by, for example,
intravenous injection, local administration (see U.S. Pat. No.
5,328,470) or by stereotactic injection (see e.g., Chen et al.
(1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical
preparation of the gene therapy vector can include the gene therapy
vector in an acceptable diluent, or can comprise 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.
[0219] Effects of SCAP RNAi on the Genes Involved in Fatty Acid and
Cholesterol Synthesis in Primary Hepatocytes and in Livers from In
Vivo Experiments
Single Dose Screen in Primary Hamster Hepatocytes
[0220] Hepatocytes were isolated from a hamster liver and plated on
60-mm dishes at a density of 1.2.times.10.sup.6 cells/dish. After a
2-h attachment period, cells were transfected with 200 nM of the
indicated siRNA using oligofectamine. Total RNA was isolated from
the cells 24-h after transfection using RNA STAT-60 solution
(Tel-Test Inc., Friendswood, Tex., USA). Ten .mu.g of RNA each dish
was treated with DNase I (DNA-free; Ambion Inc., Austin, Tex.,
USA). First-strand cDNA was synthesized from 2 .mu.g of DNase
I-treated total RNA with random hexamer primers using the ABI cDNA
synthesis kit (N808-0234; PE Biosystems, Foster City, Calif., USA).
The following specific primers for each gene were designed using
Primer Express software (PE Biosystems): .beta.-actin, 5' primer,
5'-GGCTCCCAGCACCATGAA-3', 3' primer, 5'-GCCACCGATCCACACAGAGT-3';
SCAP, 5' primer, 5'-GTACCTGCAGATGATGTCCATTG-3', 3' primer,
5'-CTGCCATCCCGGAAAGTG-3' .beta.-actin was used as the invariant
control. The real-time RT-PCR reaction was set up in a final volume
of 20 .mu.l containing 20 ng of reverse-transcribed total RNA, 167
nM of the forward and reverse primers, and 10 .mu.l of 2.times.SYBR
Green PCR Master Mix (4312704; PE Biosystems). PCR reactions were
carried out in 384-well plates using the ABI PRISM 7900HT Sequence
Detection System (PE Biosystems). All reactions were done in
triplicate. The relative amount of all mRNAs was calculated using
the comparative threshold cycle (C.sub.T) method. Hamster
.beta.-actin mRNAs was used as the invariant controls. Values
represent the amount of mRNA relative to that in untransfected
cells, which is defined as 1 (n=1 plate).
TABLE-US-00004 TABLE 4 Screening siRNAs specific for inhibition of
SCAP in mice and hamsters in hamster primary hepatocytes Duplex
identifier remaining SCAP mRNA Mock transfection 1,0 (by
definition) AL-DP-6054 1.06 AL-DP-6055 1.04 AL-DP-6056 1.59
AL-DP-6057 1.32 AL-DP-6058 1.06 AL-DP-6059 0.62 AL-DP-6061 1.21
AL-DP-6062 0.22 AL-DP-6063 0.89 AL-DP-6064 0.81 AL-DP-6065 0.31
TABLE-US-00005 TABLE 5 Screening human crossreactive siRNAs for
inhibition of SCAP in hamster primary hepatocytes Duplex identifier
remaining SCAP mRNA Mock transfection 1.0 (by definition)
AL-DP-6062 0.37 AD-9505 0.21 AD-9498 0.22 AD-9512 0.23 AD-9490 0.27
AD-9495 0.27 AD-9503 0.27 AD-9494 0.28 AD-9500 0.28 AD-9492 0.31
AD-9499 0.33 AD-9496 0.34 AD-9510 0.37 AD-9511 0.38 AD-9491 0.41
AD-9506 0.42 AD-9508 0.44 AD-9502 0.45 AD-9504 0.51 AD-9507 0.53
AD-9493 0.61 AD-9501 0.62 AD-9497 0.66 AD-9509 0.69 AD-9513
0.78
Effects of SCAP RNAi on the Genes Involved in Fatty Acid and
Cholesterol Synthesis in Hamsters In Vivo
[0221] For in vivo RNAi experiments, AL-DP-6062 formulated with
liposomes was injected (4 mg/kg) into 6 hamsters via the jugular
vein. Three days after injection, the animals were sacrificed, and
total RNA was prepared from livers using established
procedures.
[0222] Total RNA was prepared from the hepatocytes using an RNeasy
kit from QIAGEN (Valencia, Calif.). Ten .mu.g of RNA from each
hamster liver was treated with DNase I (DNA-free; Ambion Inc.,
Austin, Tex., USA). First-strand cDNA was synthesized from 2 .mu.g
of DNase I-treated total RNA with random hexamer primers using the
ABI cDNA synthesis kit (N808-0234; PE Biosystems, Foster City,
Calif., USA). Equal amounts of cDNA from 6 hamsters were pooled.
Specific primers for each gene were designed using Primer Express
software (PE Biosystems) for the following genes: .beta.-actin, 5'
primer, 5'-GGCTCCCAGCACCATGAA-3', 3' primer,
5'-GCCACCGATCCACACAGAGT-3'; SCAP, 5' primer,
5'-GTACCTGCAGATGATGTCCATTG-3', 3' primer, 5'-CTGCCATCCCGGAAAGTG-3';
SREBP-1c, 5' primer, 5'-ACGCAGTCTGGGCAACAA-3', 3' primer,
5'-GAGCTGGAGCATGTCTTCAAAC-3'; SREBP-2, 5' primer,
5'-GTCAGCAATCAAGTGGGAGAGT-3', 3' primer,
5'-CTACCACCACCAGGGAAGGA-3'; Fatty acid synthase (FAS), 5' primer,
5'-AACAAGCAATGCCAGCTCACT-3', 3' primer, 5'-AACAGGCCCAAGCTTTGTTG-3';
stearoyl-CoA desaturase-1 (SCD-1), 5' primer,
5'-CAGAATGGACGGGAGAAGCA-3', 3' primer, 5'-TCATTTCAGGGCGGATGTC-3';
HMG-CoA synthase, 5' primer, 5'-CCTATGACTGCATTGGGCG-3', 3' primer,
5'-CCCAGACTCCTCAAACAGCTG-3'; HMG-CoA reductase, 5' primer,
5'-ACCATCTGTATGATGTCAATGAACA-3', 3' primer,
5'-GCTCAATACGTCCTCTTCAAATTT-3'. The real-time RT-PCR reaction was
set up as described above. Hamster .beta.-actin was used as the
invariant control. Values represent the amount of mRNA relative to
the amount of mRNA in livers of the hamsters injected with saline,
which was defined as 1.
[0223] Protein Expression of SCAP and SREBPs in Livers of the
Hamsters Injected with siRNA
[0224] Membrane and nuclear proteins were prepared from frozen
liver as described previously (Engelking et. al., J. Clin. Invest.
113: 1168-1175, 2004). Equal amounts of protein were subjected to
SDS-PAGE on 8% gels and transferred to Hybond ECL membrane
(Amersham). Immunoblot analyses were performed using polyclonal
anti-hamster SREBP-1 and SREBP-2 antibodies (Shimomura et al.,
PNAS, 94: 12354-12359, 1997). Antibody-bound bands were detected
using the SuperSignal CL-HRP substrate system (Pierce Biotechnology
Inc., Rockford, Ill.). Anti-CREB (cAMP response element binding
protein) and anti-RAP (receptor associated protein) were used as
loading controls for hepatic nuclear and membrane proteins,
respectively. Signals were quantified using Image J program
available from the Research Services Branch, National Institute of
Mental Health (Bethesda, Md.) and values represent the amount of
protein relative to those in livers of the hamsters injected with
saline which are defined as 1.
TABLE-US-00006 TABLE 6 mRNA expression of SCAP and other genes
downstream of SCAP in hamsters 3 days after treatment with 4 mg/kg
AL-DP-6062 (Hepatocytes: n = 2, Liver: pooled cDNA from 6 hamsters)
Hepatocytes Liver SCAP 0.22 0.14 SREBP-1c 0.48 0.39 SREBP-2 0.65
0.51 FAS 0.78 0.68 SCD-1 0.74 0.86 HMG-CoA synthase 0.34 0.47
HMG-CoA reductase 0.88 0.27
TABLE-US-00007 TABLE 7 mRNA and protein expression of SCAP in
livers of the hamsters injected with siRNA (pooled cDNA or pooled
protein from 6 hamsters) siRNA SCAP Mrna SCAP protein Saline 1 1
Luciferase 1.07 0.9 SCAP 0.14 0.1 SCAP-MM 0.82 0.9
TABLE-US-00008 TABLE 8 mRNA and protein expression of SREBP-1 in
livers of the hamsters injected with siRNA (pooled cDNA or pooled
protein from 6 hamsters) mRNA Protein siRNA SREBP-1c pSREBP-1
nSREBP-1 Saline 1 1 1 Luciferase 0.59 0.8 0.9 SCAP 0.38 0.4 0.4
SCAP-MM 0.8 0.9 0.8
TABLE-US-00009 TABLE 9 mRNA and protein expression of SREBP-2 in
livers of the hamsters injected with siRNA (pooled cDNA or pooled
protein from 6 hamsters) mRNA Protein siRNA SREBP-2 pSREBP-2
nSREBP-2 Saline 1 1 1 Luciferase 0.95 1 0.7 SCAP 0.5 0.2 0.5
SCAP-MM 0.81 0.9 0.6
TABLE-US-00010 TABLE 10 Cholesterol and triglyceride concentrations
in plasma and liver. (n = 6) Plasma (mg/dl) Liver (mg/g) RNAi
cholesterol triglycerides cholesterol triglycerides Saline 91 .+-.
5 166 .+-. 12 2.1 .+-. 0.1 3.6 .+-. 0.2 Luciferase 113 .+-. 8 130
.+-. 12 2.3 .+-. 0.1 3.9 .+-. 0.3 SCAP 105 .+-. 2 157 .+-. 18 2.3
.+-. 0.1 3.7 .+-. 0.1 SCAP-MM 119 .+-. 13 164 .+-. 25 2.6 .+-. 0.1
4.0 .+-. 0.3
Sequence CWU 1
1
88121DNAArtificial SequenceSynthetic primer 1gauuggcauc cugguauact
t 21221DNAArtificial SequenceSynthetic primer 2guauaccagg
augccaauct t 21321DNAArtificial SequenceSynthetic primer
3agcgccucau cauggcuggt t 21421DNAArtificial SequenceSynthetic
primer 4ccagccauga ugaggcgcut t 21521DNAArtificial
SequenceSynthetic primer 5ggccuucuac aaccaugggt t
21621DNAArtificial SequenceSynthetic primer 6cccaugguug uagaaggcct
t 21721DNAArtificial SequenceSynthetic primer 7gagguguggg
acgccauugt t 21821DNAArtificial SequenceSynthetic primer
8caauggcguc ccacaccuct t 21921DNAArtificial SequenceSynthetic
primer 9uggauuggca uccugguaut t 211021DNAArtificial
SequenceSynthetic primer 10auaccaggau gccaauccat t
211121DNAArtificial SequenceSynthetic primer 11gccauugucu
gcaacuuugt t 211221DNAArtificial SequenceSynthetic primer
12caaaguugca gacaauggct t 211321DNAArtificial SequenceSynthetic
primer 13ccaucacccu ggucuuccat t 211421DNAArtificial
SequenceSynthetic primer 14uggaagacca gggugauggt t
211521DNAArtificial SequenceSynthetic primer 15uguccuuccg
ccacuggcct t 211621DNAArtificial SequenceSynthetic primer
16ggccaguggc ggaaggacat t 211721DNAArtificial SequenceSynthetic
primer 17ccuucuacaa ccaugggcut t 211821DNAArtificial
SequenceSynthetic primer 18agcccauggu uguagaaggt t
211921DNAArtificial SequenceSynthetic primer 19gaccgcagca
caggcaucat t 212021DNAArtificial SequenceSynthetic primer
20ugaugccugu gcugcgguct t 212121DNAArtificial SequenceSynthetic
primer 21ggauuggcau ccugguauat t 212221DNAArtificial
SequenceSynthetic primer 22uauaccagga ugccaaucct t
212321DNAArtificial SequenceSynthetic primer 23aucugggacc
gcagcacagt t 212421DNAArtificial SequenceSynthetic primer
24cugugcugcg gucccagaut t 212521DNAArtificial SequenceSynthetic
primer 25ucugcaucuu agccugcugt t 212621DNAArtificial
SequenceSynthetic primer 26cagcaggcua agaugcagat t
212721DNAArtificial SequenceSynthetic primer 27agaucgacau
ggucaaguct t 212821DNAArtificial SequenceSynthetic primer
28gacuugacca ugucgaucut t 212921DNAArtificial SequenceSynthetic
primer 29caucacccug gucuuccagt t 213021DNAArtificial
SequenceSynthetic primer 30cuggaagacc agggugaugt t
213121DNAArtificial SequenceSynthetic primer 31caucuuagcc
ugcugcuact t 213221DNAArtificial SequenceSynthetic primer
32guagcagcag gcuaagaugt t 213321DNAArtificial SequenceSynthetic
primer 33ugcaucuuag ccugcugcut t 213421DNAArtificial
SequenceSynthetic primer 34agcagcaggc uaagaugcat t
213521DNAArtificial SequenceSynthetic primer 35aagaucgaca
uggucaagut t 213621DNAArtificial SequenceSynthetic primer
36acuugaccau gucgaucuut t 213721DNAArtificial SequenceSynthetic
primer 37agguguggga cgccauugat t 213821DNAArtificial
SequenceSynthetic primer 38ucaauggcgu cccacaccut t
213921DNAArtificial SequenceSynthetic primer 39cagcgccuca
ucauggcugt t 214021DNAArtificial SequenceSynthetic primer
40cagccaugau gaggcgcugt t 214121DNAArtificial SequenceSynthetic
primer 41ggaccgcagc acaggcauct t 214221DNAArtificial
SequenceSynthetic primer 42gaugccugug cugcggucct t
214321DNAArtificial SequenceSynthetic primer 43cugccauugu
cugcaacuut t 214421DNAArtificial SequenceSynthetic primer
44aaguugcaga caauggcagt t 214521DNAArtificial SequenceSynthetic
primer 45cugcaucuua gccugcugct t 214621DNAArtificial
SequenceSynthetic primer 46gcagcaggcu aagaugcagt t
214721DNAArtificial SequenceSynthetic primer 47ucuuagccug
cugcuaccct t 214821DNAArtificial SequenceSynthetic primer
48ggguagcagc aggcuaagat t 214921DNAArtificial SequenceSynthetic
primer 49ggcgacauua ccuuguacat t 215021DNAArtificial
SequenceSynthetic primer 50uguacaaggu aaugucgcct t
215121DNAArtificial SequenceSynthetic primer 51guccugucga
ucgacauuct t 215221DNAArtificial SequenceSynthetic primer
52gaaugucgau cgacaggact t 215321DNAArtificial SequenceSynthetic
primer 53cacucaaugg cggugagaut t 215421DNAArtificial
SequenceSynthetic primer 54aucucaccgc cauugagugt t
215521DNAArtificial SequenceSynthetic primer 55uccugucgau
cgacauucgt t 215621DNAArtificial SequenceSynthetic primer
56cgaaugucga ucgacaggat t 215721DNAArtificial SequenceSynthetic
primer 57gagugucugg cuagcgaugt t 215821DNAArtificial
SequenceSynthetic primer 58caucgcuagc cagacacuct t
215921DNAArtificial SequenceSynthetic primer 59cucaccugcu
uaaucgacat t 216021DNAArtificial SequenceSynthetic primer
60ugucgauuaa gcaggugagt t 216121DNAArtificial SequenceSynthetic
primer 61ggauuguagc ugcucggcut t 216221DNAArtificial
SequenceSynthetic primer 62agccgagcag cuacaaucct t
216321DNAArtificial SequenceSynthetic primer 63uuguagcugc
ucggcuuaat t 216421DNAArtificial SequenceSynthetic primer
64uuaagccgag cagcuacaat t 216521DNAArtificial SequenceSynthetic
primer 65gcuuaauggu ucccuugaut t 216621DNAArtificial
SequenceSynthetic primer 66aucaagggaa ccauuaagct t
216721DNAArtificial SequenceSynthetic primer 67acacucaaug
gcggugagat t 216821DNAArtificial SequenceSynthetic primer
68ucucaccgcc auugagugut t 216921DNAArtificial SequenceSynthetic
primer 69accucaccug cuuaaucgat t 217021DNAArtificial
SequenceSynthetic primer 70ucgauuaagc aggugaggut t
217121DNAArtificial SequenceSynthetic primer 71gaggugaagc
uucggauugt t 217221DNAArtificial SequenceSynthetic primer
72caauccgaag cuucaccuct t 217318DNAArtificial SequenceSynthetic
primer 73ggctcccagc accatgaa 187420DNAArtificial SequenceSynthetic
primer 74gccaccgatc cacacagagt 207523DNAArtificial
SequenceSynthetic primer 75gtacctgcag atgatgtcca ttg
237618DNAArtificial SequenceSynthetic primer 76ctgccatccc ggaaagtg
187718DNAArtificial SequenceSynthetic primer 77acgcagtctg ggcaacaa
187822DNAArtificial SequenceSynthetic primer 78gagctggagc
atgtcttcaa ac 227922DNAArtificial SequenceSynthetic primer
79gtcagcaatc aagtgggaga gt 228020DNAArtificial SequenceSynthetic
primer 80ctaccaccac cagggaagga 208121DNAArtificial
SequenceSynthetic primer 81aacaagcaat gccagctcac t
218220DNAArtificial SequenceSynthetic primer 82aacaggccca
agctttgttg 208320DNAArtificial SequenceSynthetic primer
83cagaatggac gggagaagca 208419DNAArtificial SequenceSynthetic
primer 84tcatttcagg gcggatgtc 198519DNAArtificial SequenceSynthetic
primer 85cctatgactg cattgggcg 198621DNAArtificial SequenceSynthetic
primer 86cccagactcc tcaaacagct g 218725DNAArtificial
SequenceSynthetic primer 87accatctgta tgatgtcaat gaaca
258824DNAArtificial SequenceSynthetic primer 88gctcaatacg
tcctcttcaa attt 24
* * * * *
References