U.S. patent application number 10/224005 was filed with the patent office on 2003-07-31 for rna interference mediated inhibition of adenosine a1 receptor (adora1) gene expression using short interfering rna.
Invention is credited to Fosnaugh, Kathy, McSwiggen, James A..
Application Number | 20030143732 10/224005 |
Document ID | / |
Family ID | 40293860 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143732 |
Kind Code |
A1 |
Fosnaugh, Kathy ; et
al. |
July 31, 2003 |
RNA interference mediated inhibition of adenosine A1 receptor
(ADORA1) gene expression using short interfering RNA
Abstract
The present invention concerns methods and reagents useful in
modulating adenosine A1 receptor (ADORA1) gene expression in a
variety of applications, including use in therapeutic, diagnostic,
target validation, and genomic discovery applications.
Specifically, the invention relates to small interfering RNA
(siRNA) molecules capable of mediating RNA interference (RNAi)
against ADORA1 and related receptors.
Inventors: |
Fosnaugh, Kathy; (Longmont,
CO) ; McSwiggen, James A.; (Boulder, CO) |
Correspondence
Address: |
MCDONNELL BOEHNEN HULBERT & BERGHOFF
300 SOUTH WACKER DRIVE
SUITE 3200
CHICAGO
IL
60606
US
|
Family ID: |
40293860 |
Appl. No.: |
10/224005 |
Filed: |
August 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60315315 |
Aug 28, 2001 |
|
|
|
Current U.S.
Class: |
435/325 ;
435/320.1; 536/23.1 |
Current CPC
Class: |
C12N 15/113 20130101;
C12N 2310/121 20130101; C12N 2310/13 20130101; C12N 15/1138
20130101; C12N 2310/3521 20130101; A61K 38/00 20130101; C12N
15/1137 20130101; C12N 2310/12 20130101; C12N 2310/321 20130101;
C12N 2310/346 20130101; C12N 2310/18 20130101; C12N 2310/315
20130101; C12N 2310/14 20130101; C12N 2310/332 20130101; C12N
2310/317 20130101; C12N 2310/321 20130101 |
Class at
Publication: |
435/325 ;
435/320.1; 536/23.1 |
International
Class: |
C12N 005/06; C07H
021/02 |
Claims
What we claim is:
1. A short interfering RNA (siRNA) molecule that down regulates
expression of an ADORA1 gene by RNA interference.
2. The siRNA molecule of claim 1, wherein said siRNA molecule is
adapted for use to treat asthma.
3. The siRNA molecule of claim 1, wherein said siRNA molecule
comprises a sense region and an antisense region and wherein said
antisense region comprises sequence complementary to an RNA
sequence encoding ADORA1 and the sense region comprises sequence
complementary to the antisense region.
4. The siRNA molecule of claim 3, wherein said siRNA molecule is
assembled from two nucleic acid fragments wherein one fragment
comprises the sense region and the second fragment comprises the
antisense region of said siRNA molecule.
5. The siRNA molecule of claim 4, wherein said sense region and
antisense region are covalently connected via a linker
molecule.
6. The siRNA molecule of claim 5, wherein said linker molecule is a
polynucleotide linker.
7. The siRNA molecule of claim 5, wherein said linker molecule is a
non-nucleotide linker.
8. The siRNA molecule of claim 3, wherein said antisense region
comprises sequence complementary to sequence having any of SEQ ID
NOs. 1-161.
9. The siRNA molecule of claim 3, wherein said antisense region
comprises sequence having any of SEQ ID NOs. 162-322, 336, 338,
340, 342, 344, or 346.
10. The siRNA molecule of claim 3, wherein said sense region
comprises sequence having any of SEQ ID NOs. 1-161, 335, 337, 339,
341, 343, or 345.
11. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 323 and said antisense region
comprises a sequence of SEQ ID NO. 324.
12. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 325 and said antisense region
comprises a sequence of SEQ ID NO. 326.
13. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 327 and said antisense region
comprises a sequence of SEQ ID NO. 328.
14. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 329 and said antisense region
comprises a sequence of SEQ ID NO. 330.
15. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 331 and said antisense region
comprises a sequence of SEQ ID NO. 332.
16. The siRNA molecule of claim 3, wherein said sense region
comprises a sequence of SEQ ID NO. 333 and said antisense region
comprises a sequence of SEQ ID NO. 334.
17. The siRNA molecule of claim 3, wherein said sense region
comprises a 3'-terminal overhang and said antisense region
comprises a 3'-terminal overhang.
18. The siRNA molecule of claim 17, wherein said 3'-terminal
overhangs each comprise about 2 nucleotides.
19. The siRNA molecule of claim 17, wherein said antisense region
3'-terminal nucleotide overhang is complementary to RNA encoding
ADORA1.
20. The siRNA molecule of claim 3, wherein said sense region
comprises one or more 2'-O-methyl modified pyrimidine
nucleotides.
21. The siRNA molecule of claim 3, wherein said sense region
comprises a terminal cap moiety at the 5'-end, 3'-end, or both 5'
and 3' ends of said sense region.
22. The siRNA molecule of claim 3, wherein said antisense region
comprises one or more 2'-deoxy-2'-fluoro modified pyrimidine
nucleotides.
23. The siRNA molecule of claim 3, wherein said antisense region
comprises a phosphorothioate internucleotide linkage at the 3' end
of said antisense region.
24. The siRNA molecule of claim 3, wherein said antisense region
comprises between about one and about five phosphorothioate
internucleotide linkages at the 5' end of said antisense
region.
25. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise ribonucleotides that are chemically
modified at a nucleic acid sugar, base, or backbone.
26. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise deoxyribonucleotides that are
chemically modified at a nucleic acid sugar, base, or backbone.
27. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise one or more universal base
ribonucleotides.
28. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise one or more acyclic nucleotides.
29. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise nucleotides comprising
internucleotide linkages having Formula I: 9wherein each R1 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, or aralkyl, and wherein W, X, Y and Z are not all
O.
30. The siRNA molecule of claim 17, wherein said 3'-terminal
nucleotide overhangs comprise nucleotides or non-nucleotides having
Formula II: 10wherein 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic base or
any other non-naturally occurring base that can be complementary or
non-complementary to ADORA1 RNA or a non-nucleosidic base or any
other non-naturally occurring universal base that can be
complementary or non-complementary to ADORA1 RNA.
31. An expression vector comprising a nucleic acid sequence
encoding at least one siRNA molecule of claim 1 in a manner that
allows expression of the nucleic acid molecule.
32. A mammalian cell comprising an expression vector of claim
31.
33. The mammalian cell of claim 32, wherein said mammalian cell is
a human cell.
34. The expression vector of claim 31, wherein said siRNA molecule
comprises a sense region and an antisense region and wherein said
antisense region comprises sequence complementary to an RNA
sequence encoding ADORA1 and the sense region comprises sequence
complementary to the antisense region.
35. The expression vector of claim 34, wherein said siRNA molecule
comprises two distinct strands having complementarity sense and
antisense regions.
36. The expression vector of claim 34, wherein said siRNA molecule
comprises a single strand having complementary sense and antisense
regions.
Description
PRIORITY
[0001] This application claims the benefit of U.S. Patent
Application 60/315,315 filed on Aug. 28, 2001.
BACKGROUND OF THE INVENTION
[0002] The present invention concerns methods and reagents useful
in modulating gene expression associated with asthma, inflammation
and allergic response in a variety of applications, including use
in therapeutic, diagnostic, target validation, and genomic
discovery applications. Specifically, the invention relates to
short interfering nucleic acid molecules (siRNA) capable of
mediating RNA interference (RNAi) against adenosine A1 receptor
gene expression.
[0003] 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.
[0004] RNA interference refers to the process of sequence-specific
post transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNA) (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 (dsRNA) 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.
[0005] 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 (siRNA) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from Dicer
activity are typically about 21-23 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
RNAs (stRNA) 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 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).
[0006] 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, Nature, 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 (Elbashir
et al., 2001, EMBO J., 20, 6877) 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 nucleotide 3'-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
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
(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).
[0007] Studies have shown that replacing the 3'-overhanging
segments of a 21-mer siRNA duplex having 2 nucleotide 3' overhangs
with deoxyribonucleotides does not have an adverse effect on RNAi
activity. Replacing up to 4 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). 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 al., International PCT Publication No. WO 01/68836
both suggest that siRNA "may include modifications to either the
phosphate-sugar back bone or the nucleoside to include at least one
of a nitrogen or sulfur heteroatom", however neither application
teaches to what extent these modifications are tolerated in siRNA
molecules nor provide any examples of such modified siRNA. Kreutzer
and Limmer, 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 or 4'-C methylene
bridge. However, Kreutzer and Limmer similarly fail to show to what
extent these modifications are tolerated in siRNA molecules nor do
they provide any examples of such modified siRNA.
[0008] Parrish et al., 2000, Molecular Cell, 6, 1977-1087, tested
certain chemical modifications targeting the unc-22 gene in C.
elegans 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 triggers (data not shown); [phosphorothioate]
modification of more than two residues greatly destabilized the
RNAs in vitro and we were not able to assay interference
activities." Id. at 1081. The authors also tested certain
modifications at the 2'-position of the nucleotide sugar in the
long siRNA transcripts and observed 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 4-thiouracil, 5-bromouracil,
5-iodouracil, 3-(aminoallyl)uracil for uracil, and inosine for
guanosine in sense and antisense strands of the siRNA, and found
that whereas 4-thiouracil and 5-bromouracil were all well
tolerated, inosine "produced a substantial decrease in interference
activity" when incorporated in either strand. Incorporation of
5-iodouracil and 3-(aminoallyl)uracil in the antisense strand
resulted in substantial decrease in RNAi activity as well.
[0009] 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, describes a Drosophila in vitro RNAi
system and the use of specific siRNA molecules for certain
functional genomic and certain therapeutic applications; although
Tuschl, 2001, Chem. Biochem., 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, describes the use of specific dsRNAs
for use in attenuating the expression of certain target genes.
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646, describes certain methods for inhibiting the expression
of particular genes in mammalian cells using certain dsRNA
molecules. Fire et al., International PCT Publication No. WO
99/32619, describes particular methods for introducing certain
dsRNA molecules into cells for use in inhibiting gene expression.
Plaetinck et al., International PCT Publication No. WO 00/01846,
describes certain methods for identifying specific genes
responsible for conferring a particular phenotype in a cell using
specific dsRNA molecules. Mello et al., International PCT
Publication No. WO 01/29058, describes the identification of
specific genes involved in dsRNA mediated RNAi. Deschamps
Depaillette et al., International PCT Publication No. WO 99/07409,
describes specific compositions consisting of particular dsRNA
molecules combined with certain anti-viral agents. Waterhouse et
al., International PCT Publication No. 99/53050, describes certain
methods for decreasing the phenotypic expression of a nucleic acid
in plant cells. Driscoll et al., International PCT Publication No.
WO 01/49844, describes specific DNA constructs for use in
facilitating gene silencing in targeted organisms. Parrish et al.,
2000, Molecular Cell, 6, 1977-1087, describes specific chemically
modified siRNA constructs targeting the unc-22 gene of C. elegans.
Grossniklaus, International PCT Publication No. WO 01/38551,
describes certain methods for regulating polycomb gene expression
in plants. Churikov et al., International PCT Publication No. WO
01/42443, describes certain methods for modifying genetic
characteristics of an organism. Cogoni et al., International PCT
Publication No. WO 01/53475, describes certain methods for
isolating a Neurospora silending gene and uses thereof. Reed et
al., International PCT Publication No. WO 01/68836, describes
certain methods for gene silencing in plants. Honer et al,
International PCT Publication No. WO 01/70944, describes certain
methods of drug screening using transgenic nematodes as Parkinson's
disease models. Deak et al., International PCT Publication No. WO
01/72774, describes certain Drosophila derived gene products. Arndt
et al., International PCT Publication No. WO 01/92513 describes
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 describes certain methods and compositions for inhibiting
the function of certain polynucleotide sequences. Echeverri et al.,
International PCT Publication No. WO 02/38805, describes certain C.
elegans genes identified via RNAi. Kreutzer et al., International
PCT Publication No. WO 02/055692 and WO 02/055693, describes
certain methods for inhibiting gene expression using RNAi.
[0010] Asthma is a chronic inflammatory disorder of the lungs
characterized by airflow obstruction, bronchial
hyper-responsiveness, and airway inflammation. T-lymphocytes that
produce TH2 cytokines and eosinophilic leukocytes infiltrate the
airways. In the airway and in bronchial alveolar lavage (BAL) fluid
of individuals with asthma, high concentrations of TH2 cytokines,
interleukin-4 (IL-4), IL-5, and IL-13, are present along with
increased levels of adenosine. In contrast to normal individuals,
asthmatics respond to adenosine challenge with marked airway
obstruction. Upon allergen challenge, mast cells are activated by
cross-linked IgE-allergen complexes. Large amounts of prostaglandin
D2 (PGD2), the major cyclooxygenase product of arachidonic acid are
released. PGD2 is generated from PGH2 via the activity of
prostaglandin D2 synthetase (PTGDS). PGD2 receptors and adenosine
A1 receptors are present in the lungs and airway along with various
other tissues in response to allergic stimuli (Howarth, 1997,
Allergy, 52, 12).
[0011] The significance of PGD2 as a mediator of allergic asthma
has been established with the development of mice deficient in the
PGD2 receptor (DP). DP is a heterotrimeric GTP-binding
protein-coupled, rhodopsin-type receptor specific for PGD2 (Hirata
et al., 1994, PNAS USA., 91, 11192). These mice fail to develop
airway hyperreactivity and have greatly reduced eosinophil
infiltration and cytokine accumulation in response to allergens.
Upon allergen challenge mice deficient in the prostaglandin D2
(PGD2) receptor (DP) did not develop airway hyperactivity.
Cytokine, lymphocyte and eosinophil accumulation in the lungs were
greatly reduced (Matsuoka et al., 2000, Science, 287, 2013). The DP
-/- mice exhibited no behavioral, anatomic, or histological
abnormalities. Primary immune response is not affected by DP
disruption.
[0012] Asthma affects more than 100 million people worldwide and
more than 17 million Americans (5% of the population). Since 1980
the incidence has more than doubled and deaths have tripled (5,000
deaths in 1995). Annual asthma-related healthcare costs in the US
alone were estimated to exceed $14.5 billion in 2000. Current
therapies such as inhalant anti-inflammatories and bronchodilators
can be used to treat symptoms, however, these therapies do not
prevent or cure asthma.
SUMMARY OF THE INVENTION
[0013] One embodiment of the invention provides a short interfering
RNA (siRNA) molecule that down regulates expression of adenosine A1
receptor (ADORA1) by RNA interference. The siRNA molecule can be
adapted for use to treat, for example allergic/inflammatory
diseases and conditions, including but not limited to asthma,
allergic rhinitis, atopic dermatitis, and any other indications
that can respond to the level of ADORA1. The siRNA molecule can
comprise a sense region and an antisense region. The antisense
region can comprise sequence complementary to an RNA sequence
encoding ADORA1 and the sense region can comprise sequence
complementary to the antisense region. An siRNA molecule of the
invention can be adapted for use to treat asthma.
[0014] An siRNA molecule can comprise a sense region and an
antisense region and wherein said antisense region comprises
sequence complementary to an RNA sequence encoding ADORA1 and the
sense region comprises sequence complementary to the antisense
region.
[0015] The siRNA molecule can be assembled from two nucleic acid
fragments wherein one fragment comprises the sense region and the
second fragment comprises the antisense region of said siRNA
molecule. The sense region and antisense region can be covalently
connected via a linker molecule. The linker molecule can be a
polynucleotide linker or a non-nucleotide linker.
[0016] The antisense region of ADORA1 siRNA constructs can comprise
a sequence complementary to sequence having any of SEQ ID NOs.
1-161. The antisense region can also comprise sequence having any
of SEQ ID NOs. 162-322, 336, 338, 340, 342, 344, or 346. The
sequences shown in SEQ ID NO: 1-346 are not limiting. A siRNA
molecule of the invention can comprise any contiguous ADORA1
sequences (e.g., about 19 contiguous ADORA1 nucleotides. The sense
region of ADORA1 siRNA constructs can comprise sequence having any
of SEQ ID NOs. 1-161, 335, 337, 339, 341, 343, or 345. The sense
region can comprise a sequence of SEQ ID NO. 323 and the antisense
region can comprise a sequence of SEQ ID NO. 324. The sense region
can comprise a sequence of SEQ ID NO. 325 and the antisense region
can comprise a sequence of SEQ ID NO. 326. The sense region can
comprise a sequence of SEQ ID NO. 327 and the antisense region can
comprise a sequence of SEQ ID NO. 328. The sense region can
comprise a sequence of SEQ ID NO. 329 and the antisense region can
comprise a sequence of SEQ ID NO. 330. The sense region can
comprise a sequence of SEQ ID NO. 331 and the antisense region can
comprise a sequence of SEQ ID NO. 332. The sense region can
comprise a sequence of SEQ ID NO. 333 and the antisense region can
comprise a sequence of SEQ ID NO. 334.
[0017] The sense region of a siRNA molecule of the invention can
comprise a 3'-terminal overhang and the antisense region can
comprise a 3'-terminal overhang. The 3'-terminal overhangs each can
comprise about 2 nucleotides. The antisense region of the
3'-terminal nucleotide overhang can be complementary to RNA
encoding ADORA1.
[0018] The sense region of a siRNA molecule can comprise one or
more (e.g., about 1, 2, 3, 4, 5, or more) 2'-O-methyl modified
pyrimidine nucleotides. The sense region can comprise a terminal
cap moiety at the 5'-end, 3'-end, or both 5' and 3' ends of said
sense region.
[0019] The antisense region of a siRNA molecule can comprise one or
more (e.g., about 1, 2, 3, 4, 5, or more) 2'-deoxy-2'-fluoro
modified pyrimidine nucleotides. The antisense region can also
comprise a phosphorothioate internucleotide linkage at the 3' end
of said antisense region. The antisense region can comprise between
about one and about five phosphorothioate internucleotide linkages
at the 5' end of said antisense region.
[0020] The 3'-terminal nucleotide overhangs of a siRNA molecule can
comprise ribonucleotides or deoxyribonucleotides that are
chemically modified at a nucleic acid sugar, base, or backbone. The
3'-terminal nucleotide overhangs can also comprise one or more
(e.g., about 1, 2, 3, 4, 5, or more) universal base
ribonucleotides. Additionally, the 3'-terminal nucleotide overhangs
can comprise one or more (e.g., about 1, 2, 3, 4, 5, or more)
acyclic nucleotides.
[0021] The 3'-terminal nucleotide overhangs can comprise
nucleotides comprising internucleotide linkages having Formula I:
1
[0022] wherein each R1 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, or aralkyl,
and wherein W, X, Y and Z are not all O.
[0023] The 3'-terminal nucleotide overhangs can comprise
nucleotides or non-nucleotides having Formula II: 2
[0024] 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.O, CHF, or CF2, and B is a nucleosidic base or
any other non-naturally occurring base that can be complementary or
non-complementary to ADORA1 RNA or a non-nucleosidic base or any
other non-naturally occurring universal base that can be
complementary or non-complementary to ADORA1 RNA.
[0025] Another embodiment of the invention provides an expression
vector comprising a nucleic acid sequence encoding at least one
siRNA molecule of the invention in a manner that allows expression
of the nucleic acid molecule. The expression vector can be in a
mammalian cell, such as a human cell. The siRNA molecule can
comprise a sense region and an antisense region. The antisense
region can comprise sequence complementary to an RNA sequence
encoding ADORA1 and the sense region comprises sequence
complementary to the antisense region. The siRNA molecule can
comprise two distinct strands having complementarity sense and
antisense regions or can comprise a single strand having
complementary sense and antisense regions.
[0026] Therefore, this invention relates to compounds,
compositions, and methods useful for modulating gene expression,
for example, genes associated with asthma, inflammation and
allergic response by RNA interference (RNAi) using short
interfering RNA (siRNA). In particular, the instant invention
features siRNA molecules and methods to modulate the expression of
ADORA1. The siRNA of the invention can be unmodified or chemically
modified. The siRNA 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 RNA (siRNA) molecules capable of
modulating ADORA1 gene expression/activity in cells by RNA
inference (RNAi). The use of chemically modified siRNA is expected
to improve various properties of native siRNA molecules through
increased resistance to nuclease degradation in vivo and/or
improved cellular uptake. The siRNA molecules of the instant
invention provide useful reagents and methods for a variety of
therapeutic, diagnostic, agricultural, target validation, genomic
discovery, genetic engineering and pharmacogenomic
applications.
[0027] In one embodiment, the invention features one or more siRNA
molecules and methods that independently or in combination modulate
the expression of gene(s) encoding proteins associated with asthma,
inflammation, and the allergic response. Specifically, the present
invention features siRNA molecules that modulate the expression of
ADORA1 genes such as GenBank accession No. NM.sub.--000674.
[0028] The description below of the various aspects and embodiments
is provided with reference to the exemplary gene ADORA1. However,
the various aspects and embodiments are also directed to other
genes which express other adenosine receptors (A2A, A2B, and/or
A3). Those additional genes can be analyzed for target sites using
the methods described for ADORA1. Thus, the inhibition and the
effects of such inhibition of the other genes can be performed as
described herein. Thus, the inhibition and the effects of such
inhibition of the other genes can be performed as described
herein.
[0029] In one embodiment, the invention features a siRNA molecule
that down regulates expression of an ADORA1 gene, for example,
wherein the ADORA1 gene comprises ADORA1 sequence.
[0030] In one embodiment, the invention features a siRNA molecule
having RNAi activity against ADORA1 RNA, wherein the siRNA molecule
comprises a sequence complimentary to any RNA having ADORA1
encoding sequence, such as GenBank accession No.
NM.sub.--000674.
[0031] In another embodiment, the invention features a siRNA
molecule comprising sequences selected from the group consisting of
SEQ ID NOs: 1-322. In another embodiment, the invention features an
ADORA1 siRNA molecule having an antisense region complementary to
any sequence having SEQ ID NOs: 1-161. In another embodiment, the
invention features an ADORA1 siRNA molecule having an antisense
region having any of SEQ ID NOs: 162-322, 336, 338, 340, 342, 344,
346, 348, 350, 352 or 354. In another embodiment, the invention
features an ADORA1 siRNA molecule having a sense region having any
of SEQ ID NOs. 1-161, 335, 337, 339, 341, 343, or 345, 347, 349,
351 or 353. The sense region can comprise a sequence of SEQ ID NO.
323 and the antisense region can comprise a sequence of SEQ ID NO.
324. The sense region can comprise a sequence of SEQ ID NO. 325 and
the antisense region can comprise a sequence of SEQ ID NO. 326. The
sense region can comprise a sequence of SEQ ID NO. 327 and the
antisense region can comprise a sequence of SEQ ID NO. 328. The
sense region can comprise a sequence of SEQ ID NO. 329 and the
antisense region can comprise a sequence of SEQ ID NO. 330. The
sense region can comprise a sequence of SEQ ID NO. 331 and the
antisense region can comprise a sequence of SEQ ID NO. 332. The
sense region can comprise a sequence of SEQ ID NO. 333 and the
antisense region can comprise a sequence of SEQ ID NO. 334. In yet
another embodiment, the invention features a siRNA molecule
comprising a sequence, for example the antisense sequence of the
siRNA construct, complementary to a sequence or portion of sequence
comprising GenBank accession No. NM.sub.--000674.
[0032] In one embodiment, a siRNA molecule of the invention has
RNAi activity that modulates expression of RNA encoded by an ADORA1
gene.
[0033] In one embodiment, nucleic acid molecules of the invention
that act as mediators of the RNA interference gene silencing
response are double stranded RNA molecules. In another embodiment,
the siRNA molecules of the invention consist of duplexes containing
about 19 base pairs between oligonucleotides comprising about 19 to
about 25 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, or 25).
In yet another embodiment, siRNA molecules of the invention
comprise duplexes with overhanging ends of 1-3 (e.g., 1, 2, or 3)
nucleotides, for example 21 nucleotide duplexes with 19 base pairs
and 2 nucleotide 3'-overhangs. These nucleotide overhangs in the
antisense strand are optionally complementary to the target
sequence.
[0034] In one embodiment, the invention features chemically
modified siRNA constructs having specificity for ADORA1 expressing
nucleic acid molecules. Non-limiting examples of such chemical
modifications include without limitation phosphorothioate
internucleotide linkages, 2'-O-methyl ribonucleotides,
2'-deoxy-2'-fluoro ribonucleotides, "universal base" nucleotides,
5-C-methyl nucleotides, and inverted deoxyabasic residue
incorporation. These chemical modifications, when used in various
siRNA 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 siRNA constructs. Chemical modifications of the siRNA
constructs can also be used to improve the stability of the
interaction with the target RNA sequence and to improve nuclease
resistance.
[0035] In a non-limiting example, the introduction of chemically
modified nucleotides into nucleic acid molecules will provide 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
the native molecule due to improved stability and/or delivery of
the molecule. Unlike native unmodified siRNA, chemically modified
siRNA can also minimize the possibility of activating interferon
activity in humans.
[0036] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more nucleotides comprising a backbone modified
internucleotide linkage having Formula I: 3
[0037] wherein each R1 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, or aralkyl,
and wherein W, X, Y and Z are not all O.
[0038] 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 siRNA duplex, for example in the
sense strand, antisense strand, or both strands. The siRNA
molecules of the invention can comprise one or more chemically
modified internucleotide linkages having Formula I at the 3'-end,
5'-end, or both 3' and 5'-ends of the sense strand, antisense
strand, or both strands. For example, an exemplary siRNA molecule
of the invention can comprise between about 1 and 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, antisense strand, or both strands. In another
non-limiting example, an exemplary siRNA 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,
antisense strand, or both strands. In yet another non-limiting
example, an exemplary siRNA 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, antisense strand, or
both strands. In another embodiment, a siRNA molecule of the
invention having internucleotide linkage(s) of Formula I also
comprises a chemically modified nucleotide or non-nucleotide having
any of Formulae II, III, V, or VI.
[0039] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more nucleotides or non-nucleotides having Formula
II: 4
[0040] wherein each R3, R4, R5, R6, R7, R8, R10, R 11 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.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 complementary or
non-complementary to ADORA1 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
ADORA1 RNA.
[0041] The chemically modified nucleotide or non-nucleotide of
Formula II can be present in one or both oligonucleotide strands of
the siRNA duplex, for example in the sense strand, antisense
strand, or both strands. The siRNA molecules of the invention can
comprise one or more chemically modified nucleotide or
non-nucleotide of Formula II at the 3'-end, 5'-end, or both 3' and
5'-ends of the sense strand, antisense strand, or both strands. For
example, an exemplary siRNA molecule of the invention can comprise
between about 1 and about 5 or more (e.g., about 1, 2, 3, 4, 5, or
more) chemically modified nucleotide or non-nucleotide of Formula
II at the 5'-end of the sense strand, antisense strand, or both
strands. In anther non-limiting example, an exemplary siRNA
molecule of the invention can comprise between about 1 and about 5
or more (e.g., about 1, 2, 3, 4, 5, or more) chemically modified
nucleotide or non-nucleotide of Formula II at the 3'-end of the
sense strand, antisense strand, or both strands.
[0042] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises one or more nucleotides or non-nucleotides having Formula
III: 5
[0043] 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.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 ADORA1 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 ADORA1 RNA.
[0044] The chemically modified nucleotide or non-nucleotide of
Formula III can be present in one or both oligonucleotide strands
of the siRNA duplex, for example in the sense strand, antisense
strand, or both strands. The siRNA molecules of the invention can
comprise one or more chemically modified nucleotide or
non-nucleotide of Formula III at the 3'-end, 5'-end, or both 3' and
5'-ends of the sense strand, antisense strand, or both strands. For
example, an exemplary siRNA molecule of the invention can comprise
between about 1 and 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 5'-end of the sense strand, antisense strand, or both
strands. In anther non-limiting example, an exemplary siRNA
molecule of the invention can comprise between about 1 and 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, antisense strand, or both strands.
[0045] In another embodiment, a siRNA 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 siRNA construct in a 3',3', 3'-2', 2'-3', or
5',5'configuration, such as at the 3'-end, 5'-end, or both 3' and
5' ends of one or both siRNA strands.
[0046] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises a 5'-terminal phosphate group having Formula IV: 6
[0047] wherein each X and Y is independently O, S, N, alkyl,
substituted alkyl, or alkylhalo; each Z and W is independently O,
S, N, alkyl, substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl,
or alkylhalo; and wherein W, X, Y and Z are not all O.
[0048] In one embodiment, the invention features a siRNA molecule
having a 5'-terminal phosphate group having Formula IV on the
target-complementary strand, for example a strand complementary to
ADORA1 RNA, wherein the siRNA molecule comprises an all RNA siRNA
molecule. In another embodiment, the invention features a siRNA
molecule having a 5'-terminal phosphate group having Formula IV on
the target-complementary strand wherein the siRNA molecule also
comprises 1-3 (e.g., 1, 2, or 3) nucleotide 3'-overhangs having
between about 1 and 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 siRNA molecule
of the invention, for example a siRNA molecule having chemical
modifications having Formula I, Formula II and/or Formula III.
[0049] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 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 RNA (siRNA) having about 1,
2, 3, 4, 5, 6, 7, 8 or more phosphorothioate internucleotide
linkages in one siRNA strand. In yet another embodiment, the
invention features a chemically modified short interfering RNA
(siRNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or more
phosphorothioate internucleotide linkages in both siRNA strands.
The phosphorothioate internucleotide linkages can be present in one
or both oligonucleotide strands of the siRNA duplex, for example in
the sense strand, antisense strand, or both strands. The siRNA
molecules of the invention can comprise one or more
phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or
both 3' and 5'-ends of the sense strand, antisense strand, or both
strands. For example, an exemplary siRNA molecule of the invention
can comprise between about 1 and 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, antisense strand, or
both strands. In another non-limiting example, an exemplary siRNA
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, antisense strand, or
both strands. In yet another non-limiting example, an exemplary
siRNA 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
phosphorothioate internucleotide linkages in the sense strand,
antisense strand, or both strands.
[0050] In one embodiment, the invention features a siRNA molecule,
wherein the sense 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 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', 5', or both 3' and 5'-ends of the sense strand; and wherein the
antisense strand comprises any of between 1 and 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, 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', 5', or both 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 siRNA stand 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', 5', or both 3' and 5'-ends, being
present in the same or different strand.
[0051] In another embodiment, the invention features a siRNA
molecule, wherein the sense strand comprises between about 1 and
about 5, specifically about 1, 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', 5', or both 3' and 5'-ends of the sense strand; and wherein the
antisense strand comprises any of between about 1 and 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, 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', 5', or both 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
siRNA stand are chemically modified with 2'-deoxy, 2'-O-methyl
and/or 2'-deoxy-2'-fluoro nucleotides, with or without between
about 1 and 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', 5', or both 3' and 5'-ends, being present
in the same or different strand.
[0052] In one embodiment, the invention features a siRNA 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 between 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', 5', or both 3' and 5'-ends of the sense strand; and
wherein the antisense strand comprises any of between about 1 and
about 10, 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, 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', 5', or both 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 siRNA stand 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', 5', or both 3' and 5'-ends, being
present in the same or different strand.
[0053] In another embodiment, the invention features a siRNA
molecule, wherein the antisense strand comprises between about 1
and 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 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', 5', or both 3' and 5'-ends of the
sense strand; and wherein the antisense strand comprises any of
between about 1 and 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, 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', 5', or both 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 siRNA stand are chemically modified
with 2'-deoxy, 2'-O-methyl and/or 2'-deoxy-2'-fluoro nucleotides,
with or without between about 1 and about 5, for example about 1,
2, 3, 4, 5 or more phosphorothioate internucleotide linkages and/or
a terminal cap molecule at the 3', 5', or both 3' and 5'-ends,
being present in the same or different strand.
[0054] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule having between
about 1 and about 5, specifically about 1, 2, 3, 4, 5 or more
phosphorothioate internucleotide linkages in each strand of the
siRNA molecule.
[0055] In another embodiment, the invention features a siRNA
molecule comprising 2'-5' internucleotide linkages. The 2'-5'
internucleotide linkage(s) can be at the 5'-end, 3'-end, or both 5'
and 3' ends of one or both siRNA sequence strands. In addition, the
2'-5' internucleotide linkage(s) can be present at various other
positions within one or both siRNA 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 siRNA 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 siRNA molecule can comprise a 2'-5' internucleotide
linkage.
[0056] In another embodiment, a chemically modified siRNA molecule
of the invention comprises a duplex having two strands, one or both
of which can be chemically modified, wherein each strand is between
about 18 and about 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25,
26, or 27) nucleotides in length, wherein the duplex has between
about 18 and about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base
pairs, and wherein the chemical modification comprises a structure
having Formula I, Formula II, Formula III and/or Formula IV. For
example, an exemplary chemically modified siRNA molecule of the
invention comprises a duplex having two strands, one or both of
which can be chemically modified with a chemical modification
having Formula I, Formula II, Formula III, and/or Formula IV,
wherein each strand consists of 21 nucleotides, each having 2
nucleotide 3'-overhangs, and wherein the duplex has 19 base
pairs.
[0057] In another embodiment, a siRNA molecule of the invention
comprises a single stranded hairpin structure, wherein the siRNA is
between about 36 and about 70 (e.g., about 36, 40, 45, 50, 55, 60,
65, or 70) nucleotides in length having between about 18 and about
23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein
the siRNA can include a chemical modification comprising a
structure having Formula I, Formula II, Formula III and/or Formula
IV. For example, an exemplary chemically modified siRNA molecule of
the invention comprises a linear oligonucleotide having between
about 42 and 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 Formula I, Formula II, Formula III, and/or
Formula IV, wherein the linear oligonucleotide forms a hairpin
structure having 19 base pairs and a 2 nucleotide 3'-overhang.
[0058] In another embodiment, a linear hairpin siRNA molecule of
the invention contains a stem loop motif, wherein the loop portion
of the siRNA molecule is biodegradable. For example, a linear
hairpin siRNA molecule of the invention is designed such that
degradation of the loop portion of the siRNA molecule in vivo can
generate a double stranded siRNA molecule with 3'-overhangs, such
as 3'-overhangs comprising about 2 nucleotides.
[0059] In another embodiment, a siRNA molecule of the invention
comprises a circular nucleic acid molecule, wherein the siRNA is
between about 38 and about 70 (e.g., about 38, 40, 45, 50, 55, 60,
65, or 70) nucleotides in length having between about 18 and about
23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, and wherein
the siRNA can include a chemical modification, which comprises a
structure having Formula I, Formula II, Formula III and/or Formula
IV. For example, an exemplary chemically modified siRNA molecule of
the invention comprises a circular oligonucleotide having between
about 42 and 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 Formula I, Formula II, Formula III, and/or
Formula IV, wherein the circular oligonucleotide forms a dumbbell
shaped structure having 19 base pairs and 2 loops.
[0060] In another embodiment, a circular siRNA molecule of the
invention contains two loop motifs, wherein one or both loop
portions of the siRNA molecule is biodegradable. For example, a
circular siRNA molecule of the invention is designed such that
degradation of the loop portions of the siRNA molecule in vivo can
generate a double stranded siRNA molecule with 3'-overhangs, such
as 3'-overhangs comprising about 2 nucleotides.
[0061] In one embodiment, a siRNA molecule of the invention
comprises at least one abasic residue, for example a compound
having Formula V: 7
[0062] wherein each R3, R4, R5, R6, R7, R8, R10, R11, 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.O, CHF, or CF2.
[0063] In one embodiment, a siRNA molecule of the invention
comprises at least one inverted abasic residue, for example a
compound having Formula VI: 8
[0064] wherein each R3, R4, R5, R6, R7, R8, R10, R11, 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, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid,
aminoacyl, ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalklylamino, substituted silyl, or group having Formula I; R9
is O, S, CH2, S.dbd.O, CHF, or CF2, and either R2, R3, R8 or R13
serve as points of attachment to the siRNA molecule of the
invention.
[0065] In another embodiment, a siRNA molecule of the invention
comprises an abasic residue having Formula II or III, wherein the
abasic residue having Formula II or III is connected to the siRNA
construct in a 3',3', 3'-2', 2'-3', or 5',5' configuration, such as
at the 3'-end, 5'-end, or both 3' and 5' ends of one or both siRNA
strands.
[0066] In one embodiment, a siRNA 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, 3'-end, 5' and 3'-end, or any combination thereof, of the
siRNA molecule.
[0067] In another embodiment, a siRNA 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, 3'-end, 5'
and 3'-end, or any combination thereof, of the siRNA molecule.
[0068] In one embodiment, the invention features a chemically
modified short interfering RNA (siRNA) molecule capable of
mediating RNA interference (RNAi) against ADORA1 inside a cell or
reconstituted in vitro system, wherein the chemical modification
comprises a conjugate covalently attached to the siRNA molecule. In
another embodiment, the conjugate is covalently attached to the
siRNA molecule via a biodegradable linker. In one embodiment, the
conjugate molecule is attached at the 3'-end of either the sense
strand, antisense strand, or both strands of the siRNA. In another
embodiment, the conjugate molecule is attached at the 5'-end of
either the sense strand, antisense strand, or both strands of the
siRNA. In yet another embodiment, the conjugate molecule is
attached both the 3'-end and 5'-end of either the sense strand,
antisense strand, or both strands of the siRNA, or any combination
thereof. In one embodiment, a conjugate molecule of the invention
comprises a molecule that facilitates delivery of a siRNA molecule
into a biological system such as a cell. In another embodiment, the
conjugate molecule attached to the siRNA is a poly ethylene 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 siRNA
molecules are described in Vargeese et al., U.S. Ser. No.
60/311,865, incorporated by reference herein.
[0069] In one embodiment, the invention features a siRNA molecule
capable of mediating RNA interference (RNAi) against ADORA1 inside
a cell or reconstituted in vitro system, wherein one or both
strands of the siRNA comprise ribonucleotides at positions withing
the siRNA that are critical for siRNA mediated RNAi in a cell. All
other positions within the siRNA can include chemically modified
nucleotides and/or non-nucleotides such as nucleotides and or
non-nucleotides having Formula I, II, III, IV, V, or VI, or any
combination thereof to the extent that the ability of the siRNA
molecule to support RNAi activity in a cell is maintained.
[0070] In one embodiment, the invention features a method for
modulating the expression of an ADORA1 gene within a cell,
comprising: (a) synthesizing a siRNA molecule of the invention,
which can be chemically modified, wherein one of the siRNA strands
includes a sequence complementary to RNA of the ADORA1 gene; and
(b) introducing the siRNA molecule into a cell under conditions
suitable to modulate the expression of the ADORA1 gene in the
cell.
[0071] In one embodiment, the invention features a method for
modulating the expression of an ADORA1 gene within a cell,
comprising: (a) synthesizing a siRNA molecule of the invention,
which can be chemically modified, wherein one of the siRNA strands
includes a sequence complementary to RNA of the ADORA1 gene and
wherein the sense strand sequence of the siRNA is identical to the
complementary sequence of the ADORA1 RNA; and (b) introducing the
siRNA molecule into a cell under conditions suitable to modulate
the expression of the ADORA1 gene in the cell.
[0072] In another embodiment, the invention features a method for
modulating the expression of more than one ADORA1 gene within a
cell, comprising: (a) synthesizing siRNA molecules of the
invention, which can be chemically modified, wherein one of the
siRNA strands includes a sequence complementary to RNA of the
ADORA1 genes; and (b) introducing the siRNA molecules into a cell
under conditions suitable to modulate the expression of the ADORA1
genes in the cell.
[0073] In another embodiment, the invention features a method for
modulating the expression of more than one ADORA1 gene within a
cell, comprising: (a) synthesizing a siRNA molecule of the
invention, which can be chemically modified, wherein one of the
siRNA strands includes a sequence complementary to RNA of the
ADORA1 gene and wherein the sense strand sequence of the siRNA is
identical to the complementary sequence of the ADORA1 RNA; and (b)
introducing the siRNA molecules into a cell under conditions
suitable to modulate the expression of the ADORA1 genes in the
cell.
[0074] In one embodiment, the invention features a method of
modulating the expression of an ADORA1 gene in a tissue explant,
comprising: (a) synthesizing a siRNA molecule of the invention,
which can be chemically modified, wherein one of the siRNA strands
includes a sequence complementary to RNA of the ADORA1 gene; (b)
introducing the siRNA molecule into a cell of the tissue explant
derived from a particular organism under conditions suitable to
modulate the expression of the ADORA1 gene in the tissue explant,
and (c) optionally 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 ADORA1 gene
in that organism.
[0075] In one embodiment, the invention features a method of
modulating the expression of an ADORA1 gene in a tissue explant,
comprising: (a) synthesizing a siRNA molecule of the invention,
which can be chemically modified, wherein one of the siRNA strands
includes a sequence complementary to RNA of the ADORA1 gene and
wherein the sense strand sequence of the siRNA is identical to the
complementary sequence of the ADORA1 RNA; (b) introducing the siRNA
molecule into a cell of the tissue explant derived from a
particular organism under conditions suitable to modulate the
expression of the ADORA1 gene in the tissue explant, and (c)
optionally 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 ADORA1 gene
in that organism.
[0076] In another embodiment, the invention features a method of
modulating the expression of more than one ADORA1 gene in a tissue
explant, comprising: (a) synthesizing siRNA molecules of the
invention, which can be chemically modified, wherein one of the
siRNA strands includes a sequence complementary to RNA of the
ADORA1 genes; (b) introducing the siRNA molecules into a cell of
the tissue explant derived from a particular organism under
conditions suitable to modulate the expression of the ADORA1 genes
in the tissue explant, and (c) optionally 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 ADORA1 genes in that organism.
[0077] In one embodiment, the invention features a method of
modulating the expression of an ADORA1 gene in an organism,
comprising: (a) synthesizing a siRNA molecule of the invention,
which can be chemically modified, wherein one of the siRNA strands
includes a sequence complementary to RNA of the ADORA1 gene; and
(b) introducing the siRNA molecule into the organism under
conditions suitable to modulate the expression of the ADORA1 gene
in the organism.
[0078] In another embodiment, the invention features a method of
modulating the expression of more than one ADORA1 gene in an
organism, comprising: (a) synthesizing siRNA molecules of the
invention, which can be chemically modified, wherein one of the
siRNA strands includes a sequence complementary to RNA of the
ADORA1 genes; and (b) introducing the siRNA molecules into the
organism under conditions suitable to modulate the expression of
the ADORA1 genes in the organism.
[0079] The siRNA molecules of the invention can be designed to
inhibit ADORA1 gene expression through RNAi targeting of a variety
of RNA molecules. In one embodiment, the siRNA 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), and/or RNA templates used for ADORA1 activity. If
alternate splicing produces a family of transcipts that are
distinguished by usage of appropriate exons, the instant invention
can be used to inhibit gene expression through the appropriate
exons 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 exon 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 siRNA
molecules of the invention. Such applications can be implemented
using known gene sequences or from partial sequences available from
an expressed sequence tag (EST).
[0080] In another embodiment, the siRNA molecules of the invention
are used to target conserved sequences corresponding to a gene
family or gene families such as checkpoint kinase genes. As such,
siRNA molecules targeting multiple checkpoint kinase targets can
provide increased therapeutic effect. In addition, siRNA can be
used to characterize pathways of gene function in a variety of
applications. For example, the present invention can be used to
inhibit the activity of target gene(s) in a pathway to determine
the function of uncharacterized gene(s) 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 development, such as prenatal development, postnatal
development and/or aging.
[0081] In one embodiment, siRNA molecule(s) and/or methods of the
invention are used to inhibit the expression of gene(s) that encode
RNA referred to by Genbank Accession number, for example genes such
as Genbank Accession No. NM.sub.--000674. Such sequences are
readily obtained using this Genbank Accession number.
[0082] In one embodiment, the invention features a method
comprising: (a) generating a randomized library of siRNA constructs
having a predetermined complexity, such as of 4.sup.N, where N
represents the number of base paired nucleotides in each of the
siRNA construct strands (eg. for a siRNA construct having 21
nucleotide sense and antisense strands with 19 base pairs, the
complexity would be 4.sup.19); and (b) assaying the siRNA
constructs of (a) above, under conditions suitable to determine
RNAi target sites within the target ADORA1 RNA sequence. In another
embodiment, the siRNA molecules of (a) have strands of a fixed
length, for example about 23 nucleotides in length. In yet another
embodiment, the siRNA molecules of (a) are of differing length, for
example having strands of about 19 to about 25 (e.g., about 19, 20,
21, 22, 23, 24, or 25) nucleotides in length. In yet another
embodiment, the assay can comprise a reconstituted in vitro siRNA
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 ADORA1 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 site(s) within the
target ADORA1 RNA sequence. In another embodiment, the target
ADORA1 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.
[0083] In another embodiment, the invention features a method
comprising: (a) analyzing the sequence of a RNA target encoded by
an ADORA1 gene; (b) synthesizing one or more sets of siRNA
molecules having sequence complementary to one or more regions of
the RNA of (a); and (c) assaying the siRNA molecules of (b) under
conditions suitable to determine RNAi targets within the target RNA
sequence. In another embodiment, the siRNA molecules of (b) have
strands of a fixed length, for example about 23 nucleotides in
length. In yet another embodiment, the siRNA molecules of (b) are
of differing length, for example having strands of about 19 to
about 25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in
length. In yet another embodiment, the assay can comprise a
reconstituted in vitro siRNA assay as described in Example 6
herein. In another embodiment, the assay can comprise a cell
culture system in which target RNA is expressed. Fragments of
ADORA1 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 site(s)
within the target ADORA1 RNA sequence. The target ADORA1 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.
[0084] By "target site" is meant a sequence within a target RNA
that is "targeted" for cleavage mediated by a siRNA construct which
contains sequences within its antisense region that are
complementary to the target sequence.
[0085] 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.
[0086] In one embodiment, the invention features a composition
comprising a siRNA 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 siRNA 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 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.
[0087] In another embodiment, the invention features a method for
validating an ADORA1 gene target, comprising: (a) synthesizing a
siRNA molecule of the invention, which can be chemically modified,
wherein one of the siRNA strands includes a sequence complementary
to RNA of an ADORA1 target gene; (b) introducing the siRNA molecule
into a cell, tissue, or organism under conditions suitable for
modulating expression of the ADORA1 target gene in the cell,
tissue, or organism; and (c) determining the function of the gene
by assaying for any phenotypic change in the cell, tissue, or
organism.
[0088] In one embodiment, the invention features a kit containing a
siRNA molecule of the invention, which can be chemically modified,
that can be used to modulate the expression of an ADORA1 target
gene in a cell, tissue, or organism. In another embodiment, the
invention features a kit containing more than one siRNA molecule of
the invention, which can be chemically modified, that can be used
to modulate the expression of more than one ADORA1 target gene in a
cell, tissue, or organism.
[0089] In one embodiment, the invention features a cell containing
one or more siRNA molecules of the invention, which can be
chemically modified. In another embodiment, the cell containing a
siRNA molecule of the invention is a mammalian cell. In yet another
embodiment, the cell containing a siRNA molecule of the invention
is a human cell.
[0090] In one embodiment, the synthesis of a siRNA molecule of the
invention, which can be chemically modified, comprises: (a)
synthesis of two complementary strands of the siRNA molecule; (b)
annealing the two complementary strands together under conditions
suitable to obtain a double stranded siRNA molecule. In another
embodiment, synthesis of the two complementary strands of the siRNA
molecule is by solid phase oligonucleotide synthesis. In yet
another embodiment, synthesis of the two complementary strands of
the siRNA molecule is by solid phase tandem oligonucleotide
synthesis.
[0091] In one embodiment, the invention features a method for
synthesizing a siRNA duplex molecule comprising: (a) synthesizing a
first oligonucleotide sequence strand of the siRNA 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
siRNA; (b) synthesizing the second oligonucleotide sequence strand
of siRNA 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 siRNA
duplex; (c) cleaving the linker molecule of (a) under conditions
suitable for the two siRNA oligonucleotide strands to hybridize and
form a stable duplex; and (d) purifying the siRNA duplex utilizing
the chemical moiety of the second oligonucleotide sequence strand.
In another embodiment, cleavage of the linker molecule in (c) above
takes place during deprotection of the oligonucleotide, for example
under hydrolysis conditions using an alkylamine base such as
methylamine. 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 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 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 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.
[0092] In a further embodiment, the method for siRNA synthesis is a
solution phase synthesis or hybrid phase synthesis wherein both
strands of the siRNA 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
siRNA sequence strands results in formation of the double stranded
siRNA molecule.
[0093] In another embodiment, the invention features a method for
synthesizing a siRNA duplex molecule comprising: (a) synthesizing
one oligonucleotide sequence strand of the siRNA 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 first sequence strand on the scaffold of
(a), wherein the second sequence comprises the other strand of the
double stranded siRNA 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 siRNA oligonucleotide strands
connected by the cleavable linker; and (d) under conditions
suitable for the two siRNA oligonucleotide strands to hybridize and
form a stable duplex. In another 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
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 another embodiment, the chemical moiety of (b)
that can used to isolate the attached oligonucleotide sequence
comprises a trityl group, for example a dimethoxytrityl group.
[0094] In another embodiment, the invention features a method for
making a double stranded siRNA 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 siRNA molecule, for example using a
trityl-on synthesis strategy as described herein.
[0095] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications, for example one or
more chemical modifications having Formula I, II, III, IV, or V,
that increases the nuclease resistance of the siRNA construct.
[0096] In another embodiment, the invention features a method for
generating siRNA molecules with increased nuclease resistance
comprising (a) introducing nucleotides having any of Formula I-VI
into a siRNA molecule, and (b) assaying the siRNA molecule of step
(a) under conditions suitable for isolating siRNA molecules having
increased nuclease resistance.
[0097] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications described herein that
modulates the binding affinity between the sense and antisense
strands of the siRNA construct.
[0098] In another embodiment, the invention features a method for
generating siRNA molecules with increased binding affinity between
the sense and antisense strands of the siRNA molecule comprising
(a) introducing nucleotides having any of Formula I-VI into a siRNA
molecule, and (b) assaying the siRNA molecule of step (a) under
conditions suitable for isolating siRNA molecules having increased
binding affinity between the sense and antisense strands of the
siRNA molecule.
[0099] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications described herein that
modulates the binding affinity between the antisense strand of the
siRNA construct and a complementary target RNA sequence within a
cell.
[0100] In another embodiment, the invention features a method for
generating siRNA molecules with increased binding affinity between
the antisense strand of the siRNA molecule and a complementary
target RNA sequence, comprising (a) introducing nucleotides having
any of Formula I-VI into a siRNA molecule, and (b) assaying the
siRNA molecule of step (a) under conditions suitable for isolating
siRNA molecules having increased binding affinity between the
antisense strand of the siRNA molecule and a complementary target
RNA sequence.
[0101] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications described herein that
modulate the polymerase activity of a cellular polymerase capable
of generating additional endogenous siRNA molecules having sequence
homology to the chemically modified siRNA construct.
[0102] In another embodiment, the invention features a method for
generating siRNA molecules capable of mediating increased
polymerase activity of a cellular polymerase capable of generating
additional endogenous siRNA molecules having sequence homology to
the chemically modified siRNA molecule comprising (a) introducing
nucleotides having any of Formula I-VI into a siRNA molecule, and
(b) assaying the siRNA molecule of step (a) under conditions
suitable for isolating siRNA molecules capable of mediating
increased polymerase activity of a cellular polymerase capable of
generating additional endogenous siRNA molecules having sequence
homology to the chemically modified siRNA molecule.
[0103] In one embodiment, the invention features chemically
modified siRNA constructs that mediate RNAi against ADORA1 in a
cell, wherein the chemical modifications do not significantly
effect the interaction of siRNA with a target RNA 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 siRNA
constructs.
[0104] In another embodiment, the invention features a method for
generating siRNA molecules with improved RNAi activity against
ADORA1, comprising (a) introducing nucleotides having any of
Formula I-VI into a siRNA molecule, and (b) assaying the siRNA
molecule of step (a) under conditions suitable for isolating siRNA
molecules having improved RNAi activity.
[0105] In yet another embodiment, the invention features a method
for generating siRNA molecules with improved RNAi activity against
an ADORA1 target RNA, comprising (a) introducing nucleotides having
any of Formula I-VI into a siRNA molecule, and (b) assaying the
siRNA molecule of step (a) under conditions suitable for isolating
siRNA molecules having improved RNAi activity against the target
RNA.
[0106] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications described herein that
modulates the cellular uptake of the siRNA construct.
[0107] In another embodiment, the invention features a method for
generating siRNA molecules against ADORA1 with improved cellular
uptake, comprising (a) introducing nucleotides having any of
Formula I-VI into a siRNA molecule, and (b) assaying the siRNA
molecule of step (a) under conditions suitable for isolating siRNA
molecules having improved cellular uptake.
[0108] In one embodiment, the invention features siRNA constructs
that mediate RNAi against ADORA1, wherein the siRNA construct
comprises one or more chemical modifications described herein that
increases the bioavailability of the siRNA construct, for example
by attaching polymeric conjugates such as polyethyleneglycol or
equivalent conjugates that improve the pharmacokinetics of the
siRNA construct, or by attaching conjugates that target specific
tissue types or cell types in vivo. Non-limiting examples of such
conjugates are described in Vargeese et al., U.S. Serial No.
60/311,865 incorporated by reference herein.
[0109] In one embodiment, the invention features a method for
generating siRNA molecules of the invention with improved
bioavailability, comprising (a) introducing a conjugate into the
structure of a siRNA molecule, and (b) assaying the siRNA molecule
of step (a) under conditions suitable for isolating siRNA 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, polyamines such as spermine or spermidine, and
others.
[0110] In another embodiment, the invention features a method for
generating siRNA molecules of the invention with improved
bioavailability, comprising (a) introducing an excipient
formulation to a siRNA molecule, and (b) assaying the siRNA
molecule of step (a) under conditions suitable for isolating siRNA
molecules having improved bioavailability. Such excipients include
polymers such as cyclodextrins, lipids, cationic lipids,
polyamines, phospholipids, and others.
[0111] In another embodiment, the invention features a method for
generating siRNA molecules of the invention with improved
bioavailability, comprising (a) introducing nucleotides having any
of Formula I-VI into a siRNA molecule, and (b) assaying the siRNA
molecule of step (a) under conditions suitable for isolating siRNA
molecules having improved bioavailability.
[0112] In another embodiment, polyethylene glycol (PEG) can be
covalently attached to siRNA compounds of the present invention.
The attached PEG can be any molecular weight, preferably from about
2,000 to about 50,000 daltons (Da).
[0113] 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 in vitro or in vivo introduction of RNA to test samples and/or
subjects. For example, preferred components of the kit include the
siRNA and a vehicle that promotes introduction of the siRNA. Such a
kit can also include instructions to allow a user of the kit to
practice the invention.
[0114] The term "short interfering RNA" or "siRNA" as used herein
refers to any nucleic acid molecule capable of mediating RNA
interference "RNAi" or gene silencing; see for example 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. Non limiting examples of siRNA molecules of the invention
are shown in FIG. 2. For example the siRNA can be a double stranded
polynucleotide molecule comprising self complementary sense and
antisense regions, wherein the antisense region comprises
complementarity to a target nucleic acid molecule. The siRNA can be
a single stranded hairpin polynucleotide having self complementary
sense and antisense regions, wherein the antisense region comprises
complementarity to a target nucleic acid molecule. The siRNA 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
complementarity to a target nucleic acid molecule, and wherein the
circular polynucleotide can be processed either in vivo or in vitro
to generate an active siRNA capable of mediating RNAi. As used
herein, siRNA molecules need not be limited to those molecules
containing only RNA, but further encompasses chemically modified
nucleotides and non-nucleotides.
[0115] 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.
[0116] By "inhibit" it is meant that the activity of a gene
expression product or level of RNAs or equivalent RNAs encoding one
or more gene products is reduced below that observed in the absence
of the nucleic acid molecule of the invention. In one embodiment,
inhibition with a siRNA molecule preferably is below that level
observed in the presence of an inactive or attenuated molecule that
is unable to mediate an RNAi response. In another embodiment,
inhibition of gene expression with the siRNA molecule of the
instant invention is greater in the presence of the siRNA molecule
than in its absence.
[0117] 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. 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.
[0118] By "ADORA1" is meant, a polypeptide comprising an adenosine
A1 receptor or polynucleotide encoding an Ets adenosine A1
receptor, for example a polynucleotide having Genbank Accession No.
NM.sub.--000674.
[0119] By "highly conserved sequence region" is meant, a nucleotide
sequence of one or more regions in a target gene does not vary
significantly from one generation to the other or from one
biological system to the other.
[0120] By "complementarity" or "complementary" is meant that a
nucleic acid can form hydrogen bond(s) with another nucleic acid
sequence by either traditional Watson-Crick or other
non-traditional types of interaction. 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. For example, the degree of complementarity
between the sense and antisense strand of the siRNA construct can
be the same or different from the degree of complementarity between
the antisense strand of the siRNA and the target RNA sequence.
Complementarity to the target sequence of less than 100% in the
antisense strand of the siRNA duplex, including point mutations, is
reported not to be tolerated when these changes are located between
the 3'-end and the middle of the antisense siRNA (completely
abolishes siRNA activity), whereas mutations near the 5'-end of the
antisense siRNA strand can exhibit a small degree of RNAi activity
(Elbashir et al., 2001, The EMBO Journal, 20, 6877-6888).
Determination of binding free energies for nucleic acid molecules
is well known in the art (see, e.g., Turner et al., 1987, CSH Symp.
Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad.
Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. 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 base pairing) with a second
nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%,
60%, 70%, 80%, 90%, and 100% complementary). "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.
[0121] The siRNA molecules of the invention represent a novel
therapeutic approach to treat a variety of allergic/inflammatory
diseases and conditions, including but not limited to asthma,
allergic rhinitis, atopic dermatitis, and other indications that
can respond to the level of ADORA1.
[0122] In one embodiment of the present invention, each sequence of
a siRNA molecule of the invention is independently about 18 to
about 24 nucleotides in length, in specific embodiments about 18,
19, 20, 21, 22, 23, or 24 nucleotides in length. In another
embodiment, the siRNA duplexes of the invention independently
comprise between about 17 and about 23 (e.g., about 17, 18, 19, 20,
21, 22, or 23) base pairs. In yet another embodiment, siRNA
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 16 to about 22 (e.g., about 16, 17, 18, 19, 20, 21, or 22)
base pairs. Exemplary siRNA molecules of the invention are shown in
Table I and III (all sequences are shown 5'-3') and/or FIGS. 4 and
5.
[0123] 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., mammals such as humans, cows, sheep, apes,
monkeys, swine, dogs, and cats. The cell can be eukaryotic (e.g., a
mammalian cell, such as a human 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.
[0124] The siRNA 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 injection, infusion pump or
stent, with or without their incorporation in biopolymers. In
particular embodiments, the nucleic acid molecules of the invention
comprise sequences shown in Table I, III and/or FIGS. 4 and 5.
Examples of such nucleic acid molecules consist essentially of
sequences defined in these tables/figures.
[0125] In another aspect, the invention provides mammalian cells
containing one or more siRNA molecules of this invention. The one
or more siRNA molecules can independently be targeted to the same
or different sites.
[0126] 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
.beta.-D-ribo-furanose moiety. The terms include double stranded
RNA, single stranded RNA, isolated RNA such as partially purified
RNA, essentially pure RNA, synthetic RNA, recombinantly 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 end(s) of the siRNA or
internally, for example at one or more nucleotides of the RNA.
Nucleotides in the RNA molecules of the instant invention can also
comprise non-standard nucleotides, such as non-naturally occurring
nucleotides or chemically synthesized nucleotides or
deoxynucleotides. These altered RNAs can be referred to as analogs
or analogs of naturally-occurring RNA.
[0127] 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. In one embodiment, a subject is
a mammal or mammalian cells. In another embodiment, a subject is a
human or human cells.
[0128] The term "phosphorothioate" as used herein refers to an
internucleotide linkage having Formula I, wherein Z and/or W
comprise a sulfur atom. Hence, the term phosphorothioate refers to
both phosphorothioate and phosphorodithioate internucleotide
linkages.
[0129] 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).
[0130] The term "acyclic nucleotide" as used herein refers to any
nucleotide having an acyclic ribose sugar, for example where any of
the ribose carbons (C1, C2, C3, C4, or C5), are independently or in
combination absent from the nucleotide.
[0131] The nucleic acid molecules of the instant invention,
individually, or in combination or in conjunction with other drugs,
can be used to treat diseases or conditions discussed herein. For
example, to treat a particular disease or condition, the siRNA
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.
[0132] In a further embodiment, the siRNA molecules can be used in
combination with other known treatments to treat conditions or
diseases discussed above. For example, the described molecules
could be used in combination with one or more known therapeutic
agents to treat a disease or condition. Non-limiting examples of
other therapeutic agents that can be readily combined with a siRNA
molecule of the invention are enzymatic nucleic acid molecules,
allosteric nucleic acid molecules, antisense, decoy, or aptamer
nucleic acid molecules, antibodies such as monoclonal antibodies,
small molecules, and other organic and/or inorganic compounds
including metals, salts and ions.
[0133] In one embodiment, the invention features an expression
vector comprising a nucleic acid sequence encoding at least one
siRNA molecule of the invention, in a manner which allows
expression of the siRNA molecule. For example, the vector can
contain sequence(s) encoding both strands of a siRNA molecule
comprising a duplex. The vector can also contain sequence(s)
encoding a single nucleic acid molecule that is self complementary
and thus forms a siRNA molecule. Non-limiting examples of such
expression vectors are described in Paul et al, 2002, Nature
Biotechnology, 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.
[0134] In another embodiment, the invention features a mammalian
cell, for example, a human cell, including an expression vector of
the invention.
[0135] In yet another embodiment, the expression vector of the
invention comprises a sequence for a siRNA molecule having
complementarity to a RNA molecule referred to by a Genbank
Accession numbers, for example genes such as Genbank Accession No.
No. NM.sub.--000674.
[0136] In one embodiment, an expression vector of the invention
comprises a nucleic acid sequence encoding two or more siRNA
molecules, which can be the same or different.
[0137] In another aspect of the invention, siRNA 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. siRNA
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 siRNA
molecules can be delivered as described herein, and persist in
target cells. Alternatively, viral vectors can be used that provide
for transient expression of siRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the siRNA
molecules bind and down-regulate gene function or expression via
RNA interference (RNAi). Delivery of siRNA 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.
[0138] By "vectors" is meant any nucleic acid- and/or viral-based
technique used to deliver a desired nucleic acid.
[0139] By "comprising" is meant including, but not limited to,
whatever follows the word "comprising". Thus, use of the term
"comprising" indicates that the listed elements are required or
mandatory, but that other elements are optional and may or may not
be present. By "consisting of" is meant including, and limited to,
whatever follows the phrase "consisting of". Thus, the phrase
"consisting of" indicates that the listed elements are required or
mandatory, and that no other elements may be present. By
"consisting essentially of" is meant including any elements listed
after the phrase, and limited to other elements that do not
interfere with or contribute to the activity or action specified in
the disclosure for the listed elements. Thus, the phrase
"consisting essentially of" indicates that the listed elements are
required or mandatory, but that other elements are optional and may
or may not be present depending upon whether or not they affect the
activity or action of the listed elements.
[0140] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0141] First the drawings will be described briefly.
DRAWINGS
[0142] FIG. 1 shows a non-limiting example of a scheme for the
synthesis of siRNA molecules. The complementary siRNA 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 and deprotection of the
oligonucleotide, the two siRNA strands spontaneously hybridize to
form a siRNA 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.
[0143] FIG. 2 shows a MALDI-TOV mass spectrum of a purified siRNA
duplex synthesized by a method of the invention. The two peaks
shown correspond to the predicted mass of the separate siRNA
sequence strands. This result demonstrates that the siRNA duplex
generated from tandem synthesis can be purified as a single entity
using a simple trityl-on purification methodology.
[0144] FIG. 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 which in turn generates siRNA duplexes having terminal
phosphate groups (P). An active siRNA 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 siRNA molecules, thereby amplifying
the RNAi response.
[0145] FIG. 4 shows non-limiting examples of chemically modified
siRNA 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
siRNA constructs. A The sense strand comprises 21 nucleotides
having four phosphorothioate 5' and 3'-terminal internucleotide
linkages, wherein the two terminal 3'-nucleotides are optionally
base paired and wherein all pyrimidine nucleotides that may be
present are 2'-O-methyl modified nucleotides except for (N N)
nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. The antisense strand
comprises 21 nucleotides, wherein the two terminal 3'-nucleotides
are optionally complimentary to the target RNA sequence, and having
one 3'-terminal phosphorothioate internucleotide linkage and four
5'-terminal phosphorothioate internucleotide linkages and wherein
all pyrimidine nucleotides that may be present are
2'-deoxy-2'-fluoro modified nucleotides except for (N N)
nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. B 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'-O-methyl modified nucleotides except for
(N N) nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. The antisense strand
comprises 21 nucleotides, wherein the two terminal 3'-nucleotides
are optionally complimentary 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, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. C 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 modified nucleotides except for (N N) nucleotides,
which can comprise naturally occurring ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. The antisense strand comprises 21 nucleotides,
wherein the two terminal 3'-nucleotides are optionally
complimentary 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, which can
comprise naturally occurring ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein.
D The sense strand comprises 21 nucleotides having five
phosphorothioate 5' and 3'-terminal internucleotide linkages,
wherein the two terminal 3'-nucleotides are optionally base paired
and wherein all nucleotides are ribonucleotides except for (N N)
nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. The antisense strand
comprises 21 nucleotides, wherein the two terminal 3'-nucleotides
are optionally complimentary to the target RNA sequence, and having
one 3'-terminal phosphorothioate internucleotide linkage and five
5'-terminal phosphorothioate internucleotide linkages and wherein
all nucleotides are ribonucleotides except for (N N) nucleotides,
which can comprise naturally occurring ribonucleotides,
deoxynucleotides, universal bases, or other chemical modifications
described herein. E 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'-O-methyl nucleotides except for (N N) nucleotides, which can
comprise naturally occurring ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein.
The antisense strand comprises 21 nucleotides all having
phosphorothioate internucleotide linkages, wherein the two terminal
3'-nucleotides are optionally complimentary to the target RNA
sequence, and wherein all nucleotides are ribonucleotides except
for (N N) nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. F 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 nucleotides except for (N N) nucleotides, which can
comprise naturally occurring ribonucleotides, deoxynucleotides,
universal bases, or other chemical modifications described herein.
The antisense strand comprises 21 nucleotides, wherein the two
terminal 3'-nucleotides are optionally complimentary to the target
RNA sequence, and having one 3'-termninal phosphorothioate
internucleotide linkage and wherein all pyrimidine nucleotides that
may be present are 2'-deoxy-2'-fluoro nucleotides except for (N N)
nucleotides, which can comprise naturally occurring
ribonucleotides, deoxynucleotides, universal bases, or other
chemical modifications described herein. The antisense strand of
constructs A-F comprise sequence complimentary to target RNA
sequence of the invention.
[0146] FIG. 5 shows non-limiting examples of specific chemically
modified siRNA sequences of the invention. A-F applies the chemical
modifications described in FIG. 4A-F to an ADORA1 siRNA
sequence.
[0147] FIG. 6 shows non-limiting examples of different siRNA
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 comprising between about 1, 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 in 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 siRNA construct 2 in vivo and/or in vitro,
which can optionally utilize another biodegradable linker to
generate the active siRNA construct 1 in vivo and/or in vitro. As
such, the stability and/or activity of the siRNA constructs can be
modulated based on the design of the siRNA construct for use in
vivo or in vitro and/or in vitro.
[0148] FIG. 7 is a diagrammatic representation of a scheme utilized
in generating an expression cassette to generate siRNA hairpin
constructs. (A) A DNA oligomer is synthesized with a 5'-restriction
site (R1) sequence followed by a region having sequence identical
(sense region of siRNA) to a predetermined ADORA1 target seqeunce,
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, between
about 3 and 10 nucleotides. (B) The synthetic construct is then
extended by DNA polymerase to generate a hairpin structure having
self complementary sequence that will result in a siRNA transcript
having specificity for an ADORA1 target sequence and having self
complementary sense and antisense regions. (C) The construct is
heated (for example to about 95.degree. 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'-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.
[0149] FIG. 8 is a diagrammatic representation of a scheme utilized
in generating an expression cassette to generate double stranded
siRNA constructs. (A) A DNA oligomer is synthesized with a
5'-restriction (R1) site sequence followed by a region having
sequence identical (sense region of siRNA) to a predetermined
ADORA1 target seqeunce, wherein the sense region comprises, for
example, about 19, 20, 21, or 22 nucleotides (N) in length, and
which is followed by a 3'-restriction site (R2) which is adjacent
to a loop sequence of defined sequence (X). (B) The synthetic
construct is then extended by DNA polymerase to generate a hairpin
structure having self complementary sequence. (C) The construct is
processed by restriction enzymes specific to R1 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 siRNA. Poly T termination sequences can be added to the
constructs to generate U overhangs in the resulting transcript.
[0150] FIG. 9 is a diagrammatic representation of a method used to
determine target sites for siRNA mediated RNAi within a particular
target nucleic acid sequence, such as messenger RNA. (A) A pool of
siRNA oligonucleotides are synthesized wherein the antisense region
of the siRNA 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
siRNA. (B) The sequences are pooled and are inserted into vectors
such that (C) transfection of a vector into cells results in the
expression of the siRNA. (D) Cells are sorted based on phenotypic
change that is associated with modulation of the target nucleic
acid sequence. (E) The siRNA is isolated from the sorted cells and
is sequenced to identify efficacious target sites within the target
nucleic acid sequence.
MECHANISM OF ACTION OF NUCLEIC ACID MOLECULES OF THE INVENTION
[0151] RNA interference refers to the process of sequence specific
post transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNA) (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 (dsRNA) 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.
[0152] 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 (siRNA) (Berstein et al., 2001,
Nature, 409, 363). Short interfering RNAs derived from Dicer
activity are typically about 21-23 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
RNAs (stRNA) 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).
[0153] 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,
describes 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, Nature, 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 nucleotide 3'-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'-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
[0154] 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 siRNA oligonucleotide
sequences or siRNA 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.
[0155] 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 Enzymology 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, Biotechnol 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 .mu.mol scale
protocol with a 2.5 min coupling step for 2'-O-methylated
nucleotides and a 45 sec coupling step for 2'-deoxy nucleotides or
2'-deoxy-2'-fluoro nucleotides. Table II outlines the amounts and
the contact times of the reagents used in the synthesis cycle.
Alternatively, syntheses at the 0.2 .mu.mol scale can be performed
on a 96-well plate synthesizer, such as the instrument produced by
Protogene (Palo Alto, Calif.) with minimal modification to the
cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of
2'-O-methyl phosphoramidite and a 105-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 22-fold excess (40 .mu.L of 0.11 M=4.4 .mu.mol) of
deoxy phosphoramidite and a 70-fold excess of S-ethyl tetrazole (40
.mu.L of 0.25 M=10 .mu.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 calorimetric 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 I.sub.2, 49 mM pyridine, 9% water in
THF (PERSEPTIVE.TM.). 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.
[0156] 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% aq. methylamine (1 mL) at 65.degree. C. for 10 min.
After cooling to -20.degree. C., the supernatant is removed from
the polymer support. The support is washed three times with 1.0 mL
of EtOH:MeCN:H2O/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.
[0157] The method of synthesis used for RNA including certain siRNA
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 example, small
scale syntheses are conducted on a 394 Applied Biosystems, Inc.
synthesizer using a 0.2 .mu.mol 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 II outlines
the amounts and the contact times of the reagents used in the
synthesis cycle. Alternatively, syntheses at the 0.2 .mu.mol scale
can be done on a 96-well plate synthesizer, such as the instrument
produced by Protogene (Palo Alto, Calif.) with minimal modification
to the cycle. A 33-fold excess (60 .mu.L of 0.11 M=6.6 .mu.mol) of
2'-O-methyl phosphoramidite and a 75-fold excess of S-ethyl
tetrazole (60 .mu.L of 0.25 M=15 .mu.mol) can be used in each
coupling cycle of 2'-O-methyl residues relative to polymer-bound
5'-hydroxyl. A 66-fold excess (120 .mu.L of 0.11 M=13.2 .mu.mol) of
alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess
of S-ethyl tetrazole (120 .mu.L of 0.25 M=30 .mu.mol) can be used
in each coupling cycle of ribo 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); oxidation solution is 16.9 mM I.sub.2, 49 mM pyridine,
9% water in THF (PERSEPTIVE.TM.). 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.
[0158] 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.degree. C. for 10 min. After cooling to -20.degree. C., the
supernatant is removed from the polymer support. The support is
washed three times with 1.0 mL of EtOH:MeCN:H2O/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 TEA/HF/NMP solution (300 .mu.L of a
solution of 1.5 mL N-methylpyrrolidinone, 750 .mu.L TEA and 1 mL
TEA.multidot.3HF to provide a 1.4 M HF concentration) and heated to
65.degree. C. After 1.5 h, the oligomer is quenched with 1.5 M
NH.sub.4HCO.sub.3.
[0159] 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.degree. C. for 15 min. The
vial is brought to r.t. TEA.multidot.3HF (0.1 mL) is added and the
vial is heated at 65.degree. C. for 15 min. The sample is cooled at
-20.degree. C. and then quenched with 1.5 M NH.sub.4HCO.sub.3.
[0160] For purification of the trityl-on oligomers, the quenched
NH.sub.4HCO.sub.3 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 min. The cartridge is then washed
again with water, salt exchanged with 1 M NaCl and washed with
water again. The oligonucleotide is then eluted with 30%
acetonitrile.
[0161] 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, all
that is important is the ratio of chemicals used in the
reaction.
[0162] 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 Chem. 8, 204), or by
hybridization following synthesis and/or deprotection.
[0163] The siRNA molecules of the invention can also be synthesized
via a tandem synthesis methodology as described in Example 1
herein, wherein both siRNA strands are synthesized as a single
contiguous oligonucleotide fragment or strand separated by a
cleavable linker which is subsequently cleaved to provide separate
siRNA fragments or strands that hybridize and permit purification
of the siRNA duplex. The linker can be a polynucleotide linker or a
non-nucleotide linker. The tandem synthesis of siRNA as described
herein can be readily adapted to both multiwell/multiplate
synthesis platforms such as 96 well or similarly larger multi-well
platforms. The tandem synthesis of siRNA as described herein can
also be readily adapted to large scale synthesis platforms
employing batch reactors, synthesis columns and the like.
[0164] A siRNA 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.
[0165] 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'-flouro, 2'-O-methyl, 2'-H (for a review see Usman and Cedergren,
1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31,
163). siRNA 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.
[0166] In another aspect of the invention, siRNA 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. siRNA 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 siRNA molecules can be delivered as described
herein, and persist in target cells. Alternatively, viral vectors
can be used that provide for transient expression of siRNA
molecules.
Optimizing Activity of the Nucleic Acid Molecule of the
Invention
[0167] 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
Biochem. Sci. 17, 334; Usman et al., International Publication No.
WO 93/15187; and Rossi et al., International Publication 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., supra; all of which are
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. 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.
[0168] 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'-flouro, 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. 31, 163;
Burgin et al., 1996, Biochemistry, 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. Nature, 1990, 344, 565-568; 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.
5,627,053; Woolf et al., International PCT Publication No. WO
98/13526; Thompson et al., U.S. Ser. No. 60/082,404 which was filed
on Apr. 20, 1998; Karpeisky et al., 1998, Tetrahedron Lett., 39,
1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic Acid Sciences),
48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,
99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 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 the siRNA nucleic acid
molecules of the instant invention so long as the ability of siRNA
to promote RNAi is cells is not significantly inhibited.
[0169] While chemical modification of oligonucleotide
internucleotide linkages with phosphorothioate, phosphorothioate,
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.
[0170] Small interfering RNA (siRNA) 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.
[0171] 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. Chem. 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 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
mythylene bicyclo nucleotide (see for example Wengel et al.,
International PCT Publication No. WO 00/66604 and WO 99/14226).
[0172] In another embodiment, the invention features conjugates
and/or complexes of siRNA molecules of the invention. Such
conjugates and/or complexes can be used to facilitate delivery of
siRNA molecules into a biological system, such as a cell. The
conjugates and complexes provided by the instant invention can
impart therapeutic activity by transferring 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,
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 multi-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.
[0173] 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 siRNA
molecule of the invention or the sense and antisense strands of a
siRNA 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 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.
[0174] The term "biodegradable" as used herein, refers to
degradation in a biological system, for example enzymatic
degradation or chemical degradation.
[0175] 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 siRNA molecules either alone or in
combination with othe molecules contemplated by the instant
invention include therapeutically active molecules such as
antibodies, 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, siRNA, 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.
[0176] 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.
[0177] Therapeutic nucleic acid molecules (e.g., siRNA molecules)
delivered exogenously optimally are stable within cells until
reverse trascription 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 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.
[0178] In yet another embodiment, siRNA 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 in vivo the activity should not be
significantly lowered.
[0179] Use of the nucleic acid-based molecules of the invention
will lead to better treatment of the disease progression by
affording the possibility of combination therapies (e.g., multiple
siRNA 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 siRNA molecules can also include
combinations of different types of nucleic acid molecules, such as
enzymatic nucleic acid molecules (ribozymes), allozymes, antisense,
2,5-A oligoadenylate, decoys, aptamers etc.
[0180] In another aspect a siRNA molecule of the invention
comprises one or more 5' and/or a 3'-cap structure, for example on
only the sense siRNA strand, antisense siRNA strand, or both siRNA
strands.
[0181] 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 is selected from the group comprising inverted
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-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.
[0182] In yet another preferred embodiment, the 3'-cap is selected
from a group comprising, 4',5'-methylene nucleotide;
1-(beta-D-erythrofuranosyl) nucleotide; 4'-thio 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 Jyer, 1993, Tetrahedron 49, 1925;
incorporated by reference herein).
[0183] 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.
[0184] 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 group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino, or SH. The term also includes alkenyl
groups that are unsaturated hydrocarbon 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 group(s) is preferably, hydroxyl, cyano, alkoxy,
.dbd.O, .dbd.S, NO.sub.2, halogen, N(CH.sub.3).sub.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 group(s) is
preferably, hydroxyl, cyano, alkoxy, .dbd.O, .dbd.S, NO.sub.2 or
N(CH.sub.3).sub.2, amino or SH.
[0185] 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.
[0186] 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 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 & Peyman, supra). By "modified
bases" in this aspect is meant nucleotide bases other than adenine,
guanine, cytosine and uracil at 1' position or their
equivalents.
[0187] In one embodiment, the invention features modified siRNA
molecules, with phosphate backbone modifications comprising one or
more phosphorothioate, phosphorodithioate, methylphosphonate,
phosphotriester, morpholino, amidate carbamate, carboxymethyl,
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 and Properties, in
Modern Synthetic Methods, VCH, 331-417, and Mesmaeker et al., 1994,
Novel Backbone Replacements for Oligonucleotides, in Carbohydrate
Modifications in Antisense Research, ACS, 24-39.
[0188] 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.
[0189] By "unmodified nucleoside" is meant one of the bases
adenine, cytosine, guanine, thymine, uracil joined to the 1' carbon
of a .beta.-D-ribo-furanose.
[0190] 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.
[0191] In connection with 2'-modified nucleotides as described for
the present invention, by "amino" is meant 2'-NH.sub.2 or
2'-O--NH.sub.2, which may 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.
[0192] Various modifications to nucleic acid siRNA 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
[0193] A siRNA molecule of the invention can be adapted for use to
treat, for example allergic/inflammatory diseases and conditions,
including but not limited to asthma, allergic rhinitis, atopic
dermatitis, and any other indications that can respond to the level
of ADORA1 in a cell or tissue, alone or in combination with other
therapies. For example, a siRNA molecule can comprise a delivery
vehicle, including liposomes, for administration to a subject,
carriers and diluents and their salts, and/or can be present in
pharmaceutically acceptable formulations. Methods for the delivery
of nucleic acid molecules are described in Akhtar et al., 1992,
Trends Cell Bio., 2, 139; Delivery Strategies for Antisense
Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,
1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999,
Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS
Symp. Ser., 752, 184-192, 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, further describes 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 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 delivery vehicles, such as hydrogels,
cyclodextrins, 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, Clin. Cancer Res., 5,
2330-2337 and Barry et al., International PCT Publication No. WO
99/31262. Many examples in the art describe CNS delivery methods of
oligonucleotides by osmotic pump, (see Chun et al., 1998,
Neuroscience Letters, 257, 135-138, D'Aldin et al., 1998, Mol.
Brain Research, 55, 151-164, Dryden et al., 1998, J. Endocrinol.,
157, 169-175, Ghimikar et al, 1998, Neuroscience Letters, 247,
21-24) or direct infusion (Broaddus et al., 1997, Neurosurg. Focus,
3, article 4). Other routes of delivery include, but are not
limited to oral (tablet or pill form) and/or intrathecal delivery
(Gold, 1997, Neuroscience, 76, 1153-1158). More detailed
descriptions of nucleic acid delivery and administration are
provided in Sullivan et al., supra, Draper et al., PCT W093/23569,
Beigelman et al., PCT W099/05094, and Klimuk et al., PCT W099/04819
all of which have been incorporated by reference herein.
[0194] In addition, the invention features the use of methods to
deliver the nucleic acid molecules of the instant invention to
hematopoietic cells, including monocytes and lymphocytes. These
methods are described in detail by Hartmann et al., 1998, J.
Phamacol. Exp. Ther., 285(2), 920-928; Kronenwett et al., 1998,
Blood, 91(3), 852-862; Filion and Phillips, 1997, Biochim. Biophys.
Acta., 1329(2), 345-356; Ma and Wei, 1996, Leuk. Res., 20(11/12),
925-930; and Bongartz et al., 1994, Nucleic Acids Research, 22(22),
4681-8. Such methods, as described above, include the use of free
oligonucleitide, cationic lipid formulations, liposome formulations
including pH sensitive liposomes and immunoliposomes, and
bioconjugates including oligonucleotides conjugated to fusogenic
peptides, for the transfection of hematopoietic cells with
oligonucleotides.
[0195] Thus, the invention features a pharmaceutical composition
comprising one or more nucleic acid(s) 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 into 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 may also
be formulated and used as tablets, capsules or elixirs for oral
administration, suppositories for rectal administration, sterile
solutions, suspensions for injectable administration, and the other
compositions known in the art.
[0196] 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.
[0197] A pharmacological composition or formulation refers to a
composition or formulation in a form suitable for administration,
e.g., systemic 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.
[0198] 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
which lead to systemic absorption include, without limitation:
intravenous, subcutaneous, intraperitoneal, inhalation, oral,
intrapulmonary and intramuscular. Each of these administration
routes expose the siRNA 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 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 may
provide enhanced delivery of the drug to target cells by taking
advantage of the specificity of macrophage and lymphocyte immune
recognition of abnormal cells, such as cancer cells.
[0199] By "pharmaceutically acceptable formulation" 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 the
forulations and conjugates described herein, as well as other
target area specific formulations including CNS formulations
including P-glycoprotein inhibitors (such as Pluronic P85), which
can enhance entry of drugs into the CNS (Jolliet-Riant and
Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26);
biodegradable polymers, such as poly (DL-lactide-coglycolide)
microspheres for sustained release delivery after intracerebral
implantation (Emerich, D F et al, 1999, Cell Transplant, 8, 47-58)
(Alkermes, Inc. Cambridge, Mass.); and loaded nanoparticles, such
as those made of polybutylcyanoacrylate, which can deliver drugs
across the blood brain barrier and can alter neuronal uptake
mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949,
1999). 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. Drug 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.
[0200] 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 carriers 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. Pharm. 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, Biochim. Biophys. 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.
[0201] The present invention also includes compositions prepared
for storage or administration, which include a pharmaceutically
effective amount of the desired compounds in a pharmaceutically
acceptable carrier or diluent. Acceptable carriers 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 may be provided. These include sodium
benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In
addition, antioxidants and suspending agents can be used.
[0202] 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.
[0203] 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 and/or vehicles. The term parenteral as used
herein includes percutaneous, subcutaneous, intravascular (e.g.,
intravenous), intramuscular, or 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.
[0204] 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.
[0205] 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.
[0206] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-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.
[0207] 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.
[0208] 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.
[0209] 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-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring 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 ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions can also contain sweetening and flavoring
agents.
[0210] 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.
[0211] 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-irritating 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.
[0212] 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.
[0213] 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 carrier materials to produce a single dosage form
varies depending 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.
[0214] 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.
[0215] 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.
[0216] The nucleic acid molecules of the present invention may 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 may increase the
beneficial effects while reducing the presence of side effects.
[0217] In one embodiment, the invention 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. Chem. 262, 4429-4432) is unique to
hepatocytes and binds branched galactose-terminal glycoproteins,
such as asialoorosomucoid (ASOR). Binding of such glycoproteins or
synthetic glycoconjugates 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. Chem., 257, 939-945). Lee and Lee, 1987,
Glycoconjugate 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 glycoproteins or
glycoconjugates (Ponpipom et al., 1981, J. Med. Chem., 24,
1388-1395). The use of galactose and galactosamine based conjugates
to transport exogenous compounds across cell membranes can provide
a targeted delivery approach to the treatment of liver disease or
hepatocellular carcinoma. The use of bioconjugates can also provide
a reduction in the required dose of therapeutic compounds required
for treatment. Furthermore, therapeutic bioavialability,
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., U.S. Ser. No. 60/311,865, filed Aug. 13, 2001; and
Matulic-Adamic et al., U.S. Ser. No. 60/362,016, filed Mar. 6,
2002.
[0218] Alternatively, certain siRNA 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, Proc. Natl. Acad. Sci. USA, 88, 10591-5; Kashani-Sabet
et al., 1992, Antisense Res. Dev., 2, 3-15; Dropulic et al., 1992,
J. Virol., 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, Gene Therapy, 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 Symp. 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 Chem., 269, 25856.
[0219] 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. siRNA 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 for example Thompson, U.S. Pat. Nos. 5,902,880 and
6,146,886). The recombinant vectors capable of expressing the siRNA
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 administered as necessary. Once expressed, the
siRNA molecule interacts with the target mRNA and generates an RNAi
response. Delivery of siRNA molecule expressing vectors can be
systemic, such as by intravenous or intra-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).
[0220] In one aspect the invention features an expression vector
comprising a nucleic acid sequence encoding at least one siRNA
molecule of the instant invention. The expression vector can encode
one or both strands of a siRNA duplex, or a single self
complementary strand that self hybridizes into a siRNA duplex. The
nucleic acid sequences encoding the siRNA molecules of the instant
invention can be operably linked in a manner that allows expression
of the siRNA molecule (see for example Paul et al., 2002, Nature
Biotechnology, 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).
[0221] 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 siRNA molecules of the instant invention; wherein said sequence
is operably linked to said initiation region and said termination
region, in a manner that allows expression and/or delivery of the
siRNA molecule. The vector can optionally include an open reading
frame (ORF) for a protein operably linked on the 5' side or the
3'-side of the sequence encoding the siRNA of the invention; and/or
an intron (intervening sequences).
[0222] Transcription of the siRNA molecule sequences can be driven
from a promoter for eukaryotic RNA polymerase I (pol 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 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. US A, 87,
6743-7; Gao and Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber
et al., 1993, Methods Enzymol., 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 siRNA 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 Ther., 4, 45; Beigelman et al., International PCT
Publication No. WO 96/18736. The above siRNA 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).
[0223] In another aspect the invention features an expression
vector comprising a nucleic acid sequence encoding at least one of
the siRNA molecules of the invention, in a manner that allows
expression of that siRNA 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 siRNA 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 siRNA molecule.
[0224] 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 least one strand of a siRNA 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
siRNA 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 siRNA 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.
[0225] 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 siRNA 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 intron, the open reading frame and the
termination region, in a manner which allows expression and/or
delivery of the siRNA molecule.
EXAMPLES
[0226] 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 siRNA Constructs
[0227] Exemplary siRNA 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 purification process that provides RNAi molecules in high
yield. This approach is highly amenable to siRNA synthesis in
support of high throughput RNAi screening, and can be readily
adapted to multi-column or multi-well synthesis platforms.
[0228] After completing a tandem synthesis of an siRNA oligo and
its compliment 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
siRNA 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 to 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 C18
cartridge.
[0229] Standard phosphoramidite synthesis chemistry is used up to
point of introducing a tandem linker, such as an inverted
deoxyabasic succinate linker (see FIG. 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 Bromotripyrrolidinophosphoniumhe-
xaflurorophosphate (PyBrOP). After the linker is coupled, standard
synthesis chemistry is utilized to complete synthesis of the second
sequence leaving the terminal the 5'-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 50 mM NaOAc or 1.5M
NH.sub.4H.sub.2CO.sub.3.
[0230] Purification of the siRNA duplex can be readily accomplished
using solid phase extraction, for example using a Waters C18 SepPak
1 g cartridge conditioned with 1 column volume (CV) of
acetonitrile, 2 CV H2O, and 2 CV 50 mM NaOAc. The sample is loaded
and then washed with 1 CV H2O or 50 mM NaOAc. Failure sequences are
eluted with 1 CV 14% ACN (Aqueous with 50 mM NaOAc and 50mM NaCl).
The column is then washed, for example with 1 CV H2O 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 allowing to stand for approx.
10 minutes. The remaining TFA solution is removed and the column
washed with H2O followed by 1 CV 1M NaCl and additional H2O. The
siRNA duplex product is then eluted, for example using 1 CV 20%
aqueous CAN.
[0231] FIG. 2 provides an example of MALDI-TOV mass spectrometry
analysis of a purified siRNA construct in which each peak
corresponds to the calculated mass of an individual siRNA strand of
the siRNA duplex. The same purified siRNA provides three peaks when
analyzed by capillary gel electrophoresis (CGE), one peak
presumably corresponding to the duplex siRNA, and two peaks
presumably corresponding to the separate siRNA sequence strands.
Ion exchange HPLC analysis of the same siRNA contract only shows a
single peak.
Example 2
Identification of Potential siRNA Target Sites in Any RNA
Sequence
[0232] The sequence of an RNA target of interest, such as a 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 siRNA targets having
complimentarity 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 siRNA molecules targeting those sites as well. 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 target
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
siRNA molecules for efficacy, for example by using in vitro RNA
cleavage assays, 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 siRNA contruct to be used. High
throughput screening assays can be developed for screening siRNA
molecules using methods known in the art, such as with multi-well
or multi-plate assays to determine efficient reduction in target
gene expression.
Example 3
Selection of siRNA Molecule Target Sites in a RNA
[0233] The following non-limiting steps can be used to carry out
the selection of siRNAs targeting a given gene sequence or
transcipt.
[0234] 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.
[0235] 2. In some instances the siRNAs correspond to more than one
target sequence; such would be the case for example in targeting
different transcipts of the same gene, targeting different
transcipts 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 indentify subsequences that
are unique to a target sequence, such as a mutant target sequence.
Such an approach would enable the use of siRNA to target
specifically the mutant sequence and not effect the expression of
the normal sequence.
[0236] 3. In some instances the siRNA subsequences are absent in
one or more sequences while present in the desired target sequence;
such would be the case if the siRNA 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.
[0237] 4. The ranked siRNA 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.
[0238] 5. The ranked siRNA 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.
[0239] 6. The ranked siRNA 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, 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.
[0240] 7. The ranked siRNA 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 siRNA molecules with terminal
TT thymidine dinucleotides.
[0241] 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 siRNA 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
siRNA duplex. 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.
[0242] 9. The siRNA molecules are screened in an in vitro, cell
culture or animal model system to identify the most active siRNA
molecule or the most preferred target site within the target RNA
sequence.
[0243] In an alternate approach, a pool of siRNA constructs
specific to an ADORA1 target sequence is used to screen for target
sites in cells expressing ADORA1 RNA, such as human lung mast
cells. The general strategy used in this approach is shown in FIG.
9. A non-limiting example of such as pool is a pool comprising
sequences having sense sequences comprising SEQ ID NOs. 1-161 and
antisense sequences comprising SEQ ID NOs. 162-322 respectively.
Human lung mast cells expressing ADORA1 are transfected with the
pool of siRNA constructs and cells that demonstrate a phenotype
associated with ADORA1 inhibition are sorted. The pool of siRNA
constructs can be expressed from transciption cassettes inserted
into appropriate vectors (see for example FIG. 7 and FIG. 8). The
siRNA from cells demonstrating a positive phenotypic change (e.g.,
decreased adenosine receptor expression, for example as determined
by a [.sup.3H]DPCPX binding assay as described in Nyce and Metzger,
1997, Nature, 385, 721-725), are sequenced to determine the most
suitable target site(s) within the target ADORA1 RNA sequence.
Example 4
ADORA1 Targeted siRNA Design
[0244] siRNA target sites were chosen by analyzing sequences of the
ADORA1 RNA target and optionally prioritizing the target sites on
the basis of folding (structure of any given sequence analyzed to
determine siRNA accessibility to the target), using a library of
siRNA molecules as described in Example 3, or alternately by using
an in vitro siRNA system as described in Example 6 herein. siRNA
molecules were designed that could bind each target and are
optionally individually analyzed by computer folding to assess
whether the siRNA molecule can interact with the target sequence.
Varying the length of the siRNA molecules can be chosen to optimize
activity. Generally, a sufficient number of complimentary
nucleotide bases are chosen to bind to, or otherwise interact with,
the target RNA, but the degree of complementarity can be modulated
to accommodate siRNA duplexes or varying length or base
composition. By using such methodologies, siRNA molecules can be
designed to target sites within any known RNA sequence, for example
those RNA sequences corresponding to the any gene transcript.
Example 5
Chemical Synthesis and Purification of siRNA
[0245] siRNA 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
siRNA molecule(s) are complementary to the target site sequences
described above. The siRNA molecules can be chemically synthesized
using methods described herein. Inactive siRNA molecules that are
used as control sequences can be synthesized by scrambling the
sequence of the siRNA molecules such that it is not complimentary
to the target sequence.
Example 6
RNAi in vitro Assay to Assess siRNA Activity
[0246] An in vitro assay that recapitulates RNAi in a cell free
system is used to evaluate siRNA constructs targeting ADORA1 RNA
targets. The assay comprises the system described by Tuschl et al.,
1999, Genes and Development, 13, 3191-3197 and Zamore et al., 2000,
Cell, 101, 25-33 adapted for use with ADORA1 target RNA. A
Drosophila extract derived from syncytial blastoderm is used to
reconstitute RNAi activity in vitro. Target RNA is generated via in
vitro transcription from an appropriate ADORA1 expressing plasmid
using T7 RNA polymerase or via chemical synthesis as described
herein. Sense and antisense siRNA strands (for example 20 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
min. at 90.degree. C. followed by 1 hour at 37.degree. C., then
diluted in lysis buffer (for example 100 mM potassium acetate, 30
mM HEPES-KOH at pH 7.4, 2 mM 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 containing 50% lysate [vol/vol],
RNA (10-50 pM final concentration), and 10% [vol/vol] lysis buffer
containing siRNA (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.degree.
C. for 10 minutes before adding RNA, then incubated at 25.degree.
C. for an additional 60 minutes. Reactions are quenched with 4
volumes of 1.25.times. 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 siRNA is
omitted from the reaction.
[0247] Alternately, internally-labeled target RNA for the assay is
prepared by in vitro transcription in the presence of [a-.sup.32P]
CTP, passed over a G 50 Sephadex column by spin chromatography and
used as target RNA without further purification. Optionally, target
RNA is 5'-.sup.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.RTM. quantitation of bands
representing intact control RNA or RNA from control reactions
without siRNA and the cleavage products generated by the assay.
[0248] In one embodiment, this assay is used to determine target
sites the ADORA1 RNA target for siRNA mediated RNAi cleavage,
wherein a plurality of siRNA constructs are screened for RNAi
mediated cleavage of the ADORA1 RNA target, for example by
analysing the assay reaction by electrophoresis of labelled target
RNA, or by northern blotting, as well as by other methodology well
known in the art.
Example 7
Nucleic Acid Inhibition of ADORA1 Target RNA in vivo
[0249] siRNA molecules targeted to the human ADORA1 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 ADORA1 RNA are given in Table I and
III.
[0250] Two formats are used to test the efficacy of siRNAs
targeting ADORA1. First, the reagents are tested on human lung
epithelial cells (e.g., A549), to determine the extent of RNA and
protein inhibition. siRNA reagents (e.g.; see Table I, and III) are
selected against the ADORA1 target. RNA inhibition is measured
after delivery of these reagents by a suitable transfection agent
to human lung epithelial cells. Relative amounts of target RNA are
measured versus actin using real-time PCR monitoring of
amplification (eg. ABI 7700 Taqman.RTM.). A comparison is made to a
mixture of oligonucleotide sequences made to unrelated targets or
to a randomized siRNA 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
chosen, a RNA time-course of inhibition is performed with the lead
siRNA molecule. In addition, a cell-plating format can be used to
determine RNA inhibition.
Delivery of siRNA to Lung Epithelial Cells
[0251] Human lung epithelial cells (e.g., A549) are seeded, for
example, at 1.times.10.sup.5 cells per well of a six well dish in
EGM-2 (BioWhittaker) the day before transfection. siRNA (final
concentration, for example 20 nM) and cationic lipid (e.g., final
concentration 2 .mu.g/ml) are complexed in EGM basal media
(Biowhittaker) at 37.degree. C. for 30 mins in polystyrene tubes.
Following vortexing, the complexed siRNA is added to each well and
incubated for the times indicated. For initial optimization
experiments, cells are seeded, for example, at 1.times.10.sup.3 in
96 well plates and siRNA complex added as described. Efficiency of
delivery of siRNA to A549 is determined using a fluorescent siRNA
complexed with lipid. A549 in 6 well dishes are incubated with
siRNA for 24 hours, rinsed with PBS and fixed in 2%
paraformaldehyde for 15 minutes at room temperature. Uptake of
siRNA is visualised using a fluorescent microscope.
Tagman and Lightcycler Quantification of mRNA
[0252] Total RNA is prepared from cells following siRNA delivery,
for example using Qiagen RNA purification kits for 6 well or Rneasy
extraction kits for 96 well assays. For Taqman analysis,
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 .mu.l reactions consisting of 10 .mu.l total RNA, 100 nM
forward primer, 900 nM reverse primer, 100 nM probe, 1.times.
TaqMan PCR reaction buffer (PE-Applied Biosystems), 5.5 mM
MgCl.sub.2, 300 .mu.M each dATP, dCTP, dGTP, and dTTP, 10U RNase
Inhibitor (Promega), 1.25U AmpliTaq Gold (PE-Applied Biosystems)
and 10U M-MLV Reverse Transcriptase (Promega). The thermal cycling
conditions can consist of 30 min at 48.degree. C., 10 min at
95.degree. C., followed by 40 cycles of 15 sec at 95.degree. C. and
1 min at 60.degree. C. Quantitation of mRNA levels are determined
relative to standards generated from serially diluted total
cellular RNA (300, 100, 33, 11 ng/rxn) and normalizing to
.beta.-actin or GAPDH mRNA in parallel TaqMan reactions. For each
gene of interest an upper and lower primer and a flourescently
labelled 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 c RNA allularnd values are
represented as relative expression to GAPDH in each sample.
Western Blotting
[0253] Nuclear extracts can be prepared using a standard
micropreparation technique (see for example Andrews and Faller,
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.degree. 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
Models Useful to Evaluate the Down-Regulation of ADORA1 Gene
Expression
Animal Models
[0254] Evaluating the efficacy of anti-ADORA-1 agents (e.g., siRNA)
in animal models is an important prerequisite to human clinical
trials. Nyce and Metzger, 1997, Nature, 385, 721-725, describe a
useful dust mite conditioned allergic rabbit model of human asthma.
Allergic rabbits treated with aerosolized siRNA are compared to
untreated controls or animals treated with a non-specific siRNA
constrol with regard to adenosine challenge. The concentration of
aerolsolized adenosine required to reduce the dynamic compliance of
the bronchial airway 50% from a baseline values is determined in
both groups of animals. Additionally, dose response studies using
this same endpoint are performed. Airway smooth muscle is
surgically dissected from the animals and is processed for
quantitative assessment of adenosine A1 receptors. As a control for
specificity, adenosine A2 receptors and/or bradykinin receptors are
quantitated as well. Adenosine A1 receptor density can be assayed
by specific binding of a [.sup.3H]DPCPX. A dose dependent reduction
in adenosine A1 receptor densitiy is indicative of a therapeutic
response This model can be used to evaluate animals that are
treated with nucleic acid molecules of the invention and can
furthermore be used as a positive control in determining the
response of animals treated with nucleic acid molecules of the
invention by using such factors as airway obstruction, lung
capacity, and bronchiolar alveolar lavage (BAL) fluid in the
evaluation.
Cell Culture
[0255] Human epithelial lung cell lines, such as NPE cells and
NCB-20 cells, can be used to express ADORA1. Cloned human ADORA1 is
therefore expressed in CHO and COS7 cells and used in various
studies. These ADORA1 expressing lung cell lines can be used in
cell culture assays to evaluate nucleic acid molecules of the
invention. A primary endpoint in these experiments would be the
RT-PCR analysis of ADORA1 ntRNA expression in ADORA1 expressing
cells. In addition, ligand binding assays can be developed where
binding of [.sup.3H]DPCPX can be evaluated in response to treatment
with nucleic acid molecules of the invention.
Example 9
Indications
[0256] The present body of knowledge in ADORA1 research indicates
the need for methods to assay ADORA1 activity and for compounds
that can regulate ADORA1 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 ADORA1 levels. In addition, the nucleic acid
molecules can be used to treat disease state related to ADORA1
levels.
[0257] Particular degenerative and disease states that can be
associated with ADORA1 levels include, but are not limited to
allergic diseases and conditions, including but not limited to
asthma, allergic rhinitis, atopic dermatitis, and any other
diseases or conditions that are related to or will respond to the
levels of ADORA1 in a cell or tissue, alone or in combination with
other therapies.
[0258] The use of anti-inflammatories, bronchodilators, adenosine
inhibitors and adenosine A1 receptor inhibitors are examples of
other treatments or therapies can be combined with the nucleic acid
molecules of the invention. Those skilled in the art will recognize
that other drug compounds and therapies can be similarly be readily
combined with the nucleic acid molecules of the instant invention
(e.g., siRNA molecules) are hence within the scope of the instant
invention.
Example 10
Diagnostic Uses
[0259] The siRNA molecules of the invention can be used in a
variety of diagnostic applications, such as in identifying
molecular targets such as RNA in a variety of applications, for
example, in clinical, industrial, environmental, agricultural
and/or research settings. Such diagnostic use of siRNA molecules
involves utilizing reconstituted RNAi systems, for example using
cellular lysates or partially purified cellular lysates. siRNA
molecules of this invention may 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 siRNA 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
siRNA molecules described in this invention, one may 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 siRNA molecules can be used to inhibit gene expression and
define the role (essentially) of specified gene products in the
progression of disease or infection. In this manner, other genetic
targets may be defined 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 siRNA molecules targeted to different genes, siRNA
molecules coupled with known small molecule inhibitors, or
intermittent treatment with combinations siRNA molecules and/or
other chemical or biological molecules). Other in vitro uses of
siRNA 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 siRNA using standard methodologies, for example fluorescence
resonance emission transfer (FRET).
[0260] In a specific example, siRNA molecules that can cleave only
wild-type or mutant forms of the target RNA are used for the assay.
The first siRNA molecules is used to identify wild-type RNA present
in the sample and the second siRNA molecules will be used to
identify mutant RNA in the sample. As reaction controls, synthetic
substrates of both wild-type and mutant RNA will be cleaved by both
siRNA molecules to demonstrate the relative siRNA efficiencies in
the reactions and the absence of cleavage of the "non-targeted" RNA
species. The cleavage products from the synthetic substrates will
also serve to generate size markers for the analysis of wild-type
and mutant RNAs in the sample population. Thus each analysis will
require two siRNA molecules, two substrates and one unknown sample
which will be combined into six reactions. The presence of cleavage
products will be 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 will be adequate and will
decrease the cost of the initial diagnosis. Higher mutant form to
wild-type ratios will be correlated with higher risk whether RNA
levels are compared qualitatively or quantitatively.
[0261] 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.
[0262] 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.
[0263] It will be readily apparent to one skilled in the art that
varying substitutions and modifications may 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.
[0264] The invention illustratively described herein suitably may
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 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 within the scope of this invention as defined by the
description and the appended claims.
[0265] 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.
1TABLE I ADORA1 target and siRNA sequences (5'-3') Seq Seq Seq Pos
Target Sequence ID UPos Upper seq ID LPos Lower seq ID 3
GAGUGUCAGAAGUGUGAAG 1 3 GAGUGUCAGAAGUGUGAAG 1 21
CUUCACACUUCUGACACUC 162 21 GGGUGCCUGUUCUGAAUCC 2 21
GGGUGCCUGUUCUGAAUCC 2 39 GGAUUCAGAACAGGCACCC 163 39
CCAGAGCCUCCUCUCCCUC 3 39 CCAGAGCCUCCUCUCCCUC 3 57
GAGGGAGAGGAGGCUCUGG 164 57 CUGUGAGGCUGGCAGGUGA 4 57
CUGUGAGGCUGGCAGGUGA 4 75 UCACCUGCCAGCCUCACAG 165 75
AGGAAGGGUUUAACCUCAC 5 75 AGGAAGGGUUUAACCUCAC 5 93
GUGAGGUUAAACCCUUCCU 166 93 CUGGAAGGAAUCCCUGGAG 6 93
CUGGAAGGAAUCCCUGGAG 6 111 CUCCAGGGAUUCCUUCCAG 167 111
GCUAGCGGCUGCUGAAGGC 7 111 GCUAGCGGCUGCUGAAGGC 7 129
GCCUUCAGCAGCCGCUAGC 168 129 CGUCGAGGUGUGGGGGCAC 8 129
CGUCGAGGUGUGGGGGCAC 8 147 GUGCCCCCACACCUCGACG 169 147
CUUGGACAGAACAGUCAGG 9 147 CUUGGACAGAACAGUCAGG 9 165
CCUGACUGUUCUGUCCAAG 170 165 GCAGCCGGGAGCUCUGCCA 10 165
GCAGCCGGGAGCUCUGCCA 10 183 UGGCAGAGCUCCCGGCUGC 171 183
AGCUUUGGUGACCUUGGGC 11 183 AGCUUUGGUGACCUUGGGC 11 201
GCCCAAGGUCACCAAAGCU 172 201 CCGGGCUGGGAGCGCUGCG 12 201
CCGGGCUGGGAGCGCUGCG 12 219 CGCAGCGCUCCCAGCCCGG 173 219
GGCGGGAGCCGGAGGACUA 13 219 GGCGGGAGCCGGAGGACUA 13 237
UAGUCCUCCGGCUCCCGCC 174 237 AUGAGCUGCCGCGCGUUGU 14 237
AUGAGCUGCCGCGCGUUGU 14 255 ACAACGCGCGGCAGCUCAU 175 255
UCCAGAGCCCAGCCCAGCC 15 255 UCCAGAGCCCAGCCCAGCC 15 273
GGCUGGGCUGGGCUCUGGA 176 273 CCUACGCGCGCGGCCCGGA 16 273
CCUACGCGCGCGGCCCGGA 16 291 UCCGGGCCGCGCGCGUAGG 177 291
AGCUCUGUUCCCUGGAACU 17 291 AGCUCUGUUCCCUGGAACU 17 309
AGUUCCAGGGAACAGAGCU 178 309 UUUGGGCACUGCCUCUGGG 18 309
UUUGGGCACUGCCUCUGGG 18 327 CCCAGAGGCAGUGCCCAAA 179 327
GACCCCUGCCGGCCAGCAG 19 327 GACCCCUGCCGGCCAGCAG 19 345
CUGCUGGCCGGCAGGGGUC 180 345 GGCAGGAUGGUGCUUGCCU 20 345
GGCAGGAUGGUGCUUGCCU 20 363 AGGCAAGCACCAUCCUGCC 181 363
UCGUGCCCCUUGGUGCCCG 21 363 UCGUGCCCCUUGGUGCCCG 21 381
CGGGCACCAAGGGGCACGA 182 381 GUCUGCUGAUGUGCCCAGC 22 381
GUCUGCUGAUGUGCCCAGC 22 399 GCUGGGCACAUCAGCAGAC 183 399
CCUGUGCCCGCCAUGCCGC 23 399 CCUGUGCCCGCCAUGCCGC 23 417
GCGGCAUGGCGGGCACAGG 184 417 CCCUCCAUCUCAGCUUUCC 24 417
CCCUCCAUCUCAGCUUUCC 24 435 GGAAAGCUGAGAUGGAGGG 185 435
CAGGCCGCCUACAUCGGCA 25 435 CAGGCCGCCUACAUCGGCA 25 453
UGCCGAUGUAGGCGGCCUG 186 453 AUCGAGGUGCUCAUCGCCC 26 453
AUCGAGGUGCUCAUCGCCC 26 471 GGGCGAUGAGCACCUCGAU 187 471
CUGGUCUCUGUGCCCGGGA 27 471 CUGGUCUCUGUGCCCGGGA 27 489
UCCCGGGCACAGAGACCAG 188 489 AACGUGCUGGUGAUCUGGG 28 489
AACGUGCUGGUGAUCUGGG 28 507 CCCAGAUCACCAGCACGUU 189 507
GCGGUGAAGGUGAACCAGG 29 507 GCGGUGAAGGUGAACCAGG 29 525
CCUGGUUCACCUUCACCGC 190 525 GCGCUGCGGGAUGCCACCU 30 525
GCGCUGCGGGAUGCCACCU 30 543 AGGUGGCAUCCCGCAGCGC 191 543
UUCUGCUUCAUCGUGUCGC 31 543 UUCUGCUUCAUCGUGUCGC 31 561
GCGACACGAUGAAGCAGAA 192 561 CUGGCGGUGGCUGAUGUGG 32 561
CUGGCGGUGGCUGAUGUGG 32 579 CCACAUCAGCCACCGCCAG 193 579
GCCGUGGGUGCCCUGGUCA 33 579 GCCGUGGGUGCCCUGGUCA 33 597
UGACCAGGGCACCCACGGC 194 597 AUCCCCCUCGCCAUCCUCA 34 597
AUCCCCCUCGCCAUCCUCA 34 615 UGAGGAUGGCGAGGGGGAU 195 615
AUCAACAUUGGGCCACAGA 35 615 AUCAACAUUGGGCCACAGA 35 633
UCUGUGGCCCAAUGUUGAU 196 633 ACCUACUUCCACACCUGCC 36 633
ACCUACUUCCACACCUGCC 36 651 GGCAGGUGUGGAAGUAGGU 197 651
CUCAUGGUUGCCUGUCCGG 37 651 CUCAUGGUUGCCUGUCCGG 37 669
CCGGACAGGCAACCAUGAG 198 669 GUCCUCAUCCUCACCCAGA 38 669
GUCCUCAUCCUCACCCAGA 38 687 UCUGGGUGAGGAUGAGGAC 199 687
AGCUCCAUCCUGGCCCUGC 39 687 AGCUCCAUCCUGGCCCUGC 39 705
GCAGGGCCAGGAUGGAGCU 200 705 CUGGCAAUUGCUGUGGACC 40 705
CUGGCAAUUGCUGUGGACC 40 723 GGUCCACAGCAAUUGCCAG 201 723
CGCUACCUCCGGGUCAAGA 41 723 CGCUACCUCCGGGUCAAGA 41 741
UCUUGACCCGGAGGUAGCG 202 741 AUCCCUCUCCGGUACAAGA 42 741
AUCCCUCUCCGGUACAAGA 42 759 UCUUGUACCGGAGAGGGAU 203 759
AUGGUGGUGACCCCCCGGA 43 759 AUGGUGGUGACCCCCCGGA 43 777
UCCGGGGGGUCACCACCAU 204 777 AGGGCGGCGGUGGCCAUAG 44 777
AGGGCGGCGGUGGCCAUAG 44 795 CUAUGGCCACCGCCGCCCU 205 795
GCCGGCUGCUGGAUCCUCU 45 795 GCCGGCUGCUGGAUCCUCU 45 813
AGAGGAUCCAGCAGCCGGC 206 813 UCCUUCGUGGUGGGACUGA 46 813
UCCUUCGUGGUGGGACUGA 46 831 UCAGUCCCACCACGAAGGA 207 831
ACCCCUAUGUUUGGCUGGA 47 831 ACCCCUAUGUUUGGCUGGA 47 849
UCCAGCCAAACAUAGGGGU 208 849 AACAAUCUGAGUGCGGUGG 48 849
AACAAUCUGAGUGCGGUGG 48 867 CCACCGCACUCAGAUUGUU 209 867
GAGCGGGCCUGGGCAGCCA 49 867 GAGCGGGCCUGGGCAGCCA 49 885
UGGCUGCCCAGGCCCGCUC 210 885 AACGGCAGCAUGGGGGAGC 50 885
AACGGCAGCAUGGGGGAGC 50 903 GCUCCCCCAUGCUGCCGUU 211 903
CCCGUGAUCAAGUGCGAGU 51 903 CCCGUGAUCAAGUGCGAGU 51 921
ACUCGCACUUGAUCACGGG 212 921 UUCGAGAAGGUCAUCAGCA 52 921
UUCGAGAAGGUCAUCAGCA 52 939 UGCUGAUGACCUUCUCGAA 213 939
AUGGAGUACAUGGUCUACU 53 939 AUGGAGUACAUGGUCUACU 53 957
AGUAGACCAUGUACUCCAU 214 957 UUCAACUUCUUUGUGUGGG 54 957
UUCAACUUCUUUGUGUGGG 54 975 CCCACACAAAGAAGUUGAA 215 975
GUGCUGCCCCCGCUUCUCC 55 975 GUGCUGCCCCCGCUUCUCC 55 993
GGAGAAGCGGGGGCAGCAC 216 993 CUCAUGGUCCUCAUCUACC 56 993
CUCAUGGUCCUCAUCUACC 56 1011 GGUAGAUGAGGACCAUGAG 217 1011
CUGGAGGUCUUCUACCUAA 57 1011 CUGGAGGUCUUCUACCUAA 57 1029
UUAGGUAGAAGACCUCCAG 218 1029 AUCCGCAAGCAGCUCAACA 58 1029
AUCCGCAAGCAGCUCAACA 58 1047 UGUUGAGCUGCUUGCGGAU 219 1047
AAGAAGGUGUCGGCCUCCU 59 1047 AAGAAGGUGUCGGCCUCCU 59 1065
AGGAGGCCGACACCUUCUU 220 1065 UCCGGCGACCCGCAGAAGU 60 1065
UCCGGCGACCCGCAGAAGU 60 1083 ACUUCUGCGGGUCGCCGGA 221 1083
UACUAUGGGAAGGAGCUGA 61 1083 UACUAUGGGAAGGAGCUGA 61 1101
UCAGCUCCUUCCCAUAGUA 222 1101 AAGAUCGCCAAGUCGCUGG 62 1101
AAGAUCGCCAAGUCGCUGG 62 1119 CCAGCGACUUGGCGAUCUU 223 1119
GCCCUCAUCCUCUUCCUCU 63 1119 GCCCUCAUCCUCUUCCUCU 63 1137
AGAGGAAGAGGAUGAGGGC 224 1137 UUUGCCCUCAGCUGGCUGC 64 1137
UUUGCCCUCAGCUGGCUGC 64 1155 GCAGCCAGCUGAGGGCAAA 225 1155
CCUUUGCACAUCCUCAACU 65 1155 CCUUUGCACAUCCUCAACU 65 1173
AGUUGAGGAUGUGCAAAGG 226 1173 UGCAUCACCCUCUUCUGCC 66 1173
UGCAUCACCCUCUUCUGCC 66 1191 GGCAGAAGAGGGUGAUGCA 227 1191
CCGUCCUGCCACAAGCCCA 67 1191 CCGUCCUGCCACAAGCCCA 67 1209
UGGGCUUGUGGCAGGACGG 228 1209 AGCAUCCUUACCUACAUUG 68 1209
AGCAUCCUUACCUACAUUG 68 1227 CAAUGUAGGUAAGGAUGCU 229 1227
GCCAUCUUCCUCACGCACG 69 1227 GCCAUCUUCCUCACGCACG 69 1245
CGUGCGUGAGGAAGAUGGC 230 1245 GGCAACUCGGCCAUGAACC 70 1245
GGCAACUCGGCCAUGAACC 70 1263 GGUUCAUGGCCGAGUUGCC 231 1263
CCCAUUGUCUAUGCCUUCC 71 1263 CCCAUUGUCUAUGCCUUCC 71 1281
GGAAGGCAUAGACAAUGGG 232 1281 CGCAUCCAGAAGUUCCGCG 72 1281
CGCAUCCAGAAGUUCCGCG 72 1299 CGCGGAACUUCUGGAUGCG 233 1299
GUCACCUUCCUUAAGAUUU 73 1299 GUCACCUUCCUUAAGAUUU 73 1317
AAAUCUUAAGGAAGGUGAC 234 1317 UGGAAUGACCAUUUCCGCU 74 1317
UGGAAUGACCAUUUCCGCU 74 1335 AGCGGAAAUGGUCAUUCCA 235 1335
UGCCAGCCUGCACCUCCCA 75 1335 UGCCAGCCUGCACCUCCCA 75 1353
UGGGAGGUGCAGGCUGGCA 236 1353 AUUGACGAGGAUCUCCCAG 76 1353
AUUGACGAGGAUCUCCCAG 76 1371 CUGGGAGAUCCUCGUCAAU 237 1371
GAAGAGAGGCCUGAUGACU 77 1371 GAAGAGAGGCCUGAUGACU 77 1389
AGUCAUCAGGCCUCUCUUC 238 1389 UAGACCCCGCCUUCCGCUC 78 1389
UAGACCCCGCCUUCCGCUC 78 1407 GAGCGGAAGGCGGGGUCUA 239 1407
CCCACCAGCCCACAUCCAG 79 1407 CCCACCAGCCCACAUCCAG 79 1425
CUGGAUGUGGGCUGGUGGG 240 1425 GUGGGGUCUCAGUCCAGUC 80 1425
GUGGGGUCUCAGUCCAGUC 80 1443 GACUGGACUGAGACCCCAC 241 1443
CCUCACAUGCCCGCUGUCC 81 1443 CCUCACAUGCCCGCUGUCC 81 1461
GGACAGCGGGCAUGUGAGG 242 1461 CCAGGGGUCUCCCUGAGCC 82 1461
CCAGGGGUCUCCCUGAGCC 82 1479 GGCUCAGGGAGACCCCUGG 243 1479
CUGCCCCAGCUGGGCUGUU 83 1479 CUGCCCCAGCUGGGCUGUU 83 1497
AACAGCCCAGCUGGGGCAG 244 1497 UGGCUGGGGGCAUGGGGGA 84 1497
UGGCUGGGGGCAUGGGGGA 84 1515 UCCCCCAUGCCCCCAGCCA 245 1515
AGGCUCUGAAGAGAUACCC 85 1515 AGGCUCUGAAGAGAUACCC 85 1533
GGGUAUCUCUUCAGAGCCU 246 1533 CACAGAGUGUGGUCCCUCC 86 1533
CACAGAGUGUGGUCCCUCC 86 1551 GGAGGGACCACACUCUGUG 247 1551
CACUAGGAGUUAACUACCC 87 1551 CACUAGGAGUUAACUACCC 87 1569
GGGUAGUUAACUCCUAGUG 248 1569 CUACACCUCUGGGCCCUGC 88 1569
CUACACCUCUGGGCCCUGC 88 1587 GCAGGGCCCAGAGGUGUAG 249 1587
CAGGAGGCCUGGGAGGGCA 89 1587 CAGGAGGCCUGGGAGGGCA 89 1605
UGCCCUCCCAGGCCUCCUG 250 1605 AAGGGUCCUACGGAGGGAC 90 1605
AAGGGUCCUACGGAGGGAC 90 1623 GUCCCUCCGUAGGACCCUU 251 1623
CCAGGUGUCUAGAGGCAAC 91 1623 CCAGGUGUCUAGAGGCAAC 91 1641
GUUGCCUCUAGACACCUGG 252 1641 CAGUGUUCUGAGCCCCCAC 92 1641
CAGUGUUCUGAGCCCCCAC 92 1659 GUGGGGGCUCAGAACACUG 253 1659
CCUGCCUGACCAUCCCAUG 93 1659 CCUGCCUGACCAUCCCAUG 93 1677
CAUGGGAUGGUCAGGCAGG 254 1677 GAGCAGUCCAGCGCUUCAG 94 1677
GAGCAGUCCAGCGCUUCAG 94 1695 CUGAAGCGCUGGACUGCUC 255 1695
GGGCUGGGCAGGUCCUGGG 95 1695 GGGCUGGGCAGGUCCUGGG 95 1713
CCCAGGACCUGCCCAGCCC 256 1713 GGAGGCUGAGACUGCAGAG 96 1713
GGAGGCUGAGACUGCAGAG 96 1731 CUCUGCAGUCUCAGCCUCC 257 1731
GGAGCCACCUGGGCUGGGA 97 1731 GGAGCCACCUGGGCUGGGA 97 1749
UCCCAGCCCAGGUGGCUCC 258 1749 AGAAGGUGCUUGGGCUUCU 98 1749
AGAAGGUGCUUGGGCUUCU 98 1767 AGAAGCCCAAGCACCUUCU 259 1767
UGCGGUGAGGCAGGGGAGU 99 1767 UGCGGUGAGGCAGGGGAGU 99 1785
ACUCCCCUGCCUCACCGCA 260 1785 UCUGCUUGUCUUAGAUGUU 100 1785
UCUGCUUGUCUUAGAUGUU 100 1803 AACAUCUAAGACAAGCAGA 261 1803
UGGUGGUGCAGCCCCAGGA 101 1803 UGGUGGUGCAGCCCCAGGA 101 1821
UCCUGGGGCUGCACCACCA 262 1821 ACCAAGCUUAAGGAGAGGA 102 1821
ACCAAGCUUAAGGAGAGGA 102 1839 UCCUCUCCUUAAGCUUGGU 263 1839
AGAGCAUCUGCUCUGAGAC 103 1839 AGAGCAUCUGCUCUGAGAC 103 1857
GUCUCAGAGCAGAUGCUCU 264 1857 CGGAUGGAAGGAGAGAGGU 104 1857
CGGAUGGAAGGAGAGAGGU 104 1875 ACCUCUCUCCUUCCAUCCG 265 1875
UUGAGGAUGCACUGGCCUG 105 1875 UUGAGGAUGCACUGGCCUG 105 1893
CAGGCCAGUGCAUCCUCAA 266 1893 GUUCUGUAGGAGAGACUGG 106 1893
GUUCUGUAGGAGAGACUGG 106 1911 CCAGUCUCUCCUACAGAAC 267 1911
GCCAGAGGCAGCUAAGGGG 107 1911 GCCAGAGGCAGCUAAGGGG 107 1929
CCCCUUAGCUGCCUCUGGC 268 1929 GCAGGAAUCAAGGAGCCUC 108 1929
GCAGGAAUCAAGGAGCCUC 108 1947 GAGGCUCCUUGAUUCCUGC 269 1947
CCGUUCCCACCUCUGAGGA 109 1947 CCGUUCCCACCUCUGAGGA 109 1965
UCCUCAGAGGUGGGAACGG 270 1965 ACUCUGGACCCCAGGCCAU 110 1965
ACUCUGGACCCCAGGCCAU 110 1983 AUGGCCUGGGGUCCAGAGU 271 1983
UACCAGGUGCUAGGGUGCC 111 1983 UACCAGGUGCUAGGGUGCC 111 2001
GGCACCCUAGCACCUGGUA 272 2001 CUGCUCUCCUUGCCCUGGG 112 2001
CUGCUCUCCUUGCCCUGGG 112 2019 CCCAGGGCAAGGAGAGCAG 273 2019
GCCAGCCCAGGAUUGUACG 113 2019 GCCAGCCCAGGAUUGUACG 113 2037
CGUACAAUCCUGGGCUGGC 274 2037 GUGGGAGAGGCAGAAAGGG 114 2037
GUGGGAGAGGCAGAAAGGG 114 2055 CCCUUUCUGCCUCUCCCAC 275 2055
GUAGGUUCAGUAAUCAUUU 115 2055 GUAGGUUCAGUAAUCAUUU 115 2073
AAAUGAUUACUGAACCUAC 276 2073 UCUGAUGAUUUGCUGGAGU 116 2073
UCUGAUGAUUUGCUGGAGU 116 2091 ACUCCAGCAAAUCAUCAGA 277 2091
UGCUGGCUCCACGCCCUGG 117 2091 UGCUGGCUCCACGCCCUGG 117 2109
CCAGGGCGUGGAGCCAGCA 278 2109 GGGAGUGAGCUUGGUGCGG 118 2109
GGGAGUGAGCUUGGUGCGG 118 2127 CCGCACCAAGCUCACUCCC 279 2127
GUAGGUGCUGGCCUCAAAC 119 2127 GUAGGUGCUGGCCUCAAAC 119 2145
GUUUGAGGCCAGCACCUAC 280 2145 CAGCCACGAGGUGGUAGCU 120 2145
CAGCCACGAGGUGGUAGCU 120 2163 AGCUACCACCUCGUGGCUG 281 2163
UCUGAGCCCUCCUUCUUGC 121 2163 UCUGAGCCCUCCUUCUUGC 121 2181
GCAAGAAGGAGGGCUCAGA 282 2181 CCCUGAGCUUUCCGGGGAG 122 2181
CCCUGAGCUUUCCGGGGAG 122 2199 CUCCCCGGAAAGCUCAGGG 283 2199
GGAGCCUGGAGUGUAAUUA 123 2199 GGAGCCUGGAGUGUAAUUA 123 2217
UAAUUACACUCCAGGCUCC 284 2217 ACCUGUCAUCUGGGCCACC 124 2217
ACCUGUCAUCUGGGCCACC 124 2235 GGUGGCCCAGAUGACAGGU 285 2235
CAGCUCCACUGGCCCCCGU 125 2235 CAGCUCCACUGGCCCCCGU 125 2253
ACGGGGGCCAGUGGAGCUG 286 2253 UUGCCGGGCCUGGACUGUC 126 2253
UUGCCGGGCCUGGACUGUC 126 2271 GACAGUCCAGGCCCGGCAA 287 2271
CCUAGGUGACCCCAUCUCU 127 2271 CCUAGGUGACCCCAUCUCU 127 2289
AGAGAUGGGGUCACCUAGG 288 2289 UGCUGCUUCUGGGCCUGAU 128 2289
UGCUGCUUCUGGGCCUGAU 128 2307 AUCAGGCCCAGAAGCAGCA 289 2307
UGGAGAGGAGAACACUAGA 129 2307 UGGAGAGGAGAACACUAGA 129 2325
UCUAGUGUUCUCCUCUCCA 290 2325 ACAUGCCAACUCGGGAGCA 130 2325
ACAUGCCAACUCGGGAGCA 130 2343 UGCUCCCGAGUUGGCAUGU 291 2343
AUUCUGCCUGCCUGGGAAC 131 2343 AUUCUGCCUGCCUGGGAAC 131 2361
GUUCCCAGGCAGGCAGAAU 292 2361 CGGGGUGGACGAGGGAGUG 132 2361
CGGGGUGGACGAGGGAGUG 132 2379 CACUCCCUCGUCCACCCCG 293 2379
GUCUGUAAGGACUCAGUGU 133 2379 GUCUGUAAGGACUCAGUGU 133 2397
ACACUGAGUCCUUACAGAC 294 2397 UUGACUGUAGGCGCCCCUG 134 2397
UUGACUGUAGGCGCCCCUG 134 2415 CAGGGGCGCCUACAGUCAA 295 2415
GGGGUGGGUUUAGCAGGCU 135 2415 GGGGUGGGUUUAGCAGGCU 135 2433
AGCCUGCUAAACCCACCCC 296 2433 UGCAGCAGGCAGAGGAGGA 136 2433
UGCAGCAGGCAGAGGAGGA 136 2451 UCCUCCUCUGCCUGCUGCA 297 2451
AGUACCCCCCUGAGAGCAU 137 2451 AGUACCCCCCUGAGAGCAU 137 2469
AUGCUCUCAGGGGGGUACU 298 2469 UGUGGGGGAAGGCCUUGCU 138 2469
UGUGGGGGAAGGCCUUGCU 138 2487 AGCAAGGCCUUCCCCCACA 299 2487
UGUCAUGUGAAUCCCUCAA 139 2487 UGUCAUGUGAAUCCCUCAA 139 2505
UUGAGGGAUUCACAUGACA 300 2505 AUACCCCUAGUAUCUGGCU 140 2505
AUACCCCUAGUAUCUGGCU 140 2523 AGCCAGAUACUAGGGGUAU 301 2523
UGGGUUUUCAGGGGCUUUG 141 2523 UGGGUUUUCAGGGGCUUUG 141 2541
CAAAGCCCCUGAAAACCCA 302 2541 GGAAGCUCUGUUGCAGGUG 142 2541
GGAAGCUCUGUUGCAGGUG 142 2559 CACCUGCAACAGAGCUUCC 303 2559
GUCCGGGGGUCUAGGACUU 143 2559 GUCCGGGGGUCUAGGACUU 143 2577
AAGUCCUAGACCCCCGGAC 304 2577 UUAGGGAUCUGGGAUCUGG 144 2577
UUAGGGAUCUGGGAUCUGG 144 2595 CCAGAUCCCAGAUCCCUAA 305 2595
GGGAAGGACCAACCCAUGC 145 2595 GGGAAGGACCAACCCAUGC 145 2613
GCAUGGGUUGGUCCUUCCC 306 2613 CCCUGCCAAGCCUGGAGCC 146 2613
CCCUGCCAAGCCUGGAGCC 146 2631 GGCUCCAGGCUUGGCAGGG 307 2631
CCCUGUGUUGGGGGGCAAG 147 2631 CCCUGUGUUGGGGGGCAAG 147 2649
CUUGCCCCCCAACACAGGG 308 2649 GGUGGGGGAGCCUGGAGCC 148 2649
GGUGGGGGAGCCUGGAGCC 148 2667 GGCUCCAGGCUCCCCCACC 309 2667
CCCUGUGUGGGAGGGCGAG 149 2667 CCCUGUGUGGGAGGGCGAG 149 2685
CUCGCCCUCCCACACAGGG 310 2685 GGCGGGGGAGCCUGGAGCC 150 2685
GGCGGGGGAGCCUGGAGCC 150 2703 GGCUCCAGGCUCCCCCGCC 311 2703
CCCUGUGUGGGAGGGCGAG 151 2703 CCCUGUGUGGGAGGGCGAG 151 2721
CUCGCCCUCCCACACAGGG 312 2721 GGCGGGGGAUCCUGGAGCC 152 2721
GGCGGGGGAUCCUGGAGCC 152 2739 GGCUCCAGGAUCCCCCGCC 313 2739
CCCUGUGUCGGGGGGCGAG 153 2739 CCCUGUGUCGGGGGGCGAG 153 2757
CUCGCCCCCCGACACAGGG 314 2757 GGGAGGGGAGGUGGCCGUC 154 2757
GGGAGGGGAGGUGGCCGUC 154 2775 GACGGCCACCUCCCCUCCC 315 2775
CGGUUGACCUUCUGAACAU 155 2775 CGGUUGACCUUCUGAACAU 155 2793
AUGUUCAGAAGGUCAACCG 316 2793 UGAGUGUCAACUCCAGGAC 156 2793
UGAGUGUCAACUCCAGGAC 156 2811 GUCCUGGAGUUGACACUCA 317 2811
CUUGCUUCCAAGCCCUUCC 157 2811 CUUGCUUCCAAGCCCUUCC 157 2829
GGAAGGGCUUGGAAGCAAG 318 2829 CCUCUGUUGGAAAUUGGGU 158 2829
CCUCUGUUGGAAAUUGGGU 158 2847 ACCCAAUUUCCAACAGAGG 319 2847
UGUGCCCUGGCUCCCAAGG 159 2847 UGUGCCCUGGCUCCCAAGG 159 2865
CCUUGGGAGCCAGGGCACA 320 2865 GGAGGCCCAUGUGACUAAU 160 2865
GGAGGCCCAUGUGACUAAU 160 2883 AUUAGUCACAUGGGCCUCC 321 2880
UAAUAAAAAACUGUGAACC 161 2880 UAAUAAAAAACUGUGAACC 161 2898
GGUUCACAGUUUUUUAUUA 322 NM_000674.vertline.ADORA1 The 3'-ends of
the Upper sequence and the Lower sequence of the siRNA construct
can include a overhang sequence, for example about 1, 2, 3, or 4
nucleotides in length, preferably 2 nucleotides in length, wherein
the overhanging sequence of the lower sequence is optionally
complementary to a portion of the target sequence. The upper
sequence is also referred to as the sense strand, whereas # the
lower sequence is also referred to as the antisense strand.
[0266]
2TABLE II Wait Time* 2'-O- Reagent Equivalents Amount Wait Time*
DNA methyl Wait Time* RNA A. 2.5 .mu.mol Synthesis Cycle ABI 394
Instrument Phosphoramidites 6.5 163 .mu.L 45 sec 2.5 min 7.5 min
S-Ethyl Tetrazole 23.8 238 .mu.L 45 sec 2.5 min 7.5 min Acetic
Anhydride 100 233 .mu.L 5 sec 5 sec 5 sec N-Methyl 186 233 .mu.L 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 .mu.L 100
sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2 .mu.mol
Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 .mu.L 45
sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 .mu.L 45 sec 233 min
465 sec Acetic Anhydride 655 124 .mu.L 5 sec 5 sec 5 sec N-Methyl
1245 124 .mu.L 5 sec 5 sec 5 sec Imidazole TCA 700 732 .mu.L 10 sec
10 sec 10 sec Iodine 20.6 244 .mu.L 15 sec 15 sec 15 sec Beaucage
7.7 232 .mu.L 100 sec 300 sec 300 sec Acetonitrile NA 2.64 mL NA NA
NA C. 0.2 .mu.mol Synthesis Cycle 96 well Instrument Equivalents:
DNA/ Amount: DNA/2'-O- Wait Time* 2'-O- Reagent 2'-O-methyl/Ribo
methyl/Ribo Wait Time* DNA methyl Wait Time* Ribo Phosphoramidites
22/33/66 40/60/120 .mu.L 60 sec 180 sec 360 sec S-Ethyl Tetrazole
70/105/210 40/60/120 .mu.L 60 sec 180 min 360 sec Acetic Anhydride
265/265/265 50/50/50 .mu.L 10 sec 10 sec 10 sec N-Methyl
502/502/502 50/50/50 .mu.L 10 sec 10 sec 10 sec Imidazole TCA
238/475/475 250/500/500 .mu.L 15 sec 15 sec 15 sec Iodine
6.8/6.8/6.8 80/80/80 .mu.L 30 sec 30 sec 30 sec Beaucage 34/51/51
80/120/120 100 sec 200 sec 200 sec Acetonitrile NA 1150/1150/1150
.mu.L NA NA NA Wait time does not include contact time during
delivery. Tandem synthesis utilizes double coupling of linker
molecule
[0267]
3TABLE III Chemically Modified siRNAs Target Pos Aliases Sequence
(5'-3') Seq ID Strand 1819 ADORA1:1821U21 siRNA stab4 B
AccAAGcuuAAGGAGAGGAGA B 347 Upper 919 ADORA1:921U21 siRNA stab4 B
uucGAGAAGGucAucAGcAuG B 349 Upper 1621 ADORA1:1623U21 siRNA stab4 B
ccAGGuGucuAGAGGcAAcAG B 351 Upper 2773 ADORA1:2775U21 siRNA stab4 B
cGGuuGAccuucuGAAcAuGA B 353 Upper 1819 ADORA1:1839L21 siRNA (1821C)
stab5 uccucuccuuAAGcuuGGuTsT 348 Lower 919 ADORA1:939L21 siRNA
(921C) stab5 uGcuGAuGAccuucucGAATsT 350 Lower 1621 ADORA1:1641L21
siRNA(1623C) stab5 GuuGccucuAGAcAceuGGTsT 352 Lower 2773
ADORA1:2793L21 siRNA (2775C) stab5 AuGuucAGAAGGucAAccGTsT 354 Lower
Uppercase = 2'-OH u,c = 2'-fluoro U, C T = deoxy T B = inverted
deoxy abasic s = phosphorothioate linkage
* * * * *