Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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RNA INTERFERENCE MEDIATED INHIBITION OF GPRA AND AAA! GENE
EXPRESSION USING SHORT INTERFERING NUCLEIC ACTD (siNA)
This application claims the benefit of U.S, Provisional Application No.
60/570,086, filed May 11, 2004. This application is a continuation-in-part of
International Patent Application No. PCT/US04/16390, filed May 24, 2004, which
is a
continuation-in-part of U.S. Patent Application No. 10/826,966, filed April
16, 2004,
which is continuation-in-part of U.S. Patent Application No. 10/757,803, filed
January
14, 2004, which is a continuation-in-part of U.S. Patent Application No.
10/720,448,
filed November 24, 2003, which is a continuation-in-part of U.S. Patent
Application No.
10/693,059, filed October 23, 2003, which is a continuation-in-part of U.S.
Patent
Application No. 10/444,853, filed May 23, 2003, which is a continuation-in-
part of
International Patent Application No. PCT/US03/05346, filed February 20, 2003,
and a
continuation-in-part of International Patent Application No. PCT/LJS03/05028,
filed
February 20, 2003, both of which claim the benefit of U.S. Provisional
Application No.
60/358,580 filed February 20, 2002, U.S. Provisional Application No.
60/363,124 filed
March 11, 2002, U.S. Provisional Application No. 60/386,782 filed June 6,
2002, U.S.
Provisional Application No. 60/406,784 filed August 29, 2002, U.S. Provisional
Application No. 60/408,378 filed September 5, 2002, U.S. Provisional
Application No.
601409,293 filed September 9, 2002, and ~U.S. Provisional Application No.
60/440,129
filed January 15, 2003. This application is also a continuation-in-part of
International
Patent Application No. PCT/US04/13456, filed April 30, 2004, which is a
continuation-
in-part of U.S. Patent Application No. 10/780,447, filed February 13, 2004,
which is a
continuation-in-part of U.S. Patent Application No. 10/427,160, filed April
30, 2003,
which is a continuation-in-part of International Patent Application No.
PCT/US02/15876
filed May 17, 2002, which claims the benefit of U.S. Provisional Application
No.
60/292,217, filed May I8, 2001, U.S. Provisional Application No. 60/362,016,
filed
March 6, 2002, U.S. Provisional Application No. 60/306,883, filed July 20,
2001, and
U.S. Provisional Application No. 60/311,865, filed August 13, 2001. This
application is
also a continuation-in-part of U.S. Patent Application No. 10/727,780 filed
December 3,
2003. This application also claims the benefit of U.S. Provisional Application
No.
60/543,480, filed February 10, 2004. The instant application claims the
benefit of all the
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listed applications, which are hereby incorporated by reference herein in
their entireties,
including the drawings.
Field Of The Invention
The present invention relates to compounds, compositions, and methods for the
study, diagnosis, and treatment of traits, diseases and conditions that
respond to the
modulation of G protein--coupled receptor for asthma susceptibility (GPRA) and
asthma-
associated alternatively spliced gene 1 (AAA1) gene expression and/or
activity. The
present invention is also directed to compounds, compositions, and methods
relating to
traits, diseases and conditions that xespond to the modulation of expression
and/or
activity of genes involved in GPRA and/or AAA1 gene expression pathways or
other
cellular processes that mediate the maintenance or development of such traits,
diseases
and conditions. Specifically, the invention relates to small nucleic acid
molecules, such
as short interfering nucleic acid (siNA), short interfering RNA (siRNA),
double-stranded
RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules
capable of mediating RNA interference (RNAi) against GPRA and/or AAA1 gene
expression. Such small nucleic acid molecules are useful, for example, in
providing
compositions for treatment of traits, diseases and conditions that can respond
to
modulation of GPRA andlor AAA1 expression in a subject, such as respiratory
and/or
inflammatory diseases, disorders, or conditions.
Background Of The Invention
The following is a discussion of relevant art pertaining to RNAi. The
discussion is
provided only for understanding of the invention that follows. The summary is
not an
admission that any of the work described below is prior art to the claimed
invention.
RNA interference refers to the process of sequence-specific post-
transcriptional
gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore
et al.,
2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al.,
1999,
Scieface, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999,
Genes &
Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). The corresponding
process in
plants (Heifetz et al., International PCT Publication No. WO 99/61631) is
commonly
referred to as post-transcriptional gene silencing or RNA silencing and is
also referred to
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as quelling in fungi. The process of post-transcriptional gene silencing is
thought to be
an evolutionarily-conserved cellular defense mechanism used to prevent the
expression
of foreign genes and is commonly shared by diverse flora and phyla (Fire et
al., 1999,
Trends Genet., 15, 358). Such protection from foreign gene expression may have
evolved in response to the production of double-stranded RNAs (dsRNAs) derived
from
viral infection or from the random integration of transposon elements into a
host genome
via a cellular response that specifically destroys homologous single-stranded
RNA or
viral genomic RNA. The presence of dsRNA in cells triggers the RNAi response
through a mechanism that has yet to be fully characterized. This mechanism
appears to
be different from other known mechanisms involving double stranded RNA-
specific
ribonucleases, such as the interferon response that results from dsRNA-
mediated
activation of protein kinase PIER and 2',5'-oligoadenylate synthetase
resulting in non-
specific cleavage of mRNA by ribonuclease L (see for example US Patent Nos.
6,107,094; 5,898,031; Clemens et al., 1997, J. Intefferon & Cytokine Res., 17,
503-524;
Adah et al., 2001, Cun~. Med. Cl2em., 8, 1189).
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease
III
enzyme referred to as dicer (Bass, 2000, Cell, 101, 235; Zamore et al., 2000,
Cell, 101,
25-33; Hammond et al., 2000, Nature, 404, 293). Dicer is involved in the
processing of
the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs)
(Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein
et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from dicer activity are
typically
about 21 to about 23 nucleotides in length and comprise about 19 base pair
duplexes
(Zamore et al., 2000, Cell, 101, 25-33; Elbashir et al., 2001, Genes Dev., 15,
188). Dicex
has also been implicated in the excision of 21- and 22-nucleotide small
temporal RNAs
(stRNAs) from precursor RNA of conserved structure that are implicated in
translational
control (Hutvagner et al., 2001, Science, 293, 834). The RNAi response also
features an
endonuclease complex, commonly referred to as an RNA-induced silencing complex
(RISC), which mediates cleavage of single-stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage of the
target RNA
takes place in the middle of the region complementary to the antisense strand
of the
siRNA duplex (Elbashir et al., 2001, Genes Dev., 15, 188).
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RNAi has been studied in a vaxiety of systems. Fire et al., 1998, Nature, 391,
806,
were the first to observe RNAi in C elegaras. Bahramian and Zarbl, 1999,
Molecular
and Cellular Biology, 19, 274-283 and Wianny and Goetz, 1999, Nature Cell
Biol., 2,
70, describe RNAi mediated by dsRNA in mammalian systems. Hammond et al.,
2000,
Nature, 404, 293, describe RNAi in Drosoplaila cells transfected with dsRNA.
Elbashir
et al., 2001, Nature, 41I, 494 and Tuschl et al., International PCT
Publication No. WO
01/75164, describe RNAi induced by introduction of duplexes of synthetic 21-
nucleotide
RNAs in cultured mammalian cells including human embryonic kidney and HeLa
cells.
Recent work in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO .L,
20, 6877
and Tuschl et al., International PCT Publication No. WO 01/75164) has revealed
certain
requirements for siRNA length, structure, chemical composition, arid sequence
that are
essential to mediate efficient RNAi activity. These studies have shown that 21-
nucleotide siRNA duplexes axe most active when containing 3'-terminal
dinucleotide
overhangs. Furthermore, complete substitution of one or both siRNA strands
with 2'-
deoxy (2'-H) or 2'-O-methyl nucleotides abolishes RNAi activity, whereas
substitution of
the 3'-terminal siRNA overhang nucleotides with 2'-deoxy nucleotides (2'-H)
was shown
to be tolerated. Single mismatch sequences in the center of the siRNA duplex
were also
shown to abolish RNAi activity. In addition, these studies also indicate that
the position
of the cleavage site in the target RNA is defined by the 5'-end of the siRNA
guide
sequence rather than the 3'-end of the guide sequence (Elbashir et al., 2001,
EMBO J.,
20, 6877). Other studies have indicated that a 5'-phosphate on the target-
complementary
strand of a siRNA duplex is required for siRNA activity and that ATP is
utilized to
maintain the 5'-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell,
107, 309).
Studies have shown that replacing the 3'-terminal nucleotide overhanging
segments
of a 21-mer siRNA duplex having two-nucleotide 3'-overhangs with
deoxyribonucleotides does not have an adverse effect on RNAi activity.
Replacing up to
four nucleotides on each end of the siRNA with deoxyribonucleotides has been
reported
to be well tolerated, whereas complete substitution with deoxyribonucleotides
results in
no RNAi activity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,
International PCT Publication No. WO 01/75164). In addition, Elbashir et al.,
supra,
also report that substitution of siRNA with 2'-O-methyl nucleotides completely
abolishes
RNAi activity. Li et al., International PCT Publication No. WO 00/44914, and
Beach et
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al., International PCT Publication No. WO 01/68836 preliminarily suggest that
siRNA
may include modifications to either the phosphate-sugar backbone or the
nucleoside to
include at least one of a nitrogen or sulfur heteroatom, however, neither
application
postulates to what extent such modifications would be tolerated in siRNA
molecules, nor
provides any further guidance or examples of such modified siRNA. Kreutzer et
al.,
Canadian Patent Application No. 2,359,180, also describe certain chemical
modifications
for use in dsRNA constructs in order to counteract activation of double-
stranded RNA-
dependent protein kinase PKR, specifically 2'-amino or 2'-O-methyl
nucleotides, and
nucleotides containing a 2'-O ox 4'-C methylene bridge. However, Kreutzer et
al.
similarly fails to provide examples or guidance as to what extent these
modifications
would be tolerated in dsRNA molecules.
Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certain chemical
modifications targeting the unc-22 gene in C. elegaras using long (>25 nt)
siRNA
transcripts. The authors describe the introduction of thiophosphate residues
into these
siRNA transcripts by incorporating thiophosphate nucleotide analogs with T7
and T3
RNA polymerase and observed that RNAs with two phosphorothioate modified bases
also had substantial decreases in effectiveness as RNAi. Further, Parrish et
al. reported
that phosphorothioate modification of more than two residues greatly
destabilized the
RNAs ifi vitro such that interference activities could not be assayed. Id. at
1081. The
authors also tested certain modifications at the 2'-position of the nucleotide
sugar in the
long siRNA transcripts and found that substituting deoxynucleotides for
ribonucleotides
produced a substantial decrease in interference activity, especially in the
case of Uridine
to Thymidine and/or Cytidine to deoxy-Cytidine substitutions. Id. In addition,
the
authors tested certain base modifications, including substituting, in sense
and antisense
strands of the siRNA, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 3-
(aminoallyl)uracil
for uracil, and inosine for guanosine. Whereas 4-thiouracil and 5-bromouracil
substitution appeared to be tolerated, Parrish reported that inosine produced
a substantial
decrease in interference activity when incorporated in either strand. Parrish
also reported
that incorporation of 5-iodouracil and 3-(aminoallyl)uracil in the antisense
strand
resulted in a substantial decrease in RNAi activity as well.
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The use of longer dsRNA has been described. For example, Beach et al.,
International PCT Publication No. WO 01/68836, describes specific methods for
attenuating gene expression using endogenously-derived dsRNA. Tuschl et al.,
International PCT Publication No. WO 01/75164, describe a Df~osoplaila ira
vitYO RNAi
system and the use of specific siRNA molecules for certain functional genomic
and
certain therapeutic applications; although Tuschl, 2001, Chem. Biochena., 2,
239-245,
doubts that RNAi can be used to cure genetic diseases or viral infection due
to the danger
of activating interferon response. Li et al., International PCT Publication
No. WO
00/44914, describe the use of specific long (141 bp-488 bp) enzymatically
synthesized or
vector expressed dsRNAs for attenuating the expression of certain target
genes.
Zernicka-Goetz et al., International PCT Publication No. WO 01/36646, describe
certain
methods for inhibiting the expression of particular genes in mammalian cells
using
certain long (550 bp-714 bp), enzymatically synthesized or vector expressed
dsRNA
molecules. Fire et al., International PCT Publication No. WO 99/32619,
describe
particular methods for introducing certain long dsRNA molecules into cells for
use in
inhibiting gene expression in nematodes. Plaetinck et al., International PCT
Publication
No. WO 00/01846, describe certain methods for identifying specific genes
responsible
for conferring a particular phenotype in a cell using specific long dsRNA
molecules.
Mello et al., International PCT Publication No. WO 01129058, describe the
identification
of specific genes involved in dsRNA-mediated RNAi. Pachuck et al.,
International PCT
Publication No. WO 00/63364, describe certain long (at least 200 nucleotide)
dsRNA
constructs. Deschamps Depaillette et al., International PCT Publication No. WO
99/07409, describe specific compositions consisting of particular dsRNA
molecules
combined with certain anti-viral agents. Waterhouse et al., International PCT
Publication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describe certain
methods
for decreasing the phenotypic expression of a nucleic acid in plant cells
using certain
dsRNAs. Driscoll et al., International PCT Publication No. WO 01/49844,
describe
specific DNA expression constructs for use in facilitating gene silencing in
targeted
organisms.
Others have reported on various RNAi and gene-silencing systems. For example,
Parnsh et al., 2000, Molecular Cell, 6, 1077-1087, describe specific
chemically-modified
dsRNA constructs targeting the unc-22 gene of C. elegafzs. Grossniklaus,
International
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PCT Publication No. WO 01/38551, describes certain methods for regulating
polycomb
gene expression in plants using certain dsRNAs. Churikov et al., International
PCT
Publication No. WO 01/42443, describe certain methods for modifying genetic
characteristics of an organism using certain dsRNAs. Cogoni et al"
International PCT
Publication No. WO 01/53475, describe certain methods for isolating a
Neurospora
silencing gene and uses thereof. Reed et al., International PCT Publication
No. WO
01/68836, describe certain methods for gene silencing in plants. Honer et al.,
International PCT Publication No. WO 01/70944, describe certain methods of
drug
screening using transgenic nematodes as Parkinson's Disease models using
certain
dsRNAs. Deak et al., International PCT Publication No. WO 01/72774, describe
certain
Drosoplaila-derived gene products that may be related to RNAi in Drosoplaila.
Arndt et
al., International PCT Publication No. WO 01/92513 describe certain methods
for
mediating gene suppression by using factors that enhance RNAi. Tuschl et al.,
International PCT Publication No. WO 02/44321, describe certain synthetic
siRNA
constructs. Pachuk et al., International PCT Publication No. WO 00/63364, and
Satishchandran et al., International PCT Publication No. WO 01/04313, describe
certain
methods and compositions for inhibiting the function of certain polynucleotide
sequences using certain long (over 250 bp), vector expressed dsRNAs. Echevern
et al.,
International PCT Publication No. WO 02/38805, describe certain C. elegayas
genes
identified via RNAi. Kreutzer et al., International PCT Publications Nos. WO
02/055692, WO 02/055693, and EP 1144623 B1 describes certain methods for
inhibiting
gene expression using dsRNA. Graham et al., International PCT Publications
Nos. WO
99/49029 and WO 01/70949, and AU 4037501 describe certain vector expressed
siRNA
molecules. Fire et al., US 6,506,559, describe certain methods for inhibiting
gene
expression in vitro using certain long dsRNA (299 bp-1033 bp) constructs that
mediate
RNAi. Martinez et al., 2002, Cell, 110, 563-574, describe certain single
stranded siRNA
constructs, including certain 5'-phosphorylated single stranded siRNAs that
mediate
RNA interference in Hela cells. Harborth et al., 2003, Antisense & Nucleic
Acid Drug
Development, 13, 83-105, describe certain chemically and structurally modified
siRNA
molecules. Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically
and
structurally modified siRNA molecules. Woolf et al., International PCT
Publication
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Nos. WO 03/064626 and WO 03/064625 describe certain chemically modified dsRNA
constructs.
SUMMARY OF THE INVENTION
This invention relates to compounds, compositions, and methods useful for
modulating G protein-coupled receptor for asthma susceptibility (GPRA) and/or
asthma-
associated alternatively spliced gene 1 (AAAl) gene expression using short
interfering
nucleic acid (siNA) molecules. This invention also relates to compounds,
compositions,
and methods useful for modulating the expression and activity of other genes
involved in
pathways of GPRA and/or AAAI gene expression and/or activity by RNA
interference
(RNAi) using small nucleic acid molecules. In particular, the instant
invention features
small nucleic acid molecules, such as short interfering nucleic acid (siNA),
short
interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and
short hairpin RNA (shRNA) molecules and methods used to modulate the
expression of
GPRA and/or AAAl genes.
A siNA of the invention can be unmodified or chemically-modified. A siNA of
the
instant invention can be chemically synthesized, expressed from a vector or
enzymatically synthesized. The instant invention also features various
chemically-
modified synthetic short interfering nucleic acid (siNA) molecules capable of
modulating
GPRA and/or AAA1 gene expression or activity in cells by RNA interference
(RNAi).
The use of chemically-modified siNA improves various properties of native siNA
molecules through increased resistance to nuclease degradation in vivo and/or
through
improved cellular uptake. Further, contrary to earlier published studies, siNA
having
multiple chemical modifications retains its RNAi activity. The siNA molecules
of the
instant invention provide useful reagents and methods for a variety of
therapeutic,
veterinary, diagnostic, target validation, genomic discovery, genetic
engineering, and
pharmacogenomic applications.
In one embodiment, the invention features one or more siNA molecules and
methods that independently or in combination modulate the expression of GPRA
and/or
AAA1 genes encoding proteins, such as proteins comprising GPRA andlor AAA1
associated with the maintenance and/or development of inflammatory and/or
respiratory
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diseases, traits, conditions and disorders, such as genes encoding sequences
comprising
those sequences referred to by GenBank Accession Nos. shown in Table I,
referred to
herein generally as GPRA and/or AAA1. The description below of the various
aspects
and embodiments of the invention is provided with reference to exemplary GPR.A
andlor
AAA1 gene. However, the various aspects and embodiments are also directed to
other
GPRA and/or AAA1 genes, such as homolog genes and transcript variants, and
polymorphisms (e.g., single nucleotide polymorphism, (SNPs)) associated with
certain
GPRA and/or AAA1 genes. As such, the various aspects and embodiments are also
directed to other genes that are involved in GPRA andlox AAAl mediated
pathways of
signal transduction or gene expression that are involved, for example, in the
the
maintenence or development of diseases, traits, or conditions described
herein. These
additional genes can be analyzed fox target sites using the methods described
for GPRA
and/or AAA1 genes herein. Thus, the modulation of other genes and the effects
of such
modulation of the other genes can be performed, determined, and measuxed as
described
herein.
In one embodiment, the invention featuxes a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA aneUor
AAAl
gene, wherein said siNA molecule comprises about 15 to about 28 base pairs.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that directs cleavage of a GPRA and/or AAAl RNA
via
RNA interfexence (RNAi), wherein the double stranded siNA molecule comprises a
first
and a second strand, each strand of the siNA molecule is about 18 to about 28
nucleotides in length, the first strand of the siNA molecule comprises
nucleotide
sequence having sufficient complementarity to the GPRA andlor AAA1 RNA for the
siNA molecule to direct cleavage of the GPRA and/or AAA1 RNA via RNA
interference, and the second strand of said siNA molecule comprises nucleotide
sequence
that is complementary to the first strand.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that directs cleavage of a GPRA and/or AAAI RNA
via
RNA interference (RNAi), wherein the double stxanded siNA molecule comprises a
first
and a second strand, each strand of the siNA molecule is about 18 to about 23
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nucleotides in length, the first strand of the siNA molecule comprises
nucleotide
sequence having sufficient complementarity to the GPRA and/or AAA1 RNA for the
siNA molecule to direct cleavage of the GPRA and/or AAA1 RNA via RNA
interference, and the second strand of said siNA molecule comprises nucleotide
sequence
that is complementary to the first strand.
In one embodiment, the invention features a chemically synthesized double
stranded short interfering nucleic acid (siNA) molecule that directs cleavage
of a GPRA
and/or AAA1 RNA via RNA interference (RNAi), wherein each strand of the siNA
molecule is about 18 to about 28 nucleotides in length; and one strand of the
siNA
molecule comprises nucleotide sequence having sufficient complementarity to
the GPRA
and/or AAA1 RNA for the siNA molecule to direct cleavage of the GPRA and/or
AAA1
RNA via RNA interference.
In one embodiment, the invention features a chemically synthesized double
stranded short interfering nucleic acid (siNA) molecule that directs cleavage
of a GPRA
and/or AAA1 RNA via RNA interference (RNAi), wherein each strand of the siNA
molecule is about 18 to about 23 nucleotides in length; and one strand of the
siNA
molecule comprises nucleotide sequence having sufficient complementarity to
the GPRA
and/or AA.A1 RNA for the siNA molecule to direct cleavage of the GPRA and/or
AAA1
RNA via RNA interference.
In one embodiment, the invention features a siNA molecule that down-regulates
expression of a GPRA and/or AAA1 gene, for example, wherein the GPRA and/or
AAA1 gene comprises GPRA and/or AAA1 encoding sequence. In one embodiment,
the invention features a siNA molecule that down-regulates expression of a
GPRA
and/or AAAl gene, for example, wherein the GPRA and/or AAA1 gene comprises
GPRA and/or AAAl non-coding sequence or regulatory elements involved in GPRA
and/or AAA1 gene expression.
In one embodiment, a siNA of the invention is used to inhibit the expression
of
GPRA and/or AAAl genes or a GPRA and/or AAA1 gene family (e.g., GPRA and/or
AAAI superfamily genes), wherein the genes or gene family sequences share
sequence
homology. Such homologous sequences can be identified as is known in the art,
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example using sequence alignments. siNA molecules can be designed to target
such
homologous sequences, for example using perfectly complementary sequences or
by
incorporating non-canonical base pairs, for example mismatches and/or wobble
base
pairs, that can provide additional target sequences. In instances where
mismatches are
identified, non-canonical base pairs (for example, mismatches and/or wobble
bases) can
be used to generate siNA molecules that target more than one gene sequence. In
a non-
limiting example, non-canonical base pairs such as LTLT and CC base pairs are
used to
generate siNA molecules that are capable of targeting sequences for differing
GPRA
and/or AAA1 targets that share sequence homology. As such, one advantage of
using
siNAs of the invention is that a single siNA can be designed to include
nucleic acid
sequence that is complementary to the nucleotide sequence that is conserved
between the
homologous genes. In this approach, a single siNA can be used to inhibit
expression of
more than one gene instead of using more than one siNA molecule to target the
different
genes.
In one embodiment, the invention features a siNA molecule having RNAi activity
against GPRA and/or AAA1 RNA, wherein the siNA molecule comprises a sequence
complementary to any RNA having GPRA and/or AAA1 encoding sequence, such as
those sequences having GenBank Accession Nos. shown in Table I. In another
embodiment, the invention features a siNA molecule having RNAi activity
against
GPRA and/or AAA1 RNA, wherein the siNA molecule comprises a sequence
complementary to an RNA having variant GPR.A and/or AAA1 encoding sequence,
for
example other mutant GPRA and/or AAA1 genes not shown in Table I but known in
the
art to be associated with the maintenance and/or development of inflammatory
and/or
respiratory diseases, disorders, and/or conditions. Chemical modifications as
shown in
Tables III and IV or otherwise described herein can be applied to any siNA
construct of
the invention. In another embodiment, a siNA molecule of the invention
includes a
nucleotide sequence that can interact with nucleotide sequence of a GPRA
and/or AAAl
gene and thereby mediate silencing of GPRA and/or AAA1 gene expression, for
example, wherein the siNA mediates regulation of GPRA and/or AAA1 gene
expression
by cellular processes that modulate the chromatin structure or methylation
patterns of the
GPRA and/or AAAI gene and prevent transcription of the GPRA and/or AAA1 gene.
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In one embodiment, siNA molecules of the invention are used to down regulate
or
inhibit the expression of GPRA and/or AAAl proteins arising from GPRA and/or
AAA1
haplotype polymorphisms that are associated with a disease or condition,
(e.g.,
inflammatory and/or respiratory diseases, disorders, andlor conditions).
Analysis of
GPRA and/or AAA1 genes, or GPRA andlor AAAI protein or RNA levels can be used
to identify subjects with such polyrnorphisms or those subjects who are at
risk of
developing traits, conditions, or diseases described herein. These subjects
are amenable
to treatment, for example, treatment with siNA molecules of the invention and
any other
composition useful in treating diseases related to GPRA and/or AAAl gene
expression.
As such, analysis of GPRA and/or AAA1 protein or RNA levels can be used to
determine treatment type and the course of therapy in treating a subject.
Monitoring of
GPRA and/or AAA1 protein or RNA levels can be used to predict treatment
outcome
and to determine the efficacy of compounds and compositions that modulate the
level
and/or activity of certain GPRA and/or AAAl proteins associated with a trait,
condition,
or disease.
In one embodiment of the invention a siNA molecule comprises an antisense
strand comprising a nucleotide sequence that is complementary to a nucleotide
sequence
or a portion thereof encoding a GPRA and/or AAA1 protein. The siNA further
comprises a sense strand, wherein said sense strand comprises a nucleotide
sequence of a
GPRA and/or AAA1 gene or a portion thereof.
In another embodiment, a siNA molecule comprises an antisense region
comprising a nucleotide sequence that is complementary to a nucleotide
sequence
encoding a GPRA and/or AAAl protein or a portion thereof. The siNA molecule
further
comprises a sense region, wherein said sense region comprises a nucleotide
sequence of
a GPRA and/or AAA1 gene or a portion thereof.
In another embodiment, the invention features a siNA molecule comprising a
nucleotide sequence in the antisense region of the siNA molecule that is
complementary
to a nucleotide sequence or portion of sequence of a GPRA and/or AAA1 gene. In
another embodiment, the invention features a siNA molecule comprising a
region, for
example, the antisense region of the siNA construct, complementary to a
sequence
comprising a GPRA and/or AAA1 gene sequence or a portion thereof.
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In one embodiment, the antisense region of GPRA siNA constructs comprises a
sequence complementary to sequence having any of SEQ ID NOs. 1-87, 175-188,
581-
588, 597-604, 613-620, 629-636, 645-652, 661-668, 789, 791, 793, 795, 796,
798, 800,
802, 804, or 805. In one embodiment, the antisense region of GPR.A and/or AAA1
constructs comprises sequence having any of SEQ ID NOs. 88-174, 189-202, 605-
612,
621-628, 637-644, 653-660, 669-692, 790, 792, 794, 797, 799, 801, 803, or 806.
In
another embodiment, the sense region of GPRA constructs comprises sequence
having
any of SEQ ID NOs. 1-87, 175-188, 581-588, 597-604, 613-620, 629-636, 645-652,
661-
668, 789, 791, 793, 795, 796, 798, 800, 802, 804, or 805.
In one embodiment, a siNA molecule of the invention comprises any of SEQ ID
NOs. 1-806. The sequences shown in SEQ ID NOs. 1-806 are not limiting. A siNA
molecule of the invention can comprise any contiguous GPR.A and/or AAA1
sequence
(e.g., about 15 to about 25 or more, or about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25
or more contiguous GPRA and/or AAA1 nucleotides).
In yet another embodiment, the invention features a siNA molecule comprising a
sequence, for example, the antisense sequence of the siNA construct,
complementary to a
sequence or portion of sequence comprising sequence represented by GenBank
Accession Nos. shown in Table I. Chemical modifications in Tables III and IV
and
described herein can be applied to any siNA construct of the invention.
In one embodiment of the invention a siNA molecule comprises an antisense
strand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25,
26, 27, 28, 29, or 30) nucleotides, wherein the antisense strand is
complementary to a
RNA sequence or a portion thereof encoding a GPR.A and/or AAA1 protein, and
wherein
said siNA further comprises a sense strand having about 15 to about 30 (e.g.,
about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,
and wherein said
sense strand and said antisense strand are distinct nucleotide sequences where
at least
about 15 nucleotides in each strand are complementary to the other strand.
In another embodiment of the invention a siNA molecule of the invention
comprises an antisense region having about 15 to about 30 (e.g., about 15, 16,
17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the
antisense region is
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complementary to a RNA sequence encoding a GPRA and/or AAA1 protein, and
wherein said siNA further comprises a sense region having about 15 to about 30
(e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides,
wherein said sense region and said antisense region are comprised in a linear
molecule
where the sense region comprises at least about 15 nucleotides that are
complementary to
the antisense region.
In one embodiment, a siNA molecule of the invention has RNAi activity that
modulates expression of RNA encoded by a GPRA and/or AAAl gene. Because GPRA
and/or AAAl (e.g., GPRA and/or AAA1 superfamily) genes can share some degree
of
sequence homology with each other, siNA molecules can be designed to target a
class of
GPRA and/or AAA1 genes or alternately specific GPRA and/or AAAI genes (e.g.,
polymorphic variants) by selecting sequences that are either shared amongst
different
GPRA and/or AAA1 targets or alternatively that are unique for a specific GPR.A
and/or
AAA1 target. Therefore, in one embodiment, the siNA molecule can be designed
to
target conserved regions of GPRA and/or AAA1 RNA sequences having homology
among several GPRA and/or AAAl gene variants so as to target a class of GPRA
and/or
AAA1 genes with one siNA molecule. Accordingly, in one embodiment, the siNA
molecule of the invention modulates the expression of one or both GPRA and/or
AAA1
alleles in a subject. In another embodiment, the siNA molecule can be designed
to target
a sequence that is unique to a specific GPRA and/or AAA1 RNA sequence (e.g., a
single
GPRA and/or AAAl allele or GPRA and/or AAAl single nucleotide polymorphism
(SNP)) due to the high degree of specificity that the siNA molecule requires
to mediate
RNAi activity.
In one embodiment, nucleic acid molecules of the invention that act as
mediators
of the RNA interference gene silencing response are double-stranded nucleic
acid
molecules. In another embodiment, the siNA molecules of the invention consist
of
duplex nucleic acid molecules containing about 15 to about 30 base pairs
between
oligonucleotides comprising about 15 to about 30 (e.g., about 15, 16, 17, 18,
19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet another embodiment,
siNA
molecules of the invention comprise duplex nucleic acid molecules with
overhanging
ends of about 1 to about 3 (e.g., about 1, 2, or 3) nucleotides, for example,
about 21-
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nucleotide duplexes with about 19 base pairs and 3'-terminal mononucleotide,
dinucleotide, or trinucleotide overhangs. In yet another embodiment, siNA
molecules of
the invention comprise duplex nucleic acid molecules with blunt ends, where
both ends
are blunt, or alternatively, where one of the ends is blunt.
In one embodiment, the invention features one or more chemically-modified siNA
constructs having specificity for GPRA and/or AAA1 expressing nucleic acid
molecules,
such as RNA encoding a GPRA and/or AAA1 protein. In one embodiment, the
invention features a RNA based siNA molecule (e.g., a siNA comprising 2'-OH
nucleotides) having specificity for GPRA and/or AAA1 expressing nucleic acid
molecules that includes one or more chemical modifications described herein.
Non-
limiting examples of such chemical modifications include without limitation
phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-
methyl
ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, "universal base"
nucleotides,
"acyclic" nucleotides, 5-C-methyl nucleotides, and terminal glyceryl and/or
inverted
deoxy abasic residue incorporation. These chemical modifications, when used in
various
siNA constructs, (e.g., RNA based siNA constructs), are shown to preserve RNAi
activity in cells while at the same time, dramatically increasing the serum
stability of
these compounds. Furthermore, contrary to the data published by Parrish et
al., supra,
applicant demonstrates that multiple (greater than one) phosphorothioate
substitutions
are well-tolerated and confer substantial increases in serum stability for
modified siNA
constructs.
In one embodiment, a siNA molecule of the invention comprises modified
nucleotides while maintaining the ability to mediate RNAi. The modif"ied
nucleotides
can be used to improve in vitf~o or in vivo characteristics such as stability,
activity, and/or
bioavailability. For example, a siNA molecule of the invention can comprise
modif"ied
nucleotides as a percentage of the total number of nucleotides present in the
siNA
molecule. As such, a siNA molecule of the invention can generally comprise
about 5%
to about 100% modified nucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%,
35%,
40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified
nucleotides). The actual percentage of modified nucleotides present in a given
siNA
molecule will depend on the total number of nucleotides present in the siNA.
If the siNA
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molecule is single stranded, the percent modification can be based upon the
total number
of nucleotides present in the single stranded siNA molecules. Likewise, if the
siNA
molecule is double stranded, the percent modification can be based upon the
total
number of nucleotides present in the sense strand, antisense strand, or both
the sense and
antisense strands.
One aspect of the invention features a double-stranded short interfering
nucleic
acid (siNA) molecule that down-regulates expression of a GPRA and/or AAA1
gene. In
one embodiment, the double stranded siNA molecule comprises one or more
chemical
modifications and each strand of the double-stranded siNA is about 21
nucleotides long.
In one embodiment, the double-stranded siNA molecule does not contain any
ribonucleotides. In another embodiment, the double-stranded siNA molecule
comprises
one or more ribonucleotides. In one embodiment, each strand of the double-
stranded
siNA molecule independently comprises about 15 to about 30 (e.g., about 15,
16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein each
strand
comprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26,
27, 28, 29, or 30) nucleotides that are complementary to the nucleotides of
the other
strand. In one embodiment, one of the strands of the double-stranded siNA
molecule
comprises a nucleotide sequence that is complementary to a nucleotide sequence
or a
portion thereof of the GPRA and/or AAA1 gene, and the second strand of the
double-
stranded siNA molecule comprises a nucleotide sequence substantially similar
to the
nucleotide sequence of the GPR.A and/or AAA1 gene or a portion thereof.
In another embodiment, the invention features a double-stranded short
interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAAl
gene comprising an antisense region, wherein the antisense region comprises a
nucleotide sequence that is complementary to a nucleotide sequence of the
GPR.A andlor
AAAl gene or a portion thereof, and a sense region, wherein the sense region
comprises
a nucleotide sequence substantially similar to the nucleotide sequence of the
GPRA
and/or AAA1 gene or a portion thereof. In one embodiment, the antisense region
and the
sense region independently comprise about 15 to about 30 (e.g. about 15, 16,
17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein the
antisense region
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comprises about 15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26,
27, 28, 29, or 30) nucleotides that are complementary to nucleotides of the
sense region.
In another embodiment, the invention features a double-stranded short
interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAA1
gene comprising a sense region and an antisense region, wherein the antisense
region
comprises a nucleotide sequence that is complementary to a nucleotide sequence
of RNA
encoded by the GPRA and/or AAA1 gene or a portion thereof and the sense region
comprises a nucleotide sequence that is complementary to the antisense region.
In one embodiment, a siNA molecule of the invention comprises blunt ends,
i.e.,
ends that do not include any overhanging nucleotides. For example, a siNA
molecule
comprising modifications described herein (e.g., comprising nucleotides having
Formulae I-VII or siNA constructs comprising "Stab 00"-"Stab 32" (Table I~ or
any
combination thereof (see Table Ice) and/or any length described herein can
comprise
blunt ends or ends with no overhanging nucleotides.
In one embodiment, any siNA molecule of the invention can comprise one or more
blunt ends, i.e. where a blunt end does not have any overhanging nucleotides.
In one
embodiment, the blunt ended siNA molecule has a number of base pairs equal to
the
number of nucleotides present in each strand of the siNA molecule. In another
embodiment, the siNA molecule comprises one blunt end, for example wherein the
5'-
end of the antisense strand and the 3'-end of the sense strand do not have any
overhanging nucleotides. In another example, the siNA molecule comprises one
blunt
end, for example wherein the 3'-end of the antisense strand and the 5'-end of
the sense
strand do not have any overhanging nucleotides. In another example, a siNA
molecule
comprises two blunt ends, for example wherein the 3'-end of the antisense
strand and the
5'-end of the sense strand as well as the 5'-end of the antisense strand and
3'-end of the
sense strand do not have any overhanging nucleotides. A blunt ended siNA
molecule
can comprise, for example, from about 15 to about 30 nucleotides (e.g., about
15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides). Other
nucleotides
present in a blunt ended siNA molecule can comprise, for example, mismatches,
bulges,
loops, or wobble base pairs to modulate the activity of the siNA molecule to
mediate
RNA interference.
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By "blunt ends" is meant symmetric termini or termini of a double stranded
siNA
molecule having no overhanging nucleotides. The two strands of a double
stranded
siNA molecule align with each other without over-hanging nucleotides at the
termini.
For example, a blunt ended siNA construct comprises terminal nucleotides that
are
complementary between the sense and antisense regions of the siNA molecule.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPR.A and/or
AAAl
gene, wherein the siNA molecule is assembled from two separate oligonucleotide
fragments wherein one fragment comprises the sense region and the second
fragment
comprises the antisense region of the siNA molecule. The sense region can be
connected
to the antisense region via a linker molecule, such as a polynucleotide linker
or a non-
nucleotide linker.
In one embodiment, the invention features double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAA1
gene, wherein the siNA molecule comprises about 15 to about 30 (e.g. about 15,
16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein
each strand
of the siNA molecule comprises one or more chemical modifications. In another
embodiment, one of the strands of the double-stranded siNA molecule comprises
a
nucleotide sequence that is complementary to a nucleotide sequence of a GPRA
and/or
AAA1 gene or a portion thereof, and the second strand of the double-stranded
siNA
molecule comprises a nucleotide sequence substantially similar to the
nucleotide
sequence or a portion thereof of the GPRA and/or AAA1 gene. In another
embodiment,
one of the strands of the double-stranded siNA molecule comprises a nucleotide
sequence that is complementary to a nucleotide sequence of a GPRA and/or AAA1
gene
or portion thereof, and the second strand of the double-stranded siNA molecule
comprises a nucleotide sequence substantially similar to the nucleotide
sequence or
portion thereof of the GPRA and/or AAAl gene. In another embodiment, each
strand of
the siNA molecule comprises about 15 to about 30 (e.g. about 15, 16, 17, 18,
19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strand comprises
at least about
15 to about 30 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30)
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nucleotides that are complementary to the nucleotides of the other strand.
The. GPRA
and/or AAA1 gene can comprise, for example, sequences referred to in Table I.
In one embodiment, a siNA molecule of the invention comprises no
ribonucleotides. In another embodiment, a siNA molecule of the invention
comprises
~ ribonucleotides.
In one embodiment, a siNA molecule of the invention comprises an antisense
region comprising a nucleotide sequence that is complementary to a nucleotide
sequence
of a GPRA and/or AAAl gene or a portion thereof, and the siNA further
comprises a
sense region comprising a nucleotide sequence substantially similar to the
nucleotide
sequence of the GPRA and/or AAA1 gene or a portion thereof. In another
embodiment,
the antisense region and the sense region each comprise about 15 to about 30
(e.g. about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
and the
antisense region comprises at least about 15 to about 30 (e.g. about 15, 16,
17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary
to
nucleotides of the sense region. The GPRA and/or AAA1 gene can comprise, for
example, sequences referred to in Table I. In another embodiment, the siNA is
a double
stranded nucleic acid molecule, where each of the two strands of the siNA
molecule
independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19,
20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40)
nucleotides, and
where one of the strands of the siNA molecule comprises at least about 15
(e.g. about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more) nucleotides that are
complementary to
the nucleic acid sequence of the GPRA and/or AAA1 gene or a portion thereof.
In one embodiment, a siNA molecule of the invention comprises a sense region
and an antisense region, wherein the antisense region comprises a nucleotide
sequence
that is complementary to a nucleotide sequence of RNA encoded by a GPRA and/or
AAAl gene, or a portion thereof, and the sense region comprises a nucleotide
sequence
that is complementary to the antisense region. In one embodiment, the siNA
molecule is
assembled from two separate oligonucleotide fragments, wherein one fragment
comprises the sense region and the second fragment comprises the antisense
region of
the siNA molecule. In another embodiment, the sense region is connected to the
antisense region via a linker molecule. In another embodiment, the sense
region is
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connected to the antisense region via a linker molecule, such as a nucleotide
or non-
nucleotide linker. The GPRA and/or AAA1 gene can comprise, for example,
sequences
referred in to Table I.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAA1
gene comprising a sense region and an antisense region, wherein the antisense
region
comprises a nucleotide sequence that is complementary to a nucleotide sequence
of RNA
encoded by the GPRA and/or AAA1 gene or a portion thereof and the sense region
comprises a nucleotide sequence that is complementary to the antisense region,
and
wherein the siNA molecule has one or more modified pyrimidine and/or purine
nucleotides. In one embodiment, the pyrimidine nucleotides in the sense region
are 2'-O-
methyl pyrimidine nucleotides or 2'-deoxy-2'-fluoro pyrimidine nucleotides and
the
purine nucleotides present in the sense region are 2'-deoxy purine
nucleotides. In
another embodiment, the pyrimidine nucleotides in the sense region are 2'-
deoxy-2'-
fluoro pyrimidine nucleotides and the purine nucleotides present in the sense
region are
2'-O-methyl purine nucleotides. In another embodiment, the pyrimidine
nucleotides in
the sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides and the purine
nucleotides
present in the sense region are 2'-deoxy purine nucleotides. In one
embodiment, the
pyrimidine nucleotides in the antisense region are 2'-deoxy-2'-fluoro
pyrimidine
nucleotides and the purine nucleotides present in the antisense region are 2'-
O-methyl or
2'-deoxy purine nucleotides. In another embodiment of any of the above-
described siNA
molecules, any nucleotides present in a non-complementary region of the sense
strand
(e.g. overhang region) are 2'-deoxy nucleotides.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAAl
gene, wherein the siNA molecule is assembled from two separate oligonucleotide
fragments wherein one fragment comprises the sense region and the second
fragment
comprises the antisense region of the siNA molecule, and wherein the fragment
comprising the sense region includes a terminal cap moiety at the 5'-end, the
3'-end, or
both of the 5' and 3' ends of the fragment. In one embodiment, the terminal
cap moiety is
an inverted deoxy abasic moiety or glyceryl moiety. In one embodiment, each of
the two
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fragments of the siNA molecule independently comprise about 15 to about 30
(e.g. about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides. In another
embodiment, each of the two fragments of the siNA molecule independently
comprise
about 15 to about 40 (e.g. about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29,
30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limiting
example, each
of the two fragments of the siNA molecule comprise about 21 nucleotides.
In one embodiment, the invention features a siNA molecule comprising at least
one
modified nucleotide, wherein the modified nucleotide is a 2'-deoxy-2'-fluoro
nucleotide.
The siNA can be, for example, about 15 to about 40 nucleotides in length. In
one
embodiment, all pyrimidine nucleotides present in the siNA are 2'-deoxy-2'-
fluoro
pyrimidine nucleotides. In one embodiment, the modified nucleotides in the
siNA
include at least one 2'-deoxy-2'-fluoro cytidine or 2'-deoxy-2'-fluoro uridine
nucleotide.
In another embodiment, the modified nucleotides in the siNA include at least
one 2'-
fluoro cytidine and at least one 2'-deoxy-2'-fluoro uridine nucleotides. In
one
embodiment, all uridine nucleotides present in the siNA are 2'-deoxy-2'-fluoro
uridine
nucleotides. In one embodiment, all cytidine nucleotides present in the siNA
are 2'-
deoxy-2'-fluoro cytidine nucleotides. In one embodiment, all adenosine
nucleotides
present in the siNA are 2'-deoxy-2'-fluoro adenosine nucleotides. In one
embodiment,
all guanosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro guanosine
nucleotides. The siNA can further comprise at least one modified
internucleotidic
linkage, such as phosphorothioate linkage. In one embodiment, the 2'-deoxy-2'-
fluoronucleotides are present at specifically selected locations in the siNA
that are
sensitive to cleavage by ribonucleases, such as locations having pyrimidine
nucleotides.
In one embodiment, the invention features a method of increasing the stability
of a
siNA molecule against cleavage by ribonucleases comprising introducing at
least one
modified nucleotide into the siNA molecule, wherein the modified nucleotide is
a 2'-
deoxy-2'-fluoro nucleotide. In one embodiment, all pyrimidine nucleotides
present in
the siNA are 2'-deoxy-2'-fluoro pyrimidine nucleotides. In one embodiment, the
modified nucleotides in the siNA include at least one 2'-deoxy-2'-fluoro
cytidine or 2'-
deoxy-2'-fluoro uridine nucleotide. In another embodiment, the modified
nucleotides in
the siNA include at least one 2'-fluoro cytidine and at least one 2'-deoxy-2'-
fluoro
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uridine nucleotides. In one embodiment, all uridine nucleotides present in the
siNA are
2'-deoxy-2'-fluoro uridine nucleotides. In one embodiment, all cytidine
nucleotides
present in the siNA are 2'-deoxy-2'-fluoro cytidine nucleotides. In one
embodiment, all
adenosine nucleotides present in the siNA are 2'-deoxy-2'-fluoro adenosine
nucleotides.
In one embodiment, all guanosine nucleotides present in the siNA are 2'-deoxy-
2'-fluoro
guanosine nucleotides. The siNA can further comprise at least one modified
internucleotidic linkage, such as phosphorothioate linkage. In one embodiment,
the 2'-
deoxy-2'-fluoronucleotides are present at specifically selected locations in
the siNA that
are sensitive to cleavage by ribonucleases, such as locations having
pyrimidine
nucleotides.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAA1
gene comprising a sense region and an antisense region, wherein the antisense
region
comprises a nucleotide sequence that is complementary to a nucleotide sequence
of RNA
encoded by the GPRA and/or AAA1 gene or a portion thereof and the sense region
comprises a nucleotide sequence that is complementary to the antisense region,
and
wherein the purine nucleotides present in the antisense region comprise 2'-
deoxy- purine
nucleotides. In an alternative embodiment, the purine nucleotides present in
the
antisense region comprise 2'-O-methyl purine nucleotides. In either of the
above
embodiments, the antisense region can comprise a phosphorothioate
internucleotide
linkage at the 3' end of the antisense region. Alternatively, in either of the
above
embodiments, the antisense region can comprise a glyceryl modification at the
3' end of
the antisense region. In another embodiment of any of the above-described siNA
molecules, any nucleotides present in a non-complementary region of the
antisense
strand (e.g. overhang region) are 2'-deoxy nucleotides.
In one embodiment, the antisense region of a siNA molecule of the invention
comprises sequence complementary to a portion of a GPRA and/or AAA1 transcript
having sequence unique to a particular GPRA and/or AAA1 disease related
allele, such
as sequence comprising a single nucleotide polymorphism (SNP) associated with
the
disease specific allele. As such, the antisense region of a siNA molecule of
the invention
can comprise sequence complementary to sequences that are unique to a
particular allele
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to provide specificity in mediating selective RNAi against the disease,
condition, or trait
related allele.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that down-regulates expression of a GPRA and/or
AAA1
gene, wherein the siNA molecule is assembled from two separate oligonucleotide
fragments wherein one fragment comprises the sense region and the second
fragment
comprises the antisense region of the siNA molecule. In another embodiment,
the siNA
molecule is a double stranded nucleic acid molecule, where each strand is
about 21
nucleotides long and where about 19 nucleotides of each fragment of the siNA
molecule
are base-paired to the complementary nucleotides of the other fragment of the
siNA
molecule, wherein at least two 3' terminal nucleotides of each fragment of the
siNA
molecule are not base-paired to the nucleotides of the other fragment of the
siNA
molecule. In another embodiment, the siNA molecule is a double stranded
nucleic acid
molecule, where each strand is about 19 nucleotide long and where the
nucleotides of
each fragment of the siNA molecule are base-paired to the complementary
nucleotides of
the other fragment of the siNA molecule to form at least about 15 (e.g., 15,
16, 17, 1 ~, or
19) base pairs, wherein one or both ends of the siNA molecule are blunt ends.
In one
embodiment, each of the two 3' terminal nucleotides of each fragment of the
siNA
molecule is a 2'-deoxy-pyrimidine nucleotide, such as a 2'-deoxy-thymidine. In
another
embodiment, all nucleotides of each fragment of the siNA molecule are base-
paired to
the complementary nucleotides of the other fragment of the siNA molecule. In
another
embodiment, the siNA molecule is a double stranded nucleic acid molecule of
about 19
to about 25 base pairs having a sense region and an antisense region, where
about 19
nucleotides of the antisense region are base-paired to the nucleotide sequence
or a
portion thereof. of the RNA encoded by the GPRA and/or AAA1 gene. In another
embodiment, about 21 nucleotides of the antisense region are base-paired to
the
nucleotide sequence or a portion thereof of the RNA encoded by the GPRA and/or
AAA1 gene. In any of the above embodiments, the 5'-end of the fragment
comprising
said antisense region can optionally include a phosphate group.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits the expression of a GPRA andlor
AAA1 RNA
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sequence (e.g., wherein said target RNA sequence is encoded by a GPRA and/or
AAA1
gene involved in the GPRA and/or AAAl pathway), wherein the siNA molecule does
not contain any ribonucleotides and wherein each strand of the double-stranded
siNA
molecule is about 15 to about 30 nucleotides. In one embodiment, the siNA
molecule is
21 nucleotides in length. Examples of non-ribonucleotide containing siNA
constructs
are combinations of stabilization chemistries shown in Table IV in any
combination of
Sense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab 18/8,
Stab 18/11,
Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab 8/19, Stab 18/19, Stab
7/20, Stab 8/20,
Stab 18/20, Stab 7/32, Stab 8/32, or Stab 18/32 (e.g., any siNA having Stab 7,
8, 11, 12,
13, 14, 15, 17, 18, 19, 20, or 32 sense or antisense strands or any
combination thereof).
In one embodiment, the invention features a chemically synthesized double
stranded RNA molecule that directs cleavage of a GPRA and/or AAAl RNA via RNA
interference, wherein each strand of said RNA molecule is about 15 to about 30
nucleotides in length; one strand of the RNA molecule comprises nucleotide
sequence
having sufficient complementarity to the GPRA and/or AAA1 RNA for the RNA
molecule to direct cleavage of the GPRA and/or AAA1 RNA via RNA interference;
and
wherein at least one strand of the RNA molecule optionally comprises one or
more
chemically modified nucleotides described herein, such as without limitation
deoxynucleotides, 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucloetides, 2'-
O-
methoxyethyl nucleotides etc.
In one embodiment, the invention features a medicament comprising a siNA
molecule of the invention.
In one embodiment, the invention features an active ingredient comprising a
siNA
molecule of the invention.
In one embodiment, the invention features the use of a double-stranded short
interfering nucleic acid (siNA) molecule to inhibit, down-regulate, or reduce
expression
of a GPRA and/or AAA1 gene, wherein the siNA molecule comprises one or more
chemical modifications and each strand of the double-stranded siNA is
independently
about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26,
27, 28, 29 or 30 or more) nucleotides long. In one embodiment, the siNA
molecule of
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the invention is a double stranded nucleic acid molecule comprising one or
more
chemical modifications, where each of the two fragments of the siNA molecule
independently comprise about 15 to about 40 (e.g. about 15, 16, 17, 18, 19,
20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40)
nucleotides and
where one of the strands comprises at least 15 nucleotides that are
complementary to
nucleotide sequence of GPR.A and/or AAAl encoding RNA or a portion thereof. In
a
non-limiting example, each of the two fragments of the siNA molecule comprise
about
21 nucleotides. In another embodiment, the siNA molecule is a double stranded
nucleic
acid molecule comprising one or more chemical modifications, where each strand
is
about 21 nucleotide long and where about 19 nucleotides of each fragment of
the siNA
molecule are base-paired to the complementary nucleotides of the other
fragment of the
siNA molecule, wherein at least two 3' terminal nucleotides of each fragment
of the
siNA molecule are not base-paired to the nucleotides of the other fragment of
the siNA
molecule. In another embodiment, the siNA molecule is a double stranded
nucleic acid
molecule comprising one or more chemical modifications, where each strand is
about 19
nucleotide long and where the nucleotides of each fragment of the siNA
molecule are
base-paired to the complementary nucleotides of the other fragment of the siNA
molecule to form at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs,
wherein one or
both ends of the siNA molecule are blunt ends. In one embodiment, each of the
two 3'
terminal nucleotides of each fragment of the siNA molecule is a 2'-deo~y-
pyrimidine
nucleotide, such as a 2'-deoxy-thymidine. In another embodiment, all
nucleotides of
each fragment of the siNA molecule are base-paired to the complementary
nucleotides of
the other fragment of the siNA molecule. In another embodiment, the siNA
molecule is
a double stranded nucleic acid molecule of about 19 to about 25 base pairs
having a
sense region and an antisense region and comprising one or more chemical
modifications, where about 19 nucleotides of the antisense region are base-
paired to the
nucleotide sequence or a portion thereof of the RNA encoded by the GPR.A
and/or
AAA1 gene. In another embodiment, about 21 nucleotides of the antisense region
are
base-paired to the nucleotide sequence or a portion thereof of the RNA encoded
by the
GPRA and/or AAA1 gene. In any of the above embodiments, the 5'-end of the
fragment
comprising said antisense region can optionally include a phosphate group.
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In one embodiment, the invention features the use of a double-stranded short
interfering nucleic acid (siNA) molecule that inhibits, down-regulates, or
reduces
expression of a GPRA and/or AAAl gene, wherein one of the strands of the
double-
stranded siNA molecule is an antisense strand which comprises nucleotide
sequence that
is complementary to nucleotide sequence of GPRA and/or AAA1 RNA or a portion
thereof, the other strand is a sense strand which comprises nucleotide
sequence that is
complementary to a nucleotide sequence of the antisense strand and wherein a
majority
of the pyrimidine nucleotides present in the double-stranded siNA molecule
comprises a
sugar modification.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits, down-regulates, or reduces
expression of a
GPRA and/or AAA1 gene, wherein one of the strands of the double-stranded siNA
molecule is an antisense strand which comprises nucleotide sequence that is
complementary to nucleotide sequence of GPRA and/or AAA1 RNA or a portion
thereof, wherein the other strand is a sense strand which comprises nucleotide
sequence
that is complementary to a nucleotide sequence of the antisense strand and
wherein a
majority of the pyrimidine nucleotides present in the double-stranded siNA
molecule
comprises a sugar modification.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits , down-regulates, or reduces
expression of a
GPRA andlor AAA1 gene, wherein one of the strands of the double-stranded siNA
molecule is an antisense strand which comprises nucleotide sequence that is
complementary to nucleotide sequence of GPRA andlor AAA1 RNA that encodes a
protein or portion thereof, the other strand is a sense strand which comprises
nucleotide
sequence that is complementary to a nucleotide sequence of the antisense
strand and
wherein a majority of the pyrimidine nucleotides present in the double-
stranded siNA
molecule comprises a sugar modification. In one embodiment, each strand of the
siNA
molecule comprises about 15 to about 30 or more (e.g., about 15, 16, 17, 18,
19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, or 30 or more) nucleotides, wherein each
strand comprises
at least about 15 nucleotides that are complementary to the nucleotides of the
other
strand. In one embodiment, the siNA molecule is assembled from two
oligonucleotide
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fragments, wherein one fragment comprises the nucleotide sequence of the
antisense
strand of the siNA molecule and a second fragment comprises nucleotide
sequence of the
sense region of the siNA molecule. In one embodiment, the sense strand is
connected to
the antisense strand via a linker molecule, such as a polynucleotide linker or
a non-
nucleotide linker. In a further embodiment, the pyrimidine nucleotides present
in the
sense strand are 2'-deoxy-2'fluoro pyrimidine nucleotides and the purine
nucleotides
present in the sense region are 2'-deoxy purine nucleotides. In another
embodiment, the
pyrimidine nucleotides present in the sense strand are 2'-deoxy-2'fluoro
pyrimidine
nucleotides and the purine nucleotides present in the sense region are 2'-O-
methyl purine
nucleotides. In still another embodiment, the pyrimidine nucleotides present
in the
antisense strand are 2'-deoxy-2'-fluoro pyrimidine nucleotides and any purine
nucleotides
present in the antisense strand are 2'-deoxy purine nucleotides. In another
embodiment,
the antisense strand comprises one or more 2'-deoxy-2'-fluoro pyrimidine
nucleotides
and one or more 2'-O-methyl purine nucleotides. In another embodiment, the
pyrimidine nucleotides present in the antisense strand are 2'-deoxy-2'-fluoro
pyrimidine
nucleotides and any purine nucleotides present in the antisense strand are 2'-
O-methyl
purine nucleotides. In a further embodiment the sense strand comprises a 3'-
end and a 5'-
end, wherein a terminal cap moiety (e.g., an inverted deoxy abasic moiety or
inverted
deoxy nucleotide moiety such as inverted thymidine) is present at the 5'-end,
the 3'-end,
or both of the 5' and 3' ends of the sense strand. In another embodiment, the
antisense
strand comprises a phosphorothioate internucleotide linkage at the 3' end of
the antisense
strand. In another embodiment, the antisense strand comprises a glyceryl
modification at
the 3' end. In another embodiment, the 5'-end of the antisense strand
optionally includes
a phosphate group.
In any of the above-described embodiments of a double-stranded short
interfering
nucleic acid (siNA) molecule that inhibits expression of a GPRA and/or AAA1
gene,
wherein a majority of the pyrimidine nucleotides present in the double-
stranded siNA
molecule comprises a sugar modification, each of the two strands of the siNA
molecule
can comprise about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 or more) nucleotides. In one embodiment, about
15 to about
30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30 or
more) nucleotides of each strand of the siNA molecule are base-paired to the
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complementary nucleotides of the other strand of the siNA molecule. In another
embodiment, about 15 to about 30 or more (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of the siNA
molecule are
base-paired to the complementary nucleotides of the other strand of the siNA
molecule,
wherein at least two 3' terminal nucleotides of each strand of the siNA
molecule are not
base-paired to the nucleotides of the other strand of the siNA molecule. In
another
embodiment, each of the two 3' terminal nucleotides of each fragment of the
siNA
molecule is a 2'-deoxy-pyrimidine, such as 2'-deoxy-thymidine. In one
embodiment,
each strand of the siNA molecule is base-paired to the complementary
nucleotides of the
other strand of the siNA molecule. In one embodiment, about 15 to about 30
(e.g., about
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides
of the
antisense strand are base-paired to the nucleotide sequence of the GPRA and/or
AAA1
RNA or a portion thereof. In one embodiment, about 18 to about 25 (e.g., about
18, 19,
20, 21, 22, 23, 24, or 25) nucleotides of the antisense strand are base-paired
to the
nucleotide sequence of the GPRA and/or AAA1 RNA or a portion thereof.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits expression of a GPRA and/or AAA1
gene,
wherein one of the strands of the double-stranded siNA molecule is an
antisense strand
which comprises nucleotide sequence that is complementary to nucleotide
sequence of
GPRA and/or AAA1 RNA or a portion thereof, the other strand is a sense strand
which
comprises nucleotide sequence that is complementary to a nucleotide sequence
of the
antisense strand and wherein a majority of the pyrimidine nucleotides present
in the
double-stranded siNA molecule comprises a sugar modification, and wherein the
5'-end
of the antisense strand optionally includes a phosphate group.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits expression of a GPRA and/or AAA1
gene,
wherein one of the strands of the double-stranded siNA molecule is an
antisense strand
which comprises nucleotide sequence that is complementary to nucleotide
sequence of
GPRA and/or AAA1 RNA or a portion thereof, the other strand is a sense strand
which
comprises nucleotide sequence that is complementary to a nucleotide sequence
of the
antisense strand and wherein a majority of the pyrimidine nucleotides present
in the
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double-stranded siNA molecule comprises a sugar modification, and wherein the
nucleotide sequence or a portion thereof of the antisense strand is
complementary to a
nucleotide sequence of the untranslated region or a portion thereof of the
GPRA and/or
AAA 1 RNA.
In one embodiment, the invention features a double-stranded short interfering
nucleic acid (siNA) molecule that inhibits expression of a GPRA and/or AAAl
gene,
wherein one of the strands of the~double-stranded siNA molecule is an
antisense strand
which comprises nucleotide sequence that is complementary to nucleotide
sequence of
GPRA and/or AAAl RNA or a portion thereof, wherein the other strand is a sense
strand
which comprises nucleotide sequence that is complementary to a nucleotide
sequence of
the antisense strand, wherein a majority of the pyrimidine nucleotides present
in the
double-stranded siNA molecule comprises a sugar modification, and wherein the
nucleotide sequence of the antisense strand is complementary to a nucleotide
sequence of
the GPRA and/or AAA1 RNA or a portion thereof that is present in the GPRA
and/or
AAAl RNA.
In one embodiment, the invention features a composition comprising a siNA
molecule of the invention in a pharmaceutically acceptable carrier or diluent.
In a non-limiting example, the introduction of chemically-modified nucleotides
into nucleic acid molecules provides a powerful tool in overcoming potential
limitations
of in vivo stability and bioavailability inherent to native RNA molecules that
are
delivered exogenously. For example, the use of chemically-modified nucleic
acid
molecules can enable a lower dose of a particular nucleic acid molecule for a
given
therapeutic effect since chemically-modified nucleic acid molecules tend to
have a
longer half life in serum. Furthermore, certain chemical modifications can
improve the
bioavailability of nucleic acid molecules by targeting particular cells or
tissues and/or
improving cellular uptake of the nucleic acid molecule. Therefore, even if the
activity of
a chemically-modified nucleic acid molecule is reduced as compared to a native
nucleic
acid molecule, for example, when compared to an all-RNA nucleic acid molecule,
the
overall activity of the modified nucleic acid molecule can be greater than
that of the
native molecule due to improved stability and/or delivery of the molecule.
Unlike native
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unmodified siNA, chemically-modified siNA can also minimize the possibility of
activating interferon activity in humans.
In any of the embodiments of siNA molecules described herein, the antisense
region of a siNA molecule of the invention can comprise a phosphorothioate
internucleotide linkage at the 3'-end of said antisense region. In any of the
embodiments
of siNA molecules described herein, the antisense region can comprise about
one to
about five phosphorothioate internucleotide linkages at the 5'-end of said
antisense
region. In any of the embodiments of siNA molecules described herein, the 3'-
terminal
nucleotide overhangs of a siNA molecule of the invention can comprise
ribonucleotides
or deoxyribonucleotides that are chemically-modified at a nucleic acid sugar,
base, or
backbone. In any of the embodiments of siNA molecules described herein, the 3'-
terminal nucleotide overhangs can comprise one or more universal base
ribonucleotides.
In any of the embodiments of siNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise one or more acyclic nucleotides.
One embodiment of the invention provides an expression vector comprising a
nucleic acid sequence encoding at least one siNA molecule of the invention in
a manner
that allows expression of the nucleic acid molecule. Another embodiment of the
invention provides a mammalian cell comprising such an expression vector. The
mammalian cell can be a human cell. The siNA molecule of the expression vector
can
comprise a sense region and an antisense region. The antisense region can
comprise
sequence complementary to a RNA or DNA sequence encoding GPRA and/or AAAl
and the sense region can comprise sequence complementary to the antisense
region. The
siNA molecule can comprise two distinct strands having complementary sense and
antisense regions. The siNA molecule can comprise a single strand having
complementary sense and antisense regions.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
against
GPRA and/or AAAl inside a cell or reconstituted in vitro system, wherein the
chemical
modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, ~, 9, 10,
or more)
nucleotides comprising a backbone modified internucleotide linkage having
Formula I:
CA 02543029 2006-04-19
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Z
~ ~ Y R
R X z
1
W
wherein each Rl and R2 is independently any nucleotide, non-nucleotide, or
polynucleotide which can be naturally-occurring or chemically-modified, each X
and Y
is independently O, S, N, alkyl, or substituted alkyl, each Z and W is
independently O, S,
N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or acetyl and
wherein W, X,
Y, and Z are optionally not all O. In another embodiment, a backbone
modification of
the invention comprises a phosphonoacetate and/or thiophosphonoacetate
internucleotide
linkage (see for example Sheehan et al., 2003, Nucleic Acids Research, 31,
4109-4118).
The chemically-modified internucleotide linkages having Formula I, for
example,
wherein any Z, W, X, and/or Y independently comprises a sulphur atom, can be
present
in one or both oligonucleotide strands of the siNA duplex, for example, in the
sense
strand, the antisense strand, or both strands. 'The siNA molecules of the
invention can
comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
chemically-
modified internucleotide linkages having Formula I at the 3'-end, the 5'-end,
or both of
the 3' and 5'-ends of the sense strand, the antisense strand, or both strands.
For example,
an exemplary siNA molecule of the invention can comprise about 1 to about 5 or
more
(e.g., about 1, 2, 3, 4, 5, or more) chemically-modified internucleotide
linkages having
Formula I at the 5'-end of the sense strand, the antisense strand, or both
strands. In
another non-limiting example, an exemplary siNA molecule of the invention can
comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)
pyrimidine
nucleotides with chemically-modified internucleotide linkages having Formula I
in the
sense strand, the antisense strand, or both strands. In yet another non-
limiting example,
an exemplary siNA molecule of the invention can comprise one or more (e.g.,
about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotides with chemically-modified
internucleotide linkages having Formula I in the sense strand, the antisense
strand, or
both strands. In another embodiment, a siNA molecule of the invention having
internucleotide linkages) of Formula I also comprises a chemically-modified
nucleotide
or non-nucleotide having any of Formulae I-VII.
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In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
against
GPR.A and/or AAA1 inside a cell or reconstituted ih vitro system, wherein the
chemical
modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more)
nucleotides or non-nucleotides having Formula II:
1
/ R9
R12
R6
Rs ~ ~ R1o
R5 R3
wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH,
alkyl,
substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl,
S-alkyl,
N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-
alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02,
N3,
NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-
aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino,
substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=O, CHF,
or CF2,
and B is a nucleosidic base such as adenine, guanine, uracil, cytosine,
thymine, 2-
aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-
naturally
occurnng base that can be complementary or non-complementary to target RNA or
a
non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole, 5-nitroindole,
nebularine,
pyridone, pyridinone, or any other non-naturally occurnng universal base that
can be
complementary or non-complementary to target RNA.
The chemically-modified nucleotide or non-nucleotide of Formula II can be
present in one or both oligonucleotide strands of the siNA duplex, for example
in the
sense strand, the antisense strand, or both strands. The siNA molecules of the
invention
can comprise one or more chemically-modified nucleotides or non-nucleotides of
Formula II at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
sense strand, the
antisense strand, or both strands. For example, an exemplary siNA molecule of
the
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invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5,
or more)
chemically-modified nucleotides or non-nucleotides of Formula II at the 5'-end
of the
sense strand, the antisense strand, or both strands. In anther non-limiting
example, an
exemplary siNA molecule of the invention can comprise about 1 to about 5 or
more (e.g.,
about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides or non-
nucleotides of
Formula II at the 3'-end of the sense strand, the antisense strand, or both
strands.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
against
GPR.A and/or AAA1 inside a cell or reconstituted ira vitro system, wherein the
chemical
modification comprises one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more)
nucleotides or non-nucleotides having Formula III:
Rio
wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independently H, OH,
alkyl,
substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl,
S-alkyl,
N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-
alkyl-OH,
O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ON02, N02,
N3,
NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-
aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino,
substituted silyl, or group having Formula I ox II; R9 is O, S, CH2, S=O, CHF,
or CF2,
and B is a nucleosidic base such as adenine, guanine, uracil, cytosine,
thymine, 2-
aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any other non-
naturally
occurring base that can be employed to be complementary or non-complementary
to
target RNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,
5-
nitroindole, nebularine, pyridone, pyridinone, or any other non-naturally
occurring
universal base that can be complementary or non-complementary to target RNA.
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The chemically-modified nucleotide or non-nucleotide of Formula III can be
present in one or both oligonucleotide strands of the siNA duplex, for
example, in the
sense strand, the antisense strand, or both strands. The siNA molecules of the
invention
can comprise one or more chemically-modified nucleotides or non-nucleotides of
Formula III at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of the
sense strand, the
antisense strand, or both strands. For example, an exemplary siNA molecule of
the
invention can comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5,
or more)
chemically-modified nucleotides) or non-nucleotides) of Formula III at the 5'-
end of
the sense strand, the antisense strand, or both strands. In anther non-
limiting example, an
exemplary siNA molecule of the invention can comprise about 1 to about 5 or
more (e.g.,
about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide or non-nucleotide
of
Formula III at the 3'-end of the sense strand, the antisense strand, or both
strands.
In another embodiment, a siNA molecule of the invention comprises a nucleotide
having Formula II or III, wherein the nucleotide having Formula II or III is
in an inverted
configuration. For example, the nucleotide having Formula II or III is
connected to the
siNA construct in a 3'-3', 3'-2', 2'-3', or 5'-5' configuration, such as at
the 3'-end, the 5'-
end, or both of the 3' and 5'-ends of one or both siNA strands.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
against
GPRA and/or AAA1 inside a cell or reconstituted iT2 vitro system, wherein the
chemical
modification comprises a 5'-terminal phosphate group having Formula IV:
Z
X
P
Y
W
wherein each X and Y is independently O, S, N, alkyl, substituted alkyl, or
alkylhalo;
wherein each Z and W is independently O, S, N, alkyl, substituted alkyl, O-
alkyl, S-
alkyl, alkaryl, aralkyl, alkylhalo, or acetyl; and wherein W, X, Y and Z are
not all O.
In one embodiment, the invention features a siNA molecule having a 5'-terminal
phosphate group having Formula IV on the target-complementary strand, for
example, a
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strand complementary to a target RNA, wherein the siNA molecule comprises an
all
RNA siNA molecule. In another embodiment, the invention features a siNA
molecule
having a 5'-terminal phosphate group having Formula IV on the target-
complementary
strand wherein the siNA molecule also comprises about 1 to about 3 (e.g.,
about 1, 2, or
3) nucleotide 3'-terminal nucleotide overhangs having about 1 to about 4
(e.g., about 1, 2,
3, or 4) deoxyribonucleotides on the 3'-end of one or both strands. In another
embodiment, a 5'-terminal phosphate group having Formula IV is present on the
target-
complementary strand of a siNA molecule of the invention, for example a siNA
molecule having chemical modifications having any of Formulae I-VII.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule capable of mediating RNA interference (RNAi)
against
GPRA and/or AAA1 inside a cell or reconstituted in vitro system, wherein the
chemical
modification comprises one or more phosphorothioate internucleotide linkages.
For
example, in a non-limiting example, the invention features a chemically-
modified short
interfering nucleic acid (siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more
phosphorothioate internucleotide linkages in one siNA strand. In yet another
embodiment, the invention features a chemically-modified short interfering
nucleic acid
(siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more
phosphorothioate
internucleotide linkages in both siNA strands. The phosphorothioate
internucleotide
linkages can be present in one or both oligonucleotide strands of the siNA
duplex, for
example in the sense strand, the antisense strand, or both strands. The siNA
molecules
of the invention can comprise one or more phosphorothioate internucleotide
linkages at
the 3'-end, the 5'-end, or both of the 3'- and 5'-ends of the sense strand,
the antisense
strand, or both strands. For example, an exemplary siNA molecule of the
invention can
comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, or more)
consecutive
phosphorothioate internucleotide linkages at the 5'-end of the sense strand,
the antisense
strand, or both strands. In another non-limiting example, an exemplary siNA
molecule
of the invention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, or
more) pyrimidine phosphorothioate internucleotide linkages in the sense
strand, the
antisense strand, or both strands. In yet another non-limiting example, an
exemplary
siNA molecule of the invention can comprise one or more (e.g., about 1, 2, 3,
4, 5, 6, 7,
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8, 9, 10, or more) purine phosphorothioate internucleotide linkages in the
sense strand,
the antisense strand, or both strands.
In one embodiment, the invention features a siNA molecule, wherein the sense
strand comprises one or more, for example, about l, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more
phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1,
2, 3, 4, 5, 6,
7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or about
one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the
3'- and 5'-ends
of the sense strand; and wherein the antisense strand comprises about 1 to
about 10 or
more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
phosphorothioate
internucleotide linkages, and/or one or more (e.g., about l, 2, 3, 4, 5, 6, 7,
8, 9, 10 or
more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g.,
about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and
optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-
ends of the
antisense strand. In another embodiment, one or more, for example about 1, 2,
3, 4, 5, 6,
7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or antisense
siNA strand are
chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro
nucleotides,
with or without one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more,
phosphorothioate internucleotide linkages and/or a terminal cap molecule at
the 3'-end,
the 5'-end, or both of the 3'- and 5'-ends, being present in the same or
different strand.
In another embodiment, the invention features a siNA molecule, wherein the
sense
strand comprises about 1 to about 5, specifically about l, 2, 3, 4, or 5
phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, or
more) 2'-deoxy,
2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4,
5, or more)
universal base modified nucleotides, and optionally a terminal cap molecule at
the 3-end,
the 5'-end, or both of the 3'- and 5'-ends of the sense strand; and wherein
the antisense
strand comprises about 1 to about 5 or more, specifically about 1, 2, 3, 4, 5,
or more
phosphorothioate internucleotide linkages, and/or one or more (e.g., about 1,
2, 3, 4, 5, 6,
7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or
more (e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the
3'- and 5'-ends
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of the antisense strand. In another embodiment, one or more, for example about
1, 2, 3,
4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides of the sense and/or
antisense siNA
strand are chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-
fluoro
nucleotides, with or without about 1 to about 5 or more, for example about 1,
2, 3, 4, 5,
or more phosphorothioate internucleotide linkages and/or a terminal cap
molecule at the
3'-end, the 5'-end, or both of the 3'- and 5'-ends, being present in the same
or different
strand.
In one embodiment, the invention features a siNA molecule, wherein the
antisense
strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more
phosphorothioate internucleotide linkages, and/or about one or more (e.g.,
about l, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the
3'- and 5'-ends
of the sense strand; and wherein the antisense strand comprises about 1 to
about 10 or
more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
phosphorothioate
internucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or
more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro, and/or one or more (e.g.,
about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides, and
optionally a
terminal cap molecule at the 3'-end, the 5'-end, or both of the 3'- and 5'-
ends of the
antisense strand. In another embodiment, one or more, for example about 1, 2,
3, 4, 5, 6,
7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisense siNA
strand are
chemically-modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro
nucleotides,
with or without one or more, for example, about l, 2, 3, 4, 5, 6, 7, 8, 9, 10
or more
phosphorothioate internucleotide linkages and/or a terminal cap molecule at
the 3'-end,
the 5'-end, or both of the 3' and 5'-ends, being present in the same or
different strand.
In another embodiment, the invention features a siNA molecule, wherein the
antisense strand comprises about 1 to about 5 or more, specifically about 1,
2, 3, 4, 5 or
more phosphorothioate internucleotide linkages, and/or one or more (e.g.,
about 1, 2, 3,
4, 5, 6, 7, 8, 9, 10 or more) 2'-deoxy, 2'-O-methyl, 2'-deoxy-2'-fluoro,
and/or one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified
nucleotides, and
optionally a terminal cap molecule at the 3'-end, the 5'-end, or both of the
3'- and 5'-ends
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of the sense strand; and wherein the antisense strand comprises about 1 to
about 5 or
more, specifically about 1, 2, 3, 4, 5 or more phosphorothioate
internucleotide linkages,
and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2'-
deoxy, 2'-O-methyl,
2'-deoxy-2'-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,
10 or more)
universal base modified nucleotides, and optionally a terminal cap molecule at
the 3'-
end, the 5'-end, or both of the 3'- and 5'-ends of the antisense strand. In
another
embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more
pyrimidine nucleotides of the sense and/or antisense siNA strand are
chemically-
modified with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides,
with or
without about 1 to about 5, for example about 1, 2, 3, 4, 5 or more
phosphorothioate
internucleotide linkages and/or a terminal cap molecule at the 3'-end, the 5'-
end, or both
of the 3'- and 5'-ends, being present in the same or different strand.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule having about 1 to about 5 or more (specifically
about 1, 2,
3, 4, 5 or more) phosphorothioate internucleotide linkages in each strand of
the siNA
molecule.
In another embodiment, the invention features a siNA molecule comprising 2'-5'
internucleotide linkages. The 2'-S' internucleotide linkages) can be at the 3'-
end, the 5'-
end, or both of the 3'- and 5'-ends of one or both siNA sequence strands. In
addition, the
2'-5' internucleotide linkages) can be present at various other positions
within one or
both siNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
or more
including every internucleotide linkage of a pyrimidine nucleotide in one or
both strands
of the siNA molecule can comprise a 2'-5' internucleotide linkage, or about 1,
2, 3, 4, 5,
6, 7, 8, 9, 10, or more including every internucleotide linkage of a purine
nucleotide in
one or both strands of the siNA molecule can comprise a 2'-5' internucleotide
linkage.
In another embodiment, a chemically-modified siNA molecule of the invention
comprises a duplex having two strands, one or both of which can be chemically-
modified, wherein each strand is independently about 15 to about 30 (e.g.,
about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in
length, wherein the
duplex has about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26,
27, 28, 29, or 30) base pairs, and wherein the chemical modification comprises
a
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structure having any of Formulae I-VII. For example, an exemplary chemically-
modified siNA molecule of the invention comprises a duplex having two strands,
one or
both of which can be chemically-modified with a chemical modification having
any of
Formulae I-VII or any combination thereof, wherein each strand consists of
about 21
nucleotides, each having a 2-nucleotide 3'-terminal nucleotide overhang, and
wherein the
duplex has about 19 base pairs. In another embodiment, a siNA molecule of the
invention comprises a single stranded hairpin structure, wherein the siNA is
about 36 to
about 70 (e.g., about 36, 40, 45, S0, 55, 60, 65, or 70) nucleotides in length
having about
to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30)
10 base pairs, and wherein the siNA can include a chemical modification
comprising a
structure having any of Formulae I-VII or any combination thereof. For
example, an
exemplary chemically-modified siNA molecule of the invention comprises a
linear
oligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45, 46,
47, 48, 49, or
50) nucleotides that is chemically-modified with a chemical modification
having any of
15 Formulae I-VII or any combination thereof, wherein the linear
oligonucleotide forms a
hairpin structure having about 19 to about 21 (e.g., 19, 20, or 21) base pairs
and a 2-
nucleotide 3'-terminal nucleotide overhang. In another embodiment, a linear
hairpin
siNA molecule of the invention contains a stem loop motif, wherein the loop
portion of
the siNA molecule is biodegradable. For example, a linear hairpin siNA
molecule of the
invention is designed such that degradation of the loop portion of the siNA
molecule in
vivo can generate a double-stranded siNA molecule with 3'-terminal overhangs,
such as
3'-terminal nucleotide overhangs comprising about 2 nucleotides.
In another embodiment, a siNA molecule of the invention comprises a hairpin
structure, wherein the siNA is about 25 to about 50 (e.g., about 25, 26, 27,
28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50)
nucleotides in
length having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein the siNA can
include one or
more chemical modifications comprising a structure having any of Formulae I-
VII or any
combination thereof. For example, an exemplary chemically-modified siNA
molecule of
the invention comprises a linear oligonucleotide having about 25 to about 35
(e.g., about
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that is chemically-
modified with
one or more chemical modifications having any of Formulae I-VII or any
combination
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thereof, wherein the linear oligonucleotide forms a hairpin structure having
about 3 to
about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22,
23, 24, or 25) base pairs and a 5'-terminal phosphate group that can be
chemically
modified as described herein (for example a 5'-terminal phosphate group having
Formula
IV). In another embodiment, a linear hairpin siNA molecule of the invention
contains a
stem loop motif, wherein the loop portion of the siNA molecule is
biodegradable. In one
embodiment, a linear hairpin siNA molecule of the invention comprises a loop
portion
comprising a non-nucleotide linker.
In another embodiment, a siNA molecule of the invention comprises an
asymmetric hairpin structure, wherein the siNA is about 25 to about 50 (e.g.,
about 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49,
or 50) nucleotides in length having about 3 to about 25 (e.g., about 3, 4, 5,
6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and
wherein the
siNA can include one or more chemical modifications comprising a structure
having any
of Formulae I-VII or any combination thereof. For example, an exemplary
chemically-
modi~ed siNA molecule of the invention comprises a linear oligonucleotide
having
about 25 to about 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or
35) nucleotides
that is chemically-modified with one or more chemical modifications having any
of
Formulae I-VII or any combination thereof, wherein the linear oligonucleotide
forms an
asymmetric hairpin structure having about 3 to about 25 (e.g., about 3, 4, 5,
6, 7, 8, 9, 10,
1 l, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and
a 5'-terminal
phosphate group that can be chemically modified as described herein (for
example a 5'-
terminal phosphate group having Formula IV). In one embodiment, an asymmetric
hairpin siNA molecule of the invention contains a stem loop motif, wherein the
loop
portion of the siNA molecule is biodegradable. In another embodiment, an
asymmetric
hairpin siNA molecule of the invention comprises a loop portion comprising a
non-
nucleotide linker.
In another embodiment, a siNA molecule of the invention comprises an
asymmetric double stranded structure having separate polynucleotide strands
comprising
sense and antisense regions, wherein the antisense region is about 15 to about
30 (e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides in
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length, wherein the sense region is about 3 to about 25 (e.g., about 3, 4, 5,
6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides in
length, wherein
the sense region and the antisense region have at least 3 complementary
nucleotides, and
wherein the siNA can include one or more chemical modifications comprising a
structure
having any of Formulae I-VII or any combination thereof. For example, an
exemplary
chemically-modified siNA molecule of the invention comprises an asymmetric
double
stranded structure having separate polynucleotide strands comprising sense and
antisense
regions, wherein the antisense region is about 18 to about 23 (e.g., about 18,
19, 20, 21,
22, or 23) nucleotides in length and wherein the sense region is about 3 to
about 15 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) nucleotides in length,
wherein the sense
region the antisense region have at least 3 complementary nucleotides, and
wherein the
siNA can include one or more chemical modifications comprising a structure
having any
of Formulae I-VII or any combination thereof. In another embodiment, the
asymmetic
double stranded siNA molecule can also have a 5'-terminal phosphate group that
can be
chemically modified as described herein (for example a 5'-terminal phosphate
group
having Formula IV).
In another embodiment, a siNA molecule of the invention comprises a circular
nucleic acid molecule, wherein the siNA is about 38 to about 70 (e.g., about
38, 40, 45,
50, 55, 60, 65, or 70) nucleotides in length having about 15 to about 30
(e.g., about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and
wherein the
siNA can include a chemical modification, which comprises a structure having
any of
Formulae I-VII or any combination thereof. For example, an exemplary
chemically-
modified siNA molecule of the invention comprises a circular oligonucleotide
having
about 42 to about 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50)
nucleotides that is
chemically-modified with a chemical modification having any of Formulae I-VII
or any
combination thereof, wherein the circular oligonucleotide forms a dumbbell
shaped
structure having about 19 base pairs and 2 loops.
In another embodiment, a circular siNA molecule of the invention contains two
loop motifs, wherein one or both loop portions of the siNA molecule is
biodegradable.
For example, a circular siNA molecule of the invention is designed such that
degradation
of the loop portions of the siNA molecule ira vivo can generate a double-
stranded siNA
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molecule with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs
comprising about 2 nucleotides.
In one embodiment, a siNA molecule of the invention comprises at least one
(e.g.,
about l, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety, for example a
compound having
Formula V:
Ran
'13
wherein each R3, R4, R5, R6, R7, R8, R10, Rl l, R12, and R13 is independently
H, OH,
alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-
alkyl, S-
alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-
OH, O-
alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl,
ON02,
N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-
aminoacid,
O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino,
substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=O, CHF,
or CF2.
In one embodiment, a siNA molecule of the invention comprises at least one
(e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasic moiety, for
example a
compound having Formula VI:
212
R~
wherein each R3, R4, R5, R6, R7, R8, R10, R1 l, R12, and R13 is independently
H, OH,
alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3, OCF3, OCN, O-
alkyl, S-
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alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-
OH, O-
alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl,
ON02,
N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-
aminoacid,
O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino,
substituted silyl, or group having Formula I or II; R9 is O, S, CH2, S=O, CHF,
or CF2,
and either R2, R3, R8 or R13 serve as points of attachment to the siNA
molecule of the
invention.
In another embodiment, a siNA molecule of the invention comprises at least one
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) substituted polyalkyl
moieties, for
example a compound having Formula VII:
R1 . . n ~ n _ R3
R2
wherein each n is independently an integer from 1 to 12, each R1, R2 and R3 is
independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br,
CN, CF3,
OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl, SO-
alkyl,
alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-
alkyl,
alkyl-O-alkyl, ON02, N02, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-
aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,
arninoalkylamino, polyalklylamino, substituted silyl, or a group having
Formula I, and
Rl, R2 or R3 serves as points of attachment to the siNA molecule of the
invention.
In another embodiment, the invention features a compound having Formula VII,
wherein Rl and R2 are hydroxyl (OH) groups, n = 1, and R3 comprises O and is
the
point of attaclunent to the 3'-end, the 5'-end, or both of the 3' and 5'-ends
of one or both
strands of a double-stranded siNA molecule of the invention or to a single-
stranded siNA
molecule of the invention. This modification is referred to herein as
"glyceryl" (for
example modification 6 in Figure 10).
In another embodiment, a chemically modified nucleoside or non-nucleoside
(e.g.
a moiety having any of Formula V, VI or VII) of the invention is at the 3'-
end, the 5'-end,
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or both of the 3' and 5'-ends of a siNA molecule of the invention. For
example,
chemically modified nucleoside or non-nucleoside (e.g., a moiety having
Formula V, VI
or VII) can be present at the 3'-end, the 5'-end, or both of the 3' and 5'-
ends of the
antisense strand, the sense strand, or both antisense and sense strands of the
siNA
molecule. In one embodiment, the chemically modified nucleoside or non-
nucleoside
I (e.g., a moiety having Formula V, VI or VII) is present at the 5'-end and 3'-
end of the
sense strand and the 3'-end of the antisense strand of a double stranded siNA
molecule of
the invention. In one embodiment, the chemically modified nucleoside or non-
nucleoside (e.g., a moiety having Formula V, VI or VII) is present at the
terminal
position of the 5'-end and 3'-end of the sense strand and the 3'-end of the
antisense
strand of a double stranded siNA molecule of the invention. In one embodiment,
the
chemically modified nucleoside or non-nucleoside (e.g., a moiety having
Formula V, VI
or VII) is present at the two terminal positions of the 5'-end and 3'-end of
the sense
strand and the 3'-end of the antisense strand of a double stranded siNA
molecule of the
invention. In one embodiment, the chemically modified nucleoside or non-
nucleoside
(e.g., a moiety having Formula V, VI or VII) is present at the penultimate
position of the
5'-end and 3'-end of the sense strand and the 3'-end of the antisense strand
of a double
stranded siNA molecule of the invention. In addition, a moiety having Formula
VII can
be present at the 3'-end or the 5'-end of a hairpin siNA molecule as described
herein.
In another embodiment, a siNA molecule of the invention comprises an abasic
residue having Formula V or VI, wherein the abasic residue having Formula VI
or VI is
connected to the siNA construct in a 3'-3', 3'-2', 2'-3', or 5'-5'
configuration, such as at the
3'-end, the 5'-end, or both of the 3' and 5'-ends of one or both siNA strands.
In one embodiment, a siNA molecule of the invention comprises one or more
(e.g.,
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleic acid (LNA)
nucleotides, for
example, at the 5'-end, the 3'-end, both of the 5' and 3'-ends, or any
combination thereof,
of the siNA molecule.
In another embodiment, a siNA molecule of the invention comprises one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclic nucleotides, for
example, at the
5'-end, the 3'-end, both of the 5' and 3'-ends, or any combination thereof, of
the siNA
molecule.
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In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising a sense region,
wherein any
(e.g., one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-
2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-
2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more
or all)
purine nucleotides present in the sense region are 2'-deoxy purine nucleotides
(e.g.,
wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately
a plurality
of purine nucleotides are 2'-deoxy purine nucleotides).
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising a sense region,
wherein any
(e.g., one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-
2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-
2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more
or all)
purine nucleotides present in the sense region are 2'-deoxy purine nucleotides
(e.g.,
wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately
a plurality
of purine nucleotides are 2'-deoxy purine nucleotides), wherein any
nucleotides
comprising a 3'-terminal nucleotide overhang that are present in said sense
region are 2'
deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising a sense region,
wherein any
(e.g., one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-
2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-
2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any (e.g., one or more
or all)
purine nucleotides present in the sense region are 2'-O-methyl purine
nucleotides (e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides).
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising a sense region,
wherein any
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(e.g., one or more or all) pyrimidine nucleotides present in the sense region
are 2'-deoxy-
2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are
2'-deoxy-
2'-fluoro pyrimidine nucleotides or alternately a plurality of pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrirnidine nucleotides), wherein any (e.g., one or more or
all) purine
nucleotides present in the sense region are 2'-O-methyl purine nucleotides
(e.g., wherein
all purine nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of
purine nucleotides are 2'-O-methyl purine nucleotides), and wherein any
nucleotides
comprising a 3'-terminal nucleotide overhang that are present in said sense
region are 2'-
deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising an antisense region,
wherein
any (e.g., one or more or all) pyrimidine nucleotides present in the antisense
region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any
(e.g., one or
more or all) purine nucleotides present in the antisense region are 2'-O-
methyl purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl purine
nucleotides).
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising an antisense region,
wherein
any (e.g., one or more or all) pyrimidine nucleotides present in the antisense
region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), wherein any (e.g.,
one or
more or all) purine nucleotides present in the antisense region are 2'-O-
methyl purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl purine
nucleotides), and
wherein any nucleotides comprising a 3'-terminal nucleotide overhang that are
present in
said antisense region are 2'-deoxy nucleotides.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising an antisense region,
wherein
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any (e.g., one or more or all) pyrimidine nucleotides present in the antisense
region are
2'-deoxy-2'-fluoro pyrirnidine nucleotides (e.g., wherein all pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any
(e.g., one or
more or all) purine nucleotides present in the antisense region are 2'-deoxy
purine
nucleotides (e.g., wherein all purine nucleotides are 2'-deoxy purine
nucleotides or
alternately a plurality of purine nucleotides are 2'-deoxy purine
nucleotides).
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention comprising an antisense region,
wherein
any (e.g., one or more or all) pyrimidine nucleotides present in the antisense
region are
2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are
2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any
(e.g., one or
more or all) purine nucleotides present in the antisense region are 2'-O-
methyl purine
nucleotides (e.g., wherein all purine nucleotides are 2'-O-methyl purine
nucleotides or
alternately a plurality of purine nucleotides are 2'-O-methyl purine
nucleotides).
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid (siNA) molecule of the invention capable of mediating RNA
interference
(RNAi) against GPRA and/or AAAl inside a cell or reconstituted in vitro system
comprising a sense region, wherein one or more pyrimidine nucleotides present
in the
sense region are 2'-deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides or alternately a
plurality of
pyrimidine nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and one
or more
purine nucleotides present in the sense region are 2'-deoxy purine nucleotides
(e.g.,
wherein all purine nucleotides are 2'-deoxy purine nucleotides or alternately
a plurality
of purine nucleotides are 2'-deoxy purine nucleotides), and an antisense
region, wherein
one or more pyrimidine nucleotides present in the antisense region are 2'-
deoxy-2'-fluoro
pyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are 2'-deoxy-
2'-fluoro
pyrimidine nucleotides or alternately a plurality of pyrimidine nucleotides
are 2'-deoxy-
2'-fluoro pyrimidine nucleotides), and one or more purine nucleotides present
in the
antisense region are 2'-O-methyl purine nucleotides (e.g., wherein all purine
nucleotides
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are 2'-O-methyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-
O-methyl purine nucleotides). The sense region and/or the antisense region can
have a
terminal cap modification, such as any modification described herein or shown
in Figure
10, that is optionally present at the 3'-end, the 5'-end, or both of the 3'
and 5'-ends of the
sense and/or antisense sequence. The sense and/or antisense region can
optionally
further comprise a 3'-terminal nucleotide overhang having about 1 to about 4
(e.g., about
1, 2, 3, or 4) 2'-deoxynucleotides. The overhang nucleotides can further
comprise one or
more (e.g., about 1, 2, 3, 4 or more) phosphorothioate, phosphonoacetate,
and/or
thiophosphonoacetate internucleotide linkages. Non-limiting examples of these
chemically-modified siNAs are shown in Figures 4 and 5 and Tables III and IV
herein.
In any of these described embodiments, the purine nucleotides present in the
sense
region are alternatively 2'-O-methyl purine nucleotides (e.g., wherein all
purine
nucleotides are 2'-O-methyl purine nucleotides or alternately a plurality of
purine
nucleotides are 2'-O-methyl purine nucleotides) and one or more purine
nucleotides
present in the antisense region are 2'-O-methyl purine nucleotides (e.g.,
wherein all
purine nucleotides are 2'-O-methyl purine nucleotides or alternately a
plurality of purine
nucleotides are 2'-O-methyl purine nucleotides). Also, in any of these
embodiments, one
or more purine nucleotides present in the sense region are alternatively
purine
ribonucleotides (e.g., wherein all purine nucleotides are purine
ribonucleotides or
alternately a plurality of purine nucleotides are purine ribonucleotides) and
any purine
nucleotides present in the antisense region are 2'-O-methyl purine nucleotides
(e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides).
Additionally, in any
of these embodiments, one or more purine nucleotides present in the sense
region and/or
present in the antisense region are alternatively selected from the group
consisting of 2'-
deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-methoxyethyl
nucleotides,
4'-thionucleotides, and 2'-O-methyl nucleotides (e.g., wherein all purine
nucleotides are
selected from the group consisting of 2'-deoxy nucleotides, locked nucleic
acid (LNA)
nucleotides, 2'-methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl
nucleotides or alternately a plurality of purine nucleotides are selected from
the group
consisting of 2'-deoxy nucleotides, locked nucleic acid (LNA) nucleotides, 2'-
methoxyethyl nucleotides, 4'-thionucleotides, and 2'-O-methyl nucleotides).
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In another embodiment, any modified nucleotides present in the siNA molecules
of
the invention, preferably in the antisense strand of the siNA molecules of the
invention,
but also optionally in the sense and/or both antisense and sense strands,
comprise
modified nucleotides having properties or characteristics similar to naturally
occurring
ribonucleotides. For example, the invention features siNA molecules including
modified
nucleotides having a Northern conformation (e.g., Northern pseudorotation
cycle, see for
example Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed.,
1984). As
such, chemically modified nucleotides present in the siNA molecules of the
invention,
preferably in the antisense strand of the siNA molecules of the invention, but
also
optionally in the sense and/or both antisense and sense strands, are resistant
to nuclease
degradation while at the same time maintaining the capacity to mediate RNAi.
Non-
limiting examples of nucleotides having a northern configuration include
locked nucleic
acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-nibofuranosyl)
nucleotides); 2'-
methoxyethoxy (MOE) nucleotides; 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro
nucleotides, 2'-deoxy-2'-chloro nucleotides, 2'-azido nucleotides, and 2'-O-
methyl
nucleotides.
In one embodiment, the sense strand of a double stranded siNA molecule of the
invention comprises a terminal cap moiety, (see for example Figure 10) such as
an
inverted deoxyabaisc moiety, at the 3'-end, 5'-end, or both 3' and 5'-ends of
the sense
strand.
In one embodiment, the invention features a chemically-modified short
interfering
nucleic acid molecule (siNA) capable of mediating RNA interference (RNAi)
against
GPRA and/or AAA1 inside a cell or reconstituted in vitro system, wherein the
chemical
modification comprises a conjugate covalently attached to the chemically-
modified siNA
molecule. Non-limiting examples of conjugates contemplated by- the invention
include
conjugates and ligands described in Vargeese et al., USSN 10/427,160, filed
April 30,
2003, incorporated by reference herein in its entirety, including the
drawings. In another
embodiment, the conjugate is covalently attached to the chemically-modified
siNA
molecule via a biodegradable linker. In one embodiment, the conjugate molecule
is
attached at the 3'-end of either the sense strand, the antisense strand, or
both strands of
the chemically-modified siNA molecule. In another embodiment, the conjugate
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molecule is attached at the 5'-end of either the sense strand, the antisense
strand, or both
strands of the chemically-modified siNA molecule. In yet another embodiment,
the
conjugate molecule is attached both the 3'-end and 5'-end of either the sense
strand, the
antisense strand, or both strands of the chemically-modified siNA molecule, or
any
combination thereof. In one embodiment, a conjugate molecule of the invention
comprises a molecule that facilitates delivery of a chemically-modified siNA
molecule
into a biological system, such as a cell. In another embodiment, the conjugate
molecule
attached to the chemically-modified siNA molecule is a polyethylene glycol,
human
serum albumin, or a ligand for a cellular receptor that can mediate cellular
uptake.
Examples of specific conjugate molecules contemplated by the instant invention
that can
be attached to chemically-modified siNA molecules are described in Vargeese et
al.,
LT.S. Serial No. 10/201,394, filed July 22, 2002 incorporated by reference
herein. The
type of conjugates used and the extent of conjugation of siNA molecules of the
invention
can be evaluated for improved pharmacokinetic profiles, bioavailability,
and/or stability
of siNA constructs while at the same time maintaining the ability of the siNA
to mediate
RNAi activity. As such, one skilled in the art can screen siNA constructs that
are
modified with various conjugates to determine whether the siNA conjugate
complex
possesses improved properties while maintaining the ability to mediate RNAi,
for
example in animal models as are generally known in the art.
In one embodiment, the invention features a short interfering nucleic acid
(siNA)
molecule of the invention, wherein the siNA further comprises a nucleotide,
non-
nucleotide, or mixed nucleotide/non-nucleotide linker that joins the sense
region of the
siNA to the antisense region of the siNA. In one embodiment, a nucleotide
linker of the
invention can be a linker of >_ 2 nucleotides in length, for example about 3,
4, 5, 6, 7, 8,
9, or 10 nucleotides in length. In another embodiment, the nucleotide linker
can be a
nucleic acid aptamer. By "aptamer" or "nucleic acid aptamer" as used herein is
meant a
nucleic acid molecule that binds specifically to a target molecule wherein the
nucleic
acid molecule has sequence that comprises a sequence recognized by the target
molecule
in its natural setting. Alternately, an aptamer can be a nucleic acid molecule
that binds to
a target molecule where the target molecule does not naturally bind to a
nucleic acid.
The target molecule can be any molecule of interest. For example, the aptamer
can be
used to bind to a ligand-binding domain of a protein, thereby preventing
interaction of
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the naturally occurring ligand with the protein. This is a non-limiting
example and those
in the art will recognize that other embodiments can be readily generated
using
techniques generally known in the art. (See, for example, Gold et al., 1995,
Annu. Rev.
Bioclaem., 64, 763; Brody and Gold, 2000, J. Biotechraol., 74, 5; Sun, 2000,
Curr. Opita.
Mol. Tlaer., 2, 100; Kusser, 2000, J. Biotechraol., 74, 27; Hermann and Patel,
2000,
Science, 287, 820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)
In yet another embodiment, a non-nucleotide linker of the invention comprises
abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,
lipid,
polyhydrocarbon, or other polymeric compounds (e.g. polyethylene glycols such
as those
having between 2 and 100 ethylene glycol units). Specific examples include
those
described by Seela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic
Acids Res.
1987, 15:3113; Cload and Schepartz, J. Am. Claem. Soc. 1991, 113:6324;
Richardson and
Schepartz, J. Ana. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res.
1993,
21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic Acids Res.
1990,
18:6353; McCurdy et al., Nucleosides & Nucleotides 1991, 10:287; Jschke et
al.,
Tetrahedron Lett. 1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold
et al.,
International Publication No. WO 89/02439; Usman et al., International
Publication No.
WO 95/06731; Dudycz et al., International Publication No. WO 95/11910 and
Ferentz
and Verdine, .I. Am. Claem. S'oc. 1991, 113:4000, all hereby incorporated by
reference
herein. A "non-nucleotide" further means any group or compound that can be
incorporated into a nucleic acid chain in the place of one or more nucleotide
units,
including either sugar and/or phosphate substitutions, and allows the
remaining bases to
exhibit their enzymatic activity. The group or compound can be abasic in that
it does not
contain a commonly recognized nucleotide base, such as adenosine, guanine,
cytosine,
uracil or thymine, for example at the C 1 position of the sugar.
In one embodiment, the invention features a short interfering nucleic acid
(siNA)
molecule capable of mediating RNA interference (RNAi) inside a cell or
reconstituted ira
vitro system, wherein one or both strands of the siNA molecule that are
assembled from
two separate oligonucleotides do not comprise any ribonucleotides. For
example, a siNA
molecule can be assembled from a single oligonculeotide where the sense and
antisense
regions of the siNA comprise separate oligonucleotides that do not have any
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ribonucleotides (e.g., nucleotides having a 2'-OH group) present in the
oligonucleotides.
In another example, a siNA molecule can be assembled from a single
oligonculeotide
where the sense and antisense regions of the siNA are linked or circularized
by a
nucleotide or non-nucleotide linker as described herein, wherein the
oligonucleotide does
not have any ribonucleotides (e.g., nucleotides having a 2'-OH group) present
in the
oligonucleotide. Applicant has surprisingly found that the presense of
ribonucleotides
(e.g., nucleotides having a 2'-hydroxyl group) within the siNA molecule is not
required
or essential to support RNAi activity. As such, in one embodiment, all
positions within
the siNA can include chemically modified nucleotides and/or non-nucleotides
such as
nucleotides and or non-nucleotides having Formula I, II, III, IV, V, VI, or
VII or any
combination thereof to the extent that the ability of the siNA molecule to
support RNAi
activity in a cell is maintained.
In one embodiment, a siNA molecule of the invention is a single stranded siNA
molecule that mediates RNAi activity in a cell or reconstituted in vitro
system
comprising a single stranded polynucleotide having complementarity to a target
nucleic
acid sequence. In another embodiment, the single stranded siNA molecule of the
invention comprises a 5'-terminal phosphate group. In another embodiment, the
single
stranded siNA molecule of the invention comprises a 5'-terminal phosphate
group and a
3'-terminal phosphate group (e.g., a 2',3'-cyclic phosphate). In another
embodiment, the
single stranded siNA molecule of the invention comprises about 15 to about 30
(e.g.,
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)
nucleotides. In yet
another embodiment, the single stranded siNA molecule of the invention
comprises one
or more chemically modified nucleotides or non-nucleotides described herein.
For
example, all the positions within the siNA molecule can include chemically-
modified
nucleotides such as nucleotides having any of Formulae I-VII, or any
combination
thereof to the extent that the ability of the siNA molecule to support RNAi
activity in a
cell is maintained.
In one embodiment, a siNA molecule of the invention is a single stranded siNA
molecule that mediates RNAi activity in a cell or reconstituted in vitro
system
comprising a single stranded polynucleotide having complementarity to a target
nucleic
acid sequence, wherein one or more pyrimidine nucleotides present in the siNA
are 2'-
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deoxy-2'-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidine
nucleotides are 2'-
deoxy-2'-fluoro pyrimidine nucleotides or alternately a plurality of
pyrimidine
nucleotides are 2'-deoxy-2'-fluoro pyrimidine nucleotides), and wherein any
purine
nucleotides present in the antisense region are 2'-O-methyl purine nucleotides
(e.g.,
wherein all purine nucleotides are 2'-O-methyl purine nucleotides or
alternately a
plurality of purine nucleotides are 2'-O-methyl purine nucleotides), and a
terminal cap
modification, such as any modification described herein or shown in Figure 10,
that is
optionally present at the 3'-end, the 5'-end, or both of the 3' and 5'-ends of
the antisense
sequence. The siNA optionally further comprises about 1 to about 4 or more
(e.g., about
l, 2, 3, 4 or more) terminal 2'-deoxynucleotides at the 3'-end of the siNA
molecule,
wherein the terminal nucleotides can further comprise one or more (e.g., 1, 2,
3, 4 or
more) phosphorothioate, phosphonoacetate, and/or thiophosphonoacetate
internucleotide
linkages, and wherein the siNA optionally further comprises a terminal
phosphate group,
such as a 5'-terminal phosphate group. In any of these embodiments, any purine
nucleotides present in the antisense region are alternatively 2'-deoxy purine
nucleotides
(e.g., wherein all purine nucleotides are 2'-deoxy purine nucleotides or
alternately a
plurality of purine nucleotides are 2'-deoxy purine nucleotides). Also, in any
of these
embodiments, any purine nucleotides present in the siNA (i.e., purine
nucleotides present
in the sense and/or antisense region) can alternatively be locked nucleic acid
(LNA)
nucleotides (e.g., wherein all purine nucleotides are LNA nucleotides or
alternately a
plurality of purine nucleotides are LNA nucleotides). Also, in any of these
embodiments, any purine nucleotides present in the siNA are alternatively 2'-
methoxyethyl purine nucleotides (e.g., wherein all purine nucleotides are 2'-
methoxyethyl purine nucleotides or alternately a plurality of purine
nucleotides are 2'-
methoxyethyl purine nucleotides). In another embodiment, any modified
nucleotides
present in the single stranded siNA molecules of the invention comprise
modified
nucleotides having properties or characteristics similar to naturally
occurring
ribonucleotides. For example, the invention features siNA molecules including
modified
nucleotides having a Northern conformation (e.g., Northern pseudorotation
cycle, see for
example Saenger, P~iraciples of Nucleic Acid Stfzccture, Springer-Verlag ed.,
1984). As
such, chemically modified nucleotides present in the single stranded siNA
molecules of
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the invention are preferably resistant to nuclease degradation while at the
same time
maintaining the capacity to mediate RNAi.
In one embodiment, a siNA molecule of the invention comprises chemically
modified nucleotides or non-nucleotides (e.g., having any of Formulae I-VII,
such as 2'-
deoxy, 2'-deoxy-2'-fluoro, or 2'-O-methyl nucleotides) at alternating
positions within
one or more strands or regions of the siNA molecule. For example, such
chemical
modifications can be introduced at every other position of a RNA based siNA
molecule,
starting at either the first or second nucleotide from the 3'-end or 5'-end of
the siNA. In
a non-limiting example, a double stranded siNA molecule of the invention in
which each
strand of the siNA is 21 nucleotides in length is featured wherein positions
1, 3, 5, 7, 9,
11, 13, 15, 17, 19 and 21 of each strand are chemically modified (e.g., with
compounds
having any of Formulae 1-VII, such as such as 2'-deoxy, 2'-deoxy-2'-fluoro, or
2'-O-
methyl nucleotides). In another non-limiting example, a double stranded siNA
molecule
of the invention in which each strand of the siNA is 21 nucleotides in length
is featured
wherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strand are
chemically
modified (e.g., with compounds having any of Formulae 1-VII, such as such as
2'-deoxy,
2'-deoxy-2'-fluoro, or 2'-O-methyl nucleotides). Such siNA molecules can
further
comprise terminal cap moieties andlor backbone modifications as described
herein.
In one embodiment, the invention features a method for modulating the
expression
of a GPRA and/or AAAl gene within a cell comprising: (a) synthesizing a siNA
molecule of the invention, which can be chemically-modified, wherein one of
the siNA
strands comprises a sequence complementary to RNA of the GPRA and/or AAA1
gene;
and (b) introducing the siNA molecule into a cell under conditions suitable to
modulate
the expression of the GPR.A and/or AAAl gene in the cell.
In one embodiment, the invention features a method for modulating the
expression
of a GPRA and/or AAAl gene within a cell comprising: (a) synthesizing a siNA
molecule of the invention, which can be chemically-modified, wherein one of
the siNA
strands comprises a sequence complementary to RNA of the GPRA and/or AAA1 gene
and wherein the sense strand sequence of the siNA comprises a sequence
identical or
substantially similar to the sequence of the target RNA; and (b) introducing
the siNA
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molecule into a cell under conditions suitable to modulate the expression of
the GPR.A
and/or AAAl gene in the cell.
In another embodiment, the invention features a method for modulating the
expression of more than one GPRA and/or AAAI gene within a cell comprising:
(a)
synthesizing siNA molecules of the invention, which can be chemically-
modified,
wherein one of the siNA strands comprises a sequence complementary to RNA of
the
GPRA and/or AAA1 genes; and (b) introducing the siNA molecules into a cell
under
conditions suitable to modulate the expression of the GPRA and/or AAA1 genes
in the
cell.
In another embodiment, the invention features a method for modulating the
expression of two or more GPRA and/or AAAl genes within a cell comprising: (a)
synthesizing one or more siNA molecules of the invention, which can be
chemically-
modified, wherein the siNA strands comprise sequences complementary to RNA of
the
GPRA and/or AAA1 genes and wherein the sense strand sequences of the siNAs
comprise sequences identical or substantially similar to the sequences of the
target
RNAs; and (b) introducing the siNA molecules into a cell under conditions
suitable to
modulate the expression of the GPRA andlor AAAl genes in the cell.
In another embodiment, the invention features a method for modulating the
expression of more than one GPRA and/or AAAl gene within a cell comprising:
(a)
synthesizing a siNA molecule of the invention, which can be chemically-
modified,
wherein one of the siNA strands comprises a sequence complementary to RNA of
the
GPRA and/or AAAl gene and wherein the sense strand sequence of the siNA
comprises
a sequence identical or substantially similar to the sequences of the target
RNAs; and (b)
introducing the siNA molecule into a cell under conditions suitable to
modulate the
expression of the GPRA andlor AAA1 genes in the cell.
In one embodiment, siNA molecules of the invention are used as reagents in ex
vivo applications. For example, siNA reagents are introduced into tissue or
cells that are
transplanted into a subject for therapeutic effect. The cells and/or tissue
can be derived
from an organism or subject that later receives the explant, or can be derived
from
another organism or subject prior to transplantation. The siNA molecules can
be used to
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modulate the expression of one or more genes in the cells or tissue, such that
the cells or
tissue obtain a desired phenotype or are able to perform a function when
transplanted in
vivo. In one embodiment, certain target cells from a patient are extracted.
These
extracted cells are contacted with siNAs targeting a specific nucleotide
sequence within
the cells under conditions suitable for uptake of the siNAs by these cells
(e.g. using
delivery reagents such as cationic lipids, liposomes and the like or using
techniques such
as electroporation to facilitate the delivery of siNAs into cells). The cells
are then
reintroduced back into the same patient or other patients. In one embodiment,
the
invention features a method of modulating the expression of a GPRA and/or AAAl
gene
in a tissue explant comprising: (a) synthesizing a siNA molecule of the
invention, which
can be chemically-modified, wherein one of the siNA strands comprises a
sequence
complementary to RNA of the GPRA and/or AAAl gene; and (b) introducing the
siNA
molecule into a cell of the tissue explant derived from a particular organism
under
conditions suitable to modulate the expression of the GPRA and/or AAAl gene in
the
tissue explant. In another embodiment, the method further comprises
introducing the
tissue explant back into the organism the tissue was derived from or into
another
organism under conditions suitable to modulate the expression of the GPRA
and/or
AAAI gene in that organism.
In one embodiment, the invention features a method of modulating the
expression
of a GPRA and/or AAA1 gene in a tissue explant comprising: (a) synthesizing a
siNA
molecule of the invention, which can be chemically-modified, wherein one of
the siNA
strands comprises a sequence complementary to RNA of the GPRA and/or AAAl gene
and wherein the sense strand sequence of the siNA comprises a sequence
identical or
substantially similar to the sequence of the target RNA; and (b) introducing
the siNA
molecule into a cell of the tissue explant derived from a particular organism
under
conditions suitable to modulate the expression of the GPRA and/or AAAl gene in
the
tissue explant. In another embodiment, the method further comprises
introducing the
tissue explant back into the organism the tissue was derived from or into
another
organism under conditions suitable to modulate the expression of the GPRA
and/or
AAA1 gene in that organism.
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In another embodiment, the invention features a method of modulating the
expression of more than one GPRA and/or AAAl gene in a tissue explant
comprising:
(a) synthesizing siNA molecules of the invention, which can be chemically-
modified,
wherein one of the siNA strands comprises a sequence complementary to RNA of
the
GPRA and/or AAA1 genes; and (b) introducing the siNA molecules into a cell of
the
tissue explant derived from a particular organism under conditions suitable to
modulate
the expression of the GPRA and/or AAAI genes in the tissue explant. In another
embodiment, the method further comprises introducing the tissue explant back
into the
organism the tissue was derived from or into another organism under conditions
suitable
to modulate the expression of the GPRA and/or AAAl genes in that organism.
In one embodiment, the invention features a method of modulating the
expression
of a GPRA and/or AAA1 gene in a subject or organism comprising: (a)
synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein one
of the
siNA strands comprises a sequence complementary to RNA of the GPRA and/or AAAl
gene; and (b) introducing the siNA molecule into the subject or organism under
conditions suitable to modulate the expression of the GPRA and/or AAAl gene in
the
subject or organism. The level of GPRA and/or AAA1 protein or RNA can be
determined using various methods well-known in the art.
In another embodiment, the invention features a method of modulating the
expression of more than one GPRA and/or AAA1 gene in a subject or organism
comprising: (a) synthesizing siNA molecules of the invention, which can be
chemically-
modified, wherein one of the siNA strands comprises a sequence complementary
to RNA
of the GPRA and/or AAA1 genes; and (b) introducing the siNA molecules into the
subject or organism under conditions suitable to modulate the expression of
the GPRA
and/or AAAl genes in the subject or organism. The level of GPRA and/or AAAl
protein or RNA can be determined as is known in the art.
In one embodiment, the invention features a method for modulating the
expression
of a GPRA and/or AAAl gene within a cell comprising: (a) synthesizing a siNA
molecule of the invention, which can be chemically-modified, wherein the siNA
comprises a single stranded sequence having complementarity to RNA of the GPRA
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and/or AAA1 gene; and (b) introducing the siNA molecule into a cell under
conditions
suitable to modulate the expression of the GPRA and/or AAA1 gene in the cell.
In another embodiment, the invention features a method for modulating the
expression of more than one GPRA and/or AAAl gene within a cell comprising:
(a)
synthesizing siNA molecules of the invention, which can be chemically-
modified,
wherein the siNA comprises a single stranded sequence having complementarity
to RNA
of the GPRA and/or AAAl gene; and (b) contacting the cell in vitro or in vivo
with the
siNA molecule under conditions suitable to modulate the expression of the GPRA
and/or
AAAl genes in the cell.
In one embodiment, the invention features a method of modulating the
expression
of a GPRA andlor AAA1 gene in a tissue explant comprising: (a) synthesizing a
siNA
molecule of the invention, which can be chemically-modified, wherein the siNA
comprises a single stranded sequence having complementarity to RNA of the GPRA
and/or AAAl gene; and (b) contacting a cell of the tissue explant derived from
a
particular subject or organism with the siNA molecule under conditions
suitable to
modulate the expression of the GPRA and/or AAA1 gene in the tissue explant. In
another embodiment, the method further comprises introducing the tissue
explant back
into the subject or organism the tissue was derived from or into another
subject or
organism under conditions suitable to modulate the expression of the GPRA
and/or
AAAl gene in that subject or organism.
In another embodiment, the invention features a method of modulating the
expression of more than one GPRA and/or AAA1 gene in a tissue explant
comprising:
(a) synthesizing siNA molecules of the invention, which can be chemically-
modified,
wherein the siNA comprises a single stranded sequence having complementarity
to RNA
of the GPRA andlor AAAl gene; and (b) introducing the siNA molecules into a
cell of
the tissue explant derived from a particular subject or organism under
conditions suitable
to modulate the expression of the GPRA and/or AAA1 genes in the tissue
explant. In
another embodiment, the method further comprises introducing the tissue
explant back
into the subject or organism the tissue was derived from or into another
subject or
organism under conditions suitable to modulate the expression of the GPR.A
and/or
AAA1 genes in that subject or organism.
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In one embodiment, the invention features a method of modulating the
expression
of a GPRA and/or AAAl gene in a subject or organism comprising: ,(a)
synthesizing a
siNA molecule of the invention, which can be chemically-modified, wherein the
siNA
comprises a single stranded sequence having complementarity to RNA of the GPRA
and/or AAAl gene; and (b) introducing the siNA molecule into the subject or
organism
under conditions suitable to modulate the expression of the GPRA and/or AAA1
gene in
the subject or organism.
In another embodiment, the invention features a method of modulating the
expression of more than one GPRA and/or AAA1 gene in a subject or organism
comprising: (a) synthesizing siNA molecules of the invention, which can be
chemically-
modified, wherein the siNA comprises a single stranded sequence having
complementarity to RNA of the GPRA and/or AAA1 gene; and (b) introducing the
siNA
molecules into the subject or organism under conditions suitable to modulate
the
expression of the GPRA and/or AAAI genes in the subject or organism.
In one embodiment, the invention features a method of modulating the
expression
of a GPRA and/or AAA1 gene in a subject or organism comprising contacting the
subject or organism with a siNA molecule of the invention under conditions
suitable to
modulate the expression of the GPRA and/or AAAl gene in the subject or
organism.
In one embodiment, the invention features a method for treating or preventing
an
inflammatory disease, disorder, or condition in a subject or organism
comprising
contacting the subject or organism with a siNA molecule of the invention under
conditions suitable to modulate the expression of the GPRA and/or AAA1 gene in
the
subject or organism.
In one embodiment, the invention features a method for treating or preventing
a
respiratory disease, disorder, and/or condition in a subject or organism
comprising
contacting the subject or organism with a siNA molecule of the invention under
conditions suitable to modulate the expression of the GPRA and/or AAAl gene in
the
subj ect or organism.
In one embodiment, the invention features a method for treating or preventing
asthma in a subject or organism comprising contacting the subject or organism
with a
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siNA molecule of the invention under conditions suitable to modulate the
expression of
the GPRA and/or AAA1 gene in the subject or organism.
In another embodiment, the invention features a method of modulating the
expression of more than one GPRA andlor AAA1 genes in a subject or organism
comprising contacting the subject or organism with one or more siNA molecules
of the
invention under conditions suitable to modulate the expression of the GPRA
and/or
AAA1 genes in the subject or organism.
The siNA molecules of the invention can be designed to down regulate or
inhibit
target (e.g., GPRA and/or AAAl) gene expression through RNAi targeting of a
variety
of RNA molecules. In one embodiment, the siNA molecules of the invention are
used to
target various RNAs corresponding to a target gene. Non-limiting examples of
such
RNAs include messenger RNA (mRNA), alternate RNA splice variants of target
gene(s),
post-transcriptionally modified RNA of target gene(s), pre-mRNA of target
gene(s),
andlor RNA templates. If alternate splicing produces a family of transcripts
that are
distinguished by usage of appropriate axons, the instant invention can be used
to inhibit
gene expression through the appropriate axons to specifically inhibit or to
distinguish
among the functions of gene family members. For example, a protein that
contains an
alternatively spliced transmembrane domain can be expressed in both membrane
bound
and secreted forms. Use of the invention to target the axon containing the
transmembrane domain can be used to determine the functional consequences of
pharmaceutical targeting of membrane bound as opposed to the secreted form of
the
protein. Non-limiting examples of applications of the invention relating to
targeting
these RNA molecules include therapeutic pharmaceutical applications,
pharmaceutical
discovery applications, molecular diagnostic and gene function applications,
and gene
mapping, for example using single nucleotide polymorphism mapping with siNA
molecules of the invention. Such applications can be implemented using known
gene
sequences or from partial sequences available from an expressed sequence tag
(EST).
In another embodiment, the siNA molecules of the invention are used to target
conserved sequences corresponding to a gene family or gene families such as
GPRA
and/or AAA1 family genes. As such, siNA molecules targeting multiple GPRA
and/or
AAA1 targets can provide increased therapeutic effect. In addition, siNA can
be used to
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characterize pathways of gene function in a variety of applications. For
example, the
present invention can be used to inhibit the activity of target genes) in a
pathway to
determine the function of uncharacterized genes) in gene function analysis,
mRNA
function analysis, or translational analysis. The invention can be used to
determine
potential target gene pathways involved in various diseases and conditions
toward
pharmaceutical development. The invention can be used to understand pathways
of gene
expression involved in, for example inflammatory and/or respiratory diseases,
disorders
and conditions.
In one embodiment, siNA molecules) and/or methods of the invention are used to
down regulate the expression of genes) that encode RNA referred to by Genbank
Accession, for example, GPRA and/or AAA1 genes encoding RNA sequences)
referred
to herein by Genbank Accession number, for example, Genbank Accession Nos.
shown
in Table I.
In one embodiment, the invention features a method comprising: (a) generating
a
library of siNA constructs having a predetermined complexity; and (b) assaying
the siNA
constructs of (a) above, under conditions suitable to determine RNAi target
sites within
the target RNA sequence. In one embodiment, the siNA molecules of (a) have
strands of
a fixed length, for example, about 23 nucleotides in length. In another
embodiment, the
siNA molecules of (a) are of differing length, for example having strands of
about 15 to
about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, or 30)
nucleotides in length. In one embodiment, the assay can comprise a
reconstituted in
vitf-o siNA assay as described herein. In another embodiment, the assay can
comprise a
cell culture system in which target RNA is expressed. In another embodiment,
fragments
of target RNA are analyzed for detectable levels of cleavage, for example by
gel
electrophoresis, northern blot analysis, or RNAse protection assays, to
determine the
most suitable target sites) within the target RNA sequence. The target RNA
sequence
can be obtained as is known in the art, for example, by cloning and/or
transcription for in
vitro systems, and by cellular expression in in vivo systems.
In one embodiment, the invention features a method comprising: (a) generating
a
randomized library of siNA constructs having a predetermined complexity, such
as of 4N,
where N represents the number of base paired nucleotides in each of the siNA
construct
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strands (eg. for a siNA construct having 21 nucleotide sense and antisense
strands with
19 base pairs, the complexity would be 419); and (b) assaying the siNA
constructs of (a)
above, under conditions suitable to determine RNAi target sites within the
target GPRA
and/or AAA1 RNA sequence. In another embodiment, the siNA molecules of (a)
have
strands of a fixed length, for example about 23 nucleotides in length. In yet
another
embodiment, the siNA molecules of (a) are of differing length, for example
having
strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26,
27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay can
comprise a
reconstituted in vitro siNA assay as described in Example 6 herein. In another
embodiment, the assay can comprise a cell culture system in which target RNA
is
expressed. In another embodiment, fragments of GPRA and/or AAAI RNA are
analyzed for detectable levels of cleavage, for example, by gel
electrophoresis, northern
blot analysis, or RNAse protection assays, to determine the most suitable
target sites)
within the target GPRA and/or AAA1 RNA sequence. The target GPRA and/or AAAl
RNA sequence can be obtained as is known in the art, for example, by cloning
and/or
transcription for in vitro systems, and by cellular expression in ih vivo
systems.
In another embodiment, the invention features a method comprising: (a)
analyzing
the sequence of a RNA target encoded by a target gene; (b) synthesizing one or
more sets
of siNA molecules having sequence complementary to one or more regions of the
RNA
of (a); and (c) assaying the siNA molecules of (b) under conditions suitable
to determine
RNAi targets within the target RNA sequence. In one embodiment, the siNA
molecules
of (b) have strands of a fixed length, for example about 23 nucleotides in
length. In
another embodiment, the siNA molecules of (b) are of differing length, for
example
having strands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,
21, 22, 23, 24,
25, 26, 27, 28, 29, or 30) nucleotides in length. In one embodiment, the assay
can
comprise a reconstituted in vitro siNA assay as described herein. In another
embodiment, the assay can comprise a cell culture system in which target RNA
is
expressed. Fragments of target RNA are analyzed for detectable levels of
cleavage, for
example by gel electrophoresis, northern blot analysis, or RNAse protection
assays, to
determine the most suitable target sites) within the target RNA sequence. The
target
RNA sequence can be obtained as is known in the art, for example, by cloning
and/or
transcription for in vitro systems, and by expression in in vivo systems.
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By "target site" is meant a sequence within a target RNA that is "targeted"
for
cleavage mediated by a siNA construct which contains sequences within its
antisense
region that are complementary to the target sequence.
By "detectable level of cleavage" is meant cleavage of target RNA (and
formation
of cleaved product RNAs) to an extent sufficient to discern cleavage products
above the
background of RNAs produced by random degradation of the target RNA.
Production of
cleavage products from 1-5% of the target RNA is sufficient to detect above
the
background for most methods of detection.
In one embodiment, the invention features a composition comprising a siNA
molecule of the invention, which can be chemically-modified, in a
pharmaceutically
acceptable carrier or diluent. In another embodiment, the invention features a
pharmaceutical composition comprising siNA molecules of the invention, which
can be
chemically-modified, targeting one or more genes in a pharmaceutically
acceptable
carrier or diluent. In another embodiment, the invention features a method for
diagnosing a disease or condition in a subject comprising administering to the
subject a
composition of the invention under conditions suitable for the diagnosis of
the disease or
condition in the subject. In another embodiment, the invention features a
method for
treating or preventing a disease or condition in a subject, comprising
administering to the
subject a composition of the invention under conditions suitable for the
treatment or
prevention of the disease or condition in the subject, alone or in conjunction
with one or
more other therapeutic compounds. In yet another embodiment, the invention
features a
method for treating or preventing inflammatory andlor respiratory diseases,
disorders and
conditions in a subject or organism comprising administering to the subject a
composition of the invention under conditions suitable for the treatment or
prevention of
inflammatory and/or respiratory diseases, disorders and conditions in the
subject or
organism.
In another embodiment, the invention features a method for validating a GPRA
andlor AAA1 gene target, comprising: (a) synthesizing a siNA molecule of the
invention, which can be chemically-modified, wherein one of the siNA strands
includes
a sequence complementary to RNA of a GPRA and/or AAAl target gene; (b)
introducing the siNA molecule into a cell, tissue, subject, or organism under
conditions
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suitable for modulating expression of the GPRA and/or AAAl target gene in the
cell,
tissue, subject, or organism; and (c) determining the function of the gene by
assaying for
any phenotypic change in the cell, tissue, subject, or organism.
In another embodiment, the invention features a method for validating a GPRA
and/or AAAl target comprising: (a) synthesizing a siNA molecule of the
invention,
which can be chemically-modified, wherein one of the siNA strands includes a
sequence
complementary to RNA of a GPRA and/or AAA1 target gene; (b) introducing the
siNA
molecule into a biological system under conditions suitable for modulating
expression of
the GPRA and/or AAAl target gene in the biological system; and (c) determining
the
function of the gene by assaying for any phenotypic change in the biological
system.
By "biological system" is meant, material, in a purified or unpurified form,
from
biological sources, including but not limited to human or animal, wherein the
system
comprises the components required for RNAi activity. The term "biological
system"
includes, for example, a cell, tissue, subject, or organism, or extract
thereof. 'The term
biological system also includes reconstituted RNAi systems that can be used in
an i~
vitro setting.
By "phenotypic change" is meant any detectable change to a cell that occurs in
response to contact or treatment with a nucleic acid molecule of the invention
(e.g.,
siNA). Such detectable changes include, but are not limited to, changes in
shape, size,
proliferation, motility, protein expression or RNA expression or other
physical or
chemical changes as can be assayed by methods known in the art. The detectable
change
can also include expression of reporter genes/molecules such as Green
Florescent Protein
(GFP) or various tags that are used to identify an expressed protein or any
other cellular
component that can be assayed.
In one embodiment, the invention features a kit containing a siNA molecule of
the
invention, which can be chemically-modified, that can be used to modulate the
expression of a GPRA and/or AAA1 target gene in a biological system,
including, for
example, in a cell, tissue, subject, or organism. In another embodiment, the
invention
features a kit containing more than one siNA molecule of the invention, which
can be
chemically-modified, that can be used to modulate the expression of more than
one
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GPRA and/or AAA1 target gene in a biological system, including, for example,
in a cell,
tissue, subject, or organism.
In one embodiment, the invention features a cell containing one or more siNA
molecules of the invention, which can be chemically-modified. In another
embodiment,
the cell containing a siNA molecule of the invention is a mammalian cell. In
yet another
embodiment, the cell containing a siNA molecule of the invention is a human
cell.
In one embodiment, the synthesis of a siNA molecule of the invention, which
can
be chemically-modified, comprises: (a) synthesis of two complementary strands
of the
siNA molecule; (b) annealing the two complementary strands together under
conditions
suitable to obtain a double-stranded siNA molecule. In another embodiment,
synthesis
of the two complementary strands of the siNA molecule is by solid phase
oligonucleotide
synthesis. In yet another embodiment, synthesis of the two complementary
strands of the
siNA molecule is by solid phase tandem oligonucleotide synthesis.
In one embodiment, the invention features a method for synthesizing a siNA
duplex molecule comprising: (a) synthesizing a first oligonucleotide sequence
strand of
the siNA molecule, wherein the first oligonucleotide sequence strand comprises
a
cleavable linker molecule that can be used as a scaffold for the synthesis of
the second
oligonucleotide sequence strand of the siNA; (b) synthesizing the second
oligonucleotide
sequence strand of siNA on the scaffold of the first oligonucleotide sequence
strand,
wherein the second oligonucleotide sequence strand further comprises a
chemical moiety
than can be used to purify the siNA duplex; (c) cleaving the linker molecule
of (a) under
conditions suitable for the two siNA oligonucleotide strands to hybridize and
form a
stable duplex; and (d) purifying the siNA duplex utilizing the chemical moiety
of the
second oligonucleotide sequence strand. In one embodiment, cleavage of the
linker
molecule in (c) above takes place during deprotection of the oligonucleotide,
for
example, under hydrolysis conditions using an alkylarnine base such as
rnethylamine. In
one embodiment, the method of synthesis comprises solid phase synthesis on a
solid
support such as controlled pore glass (CPG) or polystyrene, wherein the first
sequence of
(a) is synthesized on a cleavable linker, such as a succinyl linker, using the
solid support
as a scaffold. The cleavable linker in (a) used as a scaffold for synthesizing
the second
strand can comprise similar reactivity as the solid support derivatized
linker, such that
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cleavage of the solid support derivatized linker and the cleavable linker of
(a) takes place
concomitantly. In another embodiment, the chemical moiety of (b) that can be
used to
isolate the attached oligonucleotide sequence comprises a trityl group, for
example a
dimethoxytrityl group, which can be employed in a trityl-on synthesis strategy
as
described herein. In yet another embodiment, the chemical moiety, such as a
dimethoxytrityl group, is removed during purification, for example, using
acidic
conditions.
In a further embodiment, the method for siNA synthesis is a solution phase
synthesis or hybrid phase synthesis wherein both strands of the siNA duplex
are
synthesized in tandem using a cleavable linker attached to the first sequence
which acts a
scaffold for synthesis of the second sequence. Cleavage of the linker under
conditions
suitable for hybridization of the separate siNA sequence strands results in
formation of
the double-stranded siNA molecule.
In another embodiment, the invention features a method for synthesizing a siNA
duplex molecule comprising: (a) synthesizing one oligonucleotide sequence
strand of
the siNA molecule, wherein the sequence comprises a cleavable linker molecule
that can
be used as a scaffold for the synthesis of another oligonucleotide sequence;
(b)
synthesizing a second oligonucleotide sequence having complementarity to the
ftrst
sequence strand on the scaffold of (a), wherein the second sequence comprises
the other
strand of the double-stranded siNA molecule and wherein the second sequence
further
comprises a chemical moiety than can be used to isolate the attached
oligonucleotide
sequence; (c) purifying the product of (b) utilizing the chemical moiety of
the second
oligonucleotide sequence strand under conditions suitable for isolating the
full-length
sequence comprising both siNA oligonucleotide strands connected by the
cleavable
linker and under conditions suitable for the two siNA oligonucleotide strands
to
hybridize and form a stable duplex. In one embodiment, cleavage of the linker
molecule
in (c) above takes place during deprotection of the oligonucleotide, for
example, under
hydrolysis conditions. In another embodiment, cleavage of the linker molecule
in (c)
above takes place after deprotection of the oligonucleotide. In another
embodiment, the
method of synthesis comprises solid phase synthesis on a solid support such as
controlled
pore glass (CPG) or polystyrene, wherein the first sequence of (a) is
synthesized on a
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cleavable linker, such as a succinyl linker, using the solid support as a
scaffold. The
cleavable linker in (a) used as a scaffold for synthesizing the second strand
can comprise
similar reactivity or differing reactivity as the solid support derivatized
linker, such that
cleavage of the solid support derivatized linker and the cleavable linker of
(a) takes place
either concomitantly or sequentially. In one embodiment, the chemical moiety
of (b) that
can be used to isolate the attached oligonucleotide sequence comprises a
trityl group, for
example a dimethoxytrityl group.
In another embodiment, the invention features a method for making a double-
stranded siNA molecule in a single synthetic process comprising: (a)
synthesizing an
oligonucleotide having a first and a second sequence, wherein the first
sequence is
complementary to the second sequence, and the first oligonucleotide sequence
is linked
to the second sequence via a cleavable linker, and wherein a terminal 5'-
protecting group,
for example, a 5'-O-dimethoxytrityl group (5'-O-DMT) remains on the
oligonucleotide
having the second sequence; (b) deprotecting the oligonucleotide whereby the
deprotection results in the cleavage of the linker joining the two
oligonucleotide
sequences; and (c) purifying the product of (b) under conditions suitable for
isolating the
double-stranded siNA molecule, for example using a trityl-on synthesis
strategy as
described herein.
In another embodiment, the method of synthesis of siNA molecules of the
invention comprises the teachings of Scaringe et al., US Patent Nos.
5,889,136;
6,008,400; and 6,111,086, incorporated by reference herein in their entirety.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications, for example, one or more chemical modifications having
any of
Formulae I-VII or any combination thereof that increases the nuclease
resistance of the
siNA construct.
In another embodiment, the invention features a method for generating siNA
molecules with increased nuclease resistance comprising (a) introducing
nucleotides
having any of Formula I-VII or any combination thereof into a siNA molecule,
and (b)
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assaying the siNA molecule of step (a) under conditions suitable for isolating
siNA
molecules having increased nuclease resistance.
In another embodiment, the invention features a method for generating siNA
molecules with improved toxicologic profiles (e.g., have attenuated or no
immunstimulatory properties) comprising (a) introducing nucleotides having any
of
Formula I-VII (e.g., siNA motifs referred to in Table IV) or any combination
thereof
into a siNA molecule, and (b) assaying the siNA molecule of step (a) under
conditions
suitable for isolating siNA molecules having improved toxicologic profiles.
In another embodiment, the invention features a method for generating siNA
molecules that do not stimulate an interferon response (e.g., no interferon
response or
attenuated interferon response) in a cell, subject, or organism, comprising
(a) introducing
nucleotides having any of Formula I-VII (e.g., siNA motifs referred to in
Table IV) or
any combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of
step (a) under conditions suitable for isolating siNA molecules that do not
stimulate an
interferon response.
By "improved toxicologic profile", is meant that the chemically modified siNA
construct exhibits decreased toxicity in a cell, subject, or organism compared
to an
unmodified siNA or siNA molecule hving fewer modifications or modifications
that are
less effective in imparting improved toxicology. In a non-limiting example,
siNA
molecules with improved toxicologic profiles are associated with a decreased
or
attenuated immunostimulatory response in a cell, subject, or organism compared
to an
unmodified siNA or siNA molecule having fewer modifications or modifications
that are
less effective in imparting improved toxicology. In one embodiment, a siNA
molecule
with an improved toxicological profile comprises no ribonucleotides. In one
embodiment, a siNA molecule with an improved toxicological profile comprises
less
than 5 ribonucleotides (e.g., 1, 2, 3, or 4 ribonucleotides). In one
embodiment, a siNA
molecule with an improved toxicological profile comprises Stab 7, Stab 8, Stab
11, Stab
12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19, Stab 20, Stab 23, Stab 24,
Stab 25, Stab
26, Stab 27, Stab 28, Stab 29, Stab 30, Stab 31, Stab 32 or any combination
thereof (see
Table IV). In one embodiment, the level of immunostimulatory response
associated
with a given siNA molecule can be measured as is known in the art, for example
by
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determining the level of PKR/interferon response, proliferation, B-cell
activation, and/or
cytokine production in assays to quantitate the immunostimulatory response of
particular
siNA molecules (see, for example, Leifer et al., 2003, Jlmmunothef~. 26, 313-
9; and U.S.
Patent No. 5968909, incorporated in its entirety by reference).
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAAI, wherein the siNA construct comprises one or more
chemical modifications described herein that modulates the binding affinity
between the
sense and antisense strands of the siNA construct.
In another embodiment, the invention features a method for generating siNA
molecules with increased binding affinity between the sense and antisense
strands of the
siNA molecule comprising (a) introducing nucleotides having any of Formula I-
VII or
any combination thereof into a siNA molecule, and (b) assaying the siNA
molecule of
step (a) under conditions suitable for isolating siNA molecules having
increased binding
affinity between the sense and antisense strands of the siNA molecule.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications described herein that modulates the binding affinity
between the
antisense strand of the siNA construct and a complementary target RNA sequence
within
a cell.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications described herein that modulates the binding affinity
between the
antisense strand of the siNA construct and a complementary target DNA sequence
within
a cell.
In another embodiment, the invention features a method for generating siNA
molecules with increased binding affinity between the antisense strand of the
siNA
molecule and a complementary target RNA sequence comprising (a) introducing
nucleotides having any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable for
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isolating siNA molecules having increased binding affinity between the
antisense strand
of the siNA molecule and a complementary target RNA sequence.
In another embodiment, the invention features a method for generating siNA
molecules with increased binding affinity between the antisense strand of the
siNA
molecule and a complementary target DNA sequence comprising (a) introducing
nucleotides having any of Formula I-VII or any combination thereof into a siNA
molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable for
isolating siNA molecules having increased binding affinity between the
antisense strand
of the siNA molecule and a complementary target DNA sequence.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications described herein that modulate the polymerise activity
of a
cellular polymerise capable of generating additional endogenous siNA molecules
having
sequence homology to the chemically-modified siNA construct.
In another embodiment, the invention features a method for generating siNA
molecules capable of mediating increased polymerise activity of a cellular
polymerise
capable of generating additional endogenous siNA molecules having sequence
homology
to a chemically-modified siNA molecule comprising (a) introducing nucleotides
having
any of Formula I-VII or any combination thereof into a siNA molecule, and (b)
assaying
the siNA molecule of step (a) under conditions suitable for isolating siNA
molecules
capable of mediating increased polymerise activity of a cellular polymerise
capable of
generating additional endogenous siNA molecules having sequence homology to
the
chemically-modified siNA molecule.
In one embodiment, the invention features chemically-modified siNA constructs
that mediate RNAi against GPRA and/or AAAI in a cell, wherein the chemical
modifications do not significantly effect the interaction of siNA with a
target RNA
molecule, DNA molecule and/or proteins or other factors that are essential for
RNAi in a
manner that would decrease the efficacy of RNAi mediated by such siNA
constructs.
In another embodiment, the invention features a method for generating siNA
molecules with improved RNAi activity against GPRA and/or AAAl comprising (a)
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introducing nucleotides having any of Formula I-VII or any combination thereof
into a
siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable
for isolating siNA molecules having improved RNAi activity.
In yet another embodiment, the invention features a method for generating siNA
molecules with improved RNAi activity against GPRA and/or AAA1 target RNA
comprising (a) introducing nucleotides having any of Formula I-VII or any
combination
thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a)
under
conditions suitable for isolating siNA molecules having improved RNAi activity
against
the target RNA.
In yet another embodiment, the invention features a method for generating siNA
molecules with improved RNAi activity against GPRA and/or AAAl target DNA
comprising (a) introducing nucleotides having any of Formula I-VII or any
combination
thereof into a siNA molecule, and (b) assaying the siNA molecule of step (a)
under
conditions suitable for isolating siNA molecules having improved RNAi activity
against
the target DNA.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications described herein that modulates the cellular uptake of
the siNA
construct.
In another embodiment, the invention features a method for generating siNA
molecules against GPRA and/or AAA1 with improved cellular uptake comprising
(a)
introducing nucleotides having any of Formula I-VII or any combination thereof
into a
siNA molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable
for isolating siNA molecules having improved cellular uptake.
In one embodiment, the invention features siNA constructs that mediate RNAi
against GPRA and/or AAA1, wherein the siNA construct comprises one or more
chemical modifications described herein that increases the bioavailability of
the siNA
construct, for example, by attaching polymeric conjugates such as
polyethyleneglycol or
equivalent conjugates that improve the pharmacokinetics of the siNA construct,
or by
attaching conjugates that target specific tissue types or cell types ira vivo.
Non-limiting
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examples of such conjugates are described in Vargeese et al., U.S. Serial No.
10/201,394
incorporated by reference herein.
In one embodiment, the invention features a method for generating siNA
molecules of the invention with improved bioavailability comprising (a)
introducing a
conjugate into the structure of a siNA molecule, and (b) assaying the siNA
molecule of
step (a) under conditions suitable for isolating siNA molecules having
improved
bioavailability. Such conjugates can include ligands for cellular receptors,
such as
peptides derived from naturally occurring protein ligands; protein
localization sequences,
including cellular ZIP code sequences; antibodies; nucleic acid aptamers;
vitamins and
other co-factors, such as folate and N-acetylgalactosamine; polymers, such as
polyethyleneglycol (PEG); phospholipids; cholesterol; polyamines, such as
spermine or
spermidine; and others.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
to a target RNA sequence or a portion thereof, and a second sequence having
complementarity to said first sequence, wherein said second sequence is
chemically
modified in a manner that it can no longer act as a guide sequence for
efficiently
mediating RNA interference and/or be recognized by cellular proteins that
facilitate
RNAi.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
to a target RNA sequence or a portion thereof, and a second sequence having
complementarity to said first sequence, wherein the second sequence is
designed or
modified in a manner that prevents its entry into the RNAi pathway as a guide
sequence
or as a sequence that is complementary to a target nucleic acid (e.g., RNA)
sequence.
Such design or modifications are expected to enhance the activity of siNA
and/or
improve the specificity of siNA molecules of the invention. These
modifications are also
expected to minimize any off target effects and/or associated toxicity.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
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to a target RNA sequence or a portion thereof, and a second sequence having
complernentarity to said first sequence, wherein said second sequence is
incapable of
acting as a guide sequence for mediating RNA interference.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
to a target RNA sequence or a portion thereof, and a second sequence having
complementarity to said first sequence, wherein said second sequence does not
have a
terminal 5'-hydroxyl (5'-OH) or 5'-phosphate group.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
to a target RNA sequence or a portion thereof, and a second sequence having
complementarity to said first sequence, wherein said second sequence comprises
a
terminal cap moiety at the 5'-end of said second sequence. In one embodiment,
the
terminal cap moiety comprises an inverted abasic, inverted deoxy abasic,
inverted
nucleotide moiety, a group shown in Figure 10, an alkyl or cycloalkyl group, a
heterocycle, or any other group that prevents RNAi activity in which the
second
sequence serves as a guide sequence or template for RNAi.
In one embodiment, the invention features a double stranded short interfering
nucleic acid (siNA) molecule that comprises a first nucleotide sequence
complementary
to a target RNA sequence or a portion thereof, and a second sequence having
complementarity to said first sequence, wherein said second sequence comprises
a
terminal cap moiety at the 5'-end and 3'-end of said second sequence. In one
embodiment, each terminal cap moiety individually comprises an inverted
abasic,
inverted deoxy abasic, inverted nucleotide moiety, a group shown in Figure 10,
an alkyl
or cycloalkyl group, a heterocycle, or any other group that prevents RNAi
activity in
which the second sequence serves as a guide sequence or template for RNAi.
In one embodiment, the invention features a method for generating siNA
molecules of the invention with improved specificity for down regulating or
inhibiting
the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or
its
corresponding RNA), comprising (a) introducing one or more chemical
modifications
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into the structure of a siNA molecule, and (b) assaying the siNA molecule of
step (a)
under conditions suitable for isolating siNA molecules having improved
specificity. In
another embodiment, the chemical modification used to improve specificity
comprises
terminal cap modifications at the 5'-end, 3'-end, or both 5' and 3'-ends of
the siNA
molecule. The terminal cap modifications can comprise, for example, structures
shown
in Figure 10 (e.g. inverted deoxyabasic moieties) or any other chemical
modification
that renders a portion of the siNA molecule (e.g. the sense strand) incapable
of mediating
RNA interference against an off target nucleic acid sequence. In a non-
limiting example,
a siNA molecule is designed such that only the antisense sequence of the siNA
molecule
can serve as a guide sequence for RISC mediated degradation of a corresponding
target
RNA sequence. This can be accomplished by rendering the sense sequence of the
siNA
inactive by introducing chemical modifications to the sense strand that
preclude
recognition of the sense strand as a guide sequence by RNAi machinery. In one
embodiment, such chemical modifications comprise any chemical group at the 5'-
end of
the sense strand of the siNA, or any other group that serves to render the
sense strand
inactive as a guide sequence for mediating RNA interference. These
modifications, for
example, can result in a molecule where the S'-end of the sense strand no
longer has a
free 5'-hydroxyl (5'-OH) or a free 5'-phosphate group (e.g., phosphate,
diphosphate,
triphosphate, cyclic phosphate etc.). Non-limiting examples of such siNA
constructs are
described herein, such as "Stab 9/10", "Stab 7/8", "Stab 7/19", "Stab 17/22",
"Stab
23/24", "Stab 24/25", and "Stab 24/26" (e.g., any siNA having Stab 7, 9, 17,
23, or 24
sense strands) chemistries and variants thereof (see Table I~ wherein the 5'-
end and 3'-
end of the sense strand of the siNA do not comprise a hydroxyl group or
phosphate
group.
In one embodiment, the invention features a method for generating siNA
molecules of the invention with improved specificity for down regulating or
inhibiting
the expression of a target nucleic acid (e.g., a DNA or RNA such as a gene or
its
corresponding RNA), comprising introducing one or more chemical modifications
into
the structure of a siNA molecule that prevent a strand or portion of the siNA
molecule
from acting as a template or guide sequence for RNAi activity. In one
embodiment, the
inactive strand or sense region of the siNA molecule is the sense strand or
sense region
of the siNA molecule, i.e. the strand or region of the siNA that does not have
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complementarity to the target nucleic acid sequence. In one embodiment, such
chemical
modifications comprise any chemical group at the 5'-end of the sense strand or
region of
the siNA that does not comprise a 5'-hydroxyl (S'-OH) or 5'-phosphate group,
or any
other group that serves to render the sense strand or sense region inactive as
a guide
sequence for mediating RNA interference. Non-limiting examples of such siNA
constructs are described herein, such as "Stab 9/10", "Stab 7/8", "Stab 7/19",
"Stab
17/22", "Stab 23/24", "Stab 24125", and "Stab 24/26" (e.g., any siNA having
Stab 7, 9,
17, 23, or 24 sense strands) chemistries and variants thereof (see Table IV)
wherein the
5'-end and 3'-end of the sense strand of the siNA do not comprise a hydroxyl
group or
phosphate group.
In one embodiment, the invention features a method for screening siNA
molecules
that are active in mediating RNA interference against a target nucleic acid
sequence
comprising (a) generating a plurality of unmodified siNA molecules, (b)
screening the
siNA molecules of step (a) under conditions suitable for isolating siNA
molecules that
are active in mediating RNA interference against the target nucleic acid
sequence, and
(c) introducing chemical modifications (e.g. chemical modifications as
described herein
or as otherwise known in the art) into the active siNA molecules of (b). In
one
embodiment, the method further comprises re-screening the chemically modified
siNA
molecules of step (c) under conditions suitable for isolating chemically
modified siNA
molecules that are active in mediating RNA interference against the taxget
nucleic acid
sequence.
In one embodiment, the invention features a method for screening chemically
modified siNA molecules that are active in mediating RNA interference against
a target
nucleic acid sequence comprising (a) generating a plurality of chemically
modified siNA
molecules (e.g. siNA molecules as described herein or as otherwise known in
the art),
and (b) screening the siNA molecules of step (a) under conditions suitable for
isolating
chemically modified siNA molecules that are active in mediating RNA
interference
against the target nucleic acid sequence.
The term "ligand" refers to any compound or molecule, such as a drug, peptide,
hormone, or neurotransmitter, that is capable of interacting with another
compound, such
as a receptor, either directly or indirectly. The receptor that interacts with
a ligand can be
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present on the surface of a cell or can alternately be an intercullular
receptor. Interaction
of the ligand with the receptor can result in a biochemical reaction, or can
simply be a
physical interaction or association.
In another embodiment, the invention features a method for generating siNA
molecules of the invention with improved bioavailability comprising (a)
introducing an
excipient formulation to a siNA molecule, and (b) assaying the siNA molecule
of step (a)
under conditions suitable for isolating siNA molecules having improved
bioavailability.
Such excipients include polymers such as cyclodextrins, lipids, cationic
lipids,
polyamines, phospholipids, nanoparticles, receptors, ligands, and others.
In another embodiment, the invention features a method for generating siNA
molecules of the invention with improved bioavailability comprising (a)
introducing
nucleotides having any of Formulae I-VII or any combination thereof into a
siNA
molecule, and (b) assaying the siNA molecule of step (a) under conditions
suitable for
isolating siNA molecules having improved bioavailability.
In another embodiment, polyethylene glycol (PEG) can be covalently attached to
siNA compounds of the present invention. The attached PEG can be any molecular
weight, preferably from about 2,000 to about 50,000 daltons (Da).
The present invention can be used alone or as a component of a kit having at
least
one of the reagents necessary to carry out the iTZ vitro or in vivo
introduction of RNA to
test samples and/or subjects. For example, preferred components of the kit
include a
siNA molecule of the invention and a vehicle that promotes introduction of the
siNA into
cells of interest as described herein (e.g., using lipids and other methods of
transfection
known in the art, see for example Beigelman et al, US 6,395,713). The kit can
be used
for target validation, such as in determining gene function and/or activity,
or in drug
optimization, and in drug discovery (see for example Usman et al., USSN
60/402,996).
Such a kit can also include instructions to allow a user of the kit to
practice the invention.
The term "short interfering nucleic acid", "siNA", "short interfering RNA",
"siRNA", "short interfering nucleic acid molecule", "short interfering
oligonucleotide
molecule", or "chemically-modified short interfering nucleic acid molecule" as
used
herein refers to any nucleic acid molecule capable of inhibiting or down
regulating gene
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expression or viral replication, for example by mediating RNA interference
"RNAi" or
gene silencing in a sequence-specific manner; see for example Zamore et al.,
2000, Cell,
101, 25-33; Bass, 2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature,
411, 494-
498; and Kreutzer et al., International PCT Publication No. WO 00/44895;
Zernicka-
Goetz et al., International PCT Publication No. WO 01/36646; Fire,
International PCT
Publication No. WO 99/32619; Plaetinck et al., International PCT Publication
No. WO
00/01846; Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li
et al.,
International PCT Publication No. WO 00/44914; Allshire, 2002, Science, 297,
1818-
1819; Volpe et al., 2002, ScieJ2ce, 297, 1833-1837; Jenuwein, 2002, Science,
297, 2215-
2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore,
2002,
Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al.,
2002,
Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831).
Non
limiting examples of siNA molecules of the invention are shown in Figures 4-6,
and
Tables II and III herein. For example the siNA can be a double-stranded
polynucleotide molecule comprising self complementary sense and antisense
regions,
wherein the antisense region comprises nucleotide sequence that is
complementary to
nucleotide sequence in a target nucleic acid molecule or a portion thereof and
the sense
region having nucleotide sequence corresponding to the target nucleic acid
sequence or a
portion thereof. The siNA can be assembled from two separate oligonucleotides,
where
one strand is the sense strand and the other is the antisense strand, wherein
the antisense
and sense strands are self complementary (i.e. each strand comprises
nucleotide
sequence that is complementary to nucleotide sequence in the other strand;
such as where
the antisense strand and sense strand form a duplex or double stranded
structure, for
example wherein the double stranded region is about 15 to about 30, e.g.,
about 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the
antisense strand
comprises nucleotide sequence that is complementary to nucleotide sequence in
a target
nucleic acid molecule or a portion thereof and the sense strand comprises
nucleotide
sequence corresponding to the target nucleic acid sequence or a portion
thereof (e.g.,
about 15 to about 25 or more nucleotides of the siNA molecule are
complementary to the
target nucleic acid or a portion thereof). Alternatively, the siNA is
assembled from a
single oligonucleotide, where the self complementary sense and antisense
regions of the
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siNA are linked by means of a nucleic acid based or non-nucleic acid-based
linker(s).
The siNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or
asymmetric hairpin secondary structure, having self complementary sense and
antisense
regions, wherein the antisense region comprises nucleotide sequence that is
complementary to nucleotide sequence in a separate target nucleic acid
molecule or a
portion thereof and the sense region having nucleotide sequence corresponding
to the
target nucleic acid sequence or a portion thereof. The siNA can be a circular
single-
stranded polynucleotide having two or more loop structures and a stem
comprising self
complementary sense and antisense regions, wherein the antisense region
comprises
nucleotide sequence that is complementary to nucleotide sequence in a target
nucleic
acid molecule or a portion thereof and the sense region having nucleotide
sequence
corresponding to the target nucleic acid sequence or a portion thereof, and
wherein the
circular polynucleotide can be processed either ih vivo or in vitro to
generate an active
siNA molecule capable of mediating RNAi. The siNA can also comprise a single
1 S stranded polynucleotide having nucleotide sequence complementary to
nucleotide
sequence in a target nucleic acid molecule or a portion thereof (for example,
where such
siNA molecule does not require the presence within the siNA molecule of
nucleotide
sequence corresponding to the target nucleic acid sequence or a portion
thereof), wherein
the single stranded polynucleotide can further comprise a terminal phosphate
group, such
as a S'-phosphate (see for example Martinez et al., 2002, Cell., 110, S63-574
and
Schwarz et al., 2002, Molecular Cell, 10, S37-S68), or S',3'-diphosphate. In
certain
embodiments, the siNA molecule of the invention comprises separate sense and
antisense sequences or regions, wherein the sense and antisense regions are
covalently
linked by nucleotide or non-nucleotide linkers molecules as is known in the
art, or are
2S alternately non-covalently linked by ionic interactions, hydrogen bonding,
van der waals
interactions, hydrophobic interactions, andlor stacking interactions. In
certain
embodiments, the siNA molecules of the invention comprise nucleotide sequence
that is
complementary to nucleotide sequence of a target gene. In another embodiment,
the
siNA molecule of the invention interacts with nucleotide sequence of a target
gene in a
mariner that causes inhibition of expression of the target gene. As used
herein, siNA
molecules need not be limited to those molecules containing only RNA, but
further
encompasses chemically-modified nucleotides and non-nucleotides. In certain
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embodiments, the short interfering nucleic acid molecules of the invention
lack 2'-
hydroxy (2'-OH) containing nucleotides. Applicant describes in certain
embodiments
short interfering nucleic acids that do not require the presence of
nucleotides having a 2'-
hydroxy group for mediating RNAi and as such, short interfering nucleic acid
molecules
of the invention optionally do not include any ribonucleotides (e.g.,
nucleotides having a
2'-OH group). Such siNA molecules that do not require the presence of
ribonucleotides
within the siNA molecule to support RNAi can however have an attached linker
or
linkers or other attached or associated groups, moieties, or chains containing
one or more
nucleotides with 2'-OH groups. Optionally, siNA molecules can comprise
ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide
positions. The
modified short interfering nucleic acid molecules of the invention can also be
referred to
as short interfering modified oligonucleotides "siMON." As used herein, the
term siNA
is meant to be equivalent to other terms used to describe nucleic acid
molecules that are
capable of mediating sequence specific RNAi, for example short interfering RNA
(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA
(shRNA), short interfering oligonucleotide, short interfering nucleic acid,
short
interfering modified oligonucleotide, chemically-modified siRNA, post-
transcriptional
gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the
term RNAi
is meant to be equivalent to other terms used to describe sequence specific
RNA
interference, such as post transcriptional gene silencing, translational
inhibition, or
epigenetics. For example, siNA molecules of the invention can be used to
epigenetically
silence genes at both the post-transcriptional level or the pre-
transcriptional level. In a
non-limiting example, epigenetic regulation of gene expression by siNA
molecules of the
invention can result from siNA mediated modification of chromatin structure or
methylation pattern to alter gene expression (see, for example, Verdel et al.,
2004,
Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672;
Allshire, 2002,
Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837;
Jenuwein, 2002,
Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
In one embodiment, a siNA molecule of the invention is a duplex forming
oligonucleotide "DFO", (see for example Figures 14-15 and Vaish et al., USSN
10/727,780 filed December 3, 2003 and International PCT Application No.
US04/16390,
filed May 24, 2004).
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In one embodiment, a siNA molecule of the invention is a multifunctional siNA,
(see for example Figures 16-21 and Jadhav et al., USSN 60/543,480 filed
February 10,
2004 and International PCT Application No. US04/16390, filed May 24, 2004).
The
multifunctional siNA of the invention can comprise sequence targeting, for
example, two
regions of GPRA and/or AAA1 RNA (see for example target sequences in Tables II
and
III).
By "asymmetric hairpin" as used herein is meant a linear siNA molecule
comprising an antisense region, a loop portion that can comprise nucleotides
or non-
nucleotides, and a sense region that comprises fewer nucleotides than the
antisense
region to the extent that the sense region has enough complementary
nucleotides to base
pair with the antisense region and form a duplex with loop. For example, an
asymmetric
hairpin siNA molecule of the invention can comprise an antisense region having
length
sufficient to mediate RNAi in a cell or in vitro system (e.g. about 15 to
about 30, or
about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleotides) and a
loop region comprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10,
11, or 12)
nucleotides, and a sense region having about 3 to about 25 (e.g., about 3, 4,
5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides
that are
complementary to the antisense region. The asymmetric hairpin siNA molecule
can also
comprise a 5'-terminal phosphate group that can be chemically modified. The
loop
portion of the asymmetric hairpin siNA molecule can comprise nucleotides, non-
nucleotides, linker molecules, or conjugate molecules as described herein.
By "asymmetric duplex" as used herein is meant a siNA molecule having two
separate strands comprising a sense region and an antisense region, wherein
the sense
region comprises fewer nucleotides than the antisense region to the extent
that the sense
region has enough complementary nucleotides to base pair with the antisense
region and
form a duplex. For example, an asymmetric duplex siNA molecule of the
invention can
comprise an antisense region having length sufficient to mediate RNAi in a
cell or in
vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25,
26, 27, 28, 29, or 30 nucleotides) and a sense region having about 3 to about
25 (e.g.,
about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25)
nucleotides that are complementary to the antisense region.
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By "modulate" is meant that the expression of the gene, or level of RNA
molecule
or equivalent RNA molecules encoding one or more proteins or protein subunits,
or
activity of one or more proteins or protein subunits is up regulated or down
regulated,
such that expression, level, or activity is greater than or less than that
observed in the
absence of the modulator. For example, the term "modulate" can mean "inhibit,"
but the
use of the word "modulate" is not limited to this definition.
By "inhibit", "down-regulate", or "reduce", it is meant that the expression of
the
gene, or level of RNA molecules or equivalent RNA molecules encoding one or
more
proteins or protein subunits, or activity of one or more proteins or protein
subunits, is
reduced below that observed in the absence of the nucleic acid molecules
(e.g., siNA) of
the invention. In one embodiment, inhibition, down-regulation or reduction
with an siNA
molecule is below that level observed in the presence of an inactive or
attenuated
molecule. In another embodiment, inhibition, down-regulation, or reduction
with siNA
molecules is below that level observed in the presence of, for example, an
siNA
molecule with scrambled sequence or with mismatches. In another embodiment,
inhibition, down-regulation, or reduction of gene expression with a nucleic
acid molecule
of the instant invention is greater in the presence of the nucleic acid
molecule than in its
absence. In one embodiment, inhibition, down regulation, or reduction of gene
expression is associated with post transcriptional silencing, such as RNAi
mediated
cleavage of a target nucleic acid molecule (e.g. RNA) or inhibition of
translation. In one
embodiment, inhibition, down regulation, or reduction of gene expression is
associated
with pretranscriptional silencing.
By "gene", or "target gene", is meant a nucleic acid that encodes an RNA, for
example, nucleic acid sequences including, but not limited to, structural
genes encoding
a polypeptide. A gene or target gene can also encode a functional RNA (fRNA)
or non-
coding RNA (ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA
(snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof.
Such non-coding RNAs can serve as target nucleic acid molecules for siNA
mediated
RNA interference in modulating the activity of fRNA or ncRNA involved in
functional
or regulatory cellular processes. Abberant fRNA or ncRNA activity leading to
disease
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can therefore be modulated by siNA molecules of the invention. siNA molecules
targeting fRNA and ncRNA can also be used to manipulate or alter the genotype
or
phenotype of a subject, organism or cell, by intervening in cellular processes
such as
genetic imprinting, transcription, translation, or nucleic acid processing
(e.g.,
transamination, methylation etc.). The target gene can be a gene derived from
a cell, an
endogenous gene, a transgene, or exogenous genes such as genes of a pathogen,
for
example a virus, which is present in the cell after infection thereof. The
cell containing
the target gene can be derived from or contained in any organism, for example
a plant,
animal, protozoan, virus, bacterium, or fungus. Non-limiting examples of
plants include
monocots, dicots, or gymnosperms. Non-limiting examples of animals include
vertebrates or invertebrates. Non-limiting examples of fungi include molds or
yeasts.
For a review, see for example Snyder and Gerstein, 2003, Science, 300, 258-
260.
By "non-canonical base pair" is meant any non-Watson Crick base pair, such as
mismatches and/or wobble base pairs, inlcuding flipped mismatches, single
hydrogen
bond mismatches, trans-type mismatches, triple base interactions, and
quadruple base
interactions. Non-limiting examples of such non-canonical base pairs include,
but are
not limited to, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU
wobble,
AA N7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC 4-carbonyl-
amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, EAU reverse Watson
Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AA Nl-amino symmetric,
AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+ carbonyl-amino N7-Nl,
GG N1-carbonyl symmetric, GG N3-amino symmetric, CC carbonyl-amino symmetric,
CC N3-amino symmetric, UU 2-carbonyl-imino symmetric, UU 4-carbonyl-imino
symmetric, AA amino-N3, AA N1-amino, AC amino 2-carbonyl, AC N3-amino, AC
N7-amino, AU amino-4-carbonyl, AU N1-imino, AU N3-imino, AU N7-imino, CC
carbonyl-amino, GA amino-N1, GA amino-N7, GA carbonyl-amino, GA N3-amino, GC
amino-N3, GC carbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GG
carbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GU imino-
2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC 4-carbonyl-amino, UC imino-
carbonyl, W imino-4-carbonyl, AC C2-H-N3, GA carbonyl-C2-H, UU imino-4-
carbonyl 2 carbonyl-CS-H, AC amino(A) N3(C)-carbonyl, GC imino amino-carbonyl,
Gpsi imino-2-carbonyl amino-2- carbonyl, and GU imino amino-2-carbonyl base
pairs.
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By "G protein-coupled receptor for asthma susceptibility," "GPR154," or "GPRA"
as used herein is meant, any G protein-coupled receptor protein, peptide, or
polypeptide
having any G protein-coupled receptor activity, such as encoded by GPRA
Genbank
Accession Nos. shown in Table I. The terms "G protein-coupled receptor for
asthma
susceptibility," "GPR154," or "GPRA" also refer to nucleic acid sequences
encoding any
G protein-coupled receptor protein, peptide, or polypeptide having G protein-
coupled
receptor activity. The terms "G protein-coupled receptor for asthma
susceptibility,"
"GPR154," or "GPRA" are also meant to include other G protein-coupled receptor
encoding sequence, such as other G protein-coupled receptor isoforms, mutant G
protein-coupled receptor genes, splice variants of G protein-coupled receptor
genes, and
G protein-coupled receptor gene polymorphisms.
By "asthma-associated alternatively spliced gene 1" or "AAA1" as used herein
is
meant, any asthma-associated alternatively spliced protein, peptide, or
polypeptide
having any asthma-associated alternatively spliced gene activity, such as
encoded by
AAA1 Genbank Accession Nos. shown in Table I. The terms "asthma-associated
alternatively spliced gene 1" or "AAA1" also refer to nucleic acid sequences
encoding
any asthma-associated alternatively spliced protein, peptide, or polypeptide
having
asthma-associated alternatively spliced gene activity. The terms "asthma-
associated
;"
alternatively spliced gene 1" or "AAA1" are also meant to include other asthma-
associated alternatively spliced gene encoding sequence, such as other asthma-
associated
alternatively spliced gene isoforms, mutant asthma-associated alternatively
spliced gene
genes, splice variants of asthma-associated alternatively spliced genes, and
asthma-
associated alternatively spliced gene polymorphisms.
By "homologous sequence" is meant, a nucleotide sequence that is shared by one
or more polynucleotide sequences, such as genes, gene transcripts and/or non-
coding
polynucleotides. For example, a homologous sequence can be a nucleotide
sequence that
is shared by two or more genes encoding related but different proteins, such
as different
members of a gene family, different protein epitopes, different protein
isoforms or
completely divergent genes, such as a cytokine and its corresponding
receptors. A
homologous sequence can be a nucleotide sequence that is shared by two or more
non-
coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences,
introns,
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and sites of transcriptional control or regulation. Homologous sequences can
also
include conserved sequence regions shared by more than one polynucleotide
sequence.
Homology does not need to be perfect homology (e.g., 100%), as partially
homologous
sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%,
96%,
95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%,
80% etc.).
By "conserved sequence region" is meant, a nucleotide sequence of one or more
regions in a polynucleotide does not vary significantly between generations or
from one
biological system, subject, or organism to another biological system, subject,
or
organism. The polynucleotide can include both coding and non-coding DNA and
RNA.
By "sense region" is meant a nucleotide sequence of a siNA molecule having
complementarity to an antisense region of the siNA molecule. In addition, the
sense
region of a siNA molecule can comprise a nucleic acid sequence having homology
with
a target nucleic acid sequence.
By "antisense region" is meant a nucleotide sequence of a siNA molecule having
complementarity to a target nucleic acid sequence. In addition, the antisense
region of a
siNA molecule can optionally comprise a nucleic acid sequence having
complementarity
to a sense region of the siNA molecule.
By "target nucleic acid" is meant any nucleic acid sequence whose expression
or
activity is to be modulated. The target nucleic acid can be DNA or RNA.
By "complementarity" is meant that a nucleic acid can form hydrogen bonds)
with
another nucleic acid sequence by either traditional Watson-Crick or other non-
traditional
types. In reference to the nucleic molecules of the present invention, the
binding free
energy for a nucleic acid molecule with its complementary sequence is
sufficient to
allow the relevant function of the nucleic acid to proceed, e.g., RNAi
activity.
Determination of binding free energies for nucleic acid molecules is well
known in the
art (see, e.g., Turner et al., 1987, CSH SynZp. Quant. Biol. LII pp.123-133;
Frier et al.,
1986, Proc. Nat. Acad. Sci. USA 83:9373-9377; Turner et al., 1987, J. Arn.
Chefn. Soc.
109:3783-3785). A percent complementarity indicates the percentage of
contiguous
residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-
Crick
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base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10
nucleotides
out of a total of 10 nucleotides in the first oligonucleotide being based
paired to a second
nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%,
90%, and
100% complementary respectively). "Perfectly complementary" means that all the
contiguous residues of a nucleic acid sequence will hydrogen bond with the
same
number of contiguous residues in a second nucleic acid sequence. In one
embodiment, a
siNA molecule of the invention comprises about 15 to about 30 or more (e.g.,
about 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more)
nucleotides that are
complementary to one or more target nucleic acid molecules or a portion
thereof.
In one embodiment, siNA molecules of the invention that down regulate or
reduce
GPRA and/or AAA1 gene expression are used for preventing or treating
inflammatory
and/or respiratory diseases, disorders, and/or conditions in a subject or
organism.
In one embodiment, the siNA molecules of the invention are used to treat
inflammatory and/or respiratory diseases, disorders, and/or conditions in a
subject or
organism.
By "inflammatory disease" or "inflammatory condition" as used herein is meant
any disease, condition, trait, genotype or phenotype characterized by an
inflammatory or
allergic process as is known in the art; such as inflammation, acute
inflammation,
chronic inflammation, respiratory disease, atherosclerosis, restenosis,
asthma, allergic
rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory
bowl disease,
inflammotory pelvic disease, pain, ocular inflammatory disease, celiac
disease, Leigh
Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal
recessive
spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic
cholecystitis,
Bronchiectasis, Silicosis and other pneumoconioses, autoimmune disease, and
any other
inflammatory disease, condition, trait, genotype or phenotype that can respond
to the
modulation of disease related gene expression in a cell or tissue, alone or in
combination
with other therapies.
By "autoimmune disease" or "autoimmune condition" as used herein is meant, any
disease, condition, trait, genotype or phenotype characterized by autoimmunity
as is
known in the art, such as multiple sclerosis, diabetes mellitus, lupus, celiac
disease,
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Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms,
Goodpasture's
syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's
encephalitis,
Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis,
Addison's
disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome;
transplantation
rejection (e.g., prevention of allograft rejection) pernicious anemia,
rheumatoid arthritis,
systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus
erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome,
Grave's disease,
and any other autoimmune disease, condition, trait, genotype or phenotype that
can
respond to the modulation of disease related gene expression in a cell or
tissue, alone or
in combination with other therapies.
By "respiratory disease" is meant, any disease or condition affecting the
respiratory tract, such as asthma, chronic obstructive pulmonary disease or
"COPD",
allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation,
allergies, impeded
respiration, respiratory distress syndrome, cystic fibrosis, pulmonary
hypertension,
pulmonary vasoconstriction, emphysema, and any other respiratory disease,
condition,
trait, genotype or phenotype that can respond to the modulation of disease
related gene
expression in a cell or tissue, alone or in combination with other therapies..
In one embodiment of the present invention, each sequence of a siNA molecule
of
the invention is independently about 15 to about 30 nucleotides in length, in
specific
embodiments about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
or 30
nucleotides in length. In another embodiment, the siNA duplexes of the
invention
independently comprise about 15 to about 30 base pairs (e.g., about 15, 16,
17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In another embodiment, one or
more strands
of the siNA molecule of the invention independently comprises about 15 to
about 30
nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, or 30)
that are complementary to a target nucleic acid molecule. In yet another
embodiment,
siNA molecules of the invention comprising hairpin or circular structures are
about 35 to
about 55 (e.g., about 35, 40, 45, 50 or 55) nucleotides in length, or about 38
to about 44
(e.g., about 38, 39, 40, 41, 42, 43, or 44) nucleotides in length and
comprising about 15
to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base
pairs.
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Exemplary siNA molecules of the invention are shown in Table II. Exemplary
synthetic
siNA molecules of the invention are shown in Table III and/or Figures 4-5.
As used herein "cell" is used in its usual biological sense, and does not
refer to an
entire multicellular organism, e.g., specifically does not refer to a human.
The cell can
be present in an organism, e.g., birds, plants and mammals such as humans,
cows, sheep,
apes, monkeys, swine, dogs, and cats. The cell can be prokaryotic (e.g.,
bacterial cell) or
eukaryotic (e.g., mammalian or plant cell). The cell can be of somatic or germ
line
origin, totipotent or pluripotent, dividing or non-dividing. The cell can also
be derived
from or can comprise a gamete or embryo, a stem cell, or a fully
differentiated cell.
The siNA molecules of the invention are added directly, or can be complexed
with
cationic lipids, packaged within liposomes, or otherwise delivered to target
cells or
tissues. The nucleic acid or nucleic acid complexes can be locally
administered to
relevant tissues ex vivo, or in vivo through direct dermal application,
transdermal
application, or injection, with or without their incorporation in biopolymers.
In
particular embodiments, the nucleic acid molecules of the invention comprise
sequences
shown in Tables II-III andlor Figures 4-5. Examples of such nucleic acid
molecules
consist essentially of sequences defined in these tables and figures.
Furthermore, the
chemically modified constructs described in Table IV can be applied to any
siNA
sequence of the invention.
In another aspect, the invention provides mammalian cells containing one or
more
siNA molecules of this invention. The one or more siNA molecules can
independently
be targeted to the same or different sites.
By "RNA" is meant a molecule comprising at least one ribonucleotide residue.
By
"ribonucleotide" is meant a nucleotide with a hydroxyl group at the 2'
position of a (3-D-
ribofuranose moiety. The terms include double-stranded RNA, single-stranded
RNA,
isolated RNA such as partially purified RNA, essentially pure RNA, synthetic
RNA,
recornbinantly produced RNA, as well as altered RNA that differs from
naturally
occurring RNA by the addition, deletion, substitution and/or alteration of one
or more
nucleotides. Such alterations can include addition of non-nucleotide material,
such as to
the ends) of the siNA or internally, for example at one or more nucleotides of
the RNA.
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Nucleotides in the RNA molecules of the instant invention can also comprise
non-
standard nucleotides, such as non-naturally occurnng nucleotides or chemically
synthesized nucleotides or deoxynucleotides. These altered RNAs can be
referred to as
analogs or analogs of naturally-occurring RNA.
By "subject" is meant an organism, which is a donor or recipient of explanted
cells
or the cells themselves. "Subject" also refers to an organism to which the
nucleic acid
molecules of the invention can be administered. A subject can be a mammal or
mammalian cells, including a human ox human cells.
The term "phosphorothioate" as used herein refers to an internucleotide
linkage
having Formula I, wherein Z and/or W compxise a sulfur atom. Hence, the term
phosphorothioate refers to both phosphorothioate and phosphorodithioate
internucleotide
linkages.
The term "phosphonoacetate" as used herein refers to an internucleotide
linkage
having Formula I, wherein Z and/or W comprise an acetyl or protected acetyl
group.
The term "thiophosphonoacetate" as used herein refers to an internucleotide
linkage having Formula I, wherein Z comprises an acetyl or protected acetyl
group and
W comprises a sulfur atom or alternately W comprises an acetyl or protected
acetyl
group and Z comprises a sulfur atom.
The term "universal base" as used herein refers to nucleotide base analogs
that
form base pairs with each of the natural DNA/RNA bases with little
discrimination
between them. Non-limiting examples of universal bases include C-phenyl, C-
naphthyl
and other aromatic derivatives, inosine, azole carboxamides, and nitroazole
derivatives
such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as
known in the art
(see for example Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
25. The term "acyclic nucleotide" as used herein refers to any nucleotide
having an
acyclic ribose sugar, for example where any of the ribose carbons (Cl, C2, C3,
C4, or
CS), are independently or in combination absent from the nucleotide.
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The nucleic acid molecules of the instant invention, individually, or in
combination
or in conjunction with other drugs, can be used to for preventing or treating
inflammatory and/or respiratory diseases, conditions, or disorders in a
subject or
organism.
For example, the siNA molecules can be administered to a subject or can be
administered to other appropriate cells evident to those skilled in the art,
individually or
in combination with one or more drugs under conditions suitable for the
treatment.
In a further embodiment, the siNA molecules can be used in combination with
other known treatments to prevent or treat inflammatory and/or respiratory
diseases,
conditions, or disorders in a subject or organism. For example, the described
molecules
could be used in combination with one or more known compounds, treatments, or
procedures to prevent or treat inflammatory and/or respiratory diseases,
conditions, or
disorders in a subject or organism as are known in the art.
In one embodiment, the invention features an expression vector comprising a
nucleic acid sequence encoding at least one siNA molecule of the invention, in
a manner
which allows expression of the siNA molecule. For example, the vector can
contain
sequences) encoding both strands of a siNA molecule comprising a duplex. The
vector
can also contain sequences) encoding a single nucleic acid molecule that is
self
complementary and thus forms a siNA molecule. Non-limiting examples of such
expression vectors are described in Paul et al., 2002, Nature Bioteclaraology,
19, 505;
Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002,
Nature
Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine, advance
online
publication doi:10.1038/nm725.
In another embodiment, the invention features a mammalian cell, for example, a
human cell, including an expression vector of the invention.
In yet another embodiment, the expression vector of the invention comprises a
sequence for a siNA molecule having complementarity to a RNA molecule referred
to by
a Genbank Accession numbers, for example Genbank Accession Nos. shown in Table
I.
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In one embodiment, an expression vector of the invention comprises a nucleic
acid
sequence encoding two or more siNA molecules, which can be the same or
different.
In another aspect of the invention, siNA molecules that interact with target
RNA
molecules and down-regulate gene encoding target RNA molecules (for example
target
RNA molecules referred to by Genbank Accession numbers herein) are expressed
from
transcription units inserted into DNA or RNA vectors. The recombinant vectors
can be
DNA plasmids or viral vectors. siNA expressing viral vectors can be
constructed based
on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or
alphavirus. The
recombinant vectors capable of expressing the siNA molecules can be delivered
as
described herein, and persist in target cells. Alternatively, viral vectors
can be used that
provide for transient expression of siNA molecules. Such vectors can be
repeatedly
administered as necessary. Once expressed, the siNA molecules bind and down-
regulate
gene function or expression via RNA interference (RNAi). Delivery of siNA
expressing
vectors can be systemic, such as by intravenous or intramuscular
administration, by
administration to target cells ex-planted from a subject followed by
reintroduction into
the subject, or by any other means that would allow for introduction into the
desired
target cell.
By "vectors" is meant any nucleic acid- and/or viral-based technique used to
deliver a desired nucleic acid.
Other features and advantages of the invention will be apparent from the
following
description of the preferred embodiments thereof, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a non-limiting example of a scheme for the synthesis of siNA
molecules. The complementary siNA sequence strands, strand 1 and strand 2, are
synthesized in tandem and are connected by a cleavable linkage, such as a
nucleotide
succinate or abasic succinate, which can be the same or different from the
cleavable
linker used for solid phase synthesis on a solid support. The synthesis can be
either solid
phase or solution phase, in the example shown, the synthesis is a solid phase
synthesis.
The synthesis is performed such that a protecting group, such as a
dimethoxytrityl group,
remains intact on the terminal nucleotide of the tandem oligonucleotide. Upon
cleavage
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and deprotection of the oligonucleotide, the two siNA strands spontaneously
hybridize to
form a siNA duplex, which allows the purification of the duplex by utilizing
the
properties of the terminal protecting group, for example by applying a trityl
on
purification method wherein only duplexes/oligonucleotides with the terminal
protecting
group are isolated.
Figure 2 shows a MALDI-TOF mass spectrum of a purified siNA duplex
synthesized by a method of the invention. The two peaks shown correspond to
the
predicted mass of the separate siNA sequence strands. This result demonstrates
that the
siNA duplex generated from tandem synthesis can be purified as a single entity
using a
simple trityl-on purification methodology.
Figure 3 shows a non-limiting proposed mechanistic representation of target
RNA
degradation involved in RNAi. Double-stranded RNA (dsRNA), which is generated
by
RNA-dependent RNA polymerase (RdRP) from foreign single-stranded RNA, for
example viral, transposon, or other exogenous RNA, activates the DICER enzyme
that in
turn generates siNA duplexes. Alternately, synthetic or expressed siNA can be
introduced directly into a cell by appropriate means. An active siNA complex
forms
which recognizes a target RNA, resulting in degradation of the target RNA by
the RISC
endonuclease complex or in the synthesis of additional RNA by RNA-dependent
RNA
polymerase (RdRP), which can activate DICER and result in additional siNA
molecules,
thereby amplifying the RNAi response.
Figure 4A-F shows non-limiting examples of chemically-modified siNA
constructs of the present invention. In the figure, N stands for any
nucleotide (adenosine,
guanosine, cytosine, uridine, or optionally thymidine, for example thymidine
can be
substituted in the overhanging regions designated by parenthesis (N N).
Various
modifications are shown for the sense and antisense strands of the siNA
constructs.
Figure 4A: The sense strand comprises 21 nucleotides wherein the two terminal
3'-nucleotides are optionally base paired and wherein all nucleotides present
are
ribonucleotides except for (N N) nucleotides, which can comprise
ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications described
herein.
The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal
glyceryl
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moiety wherein the two terminal 3'-nucleotides are optionally complementary to
the
target RNA sequence, and wherein all nucleotides present are ribonucleotides
except for
(N N) nucleotides, which can comprise ribonucleotides, deoxynucleotides,
universal
bases, or other chemical modifications described herein. A modified
internucleotide
linkage, such as a phosphorothioate, phosphorodithioate or other modified
internucleotide linkage as described herein, shown as "s", optionally connects
the (N N)
nucleotides in the antisense strand.
Figure 4B: The sense strand comprises 21 nucleotides wherein the two terminal
3'-nucleotides are optionally base paired and wherein all pyrimidine
nucleotides that may
be present are 2'deoxy-2'-fluoro modified nucleotides and all purine
nucleotides that may
be present are 2'-O-methyl modified nucleotides except for (N N) nucleotides,
which can
comprise ribonucleotides, deoxynucleotides, universal bases, or other chemical
modifications described herein. The antisense strand comprises 21 nucleotides,
optionally having a 3'-terminal glyceryl moiety and wherein the two terminal
3'-
nucleotides are optionally complementary to the target RNA sequence, and
wherein all
pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro modified
nucleotides
and all purine nucleotides that may be present are 2'-O-methyl modified
nucleotides
except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides,
universal bases, or other chemical modifications described herein. A modified
internucleotidc linkage, such as a phosphorothioate, phosphorodithioate or
other
modified internucleotide linkage as described herein, shown as "s", optionally
connects
the (N N) nucleotides in the sense and antisense strand.
Figure 4C: The sense strand comprises 21 nucleotides having 5'- and 3'-
terminal
cap moieties wherein the two terminal 3'-nucleotides are optionally base
paired and
wherein all pyrimidine nucleotides that may be present are 2'-O-methyl or 2'-
deoxy-2'-
fluoro modified nucleotides except for (N N) nucleotides, which can comprise
ribonucleotides, deoxynucleotides, universal bases, or other chemical
modifications
described herein. The antisense strand comprises 21 nucleotides, optionally
having a 3'-
terminal glyceryl moiety and wherein the two terminal 3'-nucleotides are
optionally
complementary to the target RNA sequence, and wherein all pyrimidine
nucleotides that
may be present are 2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides,
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which can comprise ribonucleotides, deoxynucleotides, universal bases, or
other
chemical modifications described herein. A modified internucleotide linkage,
such as a
phosphorothioate, phosphorodithioate or other modified internucleotide linkage
as
described herein, shown as "s", optionally connects the (N N) nucleotides in
the
antisense strand.
Figure 4D: The sense strand comprises 21 nucleotides having 5'- and 3'-
terminal
cap moieties wherein the two terminal 3'-nucleotides are optionally base
paired and
wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified
nucleotides except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications described
herein and
wherein and all purine nucleotides that may be present are 2'-deoxy
nucleotides. The
antisense strand comprises 21 nucleotides, optionally having a 3'-terminal
glyceryl
moiety and wherein the two terminal 3'-nucleotides are optionally
complementary to the
target RNA sequence, wherein all pyrimidine nucleotides that may be present
are 2'-
deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be
present are
2'-O-methyl modified nucleotides except for (N N) nucleotides, which can
comprise
ribonucleotides, deoxynucleotides, universal bases, or other chemical
modifications
described herein. A modified internucleotide linkage, such as a
phosphorothioate,
phosphorodithioate or other modified internucleotide linkage as described
herein, shown
as "s", optionally connects the (N N) nucleotides in the antisense strand.
Figure 4E: The sense strand comprises 21 nucleotides having 5'- and 3'-
terminal
cap moieties wherein the two terminal 3'-nucleotides are optionally base
paired and
wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified
nucleotides except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications described
herein.
The antisense strand comprises 21 nucleotides, optionally having a 3'-terminal
glyceryl
moiety and wherein the two terminal 3'-nucleotides are optionally
complementary to the
target RNA sequence, and wherein all pyrimidine nucleotides that may be
present are 2'-
deoxy-2'-fluoro modified nucleotides and all purine nucleotides that may be
present are
2'-O-methyl modified nucleotides except for (N N) nucleotides, which can
comprise
ribonucleotides, deoxynucleotides, universal bases, or other chemical
modifications
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described herein. A modified internucleotide linkage, such as a
phosphorothioate,
phosphorodithioate or other modified internucleotide linkage as described
herein, shown
as "s", optionally connects the (N N) nucleotides in the antisense strand.
Figure 4F: The sense strand comprises 21 nucleotides having 5'- and 3'-
terminal
S cap moieties wherein the two terminal 3'-nucleotides are optionally base
paired and
wherein all pyrimidine nucleotides that may be present are 2'-deoxy-2'-fluoro
modified
nucleotides except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications described
herein and
wherein and all purine nucleotides that may be present are 2'-deoxy
nucleotides. The
antisense strand comprises 21 nucleotides, optionally having a 3'-terminal
glyceryl
moiety and wherein the two terminal 3'-nucleotides are optionally
complementary to the
target RNA sequence, and having one 3'-terminal phosphorothioate
internucleotide
linkage and wherein all pyrimidine nucleotides that may be present are 2'-
deoxy-2'-fluoro
modified nucleotides and all purine nucleotides that may be present are 2'-
deoxy
nucleotides except for (N N) nucleotides, which can comprise ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications described
herein. A
modified internucleotide linkage, such as a phosphorothioate,
phosphorodithioate or
other modified internucleotide linkage as described herein, shown as "s",
optionally
connects the (N N) nucleotides in the antisense strand. The antisense strand
of
constructs A-F comprise sequence complementary to any target nucleic acid
sequence of
the invention. Furthermore, when a glyceryl moiety (L) is present at the 3'-
end of the
antisense strand for any construct shown in Figure 4 A-F, the modified
internucleotide
linkage is optional.
Figure SA-F shows non-limiting examples of specific chemically-modified siNA
sequences of the invention. A-F applies the chemical modifications described
in Figure
4A-F to a GPRA siNA sequence. Such chemical modifications can be applied to
any
GPR.A and/or AAA1 sequence and/or GPRA and/or AAA1 polymorphism sequence.
Figure 6 shows non-limiting examples of different siNA constructs of the
invention. The examples shown (constructs 1, 2, and 3) have 19 representative
base
pairs; however, different embodiments of the invention include any number of
base pairs
described herein. Bracketed regions represent nucleotide overhangs, for
example,
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comprising about l, 2, 3, or 4 nucleotides in length, preferably about 2
nucleotides.
Constructs 1 and 2 can be used independently for RNAi activity. Construct 2
can
comprise a polynucleotide or non-nucleotide linker, which can optionally be
designed as
a biodegradable linker. In one embodiment, the loop structure shown in
construct 2 can
comprise a biodegradable linker that results in the formation of construct 1
ira vivo and/or
in vitro. In another example, construct 3 can be used to generate construct 2
under the
same principle wherein a linker is used to generate the active siNA construct
2 ifa vivo
and/or iTa vitro, which can optionally utilize another biodegradable linker to
generate the
active siNA construct 1 is vivo and/or in vitro. As such, the stability and/or
activity of
the siNA constructs can be modulated based on the design of the siNA construct
for use
in vivo or ifa vitro and/or in vitro.
Figure 7A-C is a diagrammatic representation of a scheme utilized in
generating
an expression cassette to generate siNA hairpin constructs.
Figure 7A: A DNA oligomer is synthesized with a 5'-restriction site (Rl)
sequence followed by a region having sequence identical (sense region of siNA)
to a
predetermined GPRA and/or AAA1 target sequence, wherein the sense region
comprises, for example, about 19, 20, 21, or 22 nucleotides (N) in length,
which is
followed by a loop sequence of defined sequence (X), comprising, for example,
about 3
to about 10 nucleotides.
Figure 7B: The synthetic construct is then extended by DNA polymerase to
generate a hairpin structure having self complementary sequence that will
result in a
siNA transcript having specificity for a GPRA and/or AAA1 target sequence and
having
self complementary sense and antisense regions.
Figure 7C: The construct is heated (for example to about 95°C) to
linearize the
sequence, thus allowing extension of a complementary second DNA strand using a
primer to the 3'-restriction sequence of the first strand. The double-stranded
DNA is then
inserted into an appropriate vector for expression in cells. The construct can
be designed
such that a 3'-terminal nucleotide overhang results from the transcription,
for example,
by engineering restriction sites and/or utilizing a poly-U termination region
as described
in Paul et al., 2002, Nature Biotechnology, 29, 505-508.
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Figure 8A-C is a diagrammatic representation of a scheme utilized in
generating
an expression cassette to generate double-stranded siNA constructs.
Figure 8A: A DNA oligomer is synthesized with a 5'-restriction (Rl) site
sequence
followed by a region having sequence identical (sense region of siNA) to a
predetermined GPRA and/or AAA1 target sequence, wherein the sense region
comprises, for example, about 19, 20, 21, or 22 nucleotides (I~ in length, and
which is
followed by a 3'-restriction site (R2) which is adjacent to a loop sequence of
defined
sequence (X).
Figure 8B: The synthetic construct is then extended by DNA polymerase to
generate a hairpin structure having self complementary sequence.
Figure 8C: The construct is processed by restriction enzymes specific to Rl
and
R2 to generate a double-stranded DNA which is then inserted into an
appropriate vector
for expression in cells. The transcription cassette is designed such that a U6
promoter
region flanks each side of the dsDNA which generates the separate sense and
antisense
strands of the siNA. Poly T termination sequences can be added to the
constructs to
generate U overhangs in the resulting transcript.
Figure 9A-E is a diagrammatic representation of a method used to determine
target sites for siNA mediated RNAi within a particular target nucleic acid
sequence,
such as messenger RNA.
Figure 9A: A pool of siNA oligonucleotides are synthesized wherein the
antisense
region of the siNA constructs has complementarity to target sites across the
target
nucleic acid sequence, and wherein the sense region comprises sequence
complementary
to the antisense region of the siNA.
Figure 9B&C: (Figure 9B) The sequences are pooled and are inserted into
vectors such that (Figure 9C) transfection of a vector into cells results in
the expression
of the siNA.
Figure 9D: Cells are sorted based on phenotypic change that is associated with
modulation of the target nucleic acid sequence.
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Figure 9E: The siNA is isolated from the sorted cells and is sequenced to
identify
efficacious target sites within the target nucleic acid sequence.
Figure 10 shows non-limiting examples of different stabilization chemistries
(1-
10) that can be used, for example, to stabilize the 3'-end of siNA sequences
of the
invention, including (1) [3-3']-inverted deoxyribose; (2) deoxyribonucleotide;
(3) [5'-3']-
3'-deoxyribonucleotide; (4) [5'-3']-ribonucleotide; (5) [5'-3']-3'-O-methyl
ribonucleotide;
(6) 3'-glyceryl; (7) [3'-5']-3'-deoxyribonucleotide; (8) [3'-3']-
deoxyribonucleotide; (9) [5'-
2']-deoxyribonucleotide; and (10) [5-3']-dideoxyribonucleotide. In addition to
modified
and unmodified backbone chemistries indicated in the figure, these chemistries
can be
combined with different backbone modifications as described herein, for
example,
backbone modifications having Formula I. In addition, the 2'-deoxy nucleotide
shown 5'
to the terminal modifications shown can be another modified or unmodified
nucleotide
or non-nucleotide described herein, for example modifications having any of
Formulae I-
VII or any combination thereof.
Figure 11 shows a non-limiting example of a strategy used to identify
chemically
modified siNA constructs of the invention that are nuclease resistance while
preserving
the ability to mediate RNAi activity. Chemical modifications are introduced
into the
siNA construct based on educated design parameters (e.g. introducing 2'-
mofications,
base modifications, backbone modifications, terminal cap modifications etc).
The
modified construct in tested in an appropriate system (e.g. human serum for
nuclease
resistance, shown, or an animal model for PK/delivery parameters). In
parallel, the siNA
construct is tested for RNAi activity, for example in a cell culture system
such as a
luciferase reporter assay). Lead siNA constructs are then identified which
possess a
particular characteristic while maintaining ltNAi activity, and can be further
modified
and assayed once again. This same approach can be used to identify siNA-
conjugate
molecules with improved pharmacokinetic profiles, delivery, and ltNAi
activity.
Figure 12 shows non-limiting examples of phosphorylated siNA molecules of the
invention, including linear and duplex constructs and asymmetric derivatives
thereof.
Figure 13 shows non-limiting examples of chemically modified terminal
phosphate groups of the invention.
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Figure 14A shows a non-limiting example of methodology used to design self
complementary DFO constructs utilizing palidrome and/or repeat nucleic acid
sequences
that are identified in a target nucleic acid sequence. (i) A palindrome or
repeat sequence
is identified in a nucleic acid target sequence. (ii) A sequence is designed
that is
complementary to the target nucleic acid sequence and the palindrome sequence.
(iii) An
inverse repeat sequence of the non-palindrome/repeat portion of the
complementary
sequence is appended to the 3'-end of the complementary sequence to generate a
self
complementary DFO molecule comprising sequence complementary to the nucleic
acid
target. (iv) The DFO molecule can self assemble to form a double stranded
oligonucleotide. Figure 14B shows a non-limiting representative example of a
duplex
forming oligonucleotide sequence. Figure 14C shows a non-limiting example of
the self
assembly schematic of a representative duplex forming oligonucleotide
sequence.
Figure 14D shows a non-limiting example of the self assembly schematic of a
representative duplex forming oligonucleotide sequence followed by interaction
with a
target nucleic acid sequence resulting in modulation of gene expression.
Figure 15 shows a non-limiting example of the design of self complementary DFO
constructs utilizing palidrome and/or repeat nucleic acid sequences that are
incorporated
into the DFO constructs that have sequence complementary to any target nucleic
acid
sequence of interest. Incorporation of these palindrome/repeat sequences allow
the
design of DFO constructs that form duplexes in which each strand is capable of
mediating modulation of target gene expression, for example by RNAi. First,
the target
sequence is identified. A complementary sequence is then generated in which
nucleotide
or non-nucleotide modifications (shown as X or Y) are introduced into the
complementary sequence that generate an artificial palindrome (shown as XYXYXY
in
the Figure). An inverse repeat of the non-palindxome/repeat complementary
sequence is
appended to the 3'-end of the complementary sequence to generate a self
complementary
DFO comprising sequence complementary to the nucleic acid target. The DFO can
self
assemble to form a double stranded oligonucleotide.
Figure 16 shows non-limiting examples of multifunctional siNA molecules of the
invention comprising two separate polynucleotide sequences that are each
capable of
mediating RNAi directed cleavage of differing target nucleic acid sequences.
Figure
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16A shows a non-limiting example of a multifunctional siNA molecule having a
first
region that is complementary to a first target nucleic acid sequence
(complementary
region 1) and a second region that is complementary to a second target nucleic
acid
sequence (complementary region 2), wherein the first and second complementary
regions
are situated at the 3'-ends of each polynucleotide sequence in the
multifunctional siNA.
The dashed portions of each polynucleotide sequence of the multifunctional
siNA
construct have complementarily with regard to corresponding portions of the
siNA
duplex, but do not have complementarily to the target nucleic acid sequences.
Figure
16B shows a non-limiting example of a multifunctional siNA molecule having a
first
region that is complementary to a first target nucleic acid sequence
(complementary
region 1) and a second region that is complementary to a second target nucleic
acid
sequence (complementary region 2), wherein the first and second complementary
regions
are situated at the 5'-ends of each polynucleotide sequence in the
multifunctional siNA.
The dashed portions of each polynucleotide sequence of the multifunctional
siNA
construct have complementarily with regard to corresponding portions of the
siNA
duplex, but do not have complementarity to the target nucleic acid sequences.
Figure 17 shows non-limiting examples of multifunctional siNA molecules of the
invention comprising a single polynucleotide sequence comprising distinct
regions that
are each capable of mediating RNAi directed cleavage of differing target
nucleic acid
sequences. Figure 17A shows a non-limiting example of a multifunctional siNA
molecule having a first region that is complementary to a first target nucleic
acid
sequence (complementary region 1) and a second region that is complementary to
a
second target nucleic acid sequence (complementary region 2), wherein the
second
complementary region is situated at the 3'-end of the polynucleotide sequence
in the
multifunctional siNA. The dashed portions of each polynucleotide sequence of
the
multifunctional siNA construct have complementarity with regard to
corresponding
portions of the siNA duplex, but do not have complementarity to the target
nucleic acid
sequences. Figure 17B shows a non-limiting example of a multifunctional siNA
molecule having a first region that is complementary to a first target nucleic
acid
sequence (complementary region 1) and a second region that is complementary to
a
second target nucleic acid sequence (complementary region 2), wherein the
first
complementary region is situated at the 5'-end of the polynucleotide sequence
in the
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multifunctional siNA. The dashed portions of each polynucleotide sequence of
the
multifunctional siNA construct have complementarity with regard to
corresponding
portions of the siNA duplex, but do not have complementarity to the target
nucleic acid
sequences. In one embodiment, these multifunctional siNA constructs are
processed in
vivo or in vitro to generate multifunctional siNA constructs as shown in
Figure 16.
Figure 18 shows non-limiting examples of multifunctional siNA molecules of the
invention comprising two separate polynucleotide sequences that are each
capable of
mediating RNAi directed cleavage of differing target nucleic acid sequences
and wherein
the multifunctional siNA construct further comprises a self complementary,
palindrome,
or repeat region, thus enabling shorter bifuctional siNA constructs that can
mediate RNA
interference against differing target nucleic acid sequences. Figure 18A showa
a non-
limiting example of a multifunctional siNA molecule having a first region that
is
complementary to a first target nucleic acid sequence (complementary region 1)
and a
second region that is complementary to a second target nucleic acid sequence
(complementary region 2), wherein the first and second complementary regions
are
situated at the 3'-ends of each polynucleotide sequence in the multifunctional
siNA, and
wherein the first and second complementary regions further comprise a self
complementary, palindrome, or repeat region. The dashed portions of each
polynucleotide sequence of the multifunctional siNA construct have
complementarity
with regard to corresponding portions of the siNA duplex, but do not have
complementarity to the target nucleic acid sequences. Figure 18B shows a non-
limiting
example of a multifunctional siNA molecule having a first region that is
complementary
to a first target nucleic acid sequence (complementary region 1) and a second
region that
is complementary to a second target nucleic acid sequence (complementary
region 2),
wherein the first and second complementary regions are situated at the 5'-ends
of each
polynucleotide sequence in the multifunctional siNA, and wherein the first and
second
complementary regions further comprise a self complementary, palindrome, or
repeat
region. The dashed portions of each polynucleotide sequence of the
multifunctional
siNA construct have complementarity with regard to corresponding portions of
the siNA
duplex, but do not have complementarity to the target nucleic acid sequences.
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Figure 19 shows non-limiting examples of multifunctional siNA molecules of the
invention comprising a single polynucleotide sequence comprising distinct
regions that
are each capable of mediating RNAi directed cleavage of differing target
nucleic acid
sequences and wherein the multifunctional siNA construct further comprises a
self
complementary, palindrome, or repeat region, thus enabling shorter bifuctional
siNA
constructs that can mediate RNA interference against differing target nucleic
acid
sequences. Figure 19A shows a non-limiting example of a multifunctional siNA
molecule having a first region that is complementary to a first target nucleic
acid
sequence (complementary region 1) and a second region that is complementary to
a
second target nucleic acid sequence (complementary region 2), wherein the
second
complementary region is situated at the 3'-end of the polynucleotide sequence
in the
multifunctional siNA, and wherein the first and second complementary regions
further
comprise a self complementary, palindrome, or repeat region. The dashed
portions of
each polynucleotide sequence of the multifunctional siNA construct have
complementarity with regard to corresponding portions of the siNA duplex, but
do not
have complementarity to the target nucleic acid sequences. Figure 19S shows a
non-
limiting example of a multifunctional siNA molecule having a first region that
is
complementary to a first target nucleic acid sequence (complementary region 1)
and a
second region that is complementary to a second target nucleic acid sequence
(complementary region 2), wherein the first complementary region is situated
at the 5'-
end of the polynucleotide sequence in the multifunctional siNA, and wherein
the first
and second complementary regions further comprise a self complementary,
palindrome,
or repeat region. The dashed portions of each polynucleotide sequence of the
multifunctional siNA construct have complementarity with regard to
corresponding
portions of the siNA duplex, but do not have complementarity to the target
nucleic acid
sequences. In one embodiment, these multifunctional siNA constructs are
processed in
vivo or in vitro to generate multifunctional siNA constructs as shown in
Figure 18.
Figure 20 shows a non-limiting example of how multifunctional siNA molecules
of the invention can target two separate target nucleic acid molecules, such
as separate
RNA molecules encoding differing proteins, for example, a cytokine and its
corresponding receptor, differing viral strains, a virus and a cellular
protein involved in
viral infection or replication, or differing proteins involved in a common or
divergent
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biologic pathway that is implicated in the maintenance of progression of
disease. Each
strand of the multifunctional siNA construct comprises a region having
complementarity
to separate target nucleic acid molecules. The multifunctional siNA molecule
is
designed such that each strand of the siNA can be utilized by the RISC complex
to
initiate RNA interference mediated cleavage of its corresponding target. These
design
parameters can include destabilization of each end of the siNA construct (see
for
example Schwarz et al., 2003, Cell; 115, 199-208). Such destabilization can be
accomplished for example by using guanosine-cytidine base pairs, alternate
base pairs
(e.g., wobbles), or destabilizing chemically modified nucleotides at terminal
nucleotide
positions as is known in the art.
Figure 21 shows a non-limiting example of how multifunctional siNA molecules
of the invention can target two separate target nucleic acid sequences within
the same
target nucleic acid molecule, such as alternate coding regions of a RNA,
coding and non-
coding regions of a RNA, or alternate splice variant regions of a RNA. Each
strand of
the multifunctional siNA construct comprises a region having complementarity
to the
separate regions of the target nucleic acid molecule. The multifunctional siNA
molecule
is designed such that each strand of the siNA can be utilized by the RISC
complex to
initiate RNA interference mediated cleavage of its corresponding target
region. These
design parameters can include destabilization of each end of the siNA
construct (see for
example Schwarz et al., 2003, Cell, 115, 199-208). Such destabilization can be
accomplished for example by using guanosine-cytidine base pairs, alternate
base pairs
(e.g., wobbles), or destabilizing chemically modified nucleotides at terminal
nucleotide
positions as is known in the art.
DETAILED DESCRIPTION OF THE INVENTION
Mechanism of Action of Nucleic Acid Molecules of the Invention
The discussion that follows discusses the proposed mechanism of RNA
interference mediated by short interfering RNA as is presently known, and is
not meant
to be limiting and is not an admission of prior art. Applicant demonstrates
herein that
chemically-modified short interfering nucleic acids possess similar or
improved capacity
to mediate RNAi as do siRNA molecules and are expected to possess improved
stability
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and activity in vivo; therefore, this discussion is not meant to be limiting
only to siRNA
and can be applied to siNA as a whole. By "improved capacity to mediate RNAi"
or
"improved RNAi activity" is meant to include RNAi activity measured in vitro
and/or in
vivo where the RNAi activity is a reflection of both the ability of the siNA
to mediate
RNAi and the stability of the siNAs of the invention. In this invention, the
product of
these activities can be increased in vitf°o and/or in vivo compared to
an all RNA siRNA
or a siNA containing a plurality of ribonucleotides. In some cases, the
activity or stability
of the siNA molecule can be decreased (i.e., less than ten-fold), but the
overall activity of
the siNA molecule is enhanced in vitf°o and/or in vivo.
RNA interference refers to the process of sequence specific post-
transcriptional
gene silencing in animals mediated by short interfering RNAs (siRNAs) (Fire et
al.,
1998, Nature, 391, 806). The corresponding process in plants is commonly
referred to as
post-transcriptional gene silencing or RNA silencing and is also referred to
as quelling in
fungi. The process of post-transcriptional gene silencing is thought to be an
evolutionarily-conserved cellular defense mechanism used to prevent the
expression of
foreign genes which is commonly shared by diverse flora and phyla (Fire et
al., 1999,
Trends Genet., 15, 358). Such protection from foreign gene expression may have
evolved in response to the production of double-stranded RNAs (dsRNAs) derived
from
viral infection or the random integration of transposon elements into a host
genome via a
cellular response that specifically destroys homologous single-stranded RNA or
viral
genomic RNA. The presence of dsRNA in cells triggers the RNAi response though
a
mechanism that has yet to be fully characterized. This mechanism appears to be
different from the interferon response that results from dsRNA-mediated
activation of
protein kinase PKR and 2', 5'-oligoadenylate synthetase resulting in non-
specific
cleavage of mRNA by ribonuclease L.
The presence of long dsRNAs in cells stimulates the activity of a ribonuclease
III
enzyme referred to as Dicer. Dicer is involved in the processing of the dsRNA
into short
pieces of dsRNA known as short interfering RNAs (siRNAs) (Berstein et al.,
2001,
Nature, 409, 363). Short interfering RNAs derived from Dicer activity are
typically
about 21 to about 23 nucleotides in length and comprise about 19 base pair
duplexes.
Dicer has also been implicated in the excision of 21- and 22-nucleotide small
temporal
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RNAs (stRNAs) from precursor RNA of conserved structure that are implicated in
translational control (Hutvagner et al., 2001, Science, 293, 834). The RNAi
response
also features an endonuclease complex containing a siRNA, commonly referred to
as an
RNA-induced silencing complex (RISC), which mediates cleavage of single-
stranded
RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes
place in the middle of the region complementary to the guide sequence of the
siRNA
duplex (Elbashir et al., 2001, Genes Dev., 15, 188). In addition, RNA
interference can
also involve small RNA (e.g., micro-RNA or miRNA) mediated gene silencing,
presumably though cellular mechanisms that regulate chromatin structure and
thereby
prevent transcription of target gene sequences (see for example Allshire,
2002, Science,
297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002,
Science,
297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237). As such, siNA
molecules of the invention can be used to mediate gene silencing via
interaction with
RNA transcripts or alternately by interaction with particular gene sequences,
wherein
such interaction results in gene silencing either at the transcriptional level
or post-
transcriptional level.
RNAi has been studied in a variety of systems. Fire et al., 1998, Nature, 391,
806,
were the first to observe RNAi in C. elegans. Wianny and Goetz, 1999, Nature
Cell
Biol., 2, 70, describe RNAi mediated by dsRNA in mouse embryos. Hammond et
al.,
2000, Nature, 404, 293, describe RNAi in Drosophila cells transfected with
dsRNA.
Elbashir et al., 2001, Natuf°e, 411, 494, describe RNAi induced by
introduction of
duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cells including
human
embryonic kidney and HeLa cells. Recent work in Drosophila embryonic lysates
has
revealed certain requirements for siRNA length, structure, chemical
composition, and
sequence that are essential to mediate efficient RNAi activity. These studies
have shown
that 21 nucleotide siRNA duplexes are most active when containing two 2-
nucleotide 3'-
terminal nucleotide overhangs. Furthermore, substitution of one or both siRNA
strands
with 2'-deoxy or 2'-O-methyl nucleotides abolishes RNAi activity, whereas
substitution
of 3'-terminal siRNA nucleotides with deoxy nucleotides was shown to be
tolerated.
Mismatch sequences in the center of the siRNA duplex were also shown to
abolish RNAi
activity. In addition, these studies also indicate that the position of the
cleavage site in
the target RNA is defined by the 5'-end of the siRNA guide sequence rather
than the 3'-
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end (Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicated
that a 5'-
phosphate on the target-complementary strand of a siRNA duplex is required for
siRNA
activity and that ATP is utilized to maintain the 5'-phosphate moiety on the
siRNA
(Nykanen et al., 2001, Cell, 107, 309); however, siRNA molecules lacking a 5'-
phosphate are active when introduced exogenously, suggesting that 5'-
phosphorylation of
siRNA constructs may occur in vivo.
Synthesis of Nucleic Acid Molecules
Synthesis of nucleic acids greater than 100 nucleotides in length is difficult
using
automated methods, and the therapeutic cost of such molecules is prohibitive.
In this
invention, small nucleic acid motifs ("small" refers to nucleic acid motifs no
more than
100 nucleotides in length, preferably no more than 80 nucleotides in length,
and most
preferably no more than 50 nucleotides in length; e.g., individual siNA
oligonucleotide
sequences or siNA sequences synthesized in tandem) are preferably used for
exogenous
delivery. The simple structure of these molecules increases the ability of the
nucleic acid
to invade targeted regions of protein and/or RNA structure. Exemplary
molecules of the
instant invention are chemically synthesized, and others can similarly be
synthesized.
Oligonucleotides (e.g., certain modified oligonucleotides or portions of
oligonucleotides lacking ribonucleotides) are synthesized using protocols
known in the
art, for example as described in Caruthers et al., 1992, Methods in
Erazymology 211, 3-
19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et
al.,
1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.
Bio., 74,
59, Brennan et al., 1998, Biotechhol Bioeng., 61, 33-45, and Brennan, U.S.
Pat. No.
6,001,311. All of these references are incorporated herein by reference. The
synthesis of
oligonucleotides makes use of common nucleic acid protecting and coupling
groups,
such as dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In
a non-
limiting example, small scale syntheses are conducted on a 394 Applied
Biosystems, Inc.
synthesizer using a 0.2 p,mol scale protocol with a 2.5 min coupling step for
2'-O-
methylated nucleotides and a 45 second coupling step for 2'-deoxy nucleotides
or 2'-
deoxy-2'-fluoro nucleotides. Table V outlines the amounts and the contact
times of the
reagents used in the synthesis cycle. Alternatively, syntheses at the 0.2
~.mol scale can
be performed on a 96-well plate synthesizer, such as the instrument produced
by
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Protogene (Palo Alto, CA) with minimal modification to the cycle. A 33-fold
excess (60
~.L of 0.11 M = 6.6 ~.mol) of 2'-O-methyl phosphoramidite and a 105-fold
excess of S-
ethyl tetrazole (60 wL of 0.25 M = 15 ~mol) can be used in each coupling cycle
of 2'-O-
methyl residues relative to polymer-bound 5'-hydroxyl. A 22-fold excess (40 wL
of 0.11
M = 4.4 ~,mol) of deoxy phosphoramidite and a 70-fold excess of S-ethyl
tetrazole (40
~,L of 0.25 M = 10 ~,mol) can be used in each coupling cycle of deoxy residues
relative
to polymer-bound 5'-hydroxyl. Average coupling yields on the 394 Applied
Biosystems,
Inc. synthesizer, determined by colorimetric quantitation of the trityl
fractions, are
typically 97.5-99%. Other oligonucleotide synthesis reagents for the 394
Applied
Biosystems, Inc. synthesizer include the following: detritylation solution is
3% TCA in
methylene chloride (ABI); capping is performed with 16% N methyl imidazole in
THF
(ABI) and 10% acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation
solution
is 16.9 mM I2, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).
Burdick & Jackson Synthesis Grade acetonitrile is used directly from the
reagent bottle.
S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from the solid
obtained
from American International Chemical, Inc. Alternately, for the introduction
of
phosphorothioate linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-
dioxide,
0.05 M in acetonitrile) is used.
Deprotection of the DNA-based oligonucleotides is performed as follows: the
polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mL glass
screw top vial
and suspended in a solution of 40% aqueous methylamine (1 mL) at 65 °C
for 10
minutes. After cooling to -20 °C, the supernatant is removed from the
polymer support.
The support is washed three times with 1.0 mL of EtOH:MeCN:H20/3:1:1, vortexed
and
the supernatant is then added to the first supernatant. The combined
supernatants,
containing the oligoribonucleotide, are dried to a white powder.
The method of synthesis used for RNA including certain siNA molecules of the
invention follows the procedure as described in Usman et al., 1987, J. Am.
Chem. Soc.,
109, 7845; Scaringe et al., 1990, Nucleic Acids Res., 18, 5433; and Wincott et
al., 1995,
Nucleic Acids Res. 23, 2677-2684 Wincott et al., 1997, Methods Mol. Bio., 74,
59, and
makes use of common nucleic acid protecting and coupling groups, such as
dimethoxytrityl at the 5'-end, and phosphoramidites at the 3'-end. In a non-
limiting
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example, small scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 wmol scale protocol with a 7.5 min coupling step for
alkylsilyl
protected nucleotides and a 2.5 min coupling step for 2'-O-methylated
nucleotides.
Table V outlines the amounts and the contact times of the reagents used in the
synthesis
cycle. Alternatively, syntheses at the 0.2 ~.mol scale can be done on a 96-
well plate
synthesizer, such as the instrument produced by Protogene (Palo Alto, CA) with
minimal
modification to the cycle. A 33-fold excess (60 ~,L of 0.11 M = 6.6 wmol) of
2'-O-
methyl phosphoramidite and a 75-fold excess of S-ethyl tetrazole (60 ~L of
0.25 M = 15
~mol) can be used in each coupling cycle of 2'-O-methyl residues relative to
polymer-
bound 5'-hydroxyl. A 66-fold excess (120 ~L of 0.11 M = 13.2 ~,mol) of
alkylsilyl (ribo)
protected phosphoramidite and a 150-fold excess of S-ethyl tetrazole (120 ~.L
of 0.25 M
= 30 ~.mol) can be used in each coupling cycle of ribo residues relative to
polyrner-
bound 5'-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.
synthesizer, determined by colorimetric quantitation of the trityl fractions,
are typically
97.5-99%. Other oligonucleotide synthesis reagents for the 394 Applied
Biosystems,
Inc. synthesizer include the following: detritylation solution is 3% TCA in
methylene
chloride (ABI); capping is performed with 16% N methyl imidazole in THF (ABI)
and
10% acetic anhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9
mM I2,
49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.). Burdick &
Jackson
Synthesis Grade acetonitrile is used directly from the reagent bottle. S-
Ethyltetrazole
solution (0.25 M in acetonitrile) is made up from the solid obtained from
American
International Chemical, Inc. Alternately, for the introduction of
phosphorothioate
linkages, Beaucage reagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in
acetonitrile) is used.
Deprotection of the RNA is performed using either a two-pot or one-pot
protocol.
For the two-pot protocol, the polymer-bound trityl-on oligoribonucleotide is
transferred
to a 4 mL glass screw top vial and suspended in a solution of 40% aq,
methylamine (1
mL) at 65 °C for 10 min. After cooling to -20 °C, the
supernatant is removed from the
polymer support. The support is washed three times with 1.0 mL of
EtOH:MeCN:H20/3:1:1, vortexed and the supernatant is then added to the first
supernatant. The combined supernatants, containing the oligoribonucleotide,
are dried to
a white powder. The base deprotected oligoribonucleotide is resuspended in
anhydrous
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TEA/HF/NMP solution (300 ~,L of a solution of 1.5 mL N-methylpyrrolidinone,
750 ~.L
TEA and 1 mL TEA~3HF to provide a 1.4 M HF concentration) and heated to 65
°C.
After 1.5 h, the oligomer is quenched with 1.5 M NHqHC03.
Alternatively, for the one-pot protocol, the polymer-bound trityl-on
oligoribonucleotide is transferred to a 4 mL glass screw top vial and
suspended in a
solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL) at 65 °C for
15 minutes.
The vial is brought to room temperature TEA~3HF (0.1 mL) is added and the vial
is
heated at 65 °C for 15 minutes. The sample is cooled at -20 °C
and then quenched with
1.5 M NH4HC03.
For purification of the trityl-on oligomers, the quenched NHqHC03 solution is
loaded onto a C-18 containing cartridge that had been prewashed with
acetonitrile
followed by 50 mM TEAA. After washing the loaded cartridge with water, the RNA
is
detritylated with 0.5% TFA for 13 minutes. The cartridge is then washed main
with
water, salt exchanged with 1 M NaCI and washed with water again. The
oligonucleotide
is then eluted with 30% acetonitrile.
The average stepwise coupling yields are typically >98% (Wincott et al., 1995
Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in the art will
recognize that
the scale of synthesis can be adapted to be larger or smaller than the example
described
above including but not limited to 96-well format.
Alternatively, the nucleic acid molecules of the present invention can be
synthesized separately and joined together post-synthetically, for example, by
ligation
(Moore et al., 1992, Science 256, 9923; Draper et al., International PCT
publication No.
WO 93/23569; Shabarova et al., 1991, Nucleic Acids Research 19, 4247; Bellon
et al.,
1997, Nucleosides & Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate
Claern. 8,
204), or by hybridization following synthesis and/or deprotection.
The siNA molecules of the invention can also be synthesized via a tandem
synthesis methodology as described in Example 1 herein, wherein both siNA
strands are
synthesized as a single contiguous oligonucleotide fragment or strand
separated by a
cleavable linker which is subsequently cleaved to provide sepaxate siNA
fragments or
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strands that hybridize and permit purification of the siNA duplex. The linker
can be a
polynucleotide linker or a non-nucleotide linker. The tandem synthesis of siNA
as
described herein can be readily adapted to both multiwell/multiplate synthesis
platforms
such as 96 well or similarly larger mufti-well platforms. The tandem synthesis
of siNA as
described herein can also be readily adapted to large scale synthesis
platforms employing
batch reactors, synthesis columns and the like.
A siNA molecule can also be assembled from two distinct nucleic acid strands
or
fragments wherein one fragment includes the sense region and the second
fragment
includes the antisense region of the RNA molecule.
The nucleic acid molecules of the present invention can be modified
extensively to
enhance stability by modification with nuclease resistant groups, for example,
2'-amino,
2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-H (for a review see Usman and
Cedergren, 1992,
TIBS 17, 34; Usman et al., 1994, Nucleic Acids Syrnp. Ser. 31, 163). siNA
constructs can
be purified by gel electrophoresis using general methods or can be purified by
high
pressure liquid chromatography (HPLC; see Wincott et al., supra, the totality
of which is
hereby incorporated herein by reference) and re-suspended in water.
In another aspect of the invention, siNA molecules of the invention are
expressed
from transcription units inserted into DNA or RNA vectors. The recombinant
vectors can
be DNA plasmids or viral vectors. siNA expressing viral vectors can be
constructed
based on, but not limited to, adeno-associated virus, retrovirus, adenovirus,
or alphavirus.
The recombinant vectors capable of expressing the siNA molecules can be
delivered as
described herein, and persist in target cells. Alternatively, viral vectors
can be used that
provide for transient expression of siNA molecules.
Optimizin~~Activit~of the nucleic acid molecule of the invention.
Chemically synthesizing nucleic acid molecules with modifications (base, sugar
and/or phosphate) can prevent their degradation by serum ribonucleases, which
can
increase their potency (see e.g., Eckstein et al., International Publication
No. WO
92/07065; Perrault et al., 1990 Nature 344, 565; Pieken et al., 1991, Science
253, 314;
Usman and Cedergren, 1992, Trends in Biochern. Sci. 17, 334; Usman et al.,
International Publication No. WO 93/1517; and Rossi et al., International
Publication
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WO 2005/045038 PCT/US2004/027231
No. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat. No,
6,300,074;
and Burgin et al., supna; all of which axe incorporated by reference herein).
All of the
above references describe various chemical modifications that can be made to
the base,
phosphate and/or sugar moieties of the nucleic acid molecules described
herein.
S Modifications that enhance their efficacy in cells, and removal of bases
from nucleic acid
molecules to shorten oligonucleotide synthesis times and reduce chemical
requirements
are desired.
There are several examples in the art describing sugar, base and phosphate
modifications that can be introduced into nucleic acid molecules with
significant
enhancement in their nuclease stability and efficacy. For example,
oligonucleotides are
modified to enhance stability and/or enhance biological activity by
modification with
nuclease resistant groups, for example, 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-
methyl, 2'-O-
allyl, 2'-H, nucleotide base modifications (for a review see Usman and
Cedergren, 1992,
TIBS. 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 3I, 163; Burgin et
al., 1996,
Biochenzist~y, 35, 14090). Sugar modification of nucleic acid molecules have
been
extensively described in the art (see Eckstein et al., International
Publication PCT No.
WO 92/07065; Perrault et al. Natune, 1990, 344, S6S-568; Pieken et al.
Science, 1991,
253, 314-317; Usman and Cedergren, Trends in Biochem. Sci., 1992, 17, 334-339;
Usman et al. International Publication PCT No. WO 93/15187; Sproat, U.S. Pat.
No.
5,334,711 and Beigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et
al.,
International PCT publication No. WO 97/26270; Beigelman et al., U.S. Pat. No.
5,716,824; Usman et al., U.S. Pat. No. 5,627,OS3; Woolf et al., International
PCT
Publication No. WO 98/13526; Thompson et al., USSN 60/082,404 which was filed
on
April 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39, 1131; Earnshaw
and Gait,
1998, Biopolynaers (Nucleic Acid Sciences), 48, 39-SS; Verma and Eckstein,
1998, Annu.
Rev. Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., S,
1999-2010;
all of the references are hereby incorporated in their totality by reference
herein). Such
publications describe general methods and strategies to determine the location
of
incorporation of sugar, base and/or phosphate modifications and the like into
nucleic acid
molecules without modulating catalysis, and are incorporated by reference
herein. In
view of such teachings, similar modifications can be used as described herein
to modify
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the siNA nucleic acid molecules of the instant invention so long as the
ability of siNA to
promote RNAi is cells is not significantly inhibited.
While chemical modification of oligonucleotide internucleotide linkages with
phosphorothioate, phosphorodithioate, and/or 5'-methylphosphonate linkages
improves
stability, excessive modifications can cause some toxicity or decreased
activity.
Therefore, when designing nucleic acid molecules, the amount of these
internucleotide
linkages should be minimized. The reduction in the concentration of these
linkages
should lower toxicity, resulting in increased efficacy and higher specificity
of these
molecules.
Short interfering nucleic acid (siNA) molecules having chemical modifications
that
maintain or enhance activity are provided. Such a nucleic acid is also
generally more
resistant to nucleases than an unmodified nucleic acid. Accordingly, the in
vitro and/or
in vivo activity should not be significantly lowered. In cases in which
modulation is the
goal, therapeutic nucleic acid molecules delivered exogenously should
optimally be
stable within cells until translation of the target RNA has been modulated
long enough to
reduce the levels of the undesirable protein. This period of time varies
between hours to
days depending upon the disease state. Improvements in the chemical synthesis
of RNA
and DNA (Wincott et al., 1995, Nucleic Acids Res. 23, 2677; Caruthers et al.,
1992,
Methods in Enzymology 211, 3-19 (incorporated by reference herein)) have
expanded the
ability to modify nucleic acid molecules by introducing nucleotide
modifications to
enhance their nuclease stability, as described above.
In one embodiment, nucleic acid molecules of the invention include one or more
(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. A G-
clamp
nucleotide is a modified cytosine analog wherein the modifications confer the
ability to
hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine
within a duplex, see for example Lin and Matteucci, 1998, J. Am. Claem. Soc.,
120, 8531-
8532. A single G-clamp analog substitution within an oligonucleotide can
result in
substantially enhanced helical thermal stability and mismatch discrimination
when
hybridized to complementary oligonucleotides. The inclusion of such
nucleotides in
nucleic acid molecules of the invention results in both enhanced affinity and
specificity
to nucleic acid targets, complementary sequences, or template strands. In
another
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embodiment, nucleic acid molecules of the invention include one or more (e.g.,
about 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA "locked nucleic acid" nucleotides
such as a 2', 4'-
C methylene bicyclo nucleotide (see for example Wengel et al., International
PCT
Publication No. WO 00/66604 and WO 99/14226).
In another embodiment, the invention features conjugates and/or complexes of
siNA molecules of the invention. Such conjugates and/or complexes can be used
to
facilitate delivery of siNA molecules into a biological system, such as a
cell. The
conjugates and complexes provided by the instant invention can impart
therapeutic
activity by transfernng therapeutic compounds across cellular membranes,
altering the
pharmacokinetics, and/or modulating the localization of nucleic acid molecules
of the
invention. The present invention encompasses the design and synthesis of novel
conjugates and complexes for the delivery of molecules, including, but not
limited to,
small molecules, lipids, cholesterol, phospholipids, nucleosides, nucleotides,
nucleic
acids, antibodies, toxins, negatively charged polymers and other polymers, for
example
proteins, peptides, hormones, carbohydrates, polyethylene glycols, or
polyamines, across
cellular membranes. In general, the transporters described are designed to be
used either
individually or as part of a mufti-component system, with or without
degradable linkers.
These compounds are expected to improve delivery and/or localization of
nucleic acid
molecules of the invention into a number of cell types originating from
different tissues,
in the presence or absence of serum (see Sullenger and Cech, U.S. Pat. No.
5,854,038).
Conjugates of the molecules described herein can be attached to biologically
active
molecules via linkers that are biodegradable, such as biodegradable nucleic
acid linker
molecules.
The term "biodegradable linker" as used herein, refers to a nucleic acid or
non-
nucleic acid linker molecule that is designed as a biodegradable linker to
connect one
molecule to another molecule, for example, a biologically active molecule to a
siNA
molecule of the invention or the sense and antisense strands of a siNA
molecule of the
invention. The biodegradable linker is designed such that its stability can be
modulated
for a particular purpose, such as delivery to a particular tissue or cell
type. The stability
of a nucleic acid-based biodegradable linker molecule can be modulated by
using various
chemistries, for example combinations of ribonucleotides,
deoxyribonucleotides, and
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chemically-modified nucleotides, such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-
O-amino,
2'-C-allyl, 2'-O-allyl, and other 2'-modified or base modified nucleotides.
The
biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or
longer
nucleic acid molecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6,
7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, or can
comprise a single
nucleotide with a phosphorus-based linkage, for example, a phosphoramidate or
phosphodiester linkage. The biodegradable nucleic acid linker molecule can
also
comprise nucleic acid backbone, nucleic acid sugar, or nucleic acid base
modifications.
The term "biodegradable" as used herein, refers to degradation in a biological
system, for example, enzymatic degradation or chemical degradation.
The term "biologically active molecule" as used herein refers to compounds or
molecules that are capable of eliciting or modifying a biological response in
a system.
Non-limiting examples of biologically active siNA molecules either alone or in
combination with other molecules contemplated by the instant invention include
therapeutically active molecules such as antibodies, cholesterol, hormones,
antivirals,
peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors,
nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense
nucleic
acids, triplex forming oligonucleotides, 2,5-A chimeras, siNA, dsRNA,
allozymes,
aptamers, decoys and analogs thereof. Biologically active molecules of the
invention
also include molecules capable of modulating the pharmacokinetics and/or
pharmacodynamics of other biologically active molecules, for example, lipids
and
polymers such as polyamines, polyamides, polyethylene glycol and other
polyethers.
The term "phospholipid" as used herein, refers to a hydrophobic molecule
comprising at least one phosphorus group. For example, a phospholipid can
comprise a
phosphorus-containing group and saturated or unsaturated alkyl group,
optionally
substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl
groups.
Therapeutic nucleic acid molecules (e.g., siNA molecules) delivered
exogenously
optimally are stable within cells until reverse transcription of the RNA has
been
modulated long enough to reduce the levels of the RNA transcript. The nucleic
acid
molecules are resistant to nucleases in order to function as effective
intracellular
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therapeutic agents. Improvements in the chemical synthesis of nucleic acid
molecules
described in the instant invention and in the art have expanded the ability to
modify
nucleic acid molecules by introducing nucleotide modifications to enhance
their nuclease
stability as described above.
In yet another embodiment, siNA molecules having chemical modifications that
maintain or enhance enzymatic activity of proteins involved in RNAi are
provided. Such
nucleic acids are also generally more resistant to nucleases than unmodified
nucleic
acids. Thus, in vitro and/or ira vivo the activity should not be significantly
lowered.
Use of the nucleic acid-based molecules of the invention will lead to better
treatments by affording the possibility of combination therapies (e.g.,
multiple siNA
molecules targeted to different genes; nucleic acid molecules coupled with
known small
molecule modulators; or intermittent treatment with combinations of molecules,
including different motifs and/or other chemical or biological molecules). The
treatment
of subjects with siNA molecules can also include combinations of different
types of
nucleic acid molecules, such as enzymatic nucleic acid molecules (ribozymes),
allozyrnes, antisense, 2,5-A oligoadenylate, decoys, and aptamers.
In another aspect a siNA molecule of the invention comprises one or more 5'
and/or a 3'- cap structure, for example, on only the sense siNA strand, the
antisense siNA
strand, or both siNA strands.
By "cap structure" is meant chemical modifications, which have been
incorporated
at either terminus of the oligonucleotide (see, for example, Adamic et al.,
U.S. Pat. No.
5,998,203, incorporated by reference herein). These terminal modifications
protect the
nucleic acid molecule from exonuclease degradation, and may help in delivery
and/or
localization within a cell. The cap may be present at the 5'-terminus (5'-cap)
or at the 3'-
terminal (3'-cap) or may be present on both termini. In non-limiting examples,
the 5'-cap
includes, but is not limited to, glyceryl, inverted deoxy abasic residue
(moiety); 4',5'-
methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio
nucleotide;
carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-
nucleotides;
modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl
nucleotide;
acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic
3,5-
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dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted
abasic
moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-
butanediol
phosphate; 3'-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3'-
phosphate;
3'-phosphorothioate; phosphorodithioate; or bridging or non-bridging
methylphosphonate moiety. Non-limiting examples of cap moieties are shown in
Figure
10.
Non-limiting examples of the 3'-cap include, but are not limited to, glyceryl,
inverted deoxy abasic residue (moiety), 4', 5'-methylene nucleotide; 1-(beta-D-
erythrofuranosyl) nucleotide; 4'-thin nucleotide, carbocyclic nucleotide; 5'-
amino-alkyl
phosphate; 1,3-diamino-2-propyl phosphate; 3-aminopropyl phosphate; 6-
aminohexyl
phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-
anhydrohexitol
nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;
phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco
nucleotide; 3,4-
dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted
nucleotide
moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-phosphorothioate;
1,4-
butanediol phosphate; 5'-amino; bridging and/or non-bridging 5'-
phosphoramidate,
phosphorothioate and/or phosphorodithioate, bridging or non bridging
methylphosphonate and 5'-mercapto moieties (for more details see Beaucage and
Iyer,
1993, Tetralaedron 49, 1925; incorporated by reference herein).
By the term "non-nucleotide" is meant any group or compound which can be
incorporated into a nucleic acid chain in the place of one or more nucleotide
units,
including either sugar and/or phosphate substitutions, and allows the
remaining bases to
exhibit their enzymatic activity. The group or compound is abasic in that it
does not
contain a commonly recognized nucleotide base, such as adenosine, guanine,
cytosine,
uracil or thymine and therefore lacks a base at the 1'-position.
An "alkyl" group refers to a saturated aliphatic hydrocarbon, including
straight-
chain, branched-chain, and cyclic alkyl groups. Preferably, the alkyl group
has 1 to 12
carbons. More preferably, it is a lower alkyl of from 1 to 7 carbons, more
preferably 1 to
4 carbons. The alkyl group can be substituted or unsubstituted. When
substituted the
substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02 or
N(CH3)2,
amino, or SH. The term also includes alkenyl groups that are unsaturated
hydrocarbon
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groups containing at least one carbon-carbon double bond, including straight-
chain,
branched-chain, and cyclic groups. Preferably, the alkenyl group has 1 to 12
carbons.
More preferably, it is a lower alkenyl of from 1 to 7 carbons, more preferably
1 to 4
carbons. The alkenyl group may be substituted or unsubstituted. When
substituted the
substituted groups) is preferably, hydroxyl, cyano, alkoxy, =O, =S, N02,
halogen,
N(CH3)2, amino, or SH. The term "alkyl" also includes alkynyl groups that have
an
unsaturated hydrocarbon group containing at least one carbon-carbon triple
bond,
including straight-chain, branched-chain, and cyclic groups. Preferably, the
alkynyl
group has 1 to 12 carbons. More preferably, it is a lower alkynyl of from 1 to
7 carbons,
more preferably 1 to 4 carbons. The alkynyl group may be substituted or
unsubstituted.
When substituted the substituted groups) is preferably, hydroxyl, cyano,
alkoxy, =O,
=S, N02 or N(CH3)2, amino or SH.
Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,
heterocyclic
aryl, amide and ester groups. An "aryl" group refers to an aromatic group that
has at
least one ring having a conjugated pi electron system and includes carbocyclic
aryl,
heterocyclic aryl and biaryl groups, all of which may be optionally
substituted. The
preferred substituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl,
SH, OH,
cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An "alkylaryl" group
refers to
an alkyl group (as described above) covalently joined to an aryl group (as
described
above). Carbocyclic aryl groups are groups wherein the ring atoms on the
aromatic ring
are all carbon atoms. The carbon atoms are optionally substituted.
Heterocyclic aryl
groups are groups having from 1 to 3 heteroatoms as ring atoms in the aromatic
ring and
the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include
oxygen,
sulfur, and nitrogen, and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower
alkyl
pyrrolo, pyrimidyl, pyrazinyl, imidazolyl and the like, all optionally
substituted. An
"amide" refers to an -C(O)-NH-R, where R is either alkyl, aryl, alkylaryl or
hydrogen.
An "ester" refers to an -C(O)-OR', where R is either alkyl, aryl, alkylaryl or
hydrogen.
By "nucleotide" as used herein is as recognized in the art to include natural
bases
(standard), and modified bases well known in the art. Such bases are generally
located at
the 1' position of a nucleotide sugar moiety. Nucleotides generally comprise a
base,
sugar and a phosphate group. The nucleotides can be unmodified or modified at
the
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sugar, phosphate and/or base moiety, (also referred to interchangeably as
nucleotide
analogs, modified nucleotides, non-natural nucleotides, non-standard
nucleotides and
other; see, for example, Usman and McSwiggen, supra; Eckstein et al.,
International
PCT Publication No. WO 92/07065; Usman et al., International PCT Publication
No.
WO 93/15187; Uhlman & Peyman, supra, all are hereby incorporated by reference
herein). 'There are several examples of modified nucleic acid bases known in
the art as
summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of the
non-
limiting examples of base modifications that can be introduced into nucleic
acid
molecules include, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,
pseudouracil, 2,
4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,
aminophenyl,
5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,
ribothymidine),
5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines
(e.g. 6-
methyluridine), propyne, and others (Burgin et al., 1996, Biochemistry, 35,
14090;
Uhlman & Peyrnan, supra). By "modified bases" in this aspect is meant
nucleotide bases
other than adenine, guanine, cytosine and uracil at 1' position or their
equivalents.
In one embodiment, the invention features modified siNA molecules, with
phosphate backbone modifications comprising one or more phosphorothioate,
phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate
carbamate, carboxyrnethyl, acetamidate, polyamide, sulfonate, sulfonamide,
sulfamate,
formacetal, thioformacetal, and/or alkylsilyl, substitutions. For a review of
oligonucleotide backbone modifications, see Hunziker and Leumann, 1995,
Nucleic Acid
Analogues: Synthesis aytd Properties, in Moderrt Synthetic Methods, VCH, 331-
417, and
Mesmaeker et al., 1994, Novel Backbone Replacements for Oligonucleotides, in
Carbohydrate Modifications in Antisense Research, ACS, 24-39.
By "abasic" is meant sugar moieties lacking a base or having other chemical
groups in place of a base at the 1' position, see for example Adamic et al.,
U.S. Pat. No.
5,998,203.
By "unmodified nucleoside" is meant one of the bases adenine, cytosine,
guanine,
thymine, or uracil joined to the 1' carbon of (3-D-ribo-furanose.
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By "modified nucleoside" is meant any nucleotide base which contains a
modification in the chemical structure of an unmodified nucleotide base, sugar
and/or
phosphate. Non-limiting examples of modified nucleotides are shown by Formulae
I-VII
and/or other modifications described herein.
In connection with 2'-modified nucleotides as described for the present
invention,
by "amino" is meant f-NHZ or f-O- NHZ, which can be modified or unmodified.
Such
modified groups are described, for example, in Eckstein et al., U.S. Pat. No.
5,672,695
and Matulic-Adamic et al., U.S. Pat. No. 6,248,878, which are both
incorporated by
reference in their entireties.
Various modifications to nucleic acid siNA structure can be made to enhance
the
utility of these molecules. Such modifications will enhance shelf life, half
life in vitro,
stability, and ease of introduction of such oligonucleotides to the target
site, e.g., to
enhance penetration of cellular membranes, and confer the ability to recognize
and bind
to targeted cells.
Administration of Nucleic Acid Molecules
A siNA molecule of the invention can be adapted for use to prevent or treat
inflammatory and/or respiratory diseases, conditions, or disorders, and/or any
other trait,
disease, disoxder or condition that is related to or will respond to the
levels of GPRA.
and/or AAA1 in a cell or tissue, alone or in combination with other therapies.
For
example, a siNA molecule can comprise a delivery vehicle, including liposomes,
for
administration to a subject, Garners arid diluents and their salts, and/or can
be present in
pharmaceutically acceptable formulations. Methods for the delivexy of nucleic
acid
molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139;
Delivery
Sti°ategies for Ayatisense Oligotaucleotide Tlaet°apeutics, ed.
Akhtar, 1995, Maurer et al.,
1999, Mol. Membr. Biol., 16, 129-I40; Hofland and Huang, 1999, Handb. Exp.
Plaarnaacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser., 752, I84-
192, all of
which are incorporated herein by reference. Beigelman et al., U.S. Pat. No.
6,395,713
and Sullivan et al., PCT WO 94/02595 furthex describe the general methods for
delivery
of nucleic acid molecules. These protocols can be utilized for the delivery of
virtually
any nucleic acid molecule. Nucleic acid molecules can be administered to cells
by a
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variety of methods known to those of skill in the art, including, but not
restricted to,
encapsulation in liposomes, by iontophoresis, or by incorporation into other
vehicles,
such as biodegradable polymers, hydrogels, cyclodextrins (see for example
Gonzalez et
al., 1999, Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCT
publication Nos. WO 03/47518 and WO 03/46185), poly(lactic-co-glycolic)acid
(PLGA)
and PLCA microspheres (see for example US Patent 6,447,796 and US Patent
Application Publication No. US 2002130430), biodegradable nanocapsules, and
bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand,
International PCT Publication No. WO 00/53722). Alternatively, the nucleic
acid/vehicle combination is locally delivered by direct injection or by use of
an infusion
pump. Direct injection of the nucleic acid molecules of the invention, whether
subcutaneous, intramuscular, or intradermal, can take place using standard
needle and
syringe methodologies, or by needle-free technologies such as those described
in Conry
et al., 1999, Clira. Cancef~ Res., 5, 2330-2337 and Barry et al.,
International PCT
Publication No. WO 99/31262. The molecules of the instant invention can be
used as
pharmaceutical agents. Pharmaceutical agents prevent, modulate the occurrence,
or treat
(alleviate a symptom to some extent, preferably all of the symptoms) of a
disease state in
a subject.
In another embodiment, the nucleic acid molecules of the invention can also be
formulated or complexed with polyethyleneimine and derivatives thereof, such
as
polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or
polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-
triGAL)
derivatives. In one embodiment, the nucleic acid molecules of the invention
are
formulated as described in United States Patent Application Publication No.
20030077829, incorporated by reference herein in its entirety.
In one embodiment, a siNA molecule of the invention is complexed with
membrane disruptive agents such as those described in U.S. Patent Application
Publication No. 20010007666, incorporated by reference herein in its entirety
including
the drawings. In another embodiment, the membrane disruptive agent or agents
and the
siNA molecule are also complexed with a cationic lipid or helper lipid
molecule, such as
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those lipids described in U.S. Patent No. 6,235,310, incorporated by reference
herein in
its entirety including the drawings.
In one embodiment, a siNA molecule of the invention is complexed with delivery
systems as described in U.S. Patent Application Publication No. 2003077829 and
International PCT Publication Nos. WO 00/03683 and WO 02/087541, all
incorporated
by reference herein in their entirety including the drawings.
In one embodiment, the nucleic acid molecules of the invention are
administered
via pulmonary delivery, such as by inhalation of an aerosol or spray dried
formulation
administered by an inhalation device or nebulizer, providing rapid local
uptake of the
nucleic acid molecules into relevant pulmonary tissues. Solid particulate
compositions
containing respirable dry particles of micronized nucleic acid compositions
can be
prepared by grinding dried or lyophilized nucleic acid compositions, and then
passing the
rnicronized composition through, for example, a 400 mesh screen to break up or
separate
out large agglomerates. A solid particulate composition comprising the nucleic
acid
compositions of the invention can optionally contain a dispersant which serves
to
facilitate the formation of an aerosol as well as other therapeutic compounds.
A suitable
dispersant is lactose, which can be blended with the nucleic acid compound in
any
suitable ratio, such as a 1 to 1 ratio by weight.
Aerosols of liquid particles comprising a nucleic acid composition of the
invention
can be produced by any suitable means, such as with a nebulizer (see for
example US
4,501,729). Nebulizers are commercially available devices which transform
solutions or
suspensions of an active ingredient into a therapeutic aerosol mist either by
means of
acceleration of a compressed gas, typically air or oxygen, through a narrow
venturi
orifice or by means of ultrasonic agitation. Suitable formulations for use in
nebulizers
comprise the active ingredient in a liquid Garner in an amount of up to 40%
w/w
preferably less than 20% w/w of the formulation. The carrier is typically
water or a
dilute aqueous alcoholic solution, preferably made isotonic with body fluids
by the
addition of, for example, sodium chloride or other suitable salts. Optional
additives
include preservatives if the formulation is not prepared sterile, for example,
methyl
hydroxybenzoate, anti-oxidants, flavorings, volatile oils, buffering agents
and emulsifiers
and other formulation surfactants. The aerosols of solid particles comprising
the active
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composition and surfactant can likewise be produced with any solid particulate
aerosol
generator. Aerosol generators for administering solid particulate therapeutics
to a subject
produce particles which are respirable, as explained above, and generate a
volume of
aerosol containing a predetermined metered dose of a therapeutic composition
at a rate
suitable for human administration. One illustrative type of solid particulate
aerosol
generator is an insufflator. Suitable formulations for administration by
insufflation
include finely comminuted powders which can be delivered by means of an
insufflator.
In the insufflator, the powder, e.g., a metered dose thereof effective to
carry out the
treatments described herein, is contained in capsules or cartridges, typically
made of
gelatin or plastic, which are either pierced or opened in situ and the powder
delivered by
air drawn through the device upon inhalation or by means of a manually-
operated pump.
The powder employed in the insufflator consists either solely of the active
ingredient or
of a powder blend comprising the active ingredient, a suitable powder diluent,
such as
lactose, and an optional surfactant. The active ingredient typically comprises
from 0.1 to
100 w/w of the formulation. A second type of illustrative aerosol generator
comprises a
metered dose inhaler. Metered dose inhalers are pressurized aerosol
dispensers, typically
containing a suspension or solution formulation of the active ingredient in a
liquified
propellant. During use these devices discharge the formulation through a valve
adapted
to deliver a metered volume to produce a fine particle spray containing the
active
ingredient. Suitable propellants include certain chlorofluorocarbon compounds,
for
example, dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane
and mixtures thereof. The formulation can additionally contain one or more co-
solvents,
for example, ethanol, emulsifiers and other formulation surfactants, such as
oleic acid or
sorbitan trioleate, anti-oxidants and suitable flavoring agents. Other methods
for
pulmonary delivery are described in, for example US Patent Application No.
20040037780, and US Patent Nos. 6,592,904; 6,582,728; 6,565,885.
In one embodiment, delivery systems of the invention include, for example,
aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions,
liposomes,
ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon
bases and
powders, and can contain excipients such as solubilizers, permeation enhancers
(e.g.,
fatty acids, fatty acid esters, fatty alcohols and amino acids), and
hydrophilic polymers
(e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the
pharmaceutically
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acceptable carrier is a liposome or a transdermal enhancer. Examples of
liposomes which
can be used in this invention include the following: (1) CellFectin, 1:1.5
(M/M) liposome
formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-
tetrapalmit-y-
spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2)
Cytofectin
GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen
Research);
(3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)
(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of
the
polycationic lipid DOSPA and the neutral lipid DOPE (GIBCO BRL).
In one embodiment, delivery systems of the invention include patches, tablets,
suppositories, pessaries, gels and creams, and can contain excipients such as
solubilizers
and enhancers (e.g., propylene glycol, bile salts and amino acids), and other
vehicles
(e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic
polymers
such as hydroxypropylmethylcellulose and hyaluronic acid).
In one embodiment, siNA molecules of the invention are formulated or complexed
with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine
derivatives, including for example grafted PEIs such as galactose PEI,
cholesterol PEI,
antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives
thereof
(see for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al.,
2003,
Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Phramaceutical Research,
19,
810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et
al., 1999,
Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem.,
13, 845-
854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey
et al.,
1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled
Release,
60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-
19094;
Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; and Sagara, US
6,586,524,
incorporated by reference herein.
In one embodiment, a siNA molecule of the invention comprises a bioconjugate,
for example a nucleic acid conjugate as described in Vargeese et al., USSN
10/427,160,
filed April 30, 2003; US 6,528,631; US 6,335,434; US 6, 235,886; US 6,153,737;
US
5,214,136; US 5,138,045, all incorporated by reference herein.
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Thus, the invention features a pharmaceutical composition comprising one or
more
nucleic acids) of the invention in an acceptable carrier, such as a
stabilizer, buffer, and
the like. The polynucleotides of the invention can be administered (e.g., RNA,
DNA or
protein) and introduced to a subject by any standard means, with or without
stabilizers,
buffers, and the like, to form a pharmaceutical composition. When it is
desired to use a
liposome delivery mechanism, standard protocols for formation of liposomes can
be
followed. The compositions of the present invention can also be formulated and
used as
creams, gels, sprays, oils and other suitable compositions for topical,
dermal, or
transdermal administration as is known in the art.
The present invention also includes pharmaceutically acceptable formulations
of
the compounds described. These formulations include salts of the above
compounds,
e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic,
acetic acid, and
benzene sulfonic acid.
A pharmacological composition or formulation refers to a composition or
formulation in a form suitable for administration, e.g., systemic or local
administration,
into a cell or subject, including for example a human. Suitable forms, in
part, depend
upon the use or the route of entry, for example oral, transdermal, or by
injection. Such
forms should not prevent the composition or formulation from reaching a target
cell (i.e.,
a cell to which the negatively charged nucleic acid is desirable for
delivery). For
example, pharmacological compositions injected into the blood stream should be
soluble.
Other factors are known in the art, and include considerations such as
toxicity and forms
that prevent the composition or formulation from exerting its effect.
In one embodiment, siNA molecules of the invention are administered to a
subject
by systemic administration in a pharmaceutically acceptable composition or
formulation.
By "systemic administration" is meant in vivo systemic absorption or
accumulation of
drugs in the blood stream followed by distribution throughout the entire body.
Administration routes that lead to systemic absorption include, without
limitation:
intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary
and
intramuscular. Each of these administration routes exposes the siNA molecules
of the
invention to an accessible diseased tissue. The rate of entry of a drug into
the
circulation has been shown to be a function of molecular weight or size. The
use of a
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liposome or other drug carrier comprising the compounds of the instant
invention can
potentially localize the drug, for example, in certain tissue types, such as
the tissues of
the reticular endothelial system (RES). A liposome formulation that can
facilitate the
association of drug with the surface of cells, such as, lymphocytes and
macrophages is
also useful. This approach can provide enhanced delivery of the drug to target
cells by
taking advantage of the specificity of macrophage and lymphocyte immune
recognition
of abnormal cells.
By "pharmaceutically acceptable formulation" or "pharmaceutically acceptable
composition" is meant, a composition or formulation that allows for the
effective
distribution of the nucleic acid molecules of the instant invention in the
physical location
most suitable for their desired activity. Non-limiting examples of agents
suitable for
formulation with the nucleic acid molecules of the instant invention include:
P-
glycoprotein inhibitors (such as Pluronic P85),; biodegradable polymers, such
as poly
(DL-lactide-coglycolide) microspheres for sustained release delivery (Emerich,
DF et al,
1999, Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those made
of
polybutylcyanoacrylate. Other non-limiting examples of delivery strategies for
the
nucleic acid molecules of the instant invention include material described in
Boado et al.,
1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-
284;
Pardridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Df-zcg
Delivery
Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26, 4910-
4916; and
Tyler et al., 1999, PNAS USA., 96, 7053-7058.
The invention also features the use of the composition comprising surface-
modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or
long-
circulating liposomes or stealth liposomes). These formulations offer a method
for
increasing the accumulation of drugs in target tissues. This class of drug
Garners resists
opsonization and elimination by the mononuclear phagocytic system (MPS or
RES),
thereby enabling longer blood circulation times and enhanced tissue exposure
for the
encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et
al., Chem.
Pharna. Bull. 1995, 43, 1005-1011). Such liposomes have been shown to
accumulate
selectively in tumors, presumably by extravasation and capture in the
neovascularized
target tissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al.,1995,
Bioclaina.
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Bioplays. Acta, 1238, 86-90). The long-circulating liposomes enhance the
pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to
conventional cationic liposomes which are known to accumulate in tissues of
the MPS
(Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi et al., International
PCT
Publication No. WO 96/10391; Ansell et al., International PCT Publication No.
WO
96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-
circulating liposomes are also likely to protect drugs from nuclease
degradation to a
greater extent compared to cationic liposomes, based on their ability to avoid
accumulation in metabolically aggressive MPS tissues such as the liver and
spleen.
The present invention also includes compositions prepared for storage or
administration that include a pharmaceutically effective amount of the desired
compounds in a pharmaceutically acceptable carrier or diluent. Acceptable
Garners or
diluents for therapeutic use are well known in the pharmaceutical art, and are
described,
for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R.
Gennaro edit. 1985), hereby incorporated by reference herein. For example,
preservatives, stabilizers, dyes and flavoring agents can be provided. These
include
sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In addition,
antioxidants and suspending agents can be used.
A pharmaceutically effective dose is that dose required to prevent, inhibit
the
occurrence, or treat (alleviate a symptom to some extent, preferably all of
the symptoms)
of a disease state. The pharmaceutically effective dose depends on the type of
disease,
the composition used, the route of administration, the type of mammal being
treated, the
physical characteristics of the specific mammal under consideration,
concurrent
medication, and other factors that those skilled in the medical arts will
recognize.
Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active
ingredients is administered dependent upon potency of the negatively charged
polymer.
The nucleic acid molecules of the invention and formulations thereof can be
administered orally, topically, parenterally, by inhalation or spray, or
rectally in dosage
unit formulations containing conventional non-toxic pharmaceutically
acceptable
carriers, adjuvants andlor vehicles. The term parenteral as used herein
includes
percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular,
or
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intrathecal injection or infusion techniques and the like. In addition, there
is provided a
pharmaceutical formulation comprising a nucleic acid molecule of the invention
and a
pharmaceutically acceptable carrier. One or more nucleic acid molecules of the
invention can be present in association with one or more non-toxic
pharmaceutically
acceptable carriers and/or diluents and/or adjuvants, and if desired other
active
ingredients. The pharmaceutical compositions containing nucleic acid molecules
of the
invention can be in a form suitable for oral use, for example, as tablets,
troches,
lozenges, aqueous or oily suspensions, dispersible powders or granules,
emulsion, hard
or soft capsules, or syrups or elixirs.
Compositions intended for oral use can be prepared according to any method
known to the art for the manufacture of pharmaceutical compositions and such
compositions can contain one or more such sweetening agents, flavoring agents,
coloring
agents or preservative agents in order to provide pharmaceutically elegant and
palatable
preparations. Tablets contain the active ingredient in admixture with non-
toxic
pharmaceutically acceptable excipients that are suitable for the manufacture
of tablets.
These excipients can be, for example, inert diluents; such as calcium
carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, corn starch, or alginic acid; binding
agents, for
example starch, gelatin or acacia; and lubricating agents, for example
magnesium
stearate, stearic acid or talc. The tablets can be uncoated or they can be
coated by known
techniques. In some cases such coatings can be prepared by known techniques to
delay
disintegration and absorption in the gastrointestinal tract and thereby
provide a sustained
action over a longer period. For example, a time delay material such as
glyceryl
monosterate or glyceryl distearate can be employed.
Formulations for oral use can also be presented as hard gelatin capsules
wherein
the active ingredient is mixed with an inert solid diluent, for example,
calcium carbonate,
calcium phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is
mixed with water or an oil medium, for example peanut oil, liquid paraffin or
olive oil.
Aqueous suspensions contain the active materials in a mixture with excipients
suitable for the manufacture of aqueous suspensions. Such excipients are
suspending
agents, for example sodium carboxymethylcellulose, methylcellulose,
hydropropyl-
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methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum
acacia;
dispersing or wetting agents can be a naturally-occurring phosphatide, for
example,
lecithin, or condensation products of an alkylene oxide with fatty acids, for
example
polyoxyethylene stearate, or condensation products of ethylene oxide with long
chain
aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation
products
of ethylene oxide with partial esters derived from fatty acids and a hexitol
such as
polyoxyethylene sorbitol monooleate, or condensation products of ethylene
oxide with
partial esters derived from fatty acids and hexitol anhydrides, for example
polyethylene
sorbitan monooleate. The aqueous suspensions can also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more
coloring
agents, one or more flavoring agents, and one or more sweetening agents, such
as
sucrose or saccharin.
Oily suspensions can be formulated by suspending the active ingredients in a
vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil,
or in a mineral
oil such as liquid paraffin. The oily suspensions can contain a thickening
agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and
flavoring
agents can be added to provide palatable oral preparations. These compositions
can be
preserved by the addition of an anti-oxidant such as ascorbic acid
Dispersible powders and granules suitable for preparation of an aqueous
suspension by the addition of water provide the active ingredient in admixture
with a
dispersing or wetting agent, suspending agent and one or more preservatives.
Suitable
dispersing or wetting agents or suspending agents are exemplified by those
already
mentioned above. Additional excipients, for example sweetening, flavoring and
coloring
agents, can also be present.
Pharmaceutical compositions of the invention can also be in the form of oil-in-
water emulsions. The oily phase can be a vegetable oil or a mineral oil or
mixtures of
these. Suitable emulsifying agents can be naturally-occurnng gums, for example
gum
acacia or gum tragacanth, naturally-occurnng phosphatides, for example soy
bean,
lecithin, and esters or partial esters derived from fatty acids and hexitol,
anhydrides, for
example sorbitan monooleate, and condensation products of the said partial
esters with
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ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions
can
also contain sweetening and flavoring agents.
Syrups and elixirs can be formulated with sweetening agents, for example
glycerol,
propylene glycol, sorbitol, glucose or sucrose. Such formulations can also
contain a
demulcent, a preservative and flavoring and coloring agents. The
pharmaceutical
compositions can be in the form of a sterile injectable aqueous or oleaginous
suspension.
This suspension can be formulated according to the known art using those
suitable
dispersing or wetting agents and suspending agents that have been mentioned
above.
The sterile injectable preparation can also be a sterile injectable solution
or suspension in
a non-toxic parentally acceptable diluent or solvent, for example as a
solution in 1,3-
butanediol. Among the acceptable vehicles and solvents that can be employed
are water,
Ringer's solution and isotonic sodium chloride solution. In addition, sterile,
fixed oils
are conventionally employed as a solvent or suspending medium. For this
purpose, any
bland fixed oil can be employed including synthetic mono-or diglycerides. In
addition,
fatty acids such as oleic acid find use in the preparation of injectables.
The nucleic acid molecules of the invention can also be administered in the
form of
suppositories, e.g., for rectal administration of the drug. These compositions
can be
prepared by mixing the drug with a suitable non-irntating excipient that is
solid at
ordinary temperatures but liquid at the rectal temperature and will therefore
melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene
glycols.
Nucleic acid molecules of the invention can be administered parenterally in a
sterile medium. The drug, depending on the vehicle and concentration used, can
either
be suspended or dissolved in the vehicle. Advantageously, adjuvants such as
local
anesthetics, preservatives and buffering agents can be dissolved in the
vehicle.
Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram
of
body weight per day are useful in the treatment of the above-indicated
conditions (about
0.5 mg to about 7 g per subject per day). The amount of active ingredient that
can be
combined with the Garner materials to produce a single dosage form varies
depending
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upon the host treated and the particular mode of administration. Dosage unit
forms
generally contain between from about 1 mg to about 500 mg of an active
ingredient.
It is understood that the specific dose level for any particular subject
depends upon
a variety of factors including the activity of the specific compound employed,
the age,
body weight, general health, sex, diet, time of administration, route of
administration,
and rate of excretion, drug combination and the severity of the particular
disease
undergoing therapy.
For administration to non-human animals, the composition can also be added to
the
animal feed or drinking water. It can be convenient to formulate the animal
feed and
drinking water compositions so that the animal takes in a therapeutically
appropriate
quantity of the composition along with its diet. It can also be convenient to
present the
composition as a premix for addition to the feed or drinking water.
The nucleic acid molecules of the present invention can also be administered
to a
subject in combination with other therapeutic compounds to increase the
overall
therapeutic effect. The use of multiple compounds to treat an indication can
increase the
beneficial effects while reducing the presence of side effects.
In one embodiment, the invention comprises compositions suitable for
administering nucleic acid molecules of the invention to specific cell types.
For
example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu, 1987, J. Biol.
Chena.
262, 4429-4432) is unique to hepatocytes and binds branched galactose-terminal
glycoproteins, such as asialoorosomucoid (ASOR). In another example, the
folate
receptor is overexpressed in many cancer cells. Binding of such glycoproteins,
synthetic
glycoconjugates, or folates to the receptor takes place with an affinity that
strongly
depends on the degree of branching of the oligosaccharide chain, for example,
triatennary structures are bound with greater affinity than biatenarry or
monoatennary
chains (Baenziger and Fiete, 1980, Cell, 22, 611-620; Connolly et al., 1982,
J. Biol.
Claem., 257, 939-945). Lee and Lee, 1987, Glycocor jugate J., 4, 317-328,
obtained this
high specificity through the use of N-acetyl-D-galactosamine as the
carbohydrate moiety,
which has higher affinity for the receptor, compared to galactose. This
"clustering effect"
has also been described for the binding and uptake of mannosyl-terminating
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glycoproteins or glycoconjugates (Ponpipom et al., 1981, J. Med. Clzem., 24,
1388-
1395). The use of galactose, galactosamine, or folate based conjugates to
transport
exogenous compounds across cell membranes can provide a targeted delivery
approach
to, for example, the treatment of liver disease, cancers of the liver, or
other cancers. The
use of bioconjugates can also provide a reduction in the required dose of
therapeutic
compounds required for treatment. Furthermore, therapeutic bioavailability,
pharmacodynamics, and pharmacokinetic parameters can be modulated through the
use
of nucleic acid bioconjugates of the invention. Non-limiting examples of such
bioconjugates are described in Vargeese et al., USSN 10/201,394, filed August
13, 2001;
and Matulic-Adamic et al., USSN 60/362,016, filed March 6, 2002.
Alternatively, certain siNA molecules of the instant invention can be
expressed
within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985,
Science, 229,
345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon
et al.,
1991, PYOe. Natl. Acad. Sci. USA, 88, 10591-5; I~ashani-Sabet et al., 1992,
Antisense
Res. Dev., 2, 3-15; Dropulic et al., 1992, J. hiz~ol., 66, 1432-41;
Weerasinghe et al.,
1991, J. Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA,
89, 10802-
6; Chen et al., 1992, Nucleic Acids Res., 20, 4581-9; Sarver et al., 1990
Science, 247,
1222-1225; Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Good et al.,
1997,
Gerze Tlzez~apy, 4, 45. Those skilled in the art realize that any nucleic acid
can be
expressed in eukaryotic cells from the appropriate DNA/RNA vector. The
activity of
such nucleic acids can be augmented by their release from the primary
transcript by a
enzymatic nucleic acid (Draper et al., PCT WO 93/23569, and Sullivan et al.,
PCT WO
94/02595; Ohkawa et al., 1992, Nucleic Acids Syznp. Ser., 27, 15-6; Taira et
al., 1991,
Nucleic Acids Res., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,
3249-55;
Chowrira et al., 1994, J. Biol. Clzezn., 269, 25856.
In another aspect of the invention, RNA molecules of the present invention can
be
expressed from transcription units (see for example Couture et al., 1996,
TIG., 12, 510)
inserted into DNA or RNA vectors. The recombinant vectors can be DNA plasmids
or
viral vectors. siNA expressing viral vectors can be constructed based on, but
not limited
to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. In another
embodiment,
pol III based constructs are used to express nucleic acid molecules of the
invention (see
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for example Thompson, U.S. Pats. Nos. 5,902,880 and 6,146,886). The
recombinant
vectors capable of expressing the siNA molecules can be delivered as described
above,
and persist in target cells. Alternatively, viral vectors can be used that
provide for
transient expression of nucleic acid molecules. Such vectors can be repeatedly
S administered as necessary. Once expressed, the siNA molecule interacts with
the target
mRNA and generates an RNAi response. Delivery of siNA molecule expressing
vectors
can be systemic, such as by intravenous or infra-muscular administration, by
administration to target cells ex-planted from a subject followed by
reintroduction into
the subject, or by any other means that would allow for introduction into the
desired
target cell (for a review see Couture et al., 1996, TIG., 12, 510).
In one aspect the invention features an expression vector comprising a nucleic
acid
sequence encoding at least one siNA molecule of the instant invention. The
expression
vector can encode one or both strands of a siNA duplex, or a single self
complementary
strand that self hybridizes into a siNA duplex. The nucleic acid sequences
encoding the
1 S siNA molecules of the instant invention can be operably linked in a manner
that allows
expression of the siNA molecule (see for example Paul et al., 2002, Nature
Biotechnology, 19, SOS; Miyagishi and Taira, 2002, Nature Biotechnology, 19,
497; Lee
et al., 2002, Nature Biotechraology, 19, 500; and Novina et al., 2002, Nature
Medicine,
advance online publication doi:10.1038/nm725).
In another aspect, the invention features an expression vector comprising: a)
a
transcription initiation region (e.g., eukaryotic pol I, II or III initiation
region); b) a
transcription termination region (e.g., eukaryotic pol I, II or III
termination region); and
c) a nucleic acid sequence encoding at least one of the siNA molecules of the
instant
invention, wherein said sequence is operably linked to said initiation region
and said
2S termination region in a manner that allows expression and/or delivery of
the siNA
molecule. The vector can optionally include an open reading frame (ORF) for a
protein
operably linked on the S' side or the 3'-side of the sequence encoding the
siNA of the
invention; and/or an intron (intervening sequences).
Transcription of the siNA molecule sequences can be driven from a promoter for
eukaryotic RNA polymerase I (poI I), RNA polymerase II (pol II), or RNA
polymerase
III (pol III). Transcripts from pol II or pol III promoters are expressed at
high levels in
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all cells; the levels of a given pol II promoter in a given cell type depends
on the nature
of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
Prokaryotic
RNA polymerase promoters are also used, providing that the prokaryotic RNA
polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss,
1990,
Proc. Natl. Acad. Sci. U S A, 87, 6743-7; Gao and Huang 1993, Nucleic Acids
Res., 21,
2867-72; Lieber et al., 1993, Methods Erazynol., 217, 47-66; Zhou et al.,
1990, Mol.
Cell. Biol., 10, 4529-37). Several investigators have demonstrated that
nucleic acid
molecules expressed from such promoters can function in mammalian cells (e.g.
Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwang et al., 1992,
Proc.
Natl. Acad. Sci. U S A, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res.,
20, 4581-9;
Yu et al., 1993, Proc. Natl. Acad. Sci. U S A, 90, 6340-4; L'Huillier et al.,
1992, EMBO
J., 11, 4411-8; Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U. S. A, 90,
8000-4;
Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger ~ Cech, 1993,
Science,
262, 1566). More specifically, transcription units such as the ones derived
from genes
encoding U6 small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA
are
useful in generating high concentrations of desired RNA molecules such as siNA
in cells
(Thompson et al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,
1994,
Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No. 5,624,803; Good et
al., 1997,
Gene TheY., 4, 45; Beigelman et al., International PCT Publication No. WO
96/18736.
The above siNA transcription units can be incorporated into a variety of
vectors for
introduction into mammalian cells, including but not restricted to, plasmid
DNA vectors,
viral DNA vectors (such as adenovirus or adeno-associated virus vectors), or
viral RNA
vectors (such as retroviral or alphavirus vectors) (for a review see Couture
and
Stinchcomb, 1996, supra).
In another aspect the invention features an expression vector comprising a
nucleic
acid sequence encoding at least one of the siNA molecules of the invention in
a manner
that allows expression of that siNA molecule. The expression vector comprises
in one
embodiment; a) a transcription initiation region; b) a transcription
termination region;
and c) a nucleic acid sequence encoding at least one strand of the siNA
molecule,
wherein the sequence is operably linked to the initiation region and the
termination
region in a manner that allows expression and/or delivery of the siNA
molecule.
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In another embodiment the expression vector comprises: a) a transcription
initiation region; b) a transcription termination region; c) an open reading
frame; and d) a
nucleic acid sequence encoding at Ieast one strand of a siNA molecule, wherein
the
sequence is operably linked to the 3'-end of the open reading frame and
wherein the
sequence is operably linked to the initiation region, the open reading frame
and the
termination region in a manner that allows expression and/or delivery of the
siNA
molecule. In yet another embodiment, the expression vector comprises: a) a
transcription initiation region; b) a transcription termination region; c) an
intron; and d) a
nucleic acid sequence encoding at least one siNA molecule, wherein the
sequence is
operably linked to the initiation region, the intron and the termination
region in a manner
which allows expression and/or delivery of the nucleic acid molecule.
In another embodiment, the expression vector comprises: a) a transcription
initiation region; b) a transcription termination region; c) an intron; d) an
open reading
frame; and e) a nucleic acid sequence encoding at least one strand of a siNA
molecule,
wherein the sequence is operably linked to the 3'-end of the open xeading
frame and
wherein the sequence is operably linked to the initiation region, the intron,
the open
reading frame and the termination region in a manner which allows expression
and/or
delivery of the siNA molecule.
GPRA and AAAI Biolo~y and Biochemistry
The following discussion is adapted from the OMIM database entry for g protein-
coupled receptor for asthma susceptibility; GPRA, vasopressin receptor-related
receptor
1; vrrl, pgrl4 and asthma-associated alternatively spliced gene 1
To positionally clone genes confernng susceptibility to asthma and showing
linkage to chromosome 7p, Laitinen et al., 2004, Science, 304, 300-304, used a
hierarchical genotyping approach to identify a 133-kb risk-conferring segment
of
chromosome 7p. The segment was examined for specific genes resulting in the
identification of a gene designated GPRA for G protein-coupled receptor for
asthma
susceptibility. The 133-kb segment spans from intron 2 to intron 5 of GPR.A.
Northern
blot hybridization with a 1,285-by full-Iength GPRA cDNA probe identified a
2.4-kb
transcript in all tissues examined. GPR.A expression was much highex in the
ciliated
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cells of the respiratory epithelium from asthma patients compared with those
from
normal control patients. Asthmatic smooth muscle immunohistochemically stained
strongly positive for GPRA isoform B, in contrast to the negative Ending in
controls. In
addition, a higher level of GPRA expression was also found in mRNA from lungs
of
sensitized versus control mice after inhaled ovalbumin challenge.
Another risk confernng segment identiEed by Laitinen et al., supra, referred
to as
AAA1 for asthma-associated alternatively spliced gene 1, lies on the opposite
DNA
strand from GPRA and showed only weak homologies to known proteins. AAA1
exhibits complex alternative splicing. Laitinen et al., supra concluded that
several lines
of evidence suggested that AAA1 may not represent a protein-coding gene,
although its
expression was modified by the haplotype. The longest open-reading frame
comprised
only 74 potential amino acids, and ira vitro translation failed to yield a
stable polypeptide.
Transiently transfected cells did not produce recombinant protein. Polyclonal
peptide
antibodies detected the antigen but no proteins in Western blots or
immunohistochemistry.
The use of small interfering nucleic acid molecules targeting GPRA and AAA1,
such as disease related alleles of GPRA and/or AAA1, therefore provides a
class of novel
therapeutic agents that can be used in the the treatment of asthma and
associated
conditions that can respond to modulation of GPR.A and/or AAA1 levels in a
cell, tissue,
or subject.
Examples:
The following are non-limiting examples showing the selection, isolation,
synthesis and activity of nucleic acids of the instant invention.
Example 1: Tandem synthesis of siNA constructs
Exemplary siNA molecules of the invention are synthesized in tandem using a
cleavable linker, for example, a succinyl-based linker. Tandem synthesis as
described
herein is followed by a one-step puriEcation process that provides RNAi
molecules in
high yield. This approach is highly amenable to siNA synthesis in support of
high
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throughput RNAi screening, and can be readily adapted to multi-column or multi-
well
synthesis platforms.
After completing a tandem synthesis of a siNA oligo and its complement in
which
the 5'-terminal dimethoxytrityl (5'-O-DMT) group remains intact (trityl on
synthesis), the
oligonucleotides are deprotected as described above. Following deprotection,
the siNA
sequence strands are allowed to spontaneously hybridize. This hybridization
yields a
duplex in which one strand has retained the 5'-O-DMT group while the
complementary
strand comprises a terminal 5'-hydroxyl. The newly formed duplex behaves as a
single
molecule during routine solid-phase extraction purification (Trityl-On
purification) even
though only one molecule has a dimethoxytrityl group. Because the strands form
a
stable duplex, this dimethoxytrityl group (or an equivalent group, such as
other trityl
groups or other hydrophobic moieties) is all that is required to purify the
pair of oligos,
for example, by using a C 18 cartridge.
Standard phosphoramidite synthesis chemistry is used up to the point of
introducing a tandem linker, such as an inverted deoxy abasic succinate or
glyceryl
succinate linker (see Figure 1) or an equivalent cleavable linker. A non-
limiting
example of linker coupling conditions that can be used includes a hindered
base such as
diisopropylethylamine (DIPA) and/or DMAP in the presence of an activator
reagent such
as Bromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After the
linker is
coupled, standard synthesis chemistry is utilized to complete synthesis of the
second
sequence leaving the terminal the S'-O-DMT intact. Following synthesis, the
resulting
oligonucleotide is deprotected according to the procedures described herein
and
quenched with a suitable buffer, for example with SOmM NaOAc or 1.SM NH4H2C03.
Purification of the siNA duplex can be readily accomplished using solid phase
extraction, for example, using a Waters C18 SepPak lg cartridge conditioned
with 1
column volume (CV) of acetonitrile, 2 CV H20, and 2 CV SOmM NaOAc. The sample
is loaded and then washed with 1 CV H20 or SOmM NaOAc. Failure sequences are
eluted with 1 CV 14% ACN (Aqueous with SOmM NaOAc and SOmM NaCI). The
column is then washed, for example with 1 CV H20 followed by on-column
detritylation, for example by passing 1 CV of 1% aqueous trifluoroacetic acid
(TFA)
over the column, then adding a second CV of 1% aqueous TFA to the column and
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allowing to stand for approximately 10 minutes. The remaining TFA solution is
removed and the column washed with H20 followed by 1 CV 1M NaCI and additional
H20. 'The siNA duplex product is then eluted, for example, using 1 CV 20%
aqueous
CAN.
Figure 2 provides an example of MALDI-TOF mass spectrometry analysis of a
purified siNA construct in which each peak corresponds to the calculated mass
of an
individual siNA strand of the siNA duplex. The same purified siNA provides
three
peaks when analyzed by capillary gel electrophoresis (CGE), one peak
presumably
corresponding to the duplex siNA, and two peaks presumably corresponding to
the
separate siNA sequence strands. Ion exchange HPLC analysis of the same siNA
contract
only shows a single peak. Testing of the purred siNA construct using a
luciferase
reporter assay described below demonstrated the same RNAi activity compared to
siNA
constructs generated from separately synthesized oligonucleotide sequence
strands.
Example 2: Identification of potential siNA target sites in any RNA sequence
The sequence of an RNA target of interest, such as a viral or human mRNA
transcript, is screened for target sites, for example by using a computer
folding
algorithm. In a non-limiting example, the sequence of a gene or RNA gene
transcript
derived from a database, such as Genbank, is used to generate siNA targets
having
complementarity to the target. Such sequences can be obtained from a database,
or can
be determined experimentally as known in the art. Target sites that are known,
for
example, those target sites determined to be effective target sites based on
studies with
other nucleic acid molecules, for example ribozymes or antisense, or those
targets known
to be associated with a disease or condition such as those sites containing
mutations or
deletions, can be used to design siNA molecules targeting those sites. Various
parameters can be used to determine which sites are the most suitable target
sites within
the target RNA sequence. These parameters include but are not limited to
secondary or
tertiary RNA structure, the nucleotide base composition of the target
sequence, the
degree of homology between various regions of the taxget sequence, or the
relative
position of the target sequence within the RNA transcript. Based on these
determinations, any number of target sites within the RNA transcript can be
chosen to
screen siNA molecules for efficacy, for example by using ira vitro RNA
cleavage assays,
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cell culture, or animal models. In a non-limiting example, anywhere from 1 to
1000
target sites are chosen within the transcript based on the size of the siNA
construct to be
used. High throughput screening assays can be developed for screening siNA
molecules
using methods known in the art, such as with mufti-well or mufti-plate assays
to
determine efficient reduction in target gene expression.
Example 3: Selection of siNA molecule target sites in a RNA
The following non-limiting steps can be used to carry out the selection of
siNAs
targeting a given gene sequence or transcript.
1. The target sequence is parsed in silico into a list of all fragments or
subsequences of a
particular length, for example 23 nucleotide fragments, contained within the
target
sequence. This step is typically carried out using a custom Perl script, but
commercial
sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin
Package can be employed as well.
2. In some instances the siNAs correspond to more than one target sequence;
such
would be the case for example in targeting different transcripts of the same
gene,
targeting different transcripts of more than one gene, or for targeting both
the human
gene and an animal homolog. In this case, a subsequence list of a particular
length is
generated for each of the targets, and then the lists are compared to find
matching
sequences in each list. The subsequences are then ranked according to the
number of
target sequences that contain the given subsequence; the goal is to find
subsequences
that are present in most or all of the target sequences. Alternately, the
ranking can
identify subsequences that are unique to a target sequence, such as a mutant
target
sequence. Such an approach would enable the use of siNA to target specifically
the
mutant sequence and not effect the expression of the normal sequence.
3. In some instances the siNA subsequences are absent in one or more sequences
while
present in the desired target sequence; such would be the case if the siNA
targets a
gene with a paralogous family member that is to remain untargeted. As in case
2
above, a subsequence list of a particular length is generated for each of the
targets,
and then the lists are compared to find sequences that are present in the
target gene
but are absent in the untargeted paralog.
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4. The ranked siNA subsequences can be further analyzed and ranked according
to GC
content. A preference can be given to sites containing 30-70% GC, with a
further
preference to sites containing 40-60% GC.
5. The ranked siNA subsequences can be further analyzed and ranked according
to self
folding and internal hairpins. Weaker internal folds are preferred; strong
hairpin
structures are to be avoided.
6. The ranked siNA subsequences can be further analyzed and ranked according
to
whether they have runs of GGG or CCC in the sequence. GGG (or even more Gs) in
either strand can make oligonucleotide synthesis problematic and can
potentially
interfere with RNAi activity, so it is avoided whenever better sequences are
available.
CCC is searched in the target strand because that will place GGG in the
antisense
strand.
7. The ranked siNA subsequences can be further analyzed and ranked according
to
whether they have the dinucleotide UU (uridine dinucleotide) on the 3'-end of
the
sequence, and/or AA on the 5'-end of the sequence (to yield 3' UU on the
antisense
sequence). These sequences allow one to design siNA molecules with terminal TT
thymidine dinucleotides.
8. Four or five target sites are chosen from the ranked list of subsequences
as described
above. For example, in subsequences having 23 nucleotides, the right 21
nucleotides
of each chosen 23-mer subsequence are then designed and synthesized for the
upper
(sense) strand of the siNA duplex, while the reverse complement of the left 21
nucleotides of each chosen 23-mer subsequence are then designed and
synthesized for
the lower (antisense) strand of the siNA duplex (see Tables II and III). If
terminal
TT residues are desired for the sequence (as described in paragraph 7), then
the two 3'
terminal nucleotides of both the sense and antisense strands are replaced by
TT prior
to synthesizing the oligos.
9. The siNA molecules are screened in an in vitro, cell culture or animal
model system
to identify the most active siNA molecule or the most preferred target site
within the
target RNA sequence.
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10. Other design considerations can be used when selecting target nucleic acid
sequences, see, for example, Reynolds et al., 2004, Nature Bioteclanology
Advanced
Online Publication, 1 February 2004, doi:10.10381nbt936 and LTi-Tei et al.,
2004,
Nucleic Acids Research, 32, doi:10.10931nar/gkh247.
In an alternate approach, a pool of siNA constructs specific to a GPRA and/or
AAA1 target sequence is used to screen for target sites in cells expressing
GPRA and/or
AAAl RNA, such as such A549. The general strategy used in this approach is
shown in
Figure 9. A non-limiting example of such is a pool comprising sequences having
any of
SEQ ID NOs. 1-806. Cells expressing GPRA and/or AAAl are transfected with the
pool of siNA constructs and cells that demonstrate a phenotype associated with
GPRA
andlor AAA1 inhibition are sorted. The pool of siNA constructs can be
expressed from
transcription cassettes inserted into appropriate vectors (see for example
Figure 7 and
Figure 8). The siNA from cells demonstrating a positive phenotypic change
(e.g.,
decreased proliferation, decreased GPRA and/or AAAl mRNA levels or decreased
GPRA and/or AAA1 protein expression), are sequenced to determine the most
suitable
target sites) within the target GPRA and/or AAA1 RNA sequence.
Example 4~ GPR.A and/or AAA1 tar,eted siNA design
siNA target sites were chosen by analyzing sequences of the GPRA and/or AAAl
RNA target and optionally prioritizing the target sites on the basis of
folding (structure of
any given sequence analyzed to determine siNA accessibility to the target), by
using a
library of siNA molecules as described in Example 3, or alternately by using
an ira vitro
siNA system as described in Example 6 herein. siNA molecules were designed
that
could bind each target and are optionally individually analyzed by computer
folding to
assess whether the siNA molecule can interact with the target sequence.
Varying the
length of the siNA molecules can be chosen to optimize activity. Generally, a
sufficient
number of complementary nucleotide bases are chosen to bind to, or otherwise
interact
with, the target RNA, but the degree of complementarity can be modulated to
accommodate siNA duplexes or varying length or base composition. By using such
methodologies, siNA molecules can be designed to target sites within any known
RNA
sequence, for example those RNA sequences corresponding to the any gene
transcript.
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Chemically modified siNA constructs are designed to provide nuclease stability
for
systemic administration in vivo and/or improved pharmacokinetic, localization,
and
delivery properties while preserving the ability to mediate RNAi activity.
Chemical
modifications as described herein are introduced synthetically using synthetic
methods
described herein and those generally known in the art. The synthetic siNA
constructs are
then assayed for nuclease stability in serum and/or cellular/tissue extracts
(e.g. liver
extracts). The synthetic siNA constructs are also tested in parallel for RNAi
activity
using an appropriate assay, such as a luciferase reporter assay as described
herein or
another suitable assay that can quantity RNAi activity. Synthetic siNA
constructs that
possess both nuclease stability and RNAi activity can be further modified and
re-
evaluated in stability and activity assays. The chemical modifications of the
stabilized
active siNA constructs can then be applied to any siNA sequence targeting any
chosen
RNA and used, for example, in target screening assays to pick lead siNA
compounds for
therapeutic development (see for example Figure 11).
Example 5: Chemical Synthesis and Purification of siNA
siNA molecules can be designed to interact with various sites in the RNA
message,
for example, target sequences within the RNA sequences described herein. The
sequence of one strand of the siNA molecules) is complementary to the target
site
sequences described above. The siNA molecules can be chemically synthesized
using
methods described herein. Inactive siNA molecules that are used as control
sequences
can be synthesized by scrambling the sequence of the siNA molecules such that
it is not
complementary to the target sequence. Generally, siNA constructs can by
synthesized
using solid phase oligonucleotide synthesis methods as described herein (see
for example
Usman et al., US Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657;
6,353,098;
. 6,362,323; 6,437,117; 6,469,158; Scaringe et al., US Patent Nos. 6,111,086;
6,008,400;
6,111,086 all incorporated by reference herein in their entirety).
In a non-limiting example, RNA oligonucleotides are synthesized in a stepwise
fashion using the phosphoramidite chemistry as is known in the art. Standard
phosphoramidite chemistry involves the use of nucleosides comprising any of 5'-
O-
dimethoxytrityl, 2'-O-tert-butyldimethylsilyl, 3'-O-2-Cyanoethyl N,N-
diisopropylphos-
phoroamidite groups, and exocyclic amine protecting groups (e.g. N6-benzoyl
adenosine,
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N4 acetyl cytidine, and N2-isobutyryl guanosine). Alternately, 2'-O-Silyl
Ethers can be
used in conjunction with acid-labile 2'-O-orthoester protecting groups in the
synthesis of
RNA as described by Scaringe supra. Differing 2' chemistries can require
different
protecting groups, for example 2'-deoxy-2'-amino nucleosides can utilize N-
phthaloyl
protection as described by Usman et al., US Patent 5,631,360, incorporated by
reference
herein in its entirety).
During solid phase synthesis, each nucleotide is added sequentially (3'- to 5'-
direction) to the solid support-bound oligonucleotide. The first nucleoside at
the 3'-end
of the chain is covalently attached to a solid support (e.g., controlled pore
glass or
polystyrene) using various linleers. The nucleotide precursor, a
ribonucleoside
phosphoramidite, and activator are combined resulting in the coupling of the
second
nucleoside phosphoramidite onto the 5'-end of the first nucleoside. The
support is then
washed and any unreacted 5'-hydroxyl groups are capped with a capping reagent
such as
acetic anhydride to yield inactive 5'-acetyl moieties. The trivalent
phosphorus linkage is
then oxidized to a more stable phosphate linkage. At the end of the nucleotide
addition
cycle, the 5 °-O-protecting group is cleaved under suitable conditions
(e.g., acidic
conditions for trityl-based groups and Fluoride for silyl-based groups). The
cycle is
repeated for each subsequent nucleotide.
Modiftcation of synthesis conditions can be used to optimize coupling
efficiency,
for example by using differing coupling times, differing
reagent/phosphoramidite
concentrations, differing contact times, differing solid supports and solid
support linker
chemistries depending on the particular chemical composition of the siNA to be
synthesized. Deprotection and purification of the siNA can be performed as is
generally
described in Usman et al., US 5,831,071, US 6,353,098, US 6,437,117, and
Bellon et al.,
US 6,054,576, US 6,162,909, US 6,303,773, or Scaringe supra, incorporated by
reference herein in their entireties. Additionally, deprotection conditions
can be
modified to provide the best possible yield and purity of siNA constructs. For
example,
applicant has observed that oligonucleotides comprising 2'-deoxy-2'-fluoro
nucleotides
can degrade under inappropriate deprotection conditions. Such oligonucleotides
are
deprotected using aqueous methylamine at about 35°C for 30 minutes. If
the 2'-deoxy-
2'-fluoro containing oligonucleotide also comprises ribonucleotides, after
deprotection
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with aqueous methylamine at about 35°C for 30 minutes, TEA-HF is added
and the
reaction maintained at about 65°C for an additional 15 minutes.
Example 6: RNAi in vitro asst to assess siNA activity
An in vitro assay that recapitulates RNAi in a cell-free system is used to
evaluate
siNA constructs targeting GPRA and/or AAAl RNA targets. The assay comprises
the
system described by Tuschl et al., 1999, Genes and Developfnent, 13, 3191-3197
and
Zamore et al., 2000, Cell, 101, 25-33 adapted for use with GPRA and/or AAA1
target
RNA. A Drosophila extract derived from syncytial blastoderm is used to
reconstitute
RNAi activity ira vitro. Target RNA is generated via in vitro transcription
from an
appropriate GPRA and/or AAAl expressing plasmid using T7 RNA polymerase or via
chemical synthesis as.described herein. Sense and antisense siNA strands (for
example
uM each) are annealed by incubation in buffer (such as 100 mM potassium
acetate, 30
mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for 1 minute at 90°C
followed by
1 hour at 37°C , then diluted in lysis buffer (for example 100 mM
potassium acetate, 30
15 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate). Annealing can be monitored
by
gel electrophoresis on an agarose gel in TBE buffer and stained with ethidium
bromide.
The Drosophila lysate is prepared using zero to two-hour-old embryos from
Oregon R
flies collected on yeasted molasses agar that are dechorionated and lysed. The
lysate is
centrifuged and the supernatant isolated. The assay comprises a reaction
mixture
20 containing 50% lysate [vol/vol], RNA (10-50 pM final concentration), and
10% [vol/vol]
lysis buffer containing siNA (10 nM final concentration). The reaction mixture
also
contains 10 mM creatine phosphate, 10 ug/ml creatine phosphokinase, 100 um
GTP, 100
uM UTP, 100 uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100
uM of each amino acid. The final concentration of potassium acetate is
adjusted to 100
mM. The reactions are pre-assembled on ice and preincubated at 25° C
for 10 minutes
before adding RNA, then incubated at 25° C for an additional 60
minutes. Reactions are
quenched with 4 volumes of 1.25 x Passive Lysis Buffer (Promega). Target RNA
cleavage is assayed by RT-PCR analysis or other methods known in the art and
are
compared to control reactions in which siNA is omitted from the reaction.
Alternately, internally-labeled target RNA for the assay is prepared by ira
vitro
transcription in the presence of [alpha-32p] CTP, passed over a G50 Sephadex
column by
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spin chromatography and used as target RNA without further purification.
Optionally,
target RNA is 5' 32P-end labeled using T4 polynucleotide kinase enzyme. Assays
are
performed as described above and target RNA and the specific RNA cleavage
products
generated by RNAi are visualized on an autoradiograph of a gel. The percentage
of
cleavage is determined by PHOSPHOR IMAGER~ (autoradiography) quantitation of
bands representing intact control RNA or RNA from control reactions without
siNA and
the cleavage products generated by the assay.
In one embodiment, this assay is used to determine target sites in the GPRA
and/or
AAA1 RNA target for siNA mediated RNAi cleavage, wherein a plurality of siNA
constructs are screened for RNAi mediated cleavage of the GPRA and/or AAA1 RNA
target, for example, by analyzing the assay reaction by electrophoresis of
labeled target
RNA, or by northern blotting, as well as by other methodology well known in
the art.
Example 7: Nucleic acid inhibition of GPRA and/or AAA1 target RNA
siNA molecules targeted to the human GPRA and/or AAA1 RNA are designed and
synthesized as described above. These nucleic acid molecules can be tested for
cleavage
activity in vivo, for example, using the following procedure. The target
sequences and
the nucleotide location within the GPRA and/or AAAl RNA are given in Tables II
and
III.
Two formats are used to test the efficacy of siNAs targeting GPRA and/or AAA1.
First, the reagents are tested in cell culture using, for example, A549 cells,
to determine
the extent of RNA and protein inhibition. siNA reagents (e.g.; see Tables II
and III) are
selected against the GPRA and/or AAA1 target as described herein. RNA
inhibition is
measured after delivery of these reagents by a suitable transfection agent to,
for example,
cultured A549 cells. Relative amounts of target RNA are measured versus actin
using
real-time PCR monitoring of amplification (eg., ABI 7700 TAQMAN~). A
comparison
is made to a mixture of oligonucleotide sequences made to unrelated targets or
to a
randomized siNA control with the same overall length and chemistry, but
randomly
substituted at each position. Primary and secondary lead reagents are chosen
for the
target and optimization performed. After an optimal transfection agent
concentration is
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chosen, a RNA time-course of inhibition is performed with the lead siNA
molecule. In
addition, a cell-plating format can be used to determine RNA inhibition.
Delivery of siNA to Cells
Cells such as A549 cells are seeded, for example, at 1x105 cells per well of a
six-
well dish in EGM-2 (BioWhittaker) the day before transfection. siNA (final
concentration, for example 20nM) and cationic lipid (e.g., final concentration
2~,g/ml)
are complexed in EGM basal media (Bio Whittaker) at 37°C for 30 minutes
in
polystyrene tubes. Following vortexing, the complexed siNA is added to each
well and
incubated for the times indicated. For initial optimization experiments, cells
are seeded,
for example, at 1x103 in 96 well plates and siNA complex added as described.
Efficiency
of delivery of siNA to cells is determined using a fluorescent siNA complexed
with lipid.
Cells in 6-well dishes are incubated with siNA for 24 hours, rinsed with PBS
and fixed in
2% paraformaldehyde for 15 minutes at room temperature. Uptake of siNA is
visualized
using a fluorescent microscope.
TAOMAN~ (real-time PCR monitorin og f amplification) and Liahtcycler
quantification
of mRNA
Total RNA is prepared from cells following siNA delivery, for example, using
Qiagen RNA purification kits for 6-well or Rneasy extraction kits for 96-well
assays. For
TAQMAN~ analysis (real-time PCR monitoring of amplification), dual-labeled
probes
are synthesized with the reporter dye, FAM or JOE, covalently linked at the 5'-
end and
the quencher dye TAMRA conjugated to the 3'-end. One-step RT-PCR
amplifications
are performed on, for example, an ABI PRISM 7700 Sequence Detector using 50
~.1
reactions consisting of 10 ~.l total RNA, 100 nM forward primer, 900 nM
reverse primer,
100 nM probe, 1X TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM
MgCl2, 300 ~M each dATP, dCTP, dGTP, and dTTP, l0U RNase Inhibitor (Promega),
1.25U AMPLITAQ GOLD~ (DNA polymerase) (PE-Applied Biosystems) and l0U M-
MLV Reverse Transcriptase (Promega). The thermal cycling conditions can
consist of
minutes at 48°C, 10 minutes at 95°C, followed by 40 cycles of 15
seconds at 95°C
and 1 minute at 60°C. Quantitation of mRNA levels is determined
relative to standards
30 generated from serially diluted total cellular RNA (300, 100, 33, 11
ng/rxn) and
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normalizing to 13-actin or GAPDH mRNA in parallel TAQMAN~ reactions (real-time
PCR monitoring of amplification). For each gene of interest an upper and lower
primer
and a fluorescently labeled probe are designed. Real time incorporation of
SYBR Green
I dye into a specific PCR product can be measured in glass capillary tubes
using a
lightcyler. A standard curve is generated for each primer pair using control
cRNA.
Values are represented as relative expression to GAPDH in each sample.
Western blotting
Nuclear extracts can be prepared using a standard micro preparation technique
(see
for example Andrews and Fallen 1991, Nucleic Acids Research, 19, 2499).
Protein
extracts from supernatants are prepared, for example using TCA precipitation.
An equal
volume of 20% TCA is added to the cell supernatant, incubated on ice for 1
hour and
pelleted by centrifugation for 5 minutes. Pellets are washed in acetone, dried
and
resuspended in water. Cellular protein extracts are run on a 10% Bis-Tris
NuPage
(nuclear extracts) or 4-12% Tris-Glycine (supernatant extracts) polyacrylamide
gel and
transferred onto nitro-cellulose membranes. Non-specific binding can be
blocked by
incubation, for example, with 5% non-fat milk for 1 hour followed by primary
antibody
for 16 hour at 4°C. Following washes, the secondary antibody is
applied, for example
(1:10,000 dilution) for 1 hour at room temperature and the signal detected
with
SuperSignal reagent (Pierce).
Example 8' Animal Models useful to evaluate the down-regulation of GPRA and/or
AAA1 gene expression
Evaluating the efficacy of anti-GPRA and/or AAAI agents in animal models is an
important prerequisite to human clinical trials. Laitinen et al., 2004,
Science, 304, 300-
304, describe a mouse model of of ovalbumin-induced lung inflammation in which
GPRA mRNA is significantly up-regulated in mouse lung after ovalbumin tests in
sensitized compared with nonsensitized mice. Using this model, ovalbumin
sensitized
mice can be treated with active and control siNA molecules of the invention
and GPRA
mRNA and/or protein levels can be assayed to identify or validate efficacious
siNA
molecules of the invention that are useful in treating asthma and other
conditions that
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respond to GPRA or AAAl. As such, this model provides an animal model for
testing
therapeutic drugs, including siNA constructs of the instant invention.
Example 9: RNAi mediated inhibition of GPRA and/or AAA1 expression
siNA constructs (Table III) are tested for efficacy in reducing GPRA and/or
AAA1 RNA expression in, for example, A549 cells. Cells are plated
approximately 24
hours before transfection in 96-well plates at 5,000-7,500 cells/well, 100
~.1/well, such
that at the time of transfection cells are 70-90% confluent. For transfection,
annealed
siNAs are mixed with the transfection reagent (Lipofectamine 2000, Invitrogen)
in a
volume of 50 ~1/well and incubated for 20 minutes at room temperature. The
siNA
transfection mixtures are added to cells to give a final siNA concentration of
25 nM in a
volume of 150 ~1. Each siNA transfection mixture is added to 3 wells for
triplicate siNA
treatments. Cells are incubated at 37° for 24 hours in the continued
presence of the siNA
transfection mixture. At 24 hours, RNA is prepared from each well of treated
cells. The
supernatants with the transfection mixtures are first removed and discarded,
then the
cells are lysed and RNA prepared from each well. Target gene expression
following
treatment is evaluated by RT-PCR for the target gene and for a control gene
(36B4, an
RNA polymerase subunit) for normalization. The triplicate data is averaged and
the
standard deviations determined for each treatment. Normalized data are graphed
and the
percent reduction of target mRNA by active siNAs in comparison to their
respective
inverted control siNAs is determined.
Example 10: Indications
The present body of lenowledge in GPRA and/or AAA1 research indicates the need
for methods to assay GPRA and/or AAA1 activity and for compounds that can
regulate
GPR.A and/or AAA1 expression for research, diagnostic, and therapeutic use. As
described herein, the nucleic acid molecules of the present invention can be
used in
assays to diagnose disease state related of GPRA and/or AAAl levels. In
addition, the
nucleic acid molecules can be used to treat disease state related to GPRA
and/or AAAl
levels.
Particular conditions and disease states that can be associated with GPRA
and/or
AAA1 expression modulation include, but are not limited to asthma, chronic
obstructive
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pulmonary disease or "COPD", allergic rhinitis, sinusitis, pulmonary
vasoconstriction,
inflammation, allergies, impeded respiration, respiratory distress syndrome,
cystic
fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, and
any
other diseases or conditions related to asthma that are related to or will
respond to the
levels of a GPRA andlor AAA1 gene in a cell or tissue, alone or in combination
with
other therapies.
The use of anticholinergic agents, anti-inflammatories, bronchodilators,
adenosine
inhibitors, adenosine A1 receptor inhibitors, non-selective M3 receptor
antagonists such
as atropine, ipratropium brominde and selective M3 receptor antagonists such
as
darifenacin and revatropate are all non-limiting examples of agents that can
be combined
with or used in conjunction with the nucleic acid molecules (e.g. siNA
molecules) of the
instant invention. Those skilled in the art will recognize that other
compounds and
therapies used to treat the diseases and conditions described herein can
similarly be
combined with the nucleic acid molecules of the instant invention (e.g. siNA
molecules)
and are hence within the scope of the instant invention.
Example 11: Diagnostic uses
The siNA molecules of the invention can be used in a variety of diagnostic
applications, such as in the identification of molecular targets (e.g., RNA)
in a variety of
applications, for example, in clinical, industrial, environmental,
agricultural and/or
research settings. Such diagnostic use of siNA molecules involves utilizing
reconstituted
RNAi systems, for example, using cellular lysates or partially purified
cellular lysates.
siNA molecules of this invention can be used as diagnostic tools to examine
genetic drift
and mutations within diseased cells or to detect the presence of endogenous or
exogenous, for example viral, RNA in a cell. The close relationship between
siNA
activity and the structure of the target RNA allows the detection of mutations
in any
region of the molecule, which alters the base-pairing and three-dimensional
structure of
the target RNA. By using multiple siNA molecules described in this invention,
one can
map nucleotide changes, which are important to RNA structure and function in
vitro, as
well as in cells and tissues. Cleavage of target RNAs with siNA molecules can
be used
to inhibit gene expression and define the role of specified gene products in
the
progression of disease or infection. In this manner, other genetic targets can
be defined
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as important mediators of the disease. These experiments will lead to better
treatment of
the disease progression by affording the possibility of combination therapies
(e.g.,
multiple siNA molecules targeted to different genes, siNA molecules coupled
with
known small molecule inhibitors, or intermittent treatment with combinations
siNA
molecules and/or other chemical or biological molecules). Other ira vitro uses
of siNA
molecules of this invention are well known in the art, and include detection
of the
presence of mRNAs associated with a disease, infection, or related condition.
Such
RNA is detected by determining the presence of a cleavage product after
treatment with a
siNA using standard methodologies, for example, fluorescence resonance
emission
transfer (FRET).
In a specific example, siNA molecules that cleave only wild-type or mutant
forms
of the target RNA are used for the assay. The first siNA molecules (i.e.,
those that
cleave only wild-type forms of target RNA) are used to identify wild-type RNA
present
in the sample and the second siNA molecules (i.e., those that cleave only
mutant forms
of target RNA) are used to identify mutant RNA in the sample. As reaction
controls,
synthetic substrates of both wild-type and mutant RNA are cleaved by both siNA
molecules to demonstrate the relative siNA efficiencies in the reactions and
the absence
of cleavage of the "non-targeted" RNA species. The cleavage products from the
synthetic substrates also serve to generate size markers for the analysis of
wild-type and
mutant RNAs in the sample population. Thus, each analysis requires two siNA
molecules, two substrates and one unknown sample, which is combined into six
reactions. The presence of cleavage products is determined using an RNase
protection
assay so that full-length and cleavage fragments of each RNA can be analyzed
in one
lane of a polyacrylamide gel. It is not absolutely required to quantify the
results to gain
insight into the expression of mutant RNAs and putative risk of the desired
phenotypic
changes in target cells. The expression of mRNA whose protein product is
implicated in
the development of the phenotype (i.e., disease related or infection related)
is adequate to
establish risk. If probes of comparable specific activity are used for both
transcripts,
then a qualitative comparison of RNA levels is adequate and decreases the cost
of the
initial diagnosis. Higher mutant form to wild-type ratios are correlated with
higher risk
whether RNA levels are compared qualitatively or quantitatively.
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All patents and publications mentioned in the specification are indicative of
the
levels of skill of those skilled in the art to which the invention pertains.
All references
cited in this disclosure are incorporated by reference to the same extent as
if each
reference had been incorporated by reference in its entirety individually.
One skilled in the art would readily appreciate that the present invention is
well
adapted to carry out the objects and obtain the ends and advantages mentioned,
as well as
those inherent therein. The methods and compositions described herein as
presently
representative of preferred embodiments are exemplary and are not intended as
limitations on the scope of the invention. Changes therein and other uses will
occur to
those skilled in the art, which are encompassed within the spirit of the
invention, are
defined by the scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions and
modifications can be made to the invention disclosed herein without departing
from the
scope and spirit of the invention. Thus, such additional embodiments are
within the
scope of the present invention and the following claims. The present invention
teaches
one skilled in the art to test various combinations and/or substitutions of
chemical
modifications described herein toward generating nucleic acid constructs with
improved
activity for mediating RNAi activity. Such improved activity can comprise
improved
stability, improved bioavailability, and/or improved activation of cellular
responses
mediating RNAi. Therefore, the specific embodiments described herein are not
limiting
and one skilled in the art can readily appreciate that specific combinations
of the
modifications described herein can be tested without undue experimentation
toward
identifying siNA molecules with improved RNAi activity.
The invention illustratively described herein suitably can be practiced in the
absence of any element or elements, limitation or limitations that are not
specifically
disclosed herein. Thus, for example, in each instance herein any of the terms
"comprising", "consisting essentially of', and "consisting of may be replaced
with either
of the other two terms. The terms and expressions which have been employed are
used
as terms of description and not of limitation, and there is no intention that
in the use of
such terms and expressions of excluding any equivalents of the features shown
and
described or portions thereof, but it is recognized that various modifications
are possible
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within the scope of the invention claimed. Thus, it should be understood that
although
the present invention has been specifically disclosed by preferred
embodiments, optional
features, modification and variation of the concepts herein disclosed may be
resorted to
by those skilled in the art, and that such modifications and variations are
considered to be
S within the scope of this invention as defined by the description and the
appended claims.
In addition, where features or aspects of the invention are described in terms
of
Markush groups or other grouping of alternatives, those skilled in the art
will recognize
that the invention is also thereby described in terms of any individual member
or
subgroup of members of the Markush group or other group.
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Table I: GPRA and AAA1 Accession Numbers
NM_207172
Homo Sapiens G protein-coupled receptor 154 (GPR154),
transcript variant 1, mRNA
gi~46395495~refINN!_207172.11[46395495]
NM_207173
Homo Sapiens G protein-coupled receptor 154 (GPR154),
transcript variant 2, mRNA
gi1463910841ref1NM__207173.11[46391084]
NM_207284
Homo Sapiens AAA1 protein (AAA1), transcript variant II,
mRNA
gi~464024931ref1NM_207284.11[46402493]
NM_207285
Homo Sapiens AAA1 protein (AAA1), transcript variant III,
mRNA
gi1464025011ref1NN!_207285.11[46402501]
NM_207286
Homo Sapiens AAA1 protein (AAA1), transcript variant IV,
mRNA
gi1464024971ref1NM__207286.11[46402497]
NM_207287
Homo Sapiens AAA1 protein (AAA1), transcript variant V,
mRNA
gi1464025051ref1NM_207287.11[46402505]
NM_207288
Homo Sapiens AAA1 protein (AAA1), transcript variant VI,
mRNA
gi1464024991ref1NM_207288.11[46402499]
NM_207289
Homo Sapiens AAA1 protein (AAA1), transcript variant VII,
mRNA
gi1464025081ref1NM__207289.11[46402508]
NM_207290
Homo Sapiens AAA1 protein (AAA1), transcript variant VIII,
mRNA
gi1464025031ref1NM__207290.11[46402503]
NM_207283
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Homo Sapiens AAA1 protein (AAA1), transcript variant IX,
mRNA
gi~46402495~ref~NM_207283.1 [46402495]
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N
N Q
0007O rN M ~ LI7COf~00~~ r N M~ Lf7COf~00~ O rN M d'~
M ~ O OO O O OO O O OO O O OO O O OO O r rr r r r
r r r rr r r rr r r rr r r r
H O
U U Q C~C9U U ~U ~ C9U~ U Q ~~ U ~ Q~ U ~ C9~ Q
CgC9U QQ U Q Q~ U Q (gU ~ C7~C~ ~ U~ ~ C~C9~ U U U
d. Q U (9(~(9~ U U(~Q U ~ ~ C9UQ (~C7U C7C9~U ~ U C~
o ~ C9Q ~ ~CJU = C9U U U ~ U ~C~ Q ~_ ~ U= U C7
= U U ~ ~
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U C9U UQ ~ U =U'U ~ C~ ~ C9QU = U ~U (9~~ C7=
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U U Q ~CO~ C~U~ ~~ = Q UQ U Q ~ C9U (9U
dU U U~ U C~CJU U U C9C7> > ~U C9 UC9Q U Q~ C9U Q
C9U(gU C9C9C U QQ U C9UU C~~ U~ U ~ UQ Q Q U
9
l~ 3U ~ U ~CQ7C9UU'~Q U ~ jQ = Q UU U ~ Q_ U ~ Q
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oU U C9~~ ~ C7C9C9U C ~~ ~ C9~C9 U U~ C~ U C9
-~C9C~C7UU U Q U C7C9~U Q Q C9U C7~~ U ~ ~~ U U C9
Q ~ COC9C7Q U ~~ = C9C9Q U C9~Q g =U V U ~_ ' V U
=
U U'
C~U ~ QQ U ~ C9~ U ~ =U U C~UC9
Q ~ U UU ~ U ~ ~ U UU U ~ U ~~ Q ~ QU ~ U Q
U U C9UC7U C9COU C9Q QQ U ~ Q~
Q C7Q >> U U ~C9U U UU ~ ~ = UC~U C9C9U
U U ~ C9U ~U > >U ~ ~Q U ~ QU
Q C7C~U ~ U
r O hLf7M r O~ ~fJM rO 1~~ Mr O I~~M r O I~
Or ~ I~InM r N ~.O ppO rM tf~I~OO N 'd'CON O r M~ f~00O
dN M ~ f~O r r rr ~-N NN N N NM M M MM M ~ ~tV'd'd'Lf7
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ri D
~r N M d'Lf)CO1'00~ ~ r NM d'Lf~COI~00~ Or N M d'L(7COf~OJ
r r rr r r rr r r NN N N NN N N N
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CA 02543029 2006-04-19
WO 2005/045038 PCT/US2004/027231
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163
CA 02543029 2006-04-19
WO 2005/045038 PCT/US2004/027231
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CA 02543029 2006-04-19
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177
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178
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Table IV
Non-limiting examples of Stabilization Chemistries for chemically modified
siNA constructs
Chemistry pyrimidine Purine Cap p=S Strand
"Stab 00" Ribo Ribo TT at S/AS
3'-
ends
"Stab 1" Ribo Ribo - 5 at 5'-end S/AS
1 at 3'-end
"Stab 2" Ribo Ribo - All linkagesUsually AS
"Stab 3" 2'-fluoro Ribo - 4 at 5'-end Usually S
4 at 3'-end
"Stab 4" 2'-fluoro Ribo 5' and - Usually S
3'-
ends
"Stab 5" 2'-fluoro Ribo - 1 at 3'-end Usually AS
"Stab 6" 2'-O-MethylRibo 5' and - Usually S
3'-
ends
"Stab 7" 2'-fluoro 2'-deoxy5' and - Usually S
3'-
ends
"Stab 8" 2'-fluoro 2'-O- - 1 at 3'-end SIAS
Methyl
"Stab 9" Ribo Ribo 5' and - Usually S
3'-
ends
"Stab 10" Ribo Ribo - 1 at 3'-end Usually AS
"Stab 11" 2'-fluoxo 2'-deoxy- 1 at 3'-end Usually AS
"Stab 12" 2'-fluoro LNA 5' and Usually S
3'-
ends
"Stab 13" 2'-fluoro LNA 1 at 3'-end Usually AS
"Stab 14" 2'-fluoro 2'-deoxy 2 at 5'-end Usually AS
1 at 3'-end
"Stab 15" 2'-deoxy 2'-deoxy 2 at 5'-end Usually AS
1 at 3'-end
"Stab 16" Ribo 2'-O- 5' and Usually S
3'-
Methyl ends
"Stab 17" 2'-O-Methyl2'-O- 5' and Usually S
3'-
Methyl ends
"Stab 18" 2'-fluoro 2'-O- 5' and Usually S
3'-
Methyl ends
"Stab 19" 2'-fluoro 2'-O- 3'-end S/AS
Methyl
"Stab 20" 2'-fluoro 2'-deoxy3'-end Usually AS
"Stab 21" 2'-fluoro Ribo 3'-end Usually AS
"Stab 22" Ribo Ribo 3'-end Usually AS
"Stab 23" 2'-fluoro* 2'-deoxy*5' and Usually S
3'-
ends
"Stab 24" 2'-fluoro* 2'-O- - 1 at 3'-end SIAS
Methyl*
"Stab 25" 2'-fluoro* 2'-O- - 1 at 3'-end S/AS
Methyl*
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"Stab 26" 2'-fluoro*2'-O- - S/AS
Methyl*
"Stab 27" 2'-fluoro*2'-O- 3'-end S/AS
Methyl*
"Stab 28" 2'-fluoro*2'-O- 3'-end S/AS
Methyl*
"Stab 29" 2'-fluoro*2'-O- 1 at 3'-end S/AS
Methyl*
"Stab 30" 2'-fluoro*2'-O- S/AS
Methyl*
"Stab 31" 2'-fluoro*2'-O- 3'-end S/AS
Methyl*
"Stab 32" 2'-fluoro 2'-O- S/AS
Methyl
CAP = any terminal cap, see for example Figure 10.
All Stab 00-32 chemistries can comprise 3'-terminal thyrnidine (TT) residues
All Stab 00-32 chemistries typically comprise about 21 nucleotides, but can
vary as described
herein.
S = sense strand
AS = antisense strand
*Stab 23 has a single ribonucleotide adjacent to 3'-CAP
*Stab 24 and Stab 28 have a single ribonucleotide at 5'-terminus
*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5'-terminus
*Stab 29, Stab 30, and Stab 31, any purine at first three nucleotide positions
from 5'-terminus
are ribonucleotides
p = phosphorothioate linkage
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Table V
A. 2.5 umol Synthesis Cycle ABI 394 Instrument
Reagent EquivalentsAmount Wait Time* Wait Time* Wait Time*RNA
DNA 2'-O-methyl
Phosphoramidites6.5 163 uL 45 sec 2.5 min 7.5 min
S-Ethyl 23.8 238 uL 45 sec 2.5 min 7.5 min
Tetrazole
Acetic 100 233 NL 5 sec 5 sec 5 sec
Anhydride
N-Methyl 186 233 NL 5 sec 5 sec 5 sec
Imidazole
TCA 176 2.3 mL 21 sec 21 sec 21 sec
Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec
Beaucage 12.9 645 uL 100 sec 300 sec 300 sec
I AcetonitrileNA I 6.67 NA I NA rNA
~ mL I
B. 0.2 pmol Synthesis Cycle ABI 394 Instrument
Reagent EquivalentsAmount Wait Time* Wait Time* 2'-O-methylWait Time*RNA
. DNA
Phosphoramidites15 31 uL 45 sec 233 sec 465 sec
S-Ethyl 38.7 31 NL 45 sec 233 min 465 sec
Tetrazole
Acetic 655 124 pL 5 sec 5 sec 5 sec
Anhydride
N-Methyl 1245 124 NL 5 sec 5 sec 5 sec
Imidazole
TCA 700 732 NL 10 sec 10 sec 10 sec
Iodine 20.6 244 NL 15 sec 15 sec 15 sec
Beaucage 7.7 232 NL 100 sec 300 sec 300 sec
AcetonitrileNA 2.64 NA NA NA
mL
C. 0.2 umol Synthesis Cycle 96 well Instrument
Reagent Equivalents:DNAIAmount: DNA/2'-O-Wait Time* Wait Time*Wait Time*
2'-O-methyl/Ribomethyl/Ribo DNA 2'-O- Ribo
methyl
Phosphoramidites22/33/66 40/60/120 60 sec 180 sec 360sec
uL
S-Ethyl 70/105121040/60/120 60 sec 180 min 360 sec
Tetrazole pL
Acetic 265/265/26550/50/50 NL 10 sec 10 sec 10 sec
Anhydride
N-Methyl 5021502/50250/50/50 uL 10 sec 10 sec 10 sec
Imidazole
TCA 238!475!475250!500!500 15 sec 15 sec 15 sec
pL
Iodine 6.8/6.8/6.880/80180 pL 30 sec 30 sec 30 sec
Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec
AcetonitrileNA 1150/1150/1150NA NA NA
NL
~ Wait time does not include contact time during delivery.
~ Tandem synthesis utilizes double coupling of linker molecule
181
