U.S. patent application number 14/724772 was filed with the patent office on 2015-09-17 for nicked or gapped nucleic acid molecules and uses thereof.
This patent application is currently assigned to MARINA BIOTECH, INC.. The applicant listed for this patent is MARINA BIOTECH, INC.. Invention is credited to Mohammad Ahmadian, James McSwiggen, Steven C. Quay, Narendra K. Vaish.
Application Number | 20150259686 14/724772 |
Document ID | / |
Family ID | 38984171 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259686 |
Kind Code |
A1 |
Quay; Steven C. ; et
al. |
September 17, 2015 |
NICKED OR GAPPED NUCLEIC ACID MOLECULES AND USES THEREOF
Abstract
The present disclosure provides meroduplex (nicked or gapped)
ribonucleic acid molecules (mdRNA) that decreases or silences
target gene expression. An mdRNA of this disclosure comprises at
least three strands that combine to form at least two
non-overlapping double-stranded regions separated by a nick or gap
wherein one strand is complementary to a target gene RNA. In
addition, the meroduplex may have one or more modifications or
substitutions, such as nucleotide base, sugar, terminal cap
structure, internucleotide linkage, or any combination of such
modifications. Also provided are methods of decreasing expression
of a target gene in a cell or in a subject to treat a disease
related to altered expression of a target gene.
Inventors: |
Quay; Steven C.;
(Woodinville, WA) ; McSwiggen; James; (Bothell,
WA) ; Vaish; Narendra K.; (Kirkland, WA) ;
Ahmadian; Mohammad; (Bothell, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MARINA BIOTECH, INC. |
New York |
NY |
US |
|
|
Assignee: |
MARINA BIOTECH, INC.
New York
NY
|
Family ID: |
38984171 |
Appl. No.: |
14/724772 |
Filed: |
May 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12445868 |
Apr 16, 2009 |
9074205 |
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PCT/US2007/081836 |
Oct 18, 2007 |
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14724772 |
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60862027 |
Oct 18, 2006 |
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60910393 |
Apr 5, 2007 |
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60955317 |
Aug 10, 2007 |
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60969136 |
Aug 30, 2007 |
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Current U.S.
Class: |
424/450 ;
424/489; 424/490; 435/375; 514/44A |
Current CPC
Class: |
C12N 2310/322 20130101;
A61K 31/713 20130101; C12N 2310/321 20130101; C12N 15/113 20130101;
A61K 47/645 20170801; A61P 29/00 20180101; A61P 31/00 20180101;
C12N 2310/15 20130101; A61P 35/00 20180101; C12N 2310/315 20130101;
A61K 38/00 20130101; A61K 47/66 20170801; C12N 15/111 20130101;
C12N 2310/3231 20130101; C12N 2330/30 20130101; C12N 2310/3513
20130101; C12N 2310/3521 20130101; C12N 2310/14 20130101; C12N
2320/51 20130101; C12N 2310/533 20130101; C12N 2320/30 20130101;
C12N 2310/321 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1.-64. (canceled)
65. A method for reducing expression of a target gene in a cell,
said method comprising: contacting a target gene expressing cell
with a composition comprising a 19 to 30 base pair double-stranded
ribonucleic acid, wherein said target gene expression is reduced as
a consequence of contacting said cell with said composition, said
ribonucleic acid comprising (a) an A strand having a length of 15
to 30 nucleotides and a nucleotide sequence that is complementary
to a nucleotide sequence in said target gene; (b) an S1 strand
having a length of 5 to 25 nucleotides, wherein said S1 strand
anneals to said A strand thereby forming a first double-stranded
region of 5 to 15 base pairs; and (c) an S2 strand having a length
of 5 to 25 nucleotides, wherein said S2 strand anneals to said A
strand thereby forming a second double-stranded region of 3 to 25
base pairs and wherein said annealed S2 strand is separated from
said annealed S1 strand by a nick or a gap.
66. The method of claim 65 wherein said composition comprises a
liposome, a hydrogel, a cyclodextrin, a biodegradable nanocapsule,
a bioadhesive microsphere, or a proteinaceous vector.
67. The method of claim 66 wherein said liposome is a
surface-modified liposome.
68. The method of claim 67 wherein said liposome comprises one or
more lipids selected from the group consisting of a non-cationic
lipid and a cationic lipid.
69. The method of claim 65 wherein said double-stranded ribonucleic
acid is combined, complexed, or conjugated with a peptide.
70. The method of claim 69 wherein said peptide facilitates
delivery of said double-stranded ribonucleic acid into said target
cell.
71. The method of claim 69 wherein said peptide is selected from
the group consisting of PN27, PN28, PN29, PN58, PN61, PN73, PN158,
PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365, PN404,
PN453, and PN509.
72. The method of claim 69 wherein said peptide comprises an
N-terminal protein transduction domain from HIV TAT.
73. The method of claim 65 wherein overexpression or inappropriate
expression of said target gene is associated with one or more
disease or disorder.
74. The method of claim 73 wherein said disease or disorder is
selected from the group consisting of a hyperproliferative disease
or disorder, an angiogenic disease or disorder, a metabolic disease
or disorder, and an inflammatory disease or disorder.
75. The method of claim 73 wherein said disease or disorder is
selected from the group consisting of a cardiac disease, a
pulmonary disease, a neovascularization, an ischemia, age-related
macular degeneration, diabetic retinopathy, glomerulonephritis,
diabetes, asthma, chronic obstructive pulmonary disease, chronic
bronchitis, lymphangiogenesis, and atherosclerosis.
76. The method of claim 74 wherein said hyperproliferative disease
or disorder is selected from the group consisting of a neoplasm, a
carcinoma, a sarcoma, a tumor, and a cancer.
77. The method of claim 74 wherein said hyperproliferative disease
or disorder is selected from the group consisting of an oral
cancer, a throat cancer, a laryngeal cancer, an esophageal cancer,
a pharyngeal cancer, a nasopharyngeal cancer, an oropharyngeal
cancer, a gastrointestinal tract cancer, a gastrointestinal stromal
tumor (GIST), a small intestine cancer, a colon cancer, a rectal
cancer, a colorectal cancer, an anal cancer, a pancreatic cancer, a
breast cancer, a cervical cancer, uterine cancer, a vulvar cancer,
vaginal cancer, urinary tract cancer, bladder cancer, kidney
cancer, adrenocortical cancer, islet cell carcinoma, gallbladder
cancer, stomach cancer, prostate cancer, ovarian cancer,
endometrial cancer, trophoblastic tumor, testicular cancer, penial
cancer, bone cancer, osteosarcoma, liver cancer, extrahepatic bile
duct cancer, skin cancer, basal cell carcinoma (BCC), lung cancer,
small cell lung cancer, non-small cell lung cancer (NSCLC), brain
cancer, melanoma, Kaposi's sarcoma, eye cancer, head and neck
cancer, squamous cell carcinoma of head and neck, tymoma, thymic
carcinoma, thyroid cancer, parathyroid cancer, Rippel-Linda
syndrome, leukemia, acute myeloid leukemia, chronic myelogenous
leukemia, acute lymphoblastic leukemia, hairy cell leukemia,
lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, T-cell
lymphoma, multiple myeloma, malignant pleural mesothelioma,
Barrett's adenocarcinoma, and Wilm's tumor.
78. The method of claim 74 wherein said inflammatory disease or
disorder is selected from the group consisting of diabetes
mellitus, rheumatoid arthritis, pannus growth in inflamed synovial
lining, collagen-induced arthritis, spondylarthritis, ankylosing
spondylitis, multiple sclerosis, encephalomyelitis, inflammatory
bowel disease, Chron's disease, psoriasis or psoriatic arthritis,
myasthenia gravis, systemic lupus erythematosis, graft-versus-host
disease, atherosclerosis, and an allergy.
Description
TECHNICAL FIELD
[0001] The present disclosure provides double-stranded nucleic acid
molecules capable of gene silencing and, more specifically, a
nicked or gapped double-stranded RNA (dsRNA) comprising at least
three strands that decreases expression of a target gene by, for
example, RNA interference and uses of such dsRNA to treat or
prevent disorders associated with expression of the target gene or
genes affected by the target gene.
BACKGROUND
[0002] RNA interference (RNAi) refers to the cellular process of
sequence specific, post-transcriptional gene silencing in animals
mediated by small inhibitory nucleic acid molecules, such as a
double-stranded RNA (dsRNA) that is homologous to a portion of a
targeted messenger RNA (Fire et al., Nature 391:806, 1998; Hamilton
et al., Science 286:950-951, 1999). RNAi has been observed in a
variety of organisms, including mammalians (Fire et al., Nature
391:806, 1998; Bahramian and Zarbl, Mol. Cell. Biol. 19:274-283,
1999; Wianny and Goetz, Nature Cell Biol. 2:70, 1999). RNAi can be
induced by introducing an exogenous synthetic 21-nucleotide RNA
duplex into cultured mammalian cells (Elbashir et al., Nature
411:494, 2001a).
[0003] The mechanism by which dsRNA mediates targeted
gene-silencing can be described as involving two steps. The first
step involves degradation of long dsRNAs by a ribonuclease III-like
enzyme, referred to as Dicer, into short interfering RNAs (siRNAs)
having from 21 to 23 nucleotides with double-stranded regions of
about 19 base pairs (Berstein et al., Nature 409:363, 2001;
Elbashir et al., Genes Dev. 15:188, 2001b; and Kim et al., Nature
Biotech. 23(2):222, 2005). The second step of RNAi gene-silencing
involves activation of a multi-component nuclease having one strand
(guide or antisense strand) from the siRNA and an Argonaute protein
to form an RNA-induced silencing complex ("RISC") (Elbashir et al.,
Genes Dev. 15:188, 2001). Argonaute initially associates with a
double-stranded siRNA and then endonucleolytically cleaves the
non-incorporated strand (passenger or sense strand) to facilitate
its release due to resulting thermodynamic instability of the
cleaved duplex (Leuschner et al., EMBO 7:314, 2006). The guide
strand in the activated RISC binds to a complementary target mRNA
and cleaves the mRNA to promote gene silencing. Cleavage of the
target RNA occurs in the middle of the target region that is
complementary to the guide strand (Elbashir et al., 2001b).
[0004] Target specific gene silencing can be achieved by
exogenously adding siRNA, but non-specific silencing of
non-targeted genes (referred to as off-target effects) can be a
challenge (see, e.g., Jackson et al., Nat. Biotechnol. 21:635,
2003; Du et al., Nucleic Acids Res. 33:1671, 2005. Hence, there
remains a need in the art for alternative dsRNA molecules and
methods to mediate gene silencing. The present disclosure meets
such needs, and further provides other related advantages.
BRIEF SUMMARY
[0005] The present disclosure provides dsRNA molecules comprising
at least three strands, designated herein as A, S1 and S2 (A:S1S2),
wherein S1 and S2 are complementary to, and form base pairs (bp)
with, non-overlapping regions of A. Thus, for siRNA molecules
described herein; the double-stranded region formed by the
annealing of S1 and A is distinct from the double-stranded region
formed by the annealing of S2 and A. An A:S1 duplex may be
separated from an A:S2 duplex by a "gap" resulting from at least
one unpaired nucleotide in the A strand that is positioned between
the A:S1 duplex and the A:S2 duplex and that is distinct from any
one or more unpaired nucleotide at the 3' end of either or both of
the A, S1, and/or S2 strand. Alternatively, an A:S1 duplex may be
separated from an A:S2 duplex by a "nick" such that there are no
unpaired nucleotides in the A strand that are positioned between
the A:S1 duplex and the A:S2 duplex such that the only unpaired
nucleotide, if any, is at the 3' end of either or both of the A,
S1, and/or S2 strand.
[0006] In one aspect, the instant disclosure provides a meroduplex
RNA (mdRNA) molecule, comprising a first strand that is
complementary to a target RNA, and a second strand and a third
strand that are each complementary to non-overlapping regions of
the first strand, wherein the second strand and third strands can
anneal with the first strand to form at least two double-stranded
regions separated by a gap of up to 10 nucleotides, and wherein (a)
at least one double-stranded region is from about 5 base pairs up
to 13 base pairs, or (b) the double-stranded regions combined total
about 15 base pairs to about 40 base pairs and the mdRNA molecule
comprises blunt ends. In certain embodiments, the first strand is
about 15 to about 40 nucleotides in length, and the second and
third strands are each, individually, about 5 to about 20
nucleotides, wherein the combined length of the second and third
strands is about 15 nucleotides to about 40 nucleotides. In other
embodiments, the mdRNA is a RISC activator (e.g., the first strand
has about 15 nucleotides to about 25 nucleotides) or a Dicer
substrate (e.g., the first strand has about 26 nucleotides to about
40 nucleotides). In some embodiments, the gap comprises at least
one to ten unpaired nucleotides in the first strand positioned
between the double-stranded regions formed by the second and third
strands when annealed to the first strand, or the gap comprises a
nick. In certain embodiments, the nick or gap is located 10
nucleotides from the 5'-end of the first (antisense) strand or at
the Argonaute cleavage site. In another embodiment, the meroduplex
nick or gap is positioned such that the thermal stability is
maximized for the first and second strand duplex and for the first
and third strand duplex as compared to the thermal stability of
such meroduplexes having a nick or gap in a different position.
[0007] In another aspect, the instant disclosure provides an mdRNA
molecule having a first strand that is complementary to target RNA,
and a second strand and a third strand that is each complementary
to non-overlapping regions of the first strand, wherein the second
strand and third strands can anneal with the first strand to form
at least two double-stranded regions separated by a gap of up to 10
nucleotides, and wherein (a) at least one double-stranded region is
from about 5 base pairs up to 13 base pairs, or (b) the
double-stranded regions combined total about 15 base pairs to about
40 base pairs and the mdRNA molecule comprises blunt ends; and
wherein at least one pyrimidine of the mdRNA comprises a pyrimidine
nucleoside according to Formula I or II:
##STR00001##
wherein R.sup.1 and R.sup.2 are each independently a --H, --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2,
--C.ident.N, or heterocyclo group; R.sup.3 and R.sup.4 are each
independently a hydroxyl, a protected hydroxyl, a phosphate, or an
internucleoside linking group; and R.sup.5 and R.sup.8 are
independently O or S. In certain embodiments, at least one
nucleoside is according to Formula I and in which R.sup.1 is methyl
and R.sup.2 is --OH. In certain related embodiments, at least one
uridine of the dsRNA molecule is replaced with a nucleoside
according to Formula I in which R.sup.1 is methyl and R.sup.2 is
--OH, or R.sup.1 is methyl, R.sup.2 is --OH, and R.sup.8 is S. In
some embodiments, the at least one R.sup.1 is a C.sub.1-C.sub.5
alkyl, such as methyl. In some embodiments, at least one R.sup.2 is
selected from 2'-O--(C.sub.1-C.sub.5) alkyl, 2'-O-methyl,
2'-OCH.sub.2OCH.sub.2CH.sub.3, 2'-OCH.sub.2CH.sub.2OCH.sub.3,
2'-O-allyl, or fluoro. In some embodiments, at least one pyrimidine
nucleoside of the mdRNA molecule is a locked nucleic acid (LNA) in
the form of a bicyclic sugar, wherein R.sup.2 is oxygen, and the
2'-O and 4'-C form an oxymethylene bridge on the same ribose ring
(e.g., a 5-methyluridine LNA) or is a G clamp. In other
embodiments, one or more of the nucleosides are according to
Formula I in which R.sup.1 is methyl and R.sup.2 is a
2'-O--(C.sub.1-C.sub.5) alkyl, such as 2'-O-methyl. In some
embodiments, the gap comprises at least one unpaired nucleotide in
the first strand positioned between the double-stranded regions
formed by the second and third strands when annealed to the first
strand, or the gap comprises a nick. In certain embodiments, the
nick or gap is located 10 nucleotides from the 5'-end of the first
strand or at the Argonaute cleavage site. In another embodiment,
the meroduplex nick or gap is positioned such that the thermal
stability is maximized for the first and second strand duplex and
for the first and third strand duplex as compared to the thermal
stability of such meroduplexes having a nick or gap in a different
position.
[0008] Compositions and methods disclosed herein are useful for
reducing expression of a target gene, or one or more genes that are
a part of the target gene family, in a cell or to treating or
preventing diseases or disorders associated with expression of one
or more target gene family members, such as hyperproliferative
disorders (e.g., cancer), inflammatory conditions (e.g.,
arthritis), respiratory disease, pulmonary disease, cardiovascular
disease, autoimmune disease, allergic disorders, neurologic
disease, infectious disease (e.g., viral infection, such as
influenza), renal disease, transplant rejection, or any other
disease or condition that responds to modulation of a target gene
or gene family.
[0009] In certain embodiments, dsRNA of the present disclosure
comprise, in sum, between about 15 base-pairs and about 40
base-pairs; or between about 18 and about 35 base-pairs; or between
about 20 and 30 base-pairs; or 21, 22, 23, 24, 25, 26, 27, 28, or
29 base-pairs. Within certain embodiments, the siRNA may,
optionally, comprise a single-strand 3'-overhang of between 1
nucleotide and 5 nucleotides. In particular embodiments, such a
single-strand 3'-overhang is 1, 2, 3, or 4 nucleotides.
[0010] In another aspect, dsRNA of the present disclosure comprise
either an A sense strand or an A antisense strand wherein the
length of the A strand is between about 15 nucleotides and about 50
nucleotides; or the length of the A strand is between about 18
nucleotides and about 40 nucleotides; or the length of the A strand
is between about 20 nucleotides and about 32 nucleotides; or the
length of the A strand is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
or 31 nucleotides.
[0011] In another aspect, siRNA of the present disclosure
additionally comprise two or more S strands, designated herein, for
example, as S1 and S2, wherein each S strand is complementary to a
non-overlapping region of a cognate A strand and wherein a first S
strand (S1) is separated from a second S strand (S2) by a nick or a
one or more nucleotide gap. Depending upon whether the cognate A
strand is a sense strand or an antisense strand, each S strands
will be either an antisense strand or a sense strand, respectively.
Each S strand (S1, S2, etc.) described herein is, independently,
between about 1 nucleotide and about 25 nucleotides in length; more
typically between about 4 nucleotides and about 20 nucleotides in
length; still more typically between about 5 nucleotides and about
16 nucleotides in length; most typically 6, 7, 8, 9, 10, 11, 12,
13, 14, or 15 nucleotides in length.
[0012] Depending upon the precise application contemplated, a first
S strand (S1) may be separated from a second S strand (S2) by a
nick or by a gap. In those embodiments wherein S1 and S2 are
separated by a gap, the gap is between about one nucleotide and
about 25 nucleotides; or between about one nucleotide and about 15
nucleotides; or between about one nucleotide and about 10
nucleotides; or the gap is 1, 2, 3, 4, 5, 6, 7, 8, or 9
nucleotide(s). Each S strand may, independently, terminate with a
5' hydroxyl (i.e., 5'-OH) or may terminate with a 5' phosphate
group (i.e., 5'-PO.sub.4).
[0013] In any of the aspects of this disclosure, there are provided
mdRNA molecules having a 5-methyluridine (ribothymidine) or a
2-thioribothymidine in place of at least one uridine on the first,
second, or third strand, or in place of each and every uridine on
the first, second, or third strand. In further embodiments, the
mdRNA may comprise any one of 5-methyluridine (ribothymidine),
2-thioribothymidine, deoxyuridine, locked nucleic acid (LNA)
molecule, sugar modified with 2'-Omethyl, or G clamp, or any
combination thereof. In certain embodiments, the mdRNA molecule
comprises a 2'-sugar substitution, such as a 2'-O-methyl,
2'-O-methoxyethyl, 2'-O-2-methoxyethyl, 2'-O-allyl, or halogen
(e.g., 2'-fluoro). In certain embodiments, the mdRNA molecule
further comprises at least one terminal cap substituent on one or
both ends of the first strand, second strand, or third strand, such
as independently an alkyl, abasic, deoxy abasic, glyceryl,
dinucleotide, acyclic nucleotide, or inverted deoxynucleotide
moiety. In other embodiments, the mdRNA molecule further comprises
at least one modified internucleoside linkage, such as
independently a phosphorothioate, chiral phosphorothioate,
phosphorodithioate, phosphotriester, aminoalkylphosphotriester,
methyl phosphonate, alkyl phosphonate, 3'-alkylene phosphonate,
5'-alkylene phosphonate, chiral phosphonate, phosphonoacetate,
thiophosphonoacetate, phosphinate, phosphoramidate, 3'-amino
phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester,
selenophosphate, or boranophosphate linkage.
[0014] In any of the aspects of this disclosure, some embodiments
provide an mdRNA comprising an overhang of one to four nucleotides
on at least one 3'-end that is not part of the gap, such as at
least one deoxyribonucleotide or two deoxyribonucleotides (e.g.,
thymidine). In some embodiments, at least one or two 5'-terminal
ribonucleotide of the second strand within the double-stranded
region comprises a 2'-sugar substitution. In related embodiments,
at least one or two 5'-terminal ribonucleotide of the first strand
within the double-stranded region comprises a 2'-sugar
substitution. In other related embodiments, at least one or two
5'-terminal ribonucleotide of the second strand and at least one or
two 5'-terminal ribonucleotide of the first strand within the
double-stranded regions comprise independent 2'-sugar
substitutions. In other embodiments, the mdRNA molecule comprises
at least three 5-methyluridines within at least one double-stranded
region. In some embodiments, the mdRNA molecule has a blunt end at
one or both ends. In other embodiments, the 5'-terminal of the
third strand is a hydroxyl or a phosphate.
[0015] It will be understood that methods of the present disclosure
do not require a priori knowledge of the nucleotide sequence of
every possible gene variant(s) targeted by the gapped or nicked
dsRNA. Initially, the nucleotide sequence of the siRNA may be
selected from a conserved region of the target gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 shows knockdown activity for RISC activator lacZ
dsRNA (21 nucleotide sense strand/21 nucleotide antisense strand;
21/21), Dicer substrate lacZ dsRNA (25 nucleotide sense strand/27
nucleotide antisense strand; 25/27), and meroduplex lacZ mdRNA (13
nucleotide sense strand and 11 nucleotide sense strand/27
nucleotide antisense strand; 13, 11/27--the sense strand is missing
one nucleotide so that a single nucleotide gap is left between the
13 nucleotide and 11 nucleotide sense strands when annealed to the
27 nucleotide antisense strand. Knockdown activities were
normalized to a Qneg control dsRNA and presented as a normalized
value of Qneg (i.e., Qneg represents 100% or "normal" gene
expression levels). A smaller value indicates a greater knockdown
effect.
[0017] FIG. 2 shows knockdown activity of a RISC activator
influenza dsRNA G1498 (21/21) and nicked dsRNA (10, 11/21) at 100
nM. The "wt" designation indicates an unsubstituted RNA molecule;
"rT" indicates RNA having each uridine substituted with a
ribothymidine; and "p" indicates that the 5'-nucleotide of that
strand was phosphorylated. The 21 nucleotide sense and antisense
strands of G1498 were individually nicked between the nucleotides
10 and 11 as measured from the 5'-end, and is referred to as 11,
10/21 and 21/10, 11, respectively. The G1498 single stranded 21
nucleotide antisense strand alone (designated AS-only) was used as
a control.
[0018] FIG. 3 shows knockdown activity of a lacZ dicer substrate
(25/27) having a nick in one of each of positions 8 to 14 and a one
nucleotide gap at position 13 of the sense strand (counted from the
5'-end). A dideoxy guanosine (ddG) was incorporated at the 5'-end
of the 3'-most strand of the nicked or gapped sense sequence at
position 13.
[0019] FIG. 4 shows knockdown activity of a dicer substrate
influenza dsRNA G1498DS (25/27) and this sequence nicked at one of
each of positions 8 to 14 of the sense strand, and shows the
activity of these nicked molecules that are also phosphorylated or
have a locked nucleic acid substitution.
[0020] FIG. 5 shows a dose dependent knockdown activity a dicer
substrate influenza dsRNA G1498DS (25/27) and this sequence nicked
at position 13 of the sense strand.
[0021] FIG. 6 shows knockdown activity of a dicer substrate
influenza dsRNA G1498DS having a nick or a gap of one to six
nucleotides that begins at any one of positions 8 to 12 of the
sense strand.
[0022] FIG. 7 shows knockdown activity of a LacZ RISC dsRNA having
a nick or a gap of one to six nucleotides that begins at any one of
positions 8 to 14 of the sense strand.
[0023] FIG. 8 shows knockdown activity of an influenza RISC dsRNA
having a nick at any one of positions 8 to 14 of the sense strand
and further having one or two locked nucleic acids per sense
strand.
[0024] FIG. 9 shows knockdown activity of a LacZ dicer substrate
dsRNA having a nick at any one of positions 8 to 14 of the sense
strand as compared to the same nicked dicer substrates but having a
locked nucleic acid substitution.
[0025] FIG. 10 shows the dose-dependent reduction in WSN influenza
viral titers using influenza specific mdRNA as measured by
TCID.sub.50.
[0026] FIG. 11 shows the percent knockdown in influenza viral
titers using influenza specific mdRNA against influenza strain
WSN.
[0027] FIG. 12 shows the in vivo reduction in PR8 influenza viral
titers using influenza specific mdRNA as measured by
TCID.sub.50.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0028] The instant disclosure provides gapped double-stranded RNA
(dsRNA) comprising at least three strands that is a suitable
substrate for Dicer or for association with RISC and, therefore,
may be advantageously employed for gene silencing via, for example,
the RNA interference (RNAi) pathway. That is, partially duplexed
dsRNA molecules described herein (also referred to as meroduplexes
or meromers having a nick or gap in at least one strand) are
capable of initiating an RNAi cascade that modifies (e.g., reduces)
expression of a target messenger RNA (mRNA) or a family of related
target mRNAs. The gene silencing functionality of such a structure
was unpredictable since the thermodynamically less stable nicked or
gapped dsRNA passenger strand (as compared to an intact dsRNA)
would be expected to fall apart before any gene silencing occurred
(see, e.g., Leuschner et al., EMBO 7:314, 2006; Bramsen et al.,
Nucleic Acids Res. 35:5886, 2007).
[0029] Meroduplex ribonucleic acid (mdRNA) molecules described
herein include a first (antisense) strand that is complementary to
a target mRNA, along with second and third strands (together
forming a gapped sense strand) that are each complementary to
non-overlapping regions of the first strand, wherein the second and
third strands can anneal with the first strand to form at least two
double-stranded regions separated by a gap, and wherein at least
one double-stranded region is from about 5 base pairs to 15 base
pairs, or the combined double-stranded regions total about 15 base
pairs to about 40 base pairs and the mdRNA is blunt-ended.
[0030] The gap can be from zero nucleotides (i.e., a nick in which
only a phosphodiester bond between two nucleotides is broken in a
polynucleotide molecule) up to about 10 nucleotides (i.e., the
first strand will have at least non-terminal unpaired nucleotide).
In certain embodiments, the nick or gap is located about 10
nucleotides from the 5'-end of the first (antisense) strand or at
the Argonaute cleavage site. In another embodiment, the meroduplex
nick or gap is positioned such that the thermal stability is
maximized for the first and second strand duplex and for the first
and third strand duplex as compared to the thermal stability of
such meroduplexes having a nick or gap in a different position.
[0031] Also provided herein are methods of using such dsRNA to
reduce expression of a target gene, or one or more genes that are a
part of the target gene family, in a cell or to treat or prevent
diseases or disorders associated with expression of one or more
target gene family members, such as hyperproliferative disorders
(e.g., cancer), inflammatory conditions (e.g., arthritis),
respiratory disease, pulmonary disease, cardiovascular disease,
autoimmune disease, allergic disorders, neurologic disease,
infectious disease (e.g., viral infection, such as influenza),
renal disease, transplant rejection, or any other disease or
condition that responds to modulation of a target gene or gene
family.
[0032] Prior to introducing more detail into this disclosure, it
may be helpful to an appreciation thereof to provide definitions of
certain terms to be used herein.
[0033] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated. Also, any
number range recited herein relating to any physical feature, such
as polymer subunits, size or thickness, are to be understood to
include any integer within the recited range, unless otherwise
indicated. As used herein, "about" or "consisting essentially of"
mean.+-.20% of the indicated range, value, or structure, unless
otherwise indicated. As used herein, the terms "include" and
"comprise" are open-ended and are used synonymously. It should be
understood that the terms "a" and "an" as used herein refer to "one
or more" of the enumerated components. The use of the alternative
(e.g., "or") should be understood to mean either one, both, or any
combination thereof of the alternatives.
[0034] As used herein, "complementary" refers to a nucleic acid
molecule that can form hydrogen bond(s) with another nucleic acid
molecule or itself by either traditional Watson-Crick base pairing
or other non-traditional types of pairing (e.g., Hoogsteen or
reversed Hoogsteen hydrogen bonding) between complementary
nucleosides or nucleotides. In reference to the nucleic molecules
of the present disclosure, the binding free energy for a nucleic
acid molecule with its complementary sequence is sufficient to
allow the relevant function of the nucleic acid molecule to
proceed, for example, RNAi activity, and there is a sufficient
degree of complementarity to avoid non-specific binding of the
nucleic acid molecule (e.g., dsRNA) to non-target sequences under
conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, or under conditions in which the assays are
performed in the case of in vitro assays (e.g., hybridization
assays). Determination of binding free energies for nucleic acid
molecules is well known in the art (see, e.g., Turner et al., CSH
Symp. Quant. Biol. LII:123, 1987; Frier et al., Proc. Nat. Acad.
Sci. USA 83:9373, 1986; Turner et al., J. Am. Chem. Soc. 109:3783,
1987). Thus, "complementary" or "specifically hybridizable" or
"specifically binds" are terms that indicate a sufficient degree of
complementarity or precise pairing such that stable and specific
binding occurs between a nucleic acid molecule (e.g., dsRNA) and a
DNA or RNA target. It is understood in the art that a nucleic acid
molecule need not be 100% complementary to a target nucleic acid
sequence to be specifically hybridizable or to specifically bind.
That is, two or more nucleic acid molecules may be less than fully
complementary and is indicated by a percentage of contiguous
residues in a nucleic acid molecule that can form hydrogen bonds
with a second nucleic acid molecule.
[0035] For example, a first nucleic acid molecule may have 10
nucleotides and a second nucleic acid molecule may have 10
nucleotides, then base pairing of 5, 6, 7, 8, 9, or 10 nucleotides
between the first and second nucleic acid molecules, which may or
may not form a contiguous double-stranded region, represents 50%,
60%, 70%, 80%, 90%, and 100% complementarity, respectively. In
certain embodiments, complementary nucleic acid molecules may have
wrongly paired bases--that is, bases that cannot form a traditional
Watson-Crick base pair or other non-traditional types of pair
(i.e., "mismatched" bases). For instance, fully complementary
nucleic acid molecules may be identified as having a certain number
of "mismatches," such as zero or about 1, about 2, about 3, about 4
or about 5.
[0036] "Perfectly" or "fully" complementary nucleic acid molecules
means those in which a certain number of nucleotides of a first
nucleic acid molecule hydrogen bond (anneal) with the same number
of residues in a second nucleic acid molecule to form a contiguous
double-stranded region. For example, two or more fully
complementary nucleic acid molecule strands can have the same
number of nucleotides (i.e., have the same length and form one
double-stranded region, with or without an overhang) or have a
different number of nucleotides (e.g., one strand may be shorter
than but fully contained within a second strand or one strand may
overhang the second strand).
[0037] As used herein, "ribonucleic acid" or "RNA" means a nucleic
acid molecule comprising at least one ribonucleotide molecule. It
should be understood that "ribonucleotide" refers to a nucleotide
with a hydroxyl group at the 2'-position of a .beta.-D-ribofuranose
moiety. The term RNA includes double-stranded (ds) RNA,
single-stranded (ss) RNA, isolated RNA (such as partially purified
RNA, essentially pure RNA, synthetic RNA, recombinantly produced
RNA), altered RNA (which differs from naturally occurring RNA by
the addition, deletion, substitution or alteration of one or more
nucleotides), or any combination thereof. For example, such altered
RNA can include addition of non-nucleotide material, such as at one
or both ends of an RNA molecule, internally at one or more
nucleotides of the RNA, or any combination thereof. Nucleotides in
RNA molecules of the instant disclosure can also comprise
non-standard nucleotides, such as naturally occurring nucleotides,
non-naturally occurring nucleotides, chemically-modified
nucleotides, deoxynucleotides, or any combination thereof. These
altered RNAs may be referred to as analogs or analogs of RNA
containing standard nucleotides (i.e., standard nucleotides, as
used herein, are considered to be adenine, cytidine, guanidine,
thymidine, and uridine).
[0038] The term "dsRNA" as used herein, which is interchangeable
with "mdRNA," refers to any nucleic acid molecule comprising at
least one ribonucleotide and is capable of inhibiting or down
regulating gene expression, for example, by promoting RNA
interference ("RNAi") or gene silencing in a sequence-specific
manner. The dsRNAs (mdRNAs) of the instant disclosure may be
suitable substrates for Dicer or for association with RISC to
mediate gene silencing by RNAi. One or both strands of the dsRNA
can further comprise a terminal phosphate group, such as a
5'-phosphate or 5',3'-diphosphate. As used herein, dsRNA molecules,
in addition to at least one ribonucleotide, can further include
substitutions, chemically-modified nucleotides, and
non-nucleotides. In certain embodiments, dsRNA molecules comprise
ribonucleotides up to about 100% of the nucleotide positions.
[0039] In addition, as used herein, the term dsRNA is meant to be
equivalent to other terms used to describe nucleic acid molecules
that are capable of mediating sequence specific RNAi, for example,
meroduplex RNA (mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA
(gdsRNA), short interfering nucleic acid (siNA), siRNA, micro-RNA
(miRNA), short hairpin RNA (shRNA), short interfering
oligonucleotide, short interfering substituted oligonucleotide,
short interfering modified oligonucleotide, chemically-modified
dsRNA, post-transcriptional gene silencing RNA (ptgsRNA), or the
like. The term "large double-stranded (ds) RNA" refers to any
double-stranded RNA longer than about 40 base pairs (bp) to about
100 bp or more, particularly up to about 300 bp to about 500 bp.
The sequence of a large dsRNA may represent a segment of an mRNA or
an entire mRNA. A double-stranded structure may be formed by
self-complementary nucleic acid molecule or by annealing of two or
more distinct complementary nucleic acid molecule strands.
[0040] In one aspect, a dsRNA comprises two separate
oligonucleotides, comprising a first strand (antisense) and a
second strand (sense), wherein the antisense and sense strands are
self-complementary (i.e., each strand comprises a nucleotide
sequence that is complementary to a nucleotide sequence in the
other strand and the two separate strands form a duplex or
double-stranded structure, for example, wherein the double-stranded
region is about 15 to about 24 or 25 base pairs or about 25 or 26
to about 40 base pairs); the antisense strand comprises a
nucleotide sequence that is complementary to a nucleotide sequence
in a target nucleic acid molecule or a portion thereof; and the
sense strand comprises a nucleotide sequence corresponding (i.e.,
homologous) to the target nucleic acid sequence or a portion
thereof (e.g., a sense strand of about 15 to about 25 nucleotides
or about 26 to about 40 nucleotides corresponds to the target
nucleic acid or a portion thereof).
[0041] In another aspect, the dsRNA is assembled from a single
oligonucleotide in which the self-complementary sense and antisense
strands of the dsRNA are linked by together by a nucleic acid
based-linker or a non-nucleic acid-based linker. In certain
embodiments, the first (antisense) and second (sense) strands of
the dsRNA molecule are covalently linked by a nucleotide or
non-nucleotide linker as described herein and known in the art. In
other embodiments, a first dsRNA molecule is covalently linked to
at least one second dsRNA molecule by a nucleotide or
non-nucleotide linker known in the art, wherein the first dsRNA
molecule can be linked to a plurality of other dsRNA molecules that
can be the same or different, or any combination thereof. In
another embodiment, the linked dsRNA may include a third strand
that forms a meroduplex with the linked dsRNA.
[0042] In still another aspect, dsRNA molecules described herein
form a meroduplex RNA (mdRNA) having three or more strands such as,
for example, an `A` (first or antisense) strand, `S1` (second)
strand, and `S2` (third) strand in which the `S1` and `S2` strands
are complementary to and form base pairs (bp) with non-overlapping
regions of the `A` strand (e.g., an mdRNA can have the form of
A:S1S2). The S1, S2, or more strands together essentially comprise
a sense strand to the `A` strand. The double-stranded region formed
by the annealing of the `S1` and `A` strands is distinct from and
non-overlapping with the double-stranded region formed by the
annealing of the `S2` and `A` strands. An mdRNA molecule is a
"gapped" molecule, meaning a "gap" ranging from 0 nucleotides up to
about 10 nucleotides. In one embodiment, the A:S1 duplex is
separated from the A:S2 duplex by a gap resulting from at least one
unpaired nucleotide (up to about 10 unpaired nucleotides) in the
`A` strand that is positioned between the A:S1 duplex and the A:S2
duplex and that is distinct from any one or more unpaired
nucleotide at the 3'-end of one or more of the `A`, `S1`, or `S2`
strands. In another embodiment, the A:S1 duplex is separated from
the A:S2 duplex by a gap of zero nucleotides (i.e., a nick in which
only a phosphodiester bond between two nucleotides is broken or
missing in the polynucleotide molecule) between the A:S1 duplex and
the A:S2 duplex--which can also be referred to as nicked dsRNA
(ndsRNA). For example, A:S1S2 may be comprised of a dsRNA having at
least two double-stranded regions that combined total about 14 base
pairs to about 40 base pairs and the double-stranded regions are
separated by a gap of about 0 to about 10 nucleotides, optionally
having blunt ends, or A:S1S2 may comprise a dsRNA having at least
two double-stranded regions separated by a gap of up to 10
nucleotides wherein at least one of the double-stranded regions
comprises between about 5 base pairs and 13 base pairs.
[0043] A dsRNA or large dsRNA may include a substitution or
modification in which the substitution or modification may be in a
phosphate backbone bond, a sugar, a base, or a nucleoside. Such
nucleoside substitutions can include natural non-standard
nucleosides (e.g., 5-methyluridine or 5-methylcytidine or a
2-thioribothymidine), and such backbone, sugar, or nucleoside
modifications can include an alkyl or heteroatom substitution or
addition, such as a methyl, alkoxyalkyl, halogen, nitrogen or
sulfur, or other modifications known in the art.
[0044] In certain embodiments, the dsRNA or mdRNA will be isolated.
As used herein, the term "isolated" means that the molecule
referred to is removed from its original environment, such as being
separated from some or all of the co-existing materials in a
natural environment (e.g., a natural environment may be a
cell).
[0045] As used herein, the term "RNAi" is meant to be equivalent to
other terms used to describe sequence specific RNA interference,
such as post transcriptional gene silencing, translational
inhibition, or epigenetics. For example, dsRNA molecules of this
disclosure can be used to epigenetically silence genes at the
post-transcriptional level or the pre-transcriptional level or any
combination thereof.
[0046] As used herein, "target nucleic acid" refers to any nucleic
acid sequence whose expression or activity is to be altered. The
target nucleic acid can be DNA, RNA, or analogs thereof, and
includes single, double, and multi-stranded forms. By "target site"
or "target sequence" is meant a sequence within a target nucleic
acid (e.g., mRNA) that is "targeted" for cleavage by RNAi and
mediated by a dsRNA construct of this disclosure containing a
sequence within the antisense strand that is complementary to the
target site or sequence.
[0047] As used herein, "off-target effect" or "off-target profile"
refers to the observed altered expression pattern of one or more
genes in a cell or other biological sample not targeted, directly
or indirectly, for gene silencing by an mdRNA or dsRNA. For
example, an off-target effect can be quantified by using a DNA
microarray to determine how many non-target genes have an
expression level altered by about 2-fold or more in the presence of
a candidate mdRNA or dsRNA, or analog thereof specific for a target
sequence, such as one or more target mRNA. A "minimal off-target
effect" means that an mdRNA or dsRNA affects expression by about
2-fold or more of about 25% to about 1% of the non-target genes
examined or it means that the off-target effect of substituted or
modified mdRNA or dsRNA (e.g., having at least one uridine
substituted with a 5-methyluridine or 2-thioribothymidine and
optionally having at least one nucleotide modified at the
2'-position), is reduced by at least about 1% to about 80% or more
as compared to the effect on non-target genes of an unsubstituted
or unmodified mdRNA or dsRNA.
[0048] By "sense region" or "sense strand" is meant one or more
nucleotide sequences of a dsRNA molecule having complementarity to
one or more antisense regions of the dsRNA molecule. In addition,
the sense region of a dsRNA molecule comprises a nucleic acid
sequence having homology or identity to a target sequence. By
"antisense region" or "antisense strand" is meant a nucleotide
sequence of a dsRNA molecule having complementarity to a target
nucleic acid sequence. In addition, the antisense region of a dsRNA
molecule can comprise a nucleic acid sequence regions having
complementarity to one or more sense strands of a dsRNA
molecule.
[0049] "Analog" as used herein refers to a compound that is
structurally similar to a parent compound (e.g., a nucleic acid
molecule), but differs slightly in composition (e.g., one atom or
functional group is different, added, or removed). The analog may
or may not have different chemical or physical properties than the
original compound and may or may not have improved biological or
chemical activity. For example, the analog may be more hydrophilic
or it may have altered activity as compared to a parent compound.
The analog may mimic the chemical or biological activity of the
parent compound (i.e., it may have similar or identical activity),
or, in some cases, may have increased or decreased activity. The
analog may be a naturally or non-naturally occurring (e.g.,
chemically-modified or recombinant) variant of the original
compound. An example of an RNA analog is an RNA molecule having a
non-standard nucleotide, such as 5-methyuridine or 5-methylcytidine
or 2-thioribothymidine, which may impart certain desirable
properties (e.g., improve stability, bioavailability, minimize
off-target effects or interferon response).
[0050] The term "pyrimidine" as used herein refers to conventional
pyrimidine bases, including standard pyrimidine bases uracil and
cytosine. In addition, the term pyrimidine is contemplated to
embrace natural non-standard pyrimidine bases or acids, such as
5-methyluracil, 2-thio-5-methyluracil, 4-thiouracil, pseudouracil,
dihydrouracil, orotate, 5-methylcytosine, or the like, as well as a
chemically-modified bases or "universal bases," which can be used
to substitute for a standard pyrimidine within nucleic acid
molecules of this disclosure.
[0051] The term "purine" as used herein refers to conventional
purine bases, including standard purine bases adenine and guanine.
In addition, the term purine is contemplated to embrace natural
non-standard purine bases or acids, such as N2-methylguanine,
inosine, or the like, as well as a chemically-modified bases or
"universal bases," which can be used to substitute for a standard
purine within nucleic acid molecules of this disclosure.
[0052] As used herein, the term "universal base" refers to
nucleotide base analogs that form base pairs with each of the
standard DNA/RNA bases with little discrimination between them, and
is recognized by intracellular enzymes (see, e.g., Loakes et al.,
J. Mol. Bio. 270:426-435, 1997). 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, e.g., Loakes, Nucleic Acids Res.
29:2437-2447, 2001).
[0053] The term "gene" as used herein, especially in the context of
"target gene" or "gene target" for RNAi, means a nucleic acid
molecule that encodes an RNA or a transcription product of such
gene, including a messenger RNA (mRNA, also referred to as
structural genes that encode for a polypeptide), an mRNA splice
variant of such gene, a functional RNA (fRNA), or non-coding RNA
(ncRNA), such as small temporal RNA (stRNA), microRNA (miRNA),
small nuclear RNA (snRNA), short interfering RNA (siRNA), small
nucleolar RNA (snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA)
and precursor RNAs thereof. Such non-coding RNAs can serve as
target nucleic acid molecules for dsRNA mediated RNAi to alter the
activity of fRNA or ncRNA involved in functional or regulatory
cellular processes. A target gene can be a gene derived from a
cell, such as an endogenous gene, a transgene, or exogenous gene,
including genes from a pathogen (e.g., a viral gene) that is
present in a cell after infection thereof. A cell containing a
target gene can be derived from or contained in any organism, for
example, a plant, animal, protozoan, virus, bacterium, or
fungus.
[0054] Furthermore, one or more dsRNA may be used to knockdown
expression of a target mRNA or a related mRNA splice variant. In
this regard, it is noted that a target gene may be transcribed into
two or more mRNA splice variants. In certain embodiments, knockdown
of one target mRNA splice variant without affecting one or more
other target mRNA splice variants may be desired, or vice versa.
Alternatively, knockdown of all transcription products of one or
more target family genes is contemplated herein.
[0055] As used herein, "gene silencing" refers to a partial or
complete loss-of-function through targeted inhibition of gene
expression in a cell, which may also be referred to as RNAi
"knockdown," "inhibition," "down-regulation," or "reduction" of
expression of a target gene. Depending on the circumstances and the
biological problem to be addressed, it may be preferable to
partially reduce gene expression. Alternatively, it might be
desirable to reduce gene expression as much as possible. The extent
of silencing may be determined by methods described herein and
known in the art, some of which are summarized in PCT Publication
No. WO 99/32619. Depending on the assay, quantification of gene
expression permits detection of various amounts of inhibition that
may be desired in certain embodiments of this disclosure, including
prophylactic and therapeutic methods, which will be capable of
knocking down target gene expression, in terms of mRNA level or
protein level or activity, for example, by equal to or greater than
10%, 30%, 50%, 75% 90%, 95% or 99% of baseline (i.e., normal) or
other control levels, including elevated expression levels as may
be associated with particular disease states or other conditions
targeted for therapy.
[0056] 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
this disclosure can be administered. In one embodiment, a subject
is a mammal or mammalian cell. In another embodiment, a subject is
a human or human cell.
[0057] As used herein, the term "therapeutically effective amount"
means an amount of dsRNA that is sufficient to result in a decrease
in severity of disease symptoms, an increase in frequency or
duration of disease symptom-free periods, or a prevention of
impairment or disability due to the disease, in the subject (e.g.,
mammal or human) to which it is administered. For example, a
therapeutically effective amount of dsRNA directed against a target
mRNA, which effectively down-regulates the target-encoding mRNA and
thereby reduces or prevents one or more target-associated
disorders, such as an infection, inflammation, metabolic disorders,
autoimmune condition(s), cancer, or the like. One of ordinary skill
in the art would be able to determine such therapeutically
effective amounts based on such factors as the subject's size, the
severity of symptoms, and the particular composition or route of
administration selected. For example, a therapeutically effective
amount of a compound can decrease tumor size or otherwise
ameliorate symptoms associated with a particular disorder in a
subject. The dsRNA molecules of the instant disclosure,
individually or in combination or in conjunction with other drugs,
can be used to treat diseases or conditions discussed herein, by
administering to a subject or by administering to particular cells
under conditions suitable for treatment.
[0058] In addition, it should be understood that the individual
compounds, or groups of compounds, derived from the various
combinations of the structures and substituents described herein,
are disclosed by the present application to the same extent as if
each compound or group of compounds was set forth individually.
Thus, selection of particular structures or particular substituents
is within the scope of the present disclosure. As described herein,
all value ranges are inclusive over the indicated range. Thus, a
range of C.sub.1-C.sub.4 will be understood to include the values
of 1, 2, 3, and 4, such that C.sub.1, C.sub.2, C.sub.3 and C.sub.4
are included.
[0059] The term "alkyl" as used herein refers to saturated
straight- or branched-chain aliphatic groups containing from 1-20
carbon atoms, preferably 1-8 carbon atoms and most preferably 1-4
carbon atoms. This definition applies as well to the alkyl portion
of alkoxy, alkanoyl and aralkyl groups. The alkyl group may be
substituted or unsubstituted. In certain embodiments, the alkyl is
a (C.sub.1-C.sub.4) alkyl or methyl.
[0060] The term "cycloalkyl" as used herein refers to a saturated
cyclic hydrocarbon ring system containing from 3 to 12 carbon atoms
that may be optionally substituted. Exemplary embodiments include,
but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl and
cyclohexyl. In certain embodiments, the cycloalkyl group is
cyclopropyl. In another embodiment, the (cycloalkyl)alkyl groups
contain from 3 to 12 carbon atoms in the cyclic portion and 1 to 6
carbon atoms in the alkyl portion. In certain embodiments, the
(cycloalkyl)alkyl group is cyclopropylmethyl. The alkyl groups are
optionally substituted with from one to three substituents selected
from the group consisting of halogen, hydroxy and amino.
[0061] The terms "alkanoyl" and "alkanoyloxy" as used herein refer,
respectively, to --C(O)-alkyl groups and --O--C(.dbd.O)-- alkyl
groups, each optionally containing 2 to 10 carbon atoms. Specific
embodiments of alkanoyl and alkanoyloxy groups are acetyl and
acetoxy, respectively.
[0062] The term "alkenyl" refers to an unsaturated branched,
straight-chain or cyclic alkyl group having 2 to 15 carbon atoms
and having at least one carbon-carbon double bond derived by the
removal of one hydrogen atom from a single carbon atom of a parent
alkene. The group may be in either the cis or trans conformation
about the double bond(s). Certain embodiments include ethenyl,
1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl,
3-butenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 4-pentenyl,
3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 1-heptenyl, 2-heptenyl,
1-octenyl, 2-octenyl, 1,3-octadienyl, 2-nonenyl, 1,3-nonadienyl,
2-decenyl, etc., or the like. The alkenyl group may be substituted
or unsubstituted.
[0063] The term "alkynyl" as used herein refers to an unsaturated
branched, straight-chain, or cyclic alkyl group having 2 to 10
carbon atoms and having at least one carbon-carbon triple bond
derived by the removal of one hydrogen atom from a single carbon
atom of a parent alkyne. Exemplary alkynyls include ethynyl,
1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,
1-pentynyl, 2-pentynyl, 4-pentynyl, 1-octynyl, 6-methyl-1-heptynyl,
2-decynyl, or the like. The alkynyl group may be substituted or
unsubstituted.
[0064] The term "hydroxyalkyl" alone or in combination, refers to
an alkyl group as previously defined, wherein one or several
hydrogen atoms, preferably one hydrogen atom has been replaced by a
hydroxyl group. Examples include hydroxymethyl, hydroxyethyl and
2-hydroxyethyl.
[0065] The term "aminoalkyl" as used herein refers to the group
--NRR', where R and R' may independently be hydrogen or
(C.sub.1-C.sub.4) alkyl.
[0066] The term "alkylaminoalkyl" refers to an alkylamino group
linked via an alkyl group (i.e., a group having the general
structure -alkyl-NH-alkyl or -alkyl-N(alkyl)(alkyl)). Such groups
include, but are not limited to, mono- and di-(C.sub.1-C.sub.8
alkyl)aminoC.sub.1-C.sub.8 alkyl, in which each alkyl may be the
same or different.
[0067] The term "dialkylaminoalkyl" refers to alkylamino groups
attached to an alkyl group. Examples include, but are not limited
to, N,N-dimethylaminomethyl, N,N-dimethylaminoethyl
N,N-dimethylaminopropyl, and the like. The term dialkylaminoalkyl
also includes groups where the bridging alkyl moiety is optionally
substituted.
[0068] The term "haloalkyl" refers to an alkyl group substituted
with one or more halo groups, for example, chloromethyl,
2-bromoethyl, 3-iodopropyl, trifluoromethyl, perfluoropropyl,
8-chlorononyl, or the like.
[0069] The term "carboxyalkyl" as used herein refers to the
substituent --R.sup.Z--COOH, wherein R.sup.10 is alkylene; and
carbalkoxyalkyl refers to --R.sup.10--C(O)OR.sup.11, wherein
R.sup.10 and R.sup.11 are alkylene and alkyl respectively. In
certain embodiments, alkyl refers to a saturated straight- or
branched-chain hydrocarbyl radical of 1 to 6 carbon atoms such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl,
2-methylpentyl, n-hexyl, and so forth. Alkylene is the same as
alkyl except that the group is divalent.
[0070] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. In one embodiment, the alkoxy group contains 1 to about 10
carbon atoms. Embodiments of alkoxy groups include, but are not
limited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and
pentoxy groups. Embodiments of substituted alkoxy groups include
halogenated alkoxy groups. In a further embodiment, the alkoxy
groups can be substituted with groups such as alkenyl, alkynyl,
halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkylamino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Exemplary halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy, and trichloromethoxy.
[0071] The term "alkoxyalkyl" refers to an alkylene group
substituted with an alkoxy group. For example, methoxyethyl
(CH.sub.3OCH.sub.2CH.sub.2--) and ethoxymethyl
(CH.sub.3CH.sub.2OCH.sub.2--) are both C.sub.3 alkoxyalkyl
groups.
[0072] The term "aryl" as used herein refers to monocyclic or
bicyclic aromatic hydrocarbon groups having from 6 to 12 carbon
atoms in the ring portion, for example, phenyl, naphthyl, biphenyl
and diphenyl groups, each of which may be substituted with, for
example, one to four substituents such as alkyl; substituted alkyl
as defined above, halogen, trifluoromethyl, trifluoromethoxy,
hydroxy, alkoxy, cycloalkyloxy, alkanoyl, alkanoyloxy, amino,
alkylamino, dialkylamino, nitro, cyano, carboxy, carboxyalkyl,
carbamyl, carbamoyl and aryloxy. Specific embodiments of aryl
groups in accordance with the present disclosure include phenyl,
substituted phenyl, naphthyl, biphenyl, and diphenyl.
[0073] The term "aroyl" as used alone or in combination herein
refers to an aryl radical derived from an aromatic carboxylic acid,
such as optionally substituted benzoic or naphthoic acids.
[0074] The term "aralkyl" as used herein refers to an aryl group
bonded to the 2-pyridinyl ring or the 4-pyridinyl ring through an
alkyl group, preferably one containing 1 to 10 carbon atoms. A
preferred aralkyl group is benzyl.
[0075] The term "carboxy" as used herein represents a group of the
formula --C(.dbd.O)OH or --C(.dbd.O)O.sup.-.
[0076] The term "carbonyl" as used herein refers to a group in
which an oxygen atom is double-bonded to a carbon atom
--C.dbd.O.
[0077] The term "trifluoromethyl" as used herein refers to
--CF.sub.3.
[0078] The term "trifluoromethoxy" as used herein refers to
--OCF.sub.3.
[0079] The term "hydroxyl" as used herein refers to --OH or
--O.sup.-.
[0080] The term "nitrile" or "cyano" as used herein refers to the
group --CN.
[0081] The term "nitro" as used herein alone or in combination
refers to a --NO.sub.2 group.
[0082] The term "amino" as used herein refers to the group
--NR.sup.9R.sup.9, wherein R.sup.9 may independently be hydrogen,
alkyl, aryl, alkoxy, or heteroaryl. The term "aminoalkyl" as used
herein represents a more detailed selection as compared to "amino"
and refers to the group --NR'R', wherein R' may independently be
hydrogen or (C.sub.1-C.sub.4) alkyl. The term "dialkylamino" refers
to an amino group having two attached alkyl groups that can be the
same or different.
[0083] The term "alkanoylamino" refers to alkyl, alkenyl or alkynyl
groups containing the group --C(.dbd.O)-- followed by --N(H)--, for
example, acetylamino, propanoylamino and butanoylamino and the
like.
[0084] The term "carbonylamino" refers to the group
--NR'--CO--CH.sub.2--R', wherein R' is independently selected from
hydrogen or (C.sub.1-C.sub.4) alkyl.
[0085] The term "carbamoyl" as used herein refers to
--O--C(O)NH.sub.2.
[0086] The term "carbamyl" as used herein refers to a functional
group in which a nitrogen atom is directly bonded to a carbonyl,
i.e., as in --NR'C(.dbd.O)R' or --C(.dbd.O)NR'R', wherein R' can be
independently hydrogen, alkyl, substituted alkyl, alkenyl,
substituted alkenyl, alkoxy, cycloalkyl, aryl, heterocyclo, or
heteroaryl.
[0087] The term "alkylsulfonylamino" refers to refers to the group
--NHS(O).sub.2R.sup.12, wherein R.sup.12 is alkyl.
[0088] The term "halogen" as used herein refers to bromine,
chlorine, fluorine or iodine. In one embodiment, the halogen is
fluorine. In another embodiment, the halogen is chlorine.
[0089] The term "heterocyclo" refers to an optionally substituted,
unsaturated, partially saturated, or fully saturated, aromatic or
nonaromatic cyclic group that is a 4 to 7 membered monocyclic, or 7
to 11 membered bicyclic ring system that has at least one
heteroatom in at least one carbon atom-containing ring. The
substituents on the heterocyclo rings may be selected from those
given above for the aryl groups. Each ring of the heterocyclo group
containing a heteroatom may have 1, 2, or 3 heteroatoms selected
from nitrogen, oxygen or sulfur. Plural heteroatoms in a given
heterocyclo ring may be the same or different.
[0090] Exemplary monocyclic heterocyclo groups include
pyrrolidinyl, pyrrolyl, indolyl, pyrazolyl, imidazolyl, oxazolyl,
isoxazolyl, thiazolyl, furyl, tetrahydrofuryl, thienyl,
piperidinyl, piperazinyl, azepinyl, pyrimidinyl, pyridazinyl,
tetrahydropyranyl, morpholinyl, dioxanyl, triazinyl and triazolyl.
Preferred bicyclic heterocyclo groups include benzothiazolyl,
benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl,
benzimidazolyl, benzofuryl, indazolyl, benzisothiazolyl,
isoindolinyl and tetrahydroquinolinyl. In more detailed embodiments
heterocyclo groups may include indolyl, imidazolyl, furyl, thienyl,
thiazolyl, pyrrolidyl, pyridyl and pyrimidyl.
[0091] "Substituted" refers to a group in which one or more
hydrogen atoms are each independently replaced with the same or
different substituent(s). Representative substituents include --X,
--R.sup.6, --O--, .dbd.O, --OR, --SR.sup.6, --S--, .dbd.S,
--NR.sup.6R.sup.6, .dbd.NR.sup.6, --CX.sub.3, --CF.sub.3, --CN,
--OCN, --SCN, --NO, --NO.sub.2, .dbd.N.sub.2, --N.sub.3,
--S(.dbd.O).sub.2O--, --S(.dbd.O).sub.2OH,
--S(.dbd.O).sub.2R.sup.6, --OS(.dbd.O).sub.2O--,
--OS(.dbd.O).sub.2OH, --OS(.dbd.O).sub.2R.sup.6,
--P(.dbd.O)(O.sup.-).sub.2, --P(.dbd.O)(OH)(O.sup.-),
--OP(.dbd.O).sub.2(O), --C(--O)R.sup.6, --C(.dbd.S)R.sup.6,
--C(.dbd.O)OR.sup.6, --C(.dbd.O)O.sup.-, --C(.dbd.S)OR.sup.6,
--NR.sup.6--C(.dbd.O)--N(R.sup.6).sub.2,
--NR.sup.6--C(.dbd.S)--N(R.sup.6).sub.2, and
--C(.dbd.NR.sup.6)NR.sup.6R.sup.6, wherein each X is independently
a halogen; and each R.sup.6 is independently hydrogen, halogen,
alkyl, aryl, arylalkyl, arylaryl, arylheteroalkyl, heteroaryl,
heteroarylalkyl, NR.sup.7R.sup.7, --C(.dbd.O)R.sup.7, and
--S(.dbd.O).sub.2R.sup.7; and each R.sup.7 is independently
hydrogen, alkyl, alkanyl, alkynyl, aryl, arylalkyl, arylheteralkyl,
arylaryl, heteroaryl or heteroarylalkyl. Aryl containing
substituents, whether or not having one or more substitutions, may
be attached in a para (p-), meta (m-) or ortho (o-) conformation,
or any combination thereof.
Gapped or Nicked dsRNA Molecules
[0092] This disclosure provides compounds, compositions, and
methods useful for altering expression or activity of a target gene
by RNA interference (RNAi) using small nucleic acid molecules. In
more detailed embodiments, this disclosure provides small nucleic
acid molecules, such as short interfering nucleic acid (siNA),
short interfering RNA (siRNA), double-stranded RNA (dsRNA), nicked
double-stranded RNA (ndsRNA), gapped double-stranded RNA (gdsRNA),
microRNA (miRNA), short hairpin RNA (shRNA) molecules, or any
combination thereof, which have a at least one nick or gap and
alter expression of a target gene or family of genes to prevent,
treat, or alleviate symptoms of a disease or disorder in a subject
(e.g., human). Within these and related therapeutic compositions
and methods, the use of nicked or gapped dsRNAs (which have been
optionally substituted or modified) will often improve properties
of the dsRNA molecules in comparison to the properties of native
dsRNA molecules, such as reduced off-target effects, reduced
interferon response, increased resistance to nuclease degradation
in vivo, improved cellular uptake, increased potency, or any
combination thereof.
[0093] In particular embodiments, there are provided methods of
treating or preventing diseases, disorders, or conditions related
to gene expression, including those related, or responsive, to the
level of a target nucleic acid molecule (e.g., mRNA) in a cell or
tissue, by administering a gapped dsRNA (mdRNA) molecule of this
disclosure, alone or in combination with an adjunctive therapy, in
an amount sufficient to activate target gene-specific RNAi. In one
embodiment, there is provided a method of treating or preventing a
disease or disorder by administering a dsRNA molecule that is
capable of target gene-specific RNAi, which dsRNA has at least one
substitution or modification as described herein and has a reduced
or minimal off-target effect.
[0094] The "percent identity" between two or more nucleic acid
sequences is a function of the number of identical positions shared
by the sequences (i.e., % identity=number of identical
positions/total number of positions.times.100), taking into account
the number of gaps, and the length of each gap that needs to be
introduced to optimize alignment of two or more sequences. The
comparison of sequences and determination of percent identity
between two or more sequences can be accomplished using a
mathematical algorithm, such as BLAST and Gapped BLAST programs at
their default parameters (e.g., Altschul et al., J. Mol. Biol.
215:403, 1990; see also BLASTN at www.ncbi.nlm.nih.gov/BLAST).
[0095] In one aspect, the instant disclosure provides a meroduplex
ribonucleic acid (mdRNA) molecule, comprising a first strand that
is complementary to a target mRNA, and a second strand and a third
strand that is each complementary to non-overlapping regions of the
first strand, wherein the second strand and third strands can
anneal with the first strand to form at least two double-stranded
regions separated by a gap of up to 10 nucleotides, and wherein (a)
at least one double-stranded region comprises from about 5 base
pairs to 13 base pairs, or (b) wherein the combined double-stranded
regions total about 15 base pairs to about 40 base pairs and the
mdRNA molecule comprises blunt ends; wherein at least one
pyrimidine of the mdRNA is substituted with a pyrimidine nucleoside
according to Formula I or II:
##STR00002##
wherein R.sup.1 and R.sup.2 are each independently a --H, --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2, or
heterocyclo group; R.sup.3 and R.sup.4 are each independently a
hydroxyl, a protected hydroxyl, a phosphate, or an internucleoside
linking group; and R.sup.5 and R.sup.8 are independently O or S. In
certain embodiments, at least one nucleoside is according to
Formula I in which R.sup.1 is methyl and R.sup.2 is --OH, or
R.sup.1 is methyl, R.sup.2 is --OH and R.sup.8 is S.
[0096] In other embodiments, the internucleoside linking group
covalently links from about 5 to about 40 nucleosides. In some
embodiments, the gap comprises at least one unpaired nucleotide in
the first strand positioned between the double-stranded regions
formed by the second and third strands when annealed to the first
strand, or the gap comprises a nick. In certain embodiments, the
nick or gap is located about 10 nucleotides from the 5'-end of the
first strand or at the Argonaute cleavage site. In another
embodiment, the meroduplex nick or gap is positioned such that the
thermal stability is maximized for the first and second strand
duplex and for the first and third strand duplex as compared to the
thermal stability of such meroduplexes having a nick or gap in a
different position--that is, the nick or gap is located in a
position wherein each of the two or more nicked or gapped strands
has a maximal melting temperature when annealed to the first strand
(i.e., T.sub.m or temperature at which 50% of one of the nicked or
gapped strands is annealed to the first strand).
[0097] As provided herein, any of the aspects or embodiments
disclosed herein would be useful in treating target gene associated
diseases or disorders, such as hyperproliferative disease (e.g.,
cervical cancer, ovarian cancer), angiogenic disorders (e.g., tumor
angiogenesis), or inflammatory disorders (e.g. rheumatoid
arthritis, rheumatoid arthritis, chronic obstructive bowel disease,
atherosclerosis), respiratory disease, pulmonary disease,
cardiovascular disease, autoimmune disease, allergic disorders,
neurologic disease, infectious disease (e.g., viral infection, such
as influenza), renal disease, transplant rejection, or any other
disease or condition that responds to modulation of a target gene
or gene family. In certain embodiments of the instant disclosure, a
single dsRNA can be used to knockdown mRNA expression of one or
more target gene family member.
[0098] In some embodiments, the dsRNA comprises at least three
strands in which the first strand comprises about 5 nucleotides to
about 40 nucleotides, and the second and third strands include
each, individually, about 5 nucleotides to about 20 nucleotides,
wherein the combined length of the second and third strands is
about 15 nucleotides to about 40 nucleotides. In other embodiments,
the dsRNA comprises at least two or three strands in which the
first strand comprises about 15 nucleotides to about 24 nucleotides
or about 25 nucleotides to about 40 nucleotides. In further
embodiments, the first strand will be complementary to a second
strand or a second and third strand or to a plurality of strands.
In further examples, the first strand and its complement(s) will be
able to form dsRNA or mdRNA molecules of this disclosure with about
19 to about 25 nucleotides of the first strand that is
complementary to at least one target gene mRNA.
[0099] For example, a Dicer substrate dsRNA can have about 25
nucleotides to about 40 nucleotides with only 19 nucleotides of the
antisense (first) strand being complementary to at least one target
gene family mRNA. In further embodiments, the first strand can have
complementarity with a target gene family mRNA in about 19
nucleotides to about 25 nucleotides and have one, two, or three
mismatches, or any combination thereof, with a target gene family
mRNA or any combination thereof, or the first strand of about 19
nucleotides to about 25 nucleotides (that, e.g., activates or is
capable of loading into or associating with RISC) can have at least
80% identity with the corresponding nucleotides found in at least
one target gene family mRNA, or any combination thereof.
Substituted and Modified Nicked or Gapped dsRNA Molecules
[0100] The introduction of substituted and modified nucleotides
into mdRNA and dsRNA molecules of this disclosure provides a tool
for overcoming potential limitations of in vivo stability and
bioavailability inherent to native RNA molecules (i.e., having
standard nucleotides) that are exogenously delivered. In certain
embodiments, the use of substituted or modified dsRNA molecules of
this disclosure can enable a lower dose of a particular nucleic
acid molecule for a given therapeutic effect since of dsRNA
molecules may be designed to have an increased melting temperature
or half-life in a subject or biological samples (e.g., serum).
Furthermore, certain substitutions or modifications can be used to
improve the bioavailability of dsRNA by targeting particular cells
or tissues or improving cellular uptake of the dsRNA molecules.
Therefore, even if the activity of a dsRNA molecule of this
disclosure is reduced as compared to a native RNA molecule, the
overall activity of the substituted or modified dsRNA molecule can
be greater than that of the native RNA molecule due to improved
stability or delivery of the molecule. The mdRNA structure may
result in a reduced interferon response, and substituted and
modified dsRNA can also minimize the possibility of activating an
interferon response in, for example, humans.
[0101] In certain embodiments, a dsRNA molecule of this disclosure
has at least one uridine, at least three uridines, or each and
every uridine (i.e., all uridines) of the first (antisense) strand
of the dsRNA substituted or replaced with 5-methyluridine or
2-thioribothymidine. In a related embodiment, the dsRNA molecule or
analog thereof of this disclosure has at least one uridine, at
least three uridines, or each and every uridine of the second
(sense) strand of the dsRNA substituted or replaced with
5-methyluridine or 2-thioribothymidine. In still another
embodiment, the dsRNA molecule or analog thereof of this disclosure
has at least one uridine, at least three uridines, or each and
every uridine of both the first (antisense) and second (sense)
strands of the dsRNA substituted or replaced with 5-methyluridine
or 2-thioribothymidine. In some embodiments, the double-stranded
region of a dsRNA molecule has at least three 5-methyluridines or
2-thioribothymidines. In certain embodiments, dsRNA molecules
comprise ribonucleotides at about 5% to about 95% of the nucleotide
positions in one strand, both strands, or any combination
thereof.
[0102] In further embodiments, a dsRNA molecule that decreases
expression of one or more target gene by RNAi according to the
instant disclosure further comprises one or more natural or
synthetic non-standard nucleoside. In related embodiments, the
non-standard nucleoside is one or more deoxyuridine, L- or D-locked
nucleic acid (LNA) molecule (e.g., a 5-methyluridine LNA) or
substituted LNA (e.g., having a pyrene), or a universal-binding
nucleotide, or a G clamp, or any combination thereof. In certain
embodiments, the universal-binding nucleotide can be C-phenyl,
C-naphthyl, inosine, azole carboxamide,
1-.beta.-D-ribofuranosyl-4-nitroindole,
1-.beta.-D-ribofuranosyl-5-nitroindole,
1-.beta.-D-ribofuranosyl-6-nitroindole, or
1-.beta.-D-ribofuranosyl-Substituted or modified nucleotides
present in dsRNA molecules, preferably in the antisense strand, but
also optionally in the sense or both the antisense and sense
strands, comprise modified or substituted nucleotides according to
this disclosure having properties or characteristics similar to
natural or standard ribonucleotides. For example, this disclosure
features dsRNA molecules including nucleotides having a Northern
conformation (e.g., Northern pseudorotation cycle, see, e.g.,
Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed.,
1984). As such, chemically modified nucleotides present in dsRNA
molecules of this disclosure, preferably in the antisense strand,
but also optionally in the sense or both the antisense and sense
strands, are resistant to nuclease degradation while at the same
time maintaining the capacity to mediate RNAi. Exemplary
nucleotides having a Northern configuration include locked nucleic
acid (LNA) nucleotides (e.g., 2'O, 4'-C-methylene-(D-ribofuranosyl)
nucleotides), 2'-methoxyethyl (MOE) nucleotides,
2'methyl-thio-ethyl, 2'deoxy-2'fluoro nucleotides, 2'deoxy-2'chloro
nucleotides, 2'azido nucleotides, 5-methyluridines, or 2'O-methyl
nucleotides. In certain embodiments, the LNA is a 5-methyluridine
LNA or 2-thioribothymidine LNA. In any of these embodiments, one or
more substituted or modified nucleotides can be a G clamp (e.g., a
cytosine analog that forms an additional hydrogen bond to guanine,
such as 9-(aminoethoxy)phenoxazine; see, e.g., Lin and Mateucci, J.
Am. Chem. Soc. 120:8531, 1998).
[0103] As described herein, the first and one or more second
strands of a dsRNA molecule or analog thereof provided by this
disclosure can anneal or hybridize together (i.e., due to
complementarity between the strands) to form at least one
double-stranded region having a length of about 4 to about 10 base
pairs, about 5 to about 13 base pairs, or about 15 to about 40 base
pairs. In some embodiments, the dsRNA has at least one
double-stranded region ranging in length from about 15 to about 24
base pairs or about 19 to about 23 base pairs. In other
embodiments, the dsRNA has at least one double-stranded region
ranging in length from about 26 to about 40 base pairs or about 27
to about 30 base pairs or about 30 to about 35 base pairs. In other
embodiments, the two or more strands of a dsRNA molecule of this
disclosure may optionally be covalently linked together by
nucleotide or non-nucleotide linker molecules.
[0104] In certain embodiments, the dsRNA molecule or analog thereof
comprises an overhang of one to four nucleotides on one or both
3'-ends of the dsRNA, such as an overhang comprising a
deoxyribonucleotide or two deoxyribonucleotides (e.g., thymidine,
adenine). In certain embodiments, the 3'-end comprising one or more
deoxyribonucleotide is in an mdRNA molecule and is either in the
gap, not in the gap, or any combination thereof. In some
embodiments, dsRNA molecules or analogs thereof have a blunt end at
one or both ends of the dsRNA. In certain embodiments, the 5'-end
of the first or second strand is phosphorylated. In any of the
embodiments of dsRNA molecules described herein, the 3'-terminal
nucleotide overhangs can comprise ribonucleotides or
deoxyribonucleotides that are chemically-modified at a nucleic acid
sugar, base, or backbone. In any of the embodiments of dsRNA
molecules described herein, the 3'-terminal nucleotide overhangs
can comprise one or more universal base ribonucleotides. In any of
the embodiments of dsRNA molecules described herein, the
3'-terminal nucleotide overhangs can comprise one or more acyclic
nucleotides. In any of the embodiments of dsRNA molecules described
herein, the dsRNA can further comprise a terminal phosphate group,
such as a 5'phosphate (see Martinez et al., Cell 110:563, 2002; and
Schwarz et al., Molec. Cell 10:537, 2002) or a 5'3'diphosphate.
[0105] As set forth herein, the terminal structure of dsRNAs of
this disclosure that decrease expression of one or more target gene
by, for example, RNAi may either have blunt ends or one or more
overhangs. In certain embodiments, the overhang may be at the 3'end
or the 5'end. The total length of dsRNAs having overhangs is
expressed as the sum of the length of the paired double-stranded
portion together with the overhanging nucleotides. For example, if
a 19 base pair dsRNA has a two nucleotide overhang at both ends,
the total length is expressed as 21-mer. Furthermore, since the
overhanging sequence may have low specificity to one or more target
gene, it is not necessarily complementary (antisense) or identical
(sense) to a target gene sequence. In further embodiments, a dsRNA
of this disclosure that decreases expression of one or more target
gene by RNAi may further comprise a low molecular weight structure
(e.g., a natural RNA molecule such as a tRNA, rRNA or viral RNA, or
an artificial RNA molecule) at, for example, one or more
overhanging portion of the dsRNA.
[0106] In further embodiments, a dsRNA molecule that decreases
expression of one or more target gene by RNAi according to the
instant disclosure comprises a 2'-sugar substitution, such as a
2'-deoxy, 2'-O-2-methoxyethyl, 2'-O-methoxyethyl, 2'-O-methyl,
halogen, 2'-fluoro, 2'-O-allyl, or the like, or any combination
thereof. In still further embodiments, a dsRNA molecule that
decreases expression of one or more target gene by RNAi according
to the instant disclosure further comprises a terminal cap
substituent on one or both ends of the first strand or one or more
second strands, such as an alkyl, abasic, deoxy abasic, glyceryl,
dinucleotide, acyclic nucleotide, inverted deoxynucleotide moiety,
or any combination thereof. In certain embodiments, at least one or
two 5'-terminal ribonucleotides of the sense strand within the
double-stranded region have a 2'-sugar substitution. In certain
other embodiments, at least one or two 5'-terminal ribonucleotides
of the antisense strand within the double-stranded region have a
2'-sugar substitution. In certain embodiments, at least one or two
5'-terminal ribonucleotides of the sense strand and the antisense
strand within the double-stranded region have a 2'-sugar
substitution.
[0107] In other embodiments, a dsRNA molecule that decreases
expression of one or more target gene by RNAi according to the
instant disclosure comprises one or more substitutions in the sugar
backbone, including any combination of ribosyl, 2'-deoxyribosyl, a
tetrofuranosyl (e.g., L-.alpha.-threofuranosyl), a hexopyranosyl
(e.g., .beta.-allopyranosyl, .beta.-altropyranosyl, and
(3-glucopyranosyl), a pentopyranosyl (e.g., .beta.-ribopyranosyl,
.alpha.-lyxopyranosyl, .beta.-xylopyranosyl, and
.alpha.-arabinopyranosyl), a carbocyclic (carbon only ring) analog,
a pyranose, a furanose, a morpholino, or analogs or derivatives
thereof.
[0108] In yet other embodiments, a dsRNA molecule that decreases
expression of one or more target gene by RNAi according to the
instant disclosure comprises at least one modified internucleoside
linkage, such as independently a phosphorothioate, chiral
phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate,
3'-alkylene phosphonate, 5'-alkylene phosphonate, chiral
phosphonate, phosphonoacetate, thiophosphonoacetate, phosphinate,
phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester,
selenophosphate, boranophosphate linkage, or any combination
thereof.
[0109] A modified internucleotide linkage, as described herein, can
be present in one or more strands of a dsRNA molecule of this
disclosure, for example, in the sense strand, the antisense strand,
both strands, or a plurality of strands (e.g., in an mdRNA). The
dsRNA molecules of this disclosure can comprise one or more
modified internucleotide linkages at the 3'end, the 5'end, or both
of the 3' and 5'ends of the sense strand or the antisense strand or
both strands. In one embodiment, a dsRNA molecule capable of
decreasing expression of one or more target gene by RNAi has one
modified internucleotide linkage at the 3'-end, such as a
phosphorothioate linkage. For example, this disclosure provides a
dsRNA molecule capable of decreasing expression of one or more
target gene by RNAi having about 1 to about 8 or more
phosphorothioate internucleotide linkages in one dsRNA strand. In
yet another embodiment, this disclosure provides a dsRNA molecule
capable of decreasing expression of one or more target gene by RNAi
having about 1 to about 8 or more phosphorothioate internucleotide
linkages in both dsRNA strands. In other embodiments, an exemplary
dsRNA molecule of this disclosure can comprise from about 1 to
about 5 or more consecutive phosphorothioate internucleotide
linkages at the 5'end of the sense strand, the antisense strand,
both strands, or a plurality of strands. In another example, an
exemplary dsRNA molecule of this disclosure can comprise one or
more pyrimidine phosphorothioate internucleotide linkages in the
sense strand, the antisense strand, two strands, or a plurality of
strands. In yet another example, an exemplary dsRNA molecule of
this disclosure can comprise one or more purine phosphorothioate
internucleotide linkages in the sense strand, the antisense strand,
two strands, or a plurality of strands.
[0110] Many exemplary modified nucleotide bases or analogs thereof
useful in the dsRNA of the instant disclosure include
5-methylcytosine; 5-hydroxymethylcytosine; xanthine; hypoxanthine;
2-aminoadenine; 6-methyl, 2-propyl, or other alkyl derivatives of
adenine and guanine; 8-substituted adenines and guanines (such as
8-aza, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, or the
like); 7-methyl, 7-deaza, and 3-deaza adenines and guanines;
2-thiouracil; 2-thiothymine; 2-thiocytosine; 5-methyl, 5-propynyl,
5-halo (such as 5-bromo or 5-fluoro), 5-trifluoromethyl, or other
5-substituted uracils and cytosines; and 6-azouracil. Further
useful nucleotide bases can be found in Kurreck, Eur. J. Biochem.
270:1628, 2003; Herdewijn, Antisense Nucleic Acid Develop. 10:297,
2000; Concise Encyclopedia of Polymer Science and Engineering,
pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990;
U.S. Pat. No. 3,687,808, and similar references.
[0111] Certain nucleotide base moieties are particularly useful for
increasing the binding affinity of the dsRNA molecules of this
disclosure to complementary targets. These include 5-substituted
pyrimidines; 6-azapyrimidines; and N-2, N-6, or O-6 substituted
purines (including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine). Further, for example, 5-methyluridine and
5-methylcytosine substitutions are known to increase nucleic acid
duplex stability, which can be combined with 2'-sugar modifications
(such as 2'-methoxy or 2'-methoxyethyl) or internucleoside linkages
(e.g., phosphorothioate) that provide the desired nuclease
resistance to the modified or substituted dsRNA.
[0112] In another aspect of the instant disclosure, there is
provided a dsRNA that decreases expression of one or more target
gene, comprising a first strand that is complementary to a target
gene mRNA, or any combination thereof, and a second strand that is
complementary to the first strand, wherein the first and second
strands form a double-stranded region of about 15 to about 40 base
pairs; wherein at least one pyrimidine of the dsRNA is substituted
with a pyrimidine nucleoside according to Formula I or II:
##STR00003##
wherein R.sup.1 and R.sup.2 are each independently a --H, --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2, or
heterocyclo group; R.sup.3 and R.sup.4 are each independently a
hydroxyl, a protected hydroxyl, or an internucleoside linking
group; and R.sup.5 and R.sup.8 are independently O or S. In certain
embodiments, at least one nucleoside is according to Formula I in
which R.sup.1 is methyl and R.sup.2 is --OH or R.sup.1 is methyl,
R.sup.2 is --OH, and R.sup.8 is S. In other embodiments, the
internucleoside linking group covalently links from about 5 to
about 40 nucleosides.
[0113] In certain embodiments, the first and one or more second
strands of a dsRNA, which decreases expression of one or more
target gene by RNAi and has at least one pyrimidine substituted
with a pyrimidine nucleoside according to Formula I or II, can
anneal or hybridize together (i.e., due to complementarity between
the strands) to form at least one double-stranded region having a
length or a combined length of about 15 to about 40 base pairs. In
some embodiments, the dsRNA has at least one double-stranded region
ranging in length from about 4 base pairs to about 10 base pairs or
about 5 to about 13 base pairs or about 15 to about 25 base pairs
or about 19 to about 23 base pairs. In other embodiments, the dsRNA
has at least one double-stranded region ranging in length from
about 26 to about 40 base pairs or about 27 to about 30 base pairs
or about 30 to about 35 base pairs. In certain embodiments, the
dsRNA molecule or analog thereof has an overhang of one to four
nucleotides on one or both 3'-ends, such as an overhang comprising
a deoxyribonucleotide or two deoxyribonucleotides (e.g.,
thymidine). In some embodiments, dsRNA molecule or analog thereof
has a blunt end at one or both ends of the dsRNA. In certain
embodiments, the 5'-end of the first or second strand is
phosphorylated.
[0114] In certain embodiments, at least one R.sup.1 is a
C.sub.1-C.sub.5 alkyl, such as methyl or ethyl. Within other
exemplary embodiments of this disclosure, compounds of Formula I
are a 5-alkyluridine (i.e., R.sup.1 is alkyl, R.sup.2 is --OH, and
R.sup.3, R.sup.4, and R.sup.5 are as defined herein) or compounds
of Formula II are a 5-alkylcytidine (i.e., R.sup.1 is alkyl,
R.sup.2 is --OH, and R.sup.3, R.sup.4, and R.sup.5 are as defined
herein). In related embodiments, the 5-alkyluridine is a
5-methyluridine (also referred to as ribothymidine or `t` or
`T.sup.r`--i.e., R.sup.1 is methyl and R.sup.2 is --OH), and the
5-alkylcytidine is a 5-methylcytidine. In other embodiments, at
least one, at least three, or all uridines of the first strand of
the dsRNA are replaced with 5-methyluridine, or at least one, at
least three, or all uridines of the second strand of the dsRNA are
replaced with 5-methyluridine, or any combination thereof (e.g.,
such changes are made on both strands). In certain embodiments, at
least one pyrimidine nucleoside of Formula I or Formula II has an
R.sup.5 that is S or R.sup.8 that is S.
[0115] In further embodiments, at least one pyrimidine nucleoside
of the dsRNA is a locked nucleic acid (LNA) in the form of a
bicyclic sugar, wherein R.sup.2 is oxygen, and the 2'O and 4'C form
an oxymethylene bridge on the same ribose ring. In a related
embodiment, the LNA comprises a base substitution, such as a
5-methyluridine LNA or 2-thio-5-methyluridine LNA. In other
embodiments, at least one, at least three, or all uridines of the
first strand of the dsRNA are replaced with 5-methyluridine or
2-thioribothymidine or 5-methyluridine LNA or
2-thio-5-methyluridine LNA, or at least one, at least three, or all
uridines of the second strand of the dsRNA are replaced with
5-methyluridine, 2-thioribothymidine, 5-methyluridine LNA,
2-thio-5-methyluridine LNA, or any combination thereof (e.g., such
changes are made on both strands, or some substitutions include
5-methyluridine only, 2-thioribothymidine only, 5-methyluridine LNA
only, 2-thio-5-methyluridine LNA only, or one or more
5-methyluridine or 2-thioribothymidine with one or more
5-methyluridine LNA or 2-thio-5-methyluridine LNA).
[0116] In further embodiments, a ribose of the pyrimidine
nucleoside or the internucleoside linkage can be optionally
modified. For example, compounds of Formula I or II are provided
wherein R.sup.2 is alkoxy, such as a 2'O-methyl substitution (e.g.,
which may be in addition to a 5-alkyluridine or a 5-alkylcytidine,
respectively). In certain embodiments, R.sup.2 is selected from
2'O--(C.sub.1-C.sub.5) alkyl, 2'O-methyl,
2'OCH.sub.2OCH.sub.2CH.sub.3, 2'OCH.sub.2CH.sub.2OCH.sub.3,
2'O-allyl, or 2'-fluoro. In further embodiments, one or more of the
pyrimidine nucleosides are according to Formula I in which R.sup.1
is methyl and R.sup.2 is a 2'O--(C.sub.1-C.sub.5) alkyl (e.g.,
2'O-methyl), or in which R.sup.1 is methyl, R.sup.2 is a
2'O--(C.sub.1-C.sub.5) alkyl (e.g., 2'O-methyl), and R.sup.5 or
R.sup.8 is S, or any combination thereof. In other embodiments, one
or more, or at least two, pyrimidine nucleosides according to
Formula I or II have an R.sup.2 that is not --H or --OH and is
incorporated at a 3'end or 5'end and not within the gap of one or
more strands within the double-stranded region of the dsRNA
molecule.
[0117] In further embodiments, a dsRNA molecule or analog thereof
comprising a pyrimidine nucleoside according to Formula I or
Formula II in which R.sup.2 is not --H or --OH and an overhang,
further comprises at least two of pyrimidine nucleosides that are
incorporated either at a 3'end or a 5'end or both of one strand or
two strands within the double-stranded region of the dsRNA
molecule. In a related embodiment, at least one of the at least two
pyrimidine nucleosides in which R.sup.2 is not --H or --OH is
located at a 3'end or a 5'end within the double-stranded region of
at least one strand of the dsRNA molecule, and wherein at least one
of the at least two pyrimidine nucleosides in which R.sup.2 is not
--H or --OH is located internally within a strand of the dsRNA
molecule. In still further embodiments, a dsRNA molecule or analog
thereof that has an overhang has a first of the two or more
pyrimidine nucleosides in which R.sup.2 is not --H or --OH that is
incorporated at a 5'end within the double-stranded region of the
sense strand of the dsRNA molecule and a second of the two or more
pyrimidine nucleosides is incorporated at a 5'end within the
double-stranded region of the antisense strand of the dsRNA
molecule. In any of these embodiments, one or more substituted or
modified nucleotides can be a G clamp (e.g., a cytosine analog that
forms an additional hydrogen bond to guanine, such as
9-(aminoethoxy)phenoxazine; see, e.g., Lin and Mateucci, 1998). In
any of these embodiments, provided the one or more pyrimidine
nucleosides are not within the gap.
[0118] In yet other embodiments, a dsRNA molecule or analog thereof
of Formula I or II according to the instant disclosure that has an
overhang comprises four or more independent pyrimidine nucleosides
or four or more independent pyrimidine nucleosides in which R.sup.2
is not --H or --OH, wherein (a) a first pyrimidine nucleoside is
incorporated into a 3'end within the double-stranded region of the
sense (second) strand of the dsRNA, (b) a second pyrimidine
nucleoside is incorporated into a 5'end within the double-stranded
region of the sense (second) strand, (c) a third pyrimidine
nucleoside is incorporated into a 3'end within the double-stranded
region of the antisense (first) strand of the dsRNA, and (d) a
fourth pyrimidine nucleoside is incorporated into a 5'end within
the double-stranded region of the antisense (first) strand. In any
of these embodiments, provided the one or more pyrimidine
nucleosides are not within the gap.
[0119] In further embodiments, a dsRNA molecule or analog thereof
comprising a pyrimidine nucleoside according to Formula I or
Formula II in which R.sup.2 is not --H or --OH and is blunt-ended,
further comprises at least two of pyrimidine nucleosides that are
incorporated either at a 3'end or a 5'end or both of one strand or
two strands of the dsRNA molecule. In a related embodiment, at
least one of the at least two pyrimidine nucleosides in which
R.sup.2 is not --H or --OH is located at a 3'end or a 5'end of at
least one strand of the dsRNA molecule, and wherein at least one of
the at least two pyrimidine nucleosides in which R.sup.2 is not --H
or --OH is located internally within a strand of the dsRNA
molecule. In still further embodiments, a dsRNA molecule or analog
thereof that is blunt-ended has a first of the two or more
pyrimidine nucleosides in which R.sup.2 is not --H or --OH that is
incorporated at a 5'end of the sense strand of the dsRNA molecule
and a second of the two or more pyrimidine nucleosides is
incorporated at a 5'end of the antisense strand of the dsRNA
molecule. In any of these embodiments, provided the one or more
pyrimidine nucleosides are not within the gap.
[0120] In yet other embodiments, a dsRNA molecule comprising a
pyrimidine nucleoside according to Formula I or II and that is
blunt-ended comprises four or more independent pyrimidine
nucleosides or four or more independent pyrimidine nucleosides in
which R.sup.2 is not --H or --OH, wherein (a) a first pyrimidine
nucleoside is incorporated into a 3'end within the double-stranded
region of the sense (second) strand of the dsRNA, (b) a second
pyrimidine nucleoside is incorporated into a 5'end within the
double-stranded region of the sense (second) strand, (c) a third
pyrimidine nucleoside is incorporated into a 3'end within the
double-stranded region of the antisense (first) strand of the
dsRNA, and (d) a fourth pyrimidine nucleoside is incorporated into
a 5'end within the double-stranded region of the antisense (first)
strand. In any of these embodiments, provided the one or more
pyrimidine nucleosides are not within the gap.
[0121] In still further embodiments, a dsRNA molecule or analog
thereof of Formula I or II according to the instant disclosure
further comprises a terminal cap substituent on one or both ends of
the first strand or second strand, such as an alkyl, abasic, deoxy
abasic, glyceryl, dinucleotide, acyclic nucleotide, inverted
deoxynucleotide moiety, or any combination thereof. In further
embodiments, one or more internucleoside linkage can be optionally
modified. For example, a dsRNA molecule or analog thereof of
Formula I or II according to the instant disclosure wherein at
least one internucleoside linkage is modified to a
phosphorothioate, chiral phosphorothioate, phosphorodithioate,
phosphotriester, aminoalkylphosphotriester, methyl phosphonate,
alkyl phosphonate, 3'-alkylene phosphonate, 5'-alkylene
phosphonate, chiral phosphonate, phosphonoacetate,
thiophosphonoacetate, phosphinate, phosphoramidate, 3'-amino
phosphoramidate, aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester,
selenophosphate, boranophosphate linkage, or any combination
thereof.
[0122] In still another embodiment, provided is a nicked or gapped
dsRNA molecule (ndsRNA or gdsRNA, respectively) that decreases
expression of one or more target gene by RNAi, which comprises a
first strand that is complementary to a target gene mRNA, or any
combination thereof, and two or more second strands that are
complementary to the first strand, wherein the first and at least
one of the second strands form a non-overlapping double-stranded
region of about 5 to about 13 base pairs. Any of the aforementioned
substitutions or modifications is contemplated within this
embodiment as well.
[0123] In another exemplary of this disclosure, the dsRNAs comprise
at least two or more substituted pyrimidine nucleosides can each be
independently selected wherein R.sup.1 comprises any chemical
modification or substitution as contemplated herein, for example,
an alkyl (e.g., methyl), halogen, hydroxy, alkoxy, nitro, amino,
trifluoromethyl, cycloalkyl, (cycloalkyl)alkyl, alkanoyl,
alkanoyloxy, aryl, aroyl, aralkyl, nitrile, dialkylamino, alkenyl,
alkynyl, hydroxyalkyl, aminoalkyl, alkylaminoalkyl,
dialkylaminoalkyl, haloalkyl, carboxyalkyl, alkoxyalkyl, carboxy,
carbonyl, alkanoylamino, carbamoyl, carbonylamino,
alkylsulfonylamino, or heterocyclo group. When two or more modified
ribonucleotides are present, each modified ribonucleotide can be
independently modified to have the same, or different, modification
or substitution at R.sup.1 or R.sup.2.
[0124] In other detailed embodiments, one or more substituted
pyrimidine nucleosides according to Formula I or II can be located
at any ribonucleotide position, or any combination of
ribonucleotide positions, on either or both of the sense and
antisense strands of a dsRNA molecule of this disclosure, including
at one or more multiple terminal positions as noted above, or at
any one or combination of multiple non-terminal ("internal")
positions. In this regard, each of the sense and antisense strands
can incorporate about 1 to about 6 or more of the substituted
pyrimidine nucleosides.
[0125] In certain embodiments, when two or more substituted
pyrimidine nucleosides are incorporated within a dsRNA of this
disclosure, at least one of the substituted pyrimidine nucleosides
will be at a 3' or 5'end of one or both strands, and in certain
embodiments at least one of the substituted pyrimidine nucleosides
will be at a 5'end of one or both strands. In other embodiments,
the substituted pyrimidine nucleosides are located at a position
corresponding to a position of a pyrimidine in an unmodified dsRNA
that is constructed as a homologous sequence for targeting a
cognate mRNA, as described herein.
[0126] In addition, the terminal structure of the dsRNAs of this
disclosure may have a stem-loop structure in which ends of one side
of the dsRNA molecule are connected by a linker nucleic acid, e.g.,
a linker RNA. The length of the double-stranded region (stem-loop
portion) can be, for example, about 15 to about 49 basepairs (bp),
about 15 to about 35 bp, or about 21 to about 30 bp long.
Alternatively, the length of the double-stranded region that is a
final transcription product of dsRNAs to be expressed in a target
cell may be, for example, approximately about 15 to about 49 bp,
about 15 to about 35 bp, or about 21 to about 30 bp long. When
linker segments are employed, there is no particular limitation in
the length of the linker as long as it does not hinder pairing of
the stem portion. For example, for stable pairing of the stem
portion and suppression of recombination between DNAs coding for
this portion, the linker portion may have a clover-leaf tRNA
structure. Even if the linker has a length that would hinder
pairing of the stem portion, it is possible, for example, to
construct the linker portion to include introns so that the introns
are excised during processing of a precursor RNA into mature RNA,
thereby allowing pairing of the stem portion. In the case of a
stem-loop dsRNA, either end (head or tail) of RNA with no loop
structure may have a low molecular weight RNA. As described above,
these low molecular weight RNAs may include a natural RNA molecule,
such as tRNA, rRNA or viral RNA, or an artificial RNA molecule.
[0127] A dsRNA molecule may be comprised of a circular nucleic acid
molecule, wherein the dsRNA is about 38 to about 70 nucleotides in
length having from about 18 to about 23 base pairs (e.g., about 19
to about 21) wherein the circular oligonucleotide forms a dumbbell
shaped structure having about 19 base pairs and two loops. In
certain embodiments, a circular dsRNA molecule contains two loop
motifs, wherein one or both loop portions of the dsRNA molecule is
biodegradable. For example, a circular dsRNA molecule of this
disclosure is designed such that degradation of the loop portions
of the dsRNA molecule in vivo can generate a double-stranded dsRNA
molecule with 3'terminal overhangs, such as 3'terminal nucleotide
overhangs comprising from about 1 to about 4 (unpaired)
nucleotides.
[0128] Substituting pyrimidine nucleosides into a dsRNA according
to this disclosure can be chosen to increase resistance to
enzymatic degradation, such as exonucleolytic degradation,
including 5'exonucleolytic or 3'exonucleolytic degradation. As
such, the dsRNAs described herein will exhibit significant
resistance to enzymatic degradation compared to a corresponding
dsRNA having standard nucleotides, and will thereby possess greater
stability, increased half-life, and greater bioavailability in
physiological environments (e.g., when introduced into a eukaryotic
target cell). In addition to increasing resistance of the
substituted or modified dsRNAs to exonucleolytic degradation, the
incorporation of one or more pyrimidine nucleosides according to
Formula I or II will render dsRNAs can make these molecules more
stable and bioavailable than otherwise identical dsRNAs that do not
include the substitutions or modifications. In related aspects of
this disclosure, dsRNA substitutions or modifications described
herein are chosen to improve stability of a modified dsRNA for use
within research, diagnostic and treatment methods wherein the
modified dsRNA is contacted with a biological sample, for example,
a mammalian cell, intracellular compartment, serum or other
extracellular fluid, tissue, or other in vitro or in vivo
physiological compartment or environment. In one embodiment,
diagnosis is performed on an isolated biological sample. In another
embodiment, the diagnostic method is performed in vitro. In a
further embodiment, the diagnostic method is not performed
(directly) on a human or animal body.
[0129] In addition to increasing stability of substituted or
modified dsRNAs, incorporation of one or more pyrimidine
nucleosides according to Formula I or II in a dsRNA designed for
gene silencing can be used to yield additional desired functional
results, including increasing a melting point of a substituted or
modified dsRNA compared to a corresponding, unmodified dsRNA. By
thus increasing a dsRNA melting point, the subject substitutions or
modifications will often block or reduce the occurrence or extent
of partial dehybridization of the substituted or modified dsRNA
(that would ordinarily occur and render the unmodified dsRNA more
vulnerable to degradation by certain exonucleases), thereby
increasing the stability of the substituted or modified dsRNA.
[0130] In another aspect of this disclosure, the mdRNA structure
can be used to reduce off-target effects, which can be improved
with substitutions or modifications described herein, when the
mdRNA are contacted with a biological sample (e.g., when introduced
into a target eukaryotic cell having specific, and non-specific
mRNA species present as potential specific and non-specific
targets). Similarly, the mdRNA structure can be used to reduce
interferon activation, which can be improved with substitutions or
modifications described herein, when the mdRNA is contacted with a
biological sample, for example, when introduced into a eukaryotic
cell. Hence, substituted or modified dsRNAs (mdRNAs) according to
this disclosure are employed in methods of gene silencing, wherein
the substituted or modified dsRNAs exhibit reduced or undetectable
off-target effects or reduced interferon response compared to a
corresponding dsRNA lack a nick or gap or modification.
[0131] In further embodiments, dsRNAs of this disclosure can
comprise one or more sense (second) strand that is homologous or
corresponds to a sequence of a target gene and an antisense (first)
strand that is complementary to the sense strand and a sequence of
the target gene. In exemplary embodiments, at least one strand of
the dsRNA incorporates one or more pyrimidines substituted
according to Formula I or II (e.g., wherein the pyrimidine is
replaced by one ore more 5-methyluridines or 2-thioribothymidines,
the ribose is modified to incorporate one or more 2'-O-methyl
substitutions, or any combination thereof). These and other
multiple substitutions or modifications according to Formula I or
II can be introduced into one or more pyrimidines, or into any
combination and up to all pyrimidines present in one or both
strands of a dsRNA, so long as the dsRNA retains RNAi activity.
[0132] In any of the embodiments described herein, the dsRNA may
include multiple modifications. For example, a dsRNA having at
least one ribothymidine or 2-thioribothymidine may further comprise
at least one LNA, 2'-methoxy, 2'-fluoro, 2'-deoxy, phosphorothioate
linkage, an inverted base terminal cap, or any combination thereof.
In certain embodiments, a dsRNA will have from one to all uridines
substituted with ribothymidine and have up to about 75% LNA
substitutions. In other embodiments, a dsRNA will have from one to
all uridines substituted with ribothymidine and have up to about
75% 2'-methoxy substitutions (and not at the Argonaute cleavage
site). In still other embodiments, a dsRNA will have from one to
all uridines substituted with ribothymidine and have up to about
100% 2'-fluoro substitutions. In further embodiments, a dsRNA will
have from one to all uridines substituted with ribothymidine and
have up to about 75% 2'-deoxy substitutions. In further
embodiments, a dsRNA will have up to about 75% LNA substitutions
and have up to about 75% 2'-methoxy substitutions. In still other
embodiments, a dsRNA will have up to about 75% LNA substitutions
and have up to about 100% 2'-fluoro substitutions. In further
embodiments, a dsRNA will have up to about 75% LNA substitutions
and have up to about 75% 2'-deoxy substitutions. In further
embodiments, a dsRNA will have up to about 75% 2'-methoxy
substitutions and have up to about 100% 2'-fluoro substitutions. In
further embodiments, a dsRNA will have up to about 75% 2'-methoxy
substitutions and have up to about 75% 2'-deoxy substitutions. In
further embodiments, a dsRNA will have up to about 100% 2'-fluoro
substitutions and have up to about 75% 2'-deoxy substitutions.
[0133] In further multiple modification embodiments, a dsRNA will
have from one to all uridines substituted with ribothymidine, up to
about 75% LNA substitutions, and up to about 75% 2'-methoxy
substitutions. In still further embodiments, a dsRNA will have from
one to all uridines substituted with ribothymidine, up to about 75%
LNA substitutions, and up to about 100% 2'-fluoro substitutions. In
further embodiments, a dsRNA will have from one to all uridines
substituted with ribothymidine, up to about 75% LNA substitutions,
and up to about 75% 2'-deoxy substitutions. In further embodiments,
a dsRNA will have from one to all uridines substituted with
ribothymidine, up to about 75% 2'-methoxy substitutions, and up to
about 75% 2'-fluoro substitutions. In further embodiments, a dsRNA
will have from one to all uridines substituted with ribothymidine,
up to about 75% 2'-methoxy substitutions, and up to about 75%
2'-deoxy substitutions. In further embodiments, a dsRNA will have
from one to all uridines substituted with ribothymidine, up to
about 100% 2'-fluoro substitutions, and up to about 75% 2'-deoxy
substitutions. In yet further embodiments, a dsRNA will have from
one to all uridines substituted with ribothymidine, up to about 75%
LNA substitutions, up to about 75% 2'-methoxy, up to about 100%
2'-fluoro, and up to about 75% 2'-deoxy substitutions. In other
embodiments, a dsRNA will have up to about 75% LNA substitutions,
up to about 75% 2'-methoxy substitutions, and up to about 100%
2'-fluoro substitutions. In further embodiments, a dsRNA will have
up to about 75% LNA substitutions, up to about 75% 2'-methoxy
substitutions, and up to about 75% 2'-deoxy substitutions. In
further embodiments, a dsRNA will have up to about 75% LNA
substitutions, up to about 100% 2'-fluoro substitutions, and up to
about 75% 2'-deoxy substitutions. In still further embodiments, a
dsRNA will have up to about 75% 2'-methoxy, up to about 100%
2'-fluoro, and up to about 75% 2'-deoxy substitutions.
[0134] In any of these multiple modification embodiments, the dsRNA
may further comprise up to 100% phosphorothioate internucleoside
linkages, from one to ten or more inverted base terminal caps, or
any combination thereof. Additionally, any of these multiple
modification embodiments may have these multiple modifications on
one strand, two strands, three strands, a plurality of strand, or
all strands. Finally, in any of these multiple modification dsRNA,
the dsRNA must retain gene silencing activity.
[0135] Within certain aspects, the present disclosure provides
dsRNA that decreases expression of one or more target gene by RNAi,
and compositions comprising one or more dsRNA, wherein at least one
dsRNA comprises one or more universal-binding nucleotide(s) in the
first, second or third position in the anti-codon of the antisense
strand of the dsRNA duplex and wherein the dsRNA is capable of
specifically binding to one or more target sequence, such as an RNA
expressed by a target cell. In cases wherein the sequence of a
target RNA includes one or more single nucleotide substitutions,
dsRNA comprising a universal-binding nucleotide retains its
capacity to specifically bind a target RNA, thereby mediating gene
silencing and, as a consequence, overcoming escape of the target
from dsRNA-mediated gene silencing. Non-limiting examples of
universal-binding nucleotides that may be suitably employed in the
compositions and methods disclosed herein include inosine,
1-.beta.-D-ribofuranosyl-5-nitroindole, and
1-.beta.-D-ribofuranosyl-3-nitropyrrole. For the purpose of the
present disclosure, a universal-binding nucleotide is a nucleotide
that can form a hydrogen bonded nucleotide pair with more than one
nucleotide type.
[0136] Non-limiting examples for the above compositions includes
modifying the anti-codons for tyrosine (AUA) or phenylalanine (AAA
or GAA), cysteine (ACA or GCA), histidine (AUG or GUG), asparagine
(AUU or GUU), isoleucine (UAU) and aspartate (AUC or GUC) within
the anti-codon of the antisense strand of the dsRNA molecule.
[0137] For example, within certain embodiments, the isoleucine
anti-codon UAU, for which AUA is the cognate codon, may be modified
such that the third-position uridine (U) nucleotide is substituted
with the universal-binding nucleotide inosine I to create the
anti-codon UAI. Inosine is an exemplary universal-binding
nucleotide that can pair with an adenosine (A), uridine (U), and
cytidine (C) nucleotide, but not guanosine (G). This modified
anti-codon UAI increases the specific-binding capacity of the dsRNA
molecule and thus permits the dsRNA to pair with mRNAs having any
one of AUA, UUA, and CUA in the corresponding position of the
coding strand thereby expanding the number of available RNA
degradation targets to which the dsRNA may specifically bind.
[0138] Alternatively, the anti-codon AUA may also or alternatively
be modified by substituting a universal-binding nucleotide in the
third or second position of the anti-codon such that the
anti-codon(s) represented by UAI (third position substitution) or
UIU (second position substitution) to generate dsRNA that are
capable of specifically binding to AUA, CUA and UUA and AAA, ACA
and AUA.
[0139] In certain aspects, dsRNA disclosed herein can include
between about 1 universal-binding nucleotide and about 10
universal-binding nucleotides. Within certain aspects, the
presently disclosed dsRNA may comprise a sense strand that is
homologous to a sequence of one or more target gene and an
antisense strand that is complementary to the sense strand, with
the proviso that at least one nucleotide of the antisense strand of
the otherwise complementary dsRNA duplex is replaced by one or more
universal-binding nucleotide.
[0140] By way of background, within the silencing complex, the
dsRNA molecule is positioned so that it can interact or bind to a
target RNA. The RISC will encounter thousands of different RNAs
that are in a typical cell at any given moment. But, the dsRNA
loaded in RISC will specifically anneal with a target RNA that has
close complementarity with the antisense of the dsRNA molecule. So,
unlike an interferon response to a viral infection, the silencing
complex is highly selective in identifying a target RNA. RISC
cleaves the captured target RNA strand and releases the two pieces
of the RNA (now rendered incapable of directing protein synthesis)
and moves on. RISC itself stays intact and is capable of finding
and cleaving additional target RNA molecules.
[0141] It will be understood that, regardless of the position at
which the one or more universal-binding nucleotide is substituted,
the dsRNA molecule is capable of binding to a target gene and one
or more variant(s) thereof thereby facilitating the degradation of
the target gene or variant thereof via Dicer or RISC. Thus, the
dsRNA of the present disclosure are suitable for introduction into
cells to mediate targeted post-transcriptional gene silencing of
one or more target gene or variants thereof. When a dsRNA is
inserted into a cell, the dsRNA duplex is then unwound, and the
antisense strand anneals with mRNA to form a Dicer substrate or the
antisense strand is loaded into an assembly of proteins to form the
RNA-induced silencing complex (RISC).
Synthesis of Gapped or Nicked dsRNA Molecules
[0142] Exemplary molecules of the instant disclosure are
recombinantly produced, chemically synthesized, or a combination
thereof. 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., Methods in Enzymol. 211:3, 1992;
Thompson et al., PCT Publication No. WO 99/54459, Wincott et al.,
Nucleic Acids Res. 23:2677, 1995; Wincott et al., Methods Mol. Bio.
74:59, 1997; Brennan et al., Biotechnot Bioeng. 61:33-45, 1998; and
Brennan, U.S. Pat. No. 6,001,311. Synthesis of RNA, including
certain dsRNA molecules and analogs thereof of this disclosure, can
be made using the procedure as described in Usman et al., J. Am.
Chem. Soc. 109:7845, 1987; Scaringe et al., Nucleic Acids Res.
18:5433, 1990; and Wincott et al., Nucleic Acids Res. 23:2677-2684,
1995; Wincott et al., Methods Mol. Bio. 74:59, 1997.
[0143] In certain embodiments, the nucleic acid molecules of the
present disclosure can be synthesized separately and joined
together post-synthetically, for example, by ligation (Moore et
al., Science 256:9923, 1992; Draper et al., PCT Publication No. WO
93/23569; Shabarova et al., Nucleic Acids Res. 19:4247, 1991;
Bellon et al., Nucleosides & Nucleotides 16:951, 1997; Bellon
et al., Bioconjugate Chem. 8:204, 1997), or by hybridization
following synthesis or deprotection.
[0144] In further embodiments, dsRNAs of this disclosure that
decrease expression of one or more target family gene by RNAi can
be made as single or multiple transcription products expressed by a
polynucleotide vector encoding one or more dsRNAs and directing
their expression within host cells. In these embodiments the
double-stranded portion of a final transcription product of the
dsRNAs to be expressed within the target cell can be, for example,
about 5 to about 40 bp, about 15 to about 24 bp, or about 25 to
about 40 bp long. Within exemplary embodiments, double-stranded
portions of dsRNAs, in which two or more strands pair up, are not
limited to completely paired nucleotide segments, and may contain
non-pairing portions due to a mismatch (the corresponding
nucleotides are not complementary), bulge (lacking in the
corresponding complementary nucleotide on one strand), overhang, or
the like. Non-pairing portions can be contained to the extent that
they do not interfere with dsRNA formation and function. In certain
embodiments, a "bulge" may comprise 1 to 2 non-pairing nucleotides,
and the double-stranded region of dsRNAs in which two strands pair
up may contain from about 1 to 7, or about 1 to 5 bulges. In
addition, "mismatch" portions contained in the double-stranded
region of dsRNAs may include from about 1 to 7, or about 1 to 5
mismatches. In other embodiments, the double-stranded region of
dsRNAs of this disclosure may contain both bulge and mismatched
portions in the approximate numerical ranges specified herein.
[0145] A dsRNA or analog thereof of this disclosure may be further
comprised of a nucleotide, non-nucleotide, or mixed
nucleotide/non-nucleotide linker that joins the sense region of the
dsRNA to the antisense region of the dsRNA. In one embodiment, a
nucleotide linker can be a linker of more than about 2 nucleotides
length up to about 10 nucleotides in length. In another embodiment,
the nucleotide linker can be a nucleic acid aptamer. By "aptamer"
or "nucleic acid aptamer" as used herein is meant a nucleic acid
molecule that binds specifically to a target molecule wherein the
nucleic acid molecule has sequence that comprises a sequence
recognized by the target molecule in its natural setting.
Alternately, an aptamer can be a nucleic acid molecule that binds
to a target molecule wherein the target molecule does not naturally
bind to a nucleic acid. The target molecule can be any molecule of
interest. For example, the aptamer can be used to bind to a
ligand-binding domain of a protein, thereby preventing interaction
of the naturally occurring ligand with the protein. This is a
non-limiting example and those in the art will recognize that other
embodiments can be readily generated using techniques generally
known in the art (see, e.g., Gold et al., Annu. Rev. Biochem.
64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr.
Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000;
Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical
Chem. 45:1628, 1999).
[0146] A non-nucleotide linker may be comprised of an abasic
nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,
lipid, polyhydrocarbon, or other polymeric compounds (e.g.,
polyethylene glycols such as those having between 2 and 100
ethylene glycol units). Specific examples include those described
by Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990; Seela and
Kaiser, Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J.
Am. Chem. Soc. 113:6324, 1991; Richardson and Schepartz, J. Am.
Chem. Soc. 113:5109, 1991; Ma et al., Nucleic Acids Res. 21:2585,
1993; Ma et al., Biochemistry 32:1751, 1993; Durand et al., Nucleic
Acids Res. 18:6353, 1990; McCurdy et al., Nucleosides &
Nucleotides 10:287, 1991; Jaschke et al., Tetrahedron Lett. 34:301,
1993; Ono et al., Biochemistry 30:9914, 1991; Arnold et al.,
International Publication No. WO 89/02439; Usman et al., PCT
Publication No. WO 95/06731; Dudycz et al., PCT Publication No. WO
95/11910; and Ferentz and Verdine, J. Am. Chem. Soc. 113:4000,
1991. The synthesis of a dsRNA molecule of this disclosure, which
can be further modified, comprises: (a) synthesis of two
complementary strands of the dsRNA molecule; and (b) annealing the
two complementary strands together under conditions suitable to
obtain a dsRNA molecule. In another embodiment, synthesis of the
two complementary strands of a dsRNA molecule is by solid phase
oligonucleotide synthesis. In yet another embodiment, synthesis of
the two complementary strands of a dsRNA molecule is by solid phase
tandem oligonucleotide synthesis.
[0147] Chemically synthesizing nucleic acid molecules with
substitutions or modifications (base, sugar, phosphate, or any
combination thereof) can prevent their degradation by serum
ribonucleases, which may lead to increased potency. See, for
example, Eckstein et al., PCT Publication No. WO 92/07065; Perrault
et al., Nature 344:565, 1990; Pieken et al., Science 253:314, 1991;
Usman and Cedergren, Trends in Biochem. Sci. 17:334, 1992; Usman et
al., Nucleic Acids Symp. Ser. 31:163, 1994; Beigelman et al., J.
Biol. Chem. 270:25702, 1995; Burgin et al., Biochemistry 35:14090,
1996; Burlina et al., Bioorg. Med. Chem. 5:1999-2010, 1997;
Thompson et al., Karpeisky et al., Tetrahedron Lett. 39:1131, 1998;
Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55,
1998; Verma and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998;
Herdewijn, Antisense Nucleic Acid Drug Dev. 10:297, 2000; Kurreck,
Eur. J. Biochem. 270:1628, 2003; Dorsett and Tuschl, Nature Rev.
Drug Discov. 3:318, 2004; Rossi et al., PCT Publication No. WO
91/03162; Usman et al., PCT Publication No. WO 93/15187; Beigelman
et al., PCT Publication No. WO 97/26270; Woolf et al., PCT
Publication No. WO 98/13526; Sproat, U.S. Pat. No. 5,334,711; Usman
et al., U.S. Pat. No. 5,627,053; Beigelman et al., U.S. Pat. No.
5,716,824; Otvos et al., U.S. Pat. No. 5,767,264; Gold et al., U.S.
Pat. No. 6,300,074. Each of the above references discloses various
substitutions and chemical modifications to the base, phosphate, or
sugar moieties of nucleic acid molecules, which can be used in the
dsRNAs described herein. For example, oligonucleotides can be
modified at the sugar moiety to enhance stability or prolong
biological activity by increasing nuclease resistance.
Representatives of such sugar modifications include 2'amino,
2'C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, or 2'-deoxy. Hence,
dsRNA molecules of the instant disclosure can be modified to
increase nuclease resistance or duplex stability while
substantially retaining or having enhanced RNAi activity as
compared to unmodified dsRNA.
[0148] 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 or enhance biological activity by modification
with nuclease resistant groups, for example, 2'amino, 2'C-allyl,
2'fluoro, 2'O-methyl, 2'O-allyl, 2'-H, nucleotide base
modifications. For a review, see Usman and Cedergren, TIBS 17:34,
1992; Usman et al., Nucleic Acids Symp. Ser. 31:163, 1994; Burgin
et al., Biochemistry 35:14090, 1996. Sugar modification of nucleic
acid molecules have been extensively described in the art (see
Eckstein et al., PCT Publication No. WO 92/07065; Perrault et al.,
Nature 344:565-568, 1990; Pieken et al., Science 253:314-317, 1991;
Usman and Cedergren, Trends in Biochem. Sci. 17:334-339, 1992;
Usman et al., PCT Publication No. WO 93/15187; Sproat, U.S. Pat.
No. 5,334,711 and Beigelman et cd., J. Biol. Chem. 270:25702, 1995;
Beigelman et al., 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., PCT Publication No. WO 98/13526; Thompson
et al., Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw
and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma
and Eckstein, Annu. Rev. Biochem. 67:99-134, 1998; and Burlina et
al., Bioorg. Med. Chem. 5:1999-2010, 1997. Such publications
describe general methods and strategies to determine the location
of incorporation of sugar, base or phosphate modifications and the
like into nucleic acid molecules without modulating catalysis. In
view of such teachings, similar modifications can be used as
described herein to modify the dsRNA molecules of the instant
disclosure so long as the ability of dsRNA molecules to promote
RNAi in cells is not significantly inhibited.
[0149] In one embodiment, this disclosure features substituted or
modified dsRNA molecules, such as phosphate backbone modifications
comprising one or more phosphorothioate, phosphorodithioate,
methylphosphonate, phosphotriester, morpholino, amidate carbamate,
carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide,
sulfamate, formacetal, thioformacetal, or alkylsilyl,
substitutions. For a review of oligonucleotide backbone
modifications, see Hunziker and Leumann, Nucleic Acid Analogues:
Synthesis and Properties, in Modern Synthetic Methods, VCH,
331-417, 1995; and Mesmaeker et al., ACS, 24-39, 1994.
[0150] In another embodiment, a conjugate molecule can be
optionally attached to a dsRNA or analog thereof that decreases
expression of one or more target gene by RNAi. For example, such
conjugate molecules may be polyethylene glycol, human serum
albumin, or a ligand for a cellular receptor that can, for example,
mediate cellular uptake. Examples of specific conjugate molecules
contemplated by the instant disclosure that can be attached to a
dsRNA or analog thereof of this disclosure are described in
Vargeese et al., U.S. Patent Application Publication No.
2003/0130186, published Jul. 10, 2003, and U.S. Patent Application
Publication No. 2004/0110296, published Jun. 10, 2004. In another
embodiment, a conjugate molecule is covalently attached to a dsRNA
or analog thereof that decreases expression of one or more target
gene by RNAi via a biodegradable linker. In certain embodiments, a
conjugate molecule can be attached at the 3'end of either the sense
strand, the antisense strand, or both strands of a dsRNA molecule
provided herein. In another embodiment, a conjugate molecule can be
attached at the 5'end of either the sense strand, the antisense
strand, or both strands of the dsRNA or analog thereof. In yet
another embodiment, a conjugate molecule is attached both the 3'end
and 5'end of either the sense strand, the antisense strand, or both
strands of a dsRNA molecule, or any combination thereof. In further
embodiments, a conjugate molecule of this disclosure comprises a
molecule that facilitates delivery of a dsRNA or analog thereof
into a biological system, such as a cell. A person of skill in the
art can screen dsRNA of this disclosure having various conjugates
to determine whether the dsRNA-conjugate complex possesses improved
properties (e.g., pharmacokinetic profile, bioavailability,
stability) while maintaining the ability to mediate RNAi in, for
example, an animal model as described herein or generally known in
the art.
Methods for Selecting dsRNA Molecules Specific for a Target
Sequence
[0151] As indicated above, the present disclosure also provides
methods for selecting dsRNA and analogs thereof capable of
specifically binding to one or more target gene while being not
specifically binding or minimally binding to non-target genes. The
selection process disclosed herein is useful, for example, in
eliminating dsRNA analogs that are cytotoxic due to non-specific
binding to, and subsequent degradation of, one or more non-target
genes.
[0152] Methods of the present disclosure do not require a priori
knowledge of the nucleotide sequence of every possible gene variant
targeted by the dsRNA or analog thereof. In one embodiment, the
nucleotide sequence of the dsRNA is selected from a conserved
region or consensus sequence of one or more target gene. In another
embodiment, the nucleotide sequence of the dsRNA may be selectively
or preferentially targeted to a certain sequence contained within
an mRNA splice variant of a target gene.
[0153] In certain embodiments, methods are provided for selecting
one or more dsRNA molecule that decreases expression of one or more
target gene by RNAi, comprising a first strand that is
complementary to a target gene mRNA, or any combination thereof,
and a second strand that is complementary to the first strand,
wherein the first and second strands form a double-stranded region
of about 15 to about 40 base pairs, and wherein at least one
uridine of the dsRNA molecule is replaced with a 5-methyluridine or
2-thioribothymidine, which methods employ "off-target" profiling
whereby one or more dsRNA provided herein is contacted with a cell,
either in vivo or in vitro, and total target mRNA is collected for
use in probing a microarray comprising oligonucleotides having one
or more nucleotide sequence from a panel of known genes, including
non-target genes (e.g., interferon). The "off-target" profile of
the dsRNA provided herein is quantified by determining the number
of non-target genes having reduced expression levels in the
presence of the candidate dsRNAs. The existence of "off target"
binding indicates a dsRNA provided herein that is capable of
specifically binding to one or more non-target gene messages. In
certain embodiments, a dsRNA as provided herein applicable to
therapeutic use will exhibit a greater stability, minimal
interferon response, and little or no "off-target" binding.
[0154] Still further embodiments provide methods for selecting more
efficacious dsRNA by using one or more reporter gene constructs
comprising a constitutive promoter, such as a cytomegalovirus (CMV)
or phosphoglycerate kinase (PGK) promoter, operably fused to, and
capable of altering the expression of one or more reporter genes,
such as a luciferase, chloramphenicol (CAT), or
.beta.-galactosidase, which, in turn, is operably fused in-frame
with a dsRNA (such as one having a length between about 15
base-pairs and about 40 base-pairs or from about 5 nucleotides to
about 24 nucleotides, or about 25 nucleotides to about 40
nucleotides) that contains one or more target sequence, as provided
herein.
[0155] Individual reporter gene expression constructs may be
co-transfected with one or more dsRNA or analog thereof. The
capacity of a given dsRNA to reduce the expression level of a
target gene may be determined by comparing the measured reporter
gene activity in cells transfected with or without a dsRNA molecule
of interest.
[0156] Certain embodiments disclosed herein provide methods for
selecting one or more modified dsRNA molecule(s) that employ the
step of predicting the stability of a dsRNA duplex. In some
embodiments, such a prediction is achieved by employing a
theoretical melting curve wherein a higher theoretical melting
curve indicates an increase in dsRNA duplex stability and a
concomitant decrease in cytotoxic effects. Alternatively, stability
of a dsRNA duplex may be determined empirically by measuring the
hybridization of a single RNA analog strand as described herein to
a complementary target gene within, for example, a polynucleotide
array. The melting temperature (i.e., the T.sub.m value) for each
modified RNA and complementary RNA immobilized on the array can be
determined, and from this T.sub.m value, the relative stability of
a substituted or modified RNA pairing with a complementary RNA
molecule can be determined.
[0157] For example, for universal-binding nucleotide, Kawase et al.
(Nucleic Acids Res. 14:7727, 1986) have described an analysis of
the nucleotide-pairing properties of Di (inosine) to A, C, G, and
T, which was achieved by measuring the hybridization of
oligonucleotides (ODNs) with Di in various positions to
complementary sets of ODNs made as an array. The relative strength
of nucleotide-pairing is I-C>I-A>I-G.apprxeq.I-T. Generally,
Di containing duplexes showed lower T.sub.m values when compared to
the corresponding WC nucleotide pair. The stabilization of Di by
pairing was in order of Dc>Da>Dg>Dt>Du. From the
thermodynamic values calculated using Van't Hoff plots according to
a two state model, Kawase et al. conclude that the sequence of
purine-pyrimidine is favored in double strand formation due to
nucleotide stacking. For instance the duplex formation of XY=AT is
a more favored formation than an XY=CG and TA. As a person of skill
in the art would understand, although universal-binding nucleotides
are used herein as an example of determining stability (i.e., the
T.sub.m value), other nucleotide substitutions (e.g.,
5-methyluridine for uridine) or further modifications (e.g., a
ribose modification at the 2'-position) can also be evaluated by
these or similar methods.
[0158] Within certain embodiments, methods disclosed herein
comprise the steps of (a) designing or synthesizing a suitable
dsRNA for RNAi gene silencing of one or more target gene, wherein
the dsRNA comprises at least three strands and optionally at least
one modification or substitution (such as a 5-methyluridine, LNA,
2'-methoxy, 2'-fluoro, phosphorothioate, or any combination
thereof); and (b) contacting a cell expressing one or more target
protein with the dsRNA, wherein the dsRNA is capable of
specifically binding to one or more target mRNA or gene, thereby
reducing expression of one or more target members.
[0159] Any of these methods of identifying dsRNA of interest can
also be used to examine a dsRNA that decreases expression of one or
more target gene by RNA interference, comprising a first strand
that is complementary to a target mRNA, or any combination thereof,
and a second and third strand that have non-overlapping
complementarity to the first strand, wherein the first and at least
one of the second or third strand form a double-stranded region of
about 5 to about 13 base pairs; wherein at least one pyrimidine of
the dsRNA comprises a pyrimidine nucleoside according to Formula I
or II:
##STR00004##
wherein R.sup.1 and R.sup.2 are each independently a --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2,
--C.ident.N, or heterocyclo group; R.sup.3 and R.sup.4 are each
independently a hydroxyl, a protected hydroxyl, or an
internucleoside linking group; and R.sup.5 and R.sup.8 are
independently O or S. In certain embodiments, at least one
nucleoside is according to Formula I in which R.sup.1 is methyl and
R.sup.2 is --OH, or R.sup.1 is methyl, R.sup.2 is --OH, and R.sup.8
is S. In other embodiments, the internucleoside linking group
covalently links from about 5 to about 40 nucleosides.
Compositions and Methods of Use
[0160] As set forth herein, dsRNA of the instant disclosure are
designed to target one or more target gene (including one or more
mRNA splice variants thereof) that is expressed at an elevated
level or continues to be expressed when it should not, and is a
causal or contributing factor associated with, for example, a
hyperproliferative, angiogenic, or inflammatory disease, state, or
adverse condition. In this context, a dsRNA or analog thereof of
this disclosure will effectively down regulate expression of one or
more target gene to levels that prevent, alleviate, or reduce the
severity or recurrence of one or more associated disease symptoms.
Alternatively, for various distinct disease models in which
expression of one or more target gene is not necessarily elevated
as a consequence or sequel of disease or other adverse condition,
down regulation of one or more target gene will nonetheless result
in a therapeutic result by lowering gene expression. Furthermore,
dsRNAs of this disclosure may be targeted to reduce expression of
one or more target gene, which can result in upregulation of a
"downstream" gene whose expression is negatively regulated,
directly or indirectly, by one or more target protein. The dsRNA
molecules of the instant disclosure comprise useful reagents and
can be used in methods for a variety of therapeutic, diagnostic,
target validation, genomic discovery, genetic engineering, and
pharmacogenomic applications.
[0161] In certain embodiments, aqueous suspensions contain dsRNA of
this disclosure in admixture with suitable excipients, such as
suspending agents or dispersing or wetting agents. Exemplary
suspending agents include sodium carboxymethylcellulose,
methylcellulose, hydropropyl-methylcellulose, sodium alginate,
polyvinylpyrrolidone, gum tragacanth and gum acacia. Representative
dispersing or wetting agents include naturally-occurring
phosphatides (e.g., lecithin), condensation products of an alkylene
oxide with fatty acids (e.g., polyoxyethylene stearate),
condensation products of ethylene oxide with long chain aliphatic
alcohols (e.g., heptadecaethyleneoxycetanol), condensation products
of ethylene oxide with partial esters derived from fatty acids and
hexitol (e.g., polyoxyethylene sorbitol monooleate), or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides (e.g., polyethylene
sorbitan monooleate). In certain embodiments, the aqueous
suspensions can optionally contain one or more preservatives (e.g.,
ethyl or n-propyl-p-hydroxybenzoate), one or more coloring agents,
one or more flavoring agents, or one or more sweetening agents
(e.g., sucrose, saccharin). In additional embodiments, dispersible
powders and granules suitable for preparation of an aqueous
suspension by the addition of water provide dsRNA of this
disclosure in admixture with a dispersing or wetting agent,
suspending agent and optionally one or more preservative, coloring
agent, flavoring agent, or sweetening agent.
[0162] The present disclosure includes dsRNA compositions prepared
for storage or administration that include a pharmaceutically
effective amount of a desired compound 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., 21.sup.st Edition, 2005.
In certain embodiments, pharmaceutical compositions of this
disclosure can optionally include preservatives, antioxidants,
stabilizers, dyes, flavoring agents, or any combination thereof.
Exemplary preservatives include sodium benzoate, esters of
p-hydroxybenzoic acid, and sorbic acid.
[0163] The dsRNA compositions of the instant disclosure can be
effectively employed as pharmaceutically-acceptable formulations.
Pharmaceutically-acceptable formulations prevent, alter the
occurrence or severity of, or treat (alleviate one or more
symptom(s) to a detectable or measurable extent) a disease state or
other adverse condition in a subject. A pharmaceutically acceptable
formulation includes salts of the above compounds, for example,
acid addition salts, such as salts of hydrochloric acid,
hydrobromic acid, acetic acid, or benzene sulfonic acid. A
pharmaceutical composition or formulation refers to a composition
or formulation in a form suitable for administration into a cell,
or a subject such as a human (e.g., systemic administration). The
formulations of the present disclosure, having an amount of dsRNA
sufficient to treat or prevent a disorder associated with target
gene expression are, for example, suitable for topical (e.g.,
creams, ointments, skin patches, eye drops, ear drops) application
or administration. Other routes of administration include oral,
parenteral, sublingual, bladder wash-out, vaginal, rectal, enteric,
suppository, nasal, and inhalation. The term parenteral, as used
herein, includes subcutaneous, intravenous, intramuscular,
intraarterial, intraabdominal, intraperitoneal, intraarticular,
intraocular or retrobulbar, intraaural, intrathecal, intracavitary,
intracelial, intraspinal, intrapulmonary or transpulmonary,
intrasynovial, and intraurethral injection or infusion techniques.
The pharmaceutical compositions of the present disclosure are
formulated to allow the dsRNA contained therein to be bioavailable
upon administration to a subject.
[0164] In further embodiments, dsRNA of this disclosure can be
formulated as oily suspensions or emulsions (e.g., oil-in-water) by
suspending dsRNA in, for example, a vegetable oil (e.g., arachis
oil, olive oil, sesame oil or coconut oil) or a mineral oil (e.g.,
liquid paraffin). Suitable emulsifying agents can be
naturally-occurring gums (e.g., gum acacia or gum tragacanth),
naturally-occurring phosphatides (e.g., soy bean, lecithin, esters
or partial esters derived from fatty acids and hexitol), anhydrides
(e.g., sorbitan monooleate), or condensation products of partial
esters with ethylene oxide (e.g., polyoxyethylene sorbitan
monooleate). In certain embodiments, the oily suspensions or
emulsions can optionally contain a thickening agent, such as
beeswax, hard paraffin or cetyl alcohol. In related embodiments,
sweetening agents and flavoring agents can optionally be added to
provide palatable oral preparations. In yet other embodiments,
these compositions can be preserved by the optionally adding an
anti-oxidant, such as ascorbic acid.
[0165] In further embodiments, dsRNA of this disclosure can be
formulated as syrups and elixirs with sweetening agents (e.g.,
glycerol, propylene glycol, sorbitol, glucose or sucrose). Such
formulations can also contain a demulcent, preservative, flavoring,
coloring agent, or any combination thereof. In other embodiments,
pharmaceutical compositions comprising dsRNA of this disclosure can
be in the form of a sterile, injectable aqueous or oleaginous
suspension. The sterile injectable preparation can also be a
sterile, injectable solution or suspension in a non-toxic
parenterally acceptable diluent or solvent (e.g., as a solution in
1,3-butanediol). Among the exemplary acceptable vehicles and
solvents useful in the compositions of this disclosure is water,
Ringer's solution, or isotonic sodium chloride solution. In
addition, sterile, fixed oils may be employed as a solvent or
suspending medium for the dsRNA of this disclosure. 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 parenteral formulations.
[0166] Within certain embodiments of this disclosure,
pharmaceutical compositions and methods are provided that feature
the presence or administration of one or more dsRNA or analogs
thereof of this disclosure, combined, complexed, or conjugated with
a polypeptide, optionally formulated with a
pharmaceutically-acceptable carrier, such as a diluent, stabilizer,
buffer, or the like. The negatively charged dsRNA molecules of this
disclosure may be administered to a patient by any standard means,
with or without stabilizers, buffers, or the like, to form a
composition suitable for treatment. When it is desired to use a
liposome delivery mechanism, standard protocols for formation of
liposomes can be followed. The compositions of the present
disclosure may also be formulated and used as a tablet, capsule or
elixir for oral administration, suppository for rectal
administration, sterile solution, or suspension for injectable
administration, either with or without other compounds known in the
art. Thus, dsRNAs of the present disclosure may be administered in
any form, such as nasally, transdermally, parenterally, or by local
injection.
[0167] In accordance with this disclosure herein, dsRNA molecules
(optionally substituted or modified or conjugated), compositions
thereof, and methods for inhibiting expression of one or more
target gene in a cell or organism are provided. In certain
embodiments, this disclosure provides methods and dsRNA
compositions for treating a subject, including a human cell, tissue
or individual, having a disease or at risk of developing a disease
caused by or associated with the expression of one or more target
gene. In one embodiment, the method includes administering a dsRNA
of this disclosure or a pharmaceutical composition containing the
dsRNA to a cell or an organism, such as a mammal, such that
expression of the target gene is silenced. Subjects (e.g.,
mammalian, human) amendable for treatment using the dsRNA molecules
(optionally substituted or modified or conjugated), compositions
thereof, and methods of the present disclosure include those
suffering from one or more disease or condition mediated, at least
in part, by overexpression or inappropriate expression of one or
more target gene, or which are amenable to treatment by reducing
expression of one or more target protein, including a
hyperproliferative (e.g., cancer), angiogenic, metabolic, or
inflammatory (e.g., arthritis) disease or disorder or
condition.
[0168] Compositions and methods disclosed herein are useful in the
treatment of a wide variety of target viruses, including
retrovirus, such as human immunodeficiency virus (HIV), Hepatitis C
Virus, Hepatitis B Virus, Coronavirus, as well as respiratory
viruses (including human Respiratory Syncytial Virus, human
Metapneumovirus, human Parainfluenza virus Rhinovirus and Influenza
virus.
[0169] In other examples, the compositions and methods of this
disclosure are useful as therapeutic tools to regulate expression
of one or more target gene to treat or prevent symptoms of, for
example, hyperproliferative disorders. Exemplary hyperproliferative
disorders include neoplasms, carcinomas, sarcomas, tumors, or
cancer. More exemplary hyperproliferative disorders include oral
cancer, throat cancer, laryngeal cancer, esophageal cancer,
pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer,
gastrointestinal tract cancer, gastrointestinal stromal tumors
(GIST), small intestine cancer, colon cancer, rectal cancer,
colorectal cancer, anal cancer, pancreatic cancer, breast cancer,
cervical cancer, uterine cancer, vulvar cancer, vaginal cancer,
urinary tract cancer, bladder cancer, kidney cancer, adrenocortical
cancer, islet cell carcinoma, gallbladder cancer, stomach cancer,
prostate cancer, ovarian cancer, endometrial cancer, trophoblastic
tumor, testicular cancer, penial cancer, bone cancer, osteosarcoma,
liver cancer, extrahepatic bile duct cancer, skin cancer, basal
cell carcinoma (BCC), lung cancer, small cell lung cancer,
non-small cell lung cancer (NSCLC), brain cancer, melanoma,
Kaposi's sarcoma, eye cancer, head and neck cancer, squamous cell
carcinoma of head and neck, tymoma, thymic carcinoma, thyroid
cancer, parathyroid cancer, Hippel-Lindau syndrome, leukemia, acute
myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic
leukemia, hairy cell leukemia, lymphoma, non-Hodgkin's lymphoma,
Burkitt's lymphoma, T-cell lymphoma, multiple myeloma, malignant
pleural mesothelioma, Barrett's adenocarcinoma, Wilm's tumor, or
the like.
[0170] In other examples, the compositions and methods of this
disclosure are useful as therapeutic tools to regulate expression
of one or more target gene to treat or prevent symptoms of, for
example, inflammatory disorders. Exemplary inflammatory disorders
include diabetes mellitus, rheumatoid arthritis, pannus growth in
inflamed synovial lining, collagen-induced arthritis,
spondylarthritis, ankylosing spondylitis, multiple sclerosis,
encephalomyelitis, inflammatory bowel disease, Chron's disease,
psoriasis or psoriatic arthritis, myasthenia gravis, systemic lupus
erythematosis, graft-versus-host disease, atherosclerosis, and
allergies.
[0171] Other exemplary disorders that can be treated with dsRNA of
the instant disclosure include metabolic disorders, cardiac
disease, pulmonary disease, neovascularization, ischemic disorders,
age-related macular degeneration, diabetic retinopathy,
glomerulonephritis, diabetes, asthma, chronic obstructive pulmonary
disease, chronic bronchitis, lymphangiogenesis, and
atherosclerosis.
[0172] In any of the methods disclosed herein, there may be used
one or more dsRNA, or substituted or modified dsRNA as described
herein, which comprises a first strand that is complementary to a
target mRNA and is fully complementary, with up to three
mismatches, to at least one other human target family mRNA, or
vice-versa, and a second strand and a third strand that is each
complementary to non-overlapping regions of the first strand,
wherein the second strand and third strands can anneal with the
first strand to form at least two double-stranded regions separated
by a gap of up to 10 nucleotides, and wherein at least one
double-stranded region is from about 5 base pairs up to 13 base
pairs. In other embodiments, subjects can be effectively treated,
prophylactically or therapeutically, by administering an effective
amount of one or more dsRNA having a first strand that is
complementary to a target mRNA and is fully complementary, with up
to three mismatches, to at least one other target family mRNA, or
vice-versa, and a second strand and a third strand that is each
complementary to non-overlapping regions of the first strand,
wherein the second strand and third strands can anneal with the
first strand to form at least two double-stranded regions separated
by a gap of up to 10 nucleotides, and wherein at least one
double-stranded region is from about 5 base pairs up to 13 base
pairs and at least one pyrimidine of the mdRNA is substituted with
a pyrimidine nucleoside according to Formula I or II:
##STR00005##
wherein R.sup.1 and R.sup.2 are each independently a --H, --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2,
--C.ident.N, or heterocyclo group; R.sup.3 and R.sup.4 are each
independently a hydroxyl, a protected hydroxyl, or an
internucleoside linking group; and R.sup.5 and R.sup.8 are
independently O or S. In certain embodiments, at least one
nucleoside is according to Formula I in which R.sup.1 is methyl and
R.sup.2 is --OH, or R.sup.1 is methyl, R.sup.2 is --OH, and R.sup.8
is S. In other embodiments, the internucleoside linking group
covalently links from about 5 to about 40 nucleosides.
[0173] In further embodiments, subjects can be effectively treated,
prophylactically or therapeutically, by administering an effective
amount of one or more dsRNA, or substituted or modified dsRNA as
described herein, having a first strand that is complementary to a
target mRNA and is fully complementary, with up to three
mismatches, to at least one other target gene mRNA, or vice-versa,
and a second strand and a third strand that is each complementary
to non-overlapping regions of the first strand, wherein the second
strand and third strands can anneal with the first strand to form
at least two double-stranded regions separated by a gap of up to 10
nucleotides, and wherein the combined double-stranded regions total
about 15 base pairs to about 40 base pairs and the mdRNA molecule
comprises blunt ends. In still further embodiments, methods
disclosed herein there may be used with one or more dsRNA that
comprises a first strand that is complementary to a target gene
mRNA and is fully complementary, with up to three mismatches, to at
least one other target family mRNA, or vice-versa, and a second
strand and a third strand that is each complementary to
non-overlapping regions of the first strand, wherein the second
strand and third strands can anneal with the first strand to form
at least two double-stranded regions separated by a gap of up to 10
nucleotides, and wherein at least one double-stranded region is
from about 5 base pairs up to 13 base pairs, the mdRNA molecule
comprises blunt ends, and at least one pyrimidine of the mdRNA is
substituted with a pyrimidine nucleoside according to Formula I or
II:
##STR00006##
wherein R.sup.1 and R.sup.2 are each independently a --H, --OH,
--OCH.sub.3, --OCH.sub.2OCH.sub.2CH.sub.3,
--OCH.sub.2CH.sub.2OCH.sub.3, halogen, substituted or unsubstituted
C.sub.1-C.sub.10 alkyl, alkoxy, alkoxyalkyl, hydroxyalkyl,
carboxyalkyl, alkylsulfonylamino, aminoalkyl, dialkylamino,
alkylaminoalkyl, dialkylaminoalkyl, haloalkyl, trifluoromethyl,
cycloalkyl, (cycloalkyl)alkyl, substituted or unsubstituted
C.sub.2-C.sub.10 alkenyl, substituted or unsubstituted --O-allyl,
--O--CH.sub.2CH.dbd.CH.sub.2, --O--CH.dbd.CHCH.sub.3, substituted
or unsubstituted C.sub.2-C.sub.10 alkynyl, carbamoyl, carbamyl,
carboxy, carbonylamino, substituted or unsubstituted aryl,
substituted or unsubstituted aralkyl, --NH.sub.2, --NO.sub.2,
--C.ident.N, or heterocyclo group; R.sup.3 and R.sup.4 are each
independently a hydroxyl, a protected hydroxyl, or an
internucleoside linking group; and R.sup.5 and R.sup.8 are
independently O or S. In certain embodiments, at least one
nucleoside is according to Formula I in which R.sup.1 is methyl and
R.sup.2 is --OH, or R.sup.1 is methyl, R.sup.2 is --OH, and R.sup.8
is S. In other embodiments, the internucleoside linking group
covalently links from about 5 to about 40 nucleosides.
[0174] Within additional aspects of this disclosure, combination
formulations and methods are provided comprising an effective
amount of one or more dsRNA of the present disclosure in
combination with one or more secondary or adjunctive active agents
that are formulated together or administered coordinately with the
dsRNA of this disclosure to control one or more target
gene-associated disease or condition as described herein. Useful
adjunctive therapeutic agents in these combinatorial formulations
and coordinate treatment methods include, for example, 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 or
inorganic compounds including metals, salts and ions, and other
drugs and active agents indicated for treating one or more target
gene-associated disease or condition, including chemotherapeutic
agents used to treat cancer, steroids, non-steroidal
anti-inflammatory drugs (NSAIDs), or the like.
[0175] Exemplary chemotherapeutic agents include alkylating agents
(e.g., cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas,
nitrogen mustards, uramustine, temozolomide), antimetabolites
(e.g., aminopterin, methotrexate, mercaptopurine, fluorouracil,
cytarabine), taxanes (e.g., paclitaxel, docetaxel), anthracyclines
(e.g., doxorubicin, daunorubicin, epirubicin, idaruicin,
mitoxantrone, valrubicin), bleomycin, mytomycin, actinomycin,
hydroxyurea, topoisomerase inhibitors (e.g., camptothecin,
topotecan, irinotecan, etoposide, teniposide), monoclonal
antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab,
panitumumab, rituximab, tositumomab, trastuzumab), vinca alkaloids
(e.g., vincristine, vinblastine, vindesine, vinorelbine),
cyclophosphamide, prednisone, leucovorin, oxaliplatin.
[0176] To practice the coordinate administration methods of this
disclosure, a dsRNA is administered simultaneously or sequentially
in a coordinated treatment protocol with one or more secondary or
adjunctive therapeutic agents described herein or known in the art.
The coordinate administration may be done in either order, and
there may be a time period while only one or both (or all) active
therapeutic agents, individually or collectively, exert their
biological activities. A distinguishing aspect of all such
coordinate treatment methods is that the dsRNA present in a
composition elicits some favorable clinical response, which may or
may not be in conjunction with a secondary clinical response
provided by the secondary therapeutic agent. For example, the
coordinate administration of the dsRNA with a secondary therapeutic
agent as contemplated herein can yield an enhanced (e.g.,
synergistic) therapeutic response beyond the therapeutic response
elicited by either or both the purified dsRNA or secondary
therapeutic agent alone.
[0177] In another embodiment, a dsRNA of this disclosure can
include a conjugate member on one or more of the nucleotides of a
dsRNA (e.g., terminal). The conjugate member can be, for example, a
lipophile, a terpene, a protein binding agent, a vitamin, a
carbohydrate, or a peptide. For example, the conjugate member can
be naproxen, nitroindole (or another conjugate that contributes to
stacking interactions), folate, ibuprofen, or a C5 pyrimidine
linker. In other embodiments, the conjugate member is a glyceride
lipid conjugate (e.g., a dialkyl glyceride derivatives), vitamin E
conjugates, or thio-cholesterols. Additional conjugate members
include peptides that function, when conjugated to a modified dsRNA
of this disclosure, to facilitates delivery of the dsRNA into a
target cell, or otherwise enhance delivery, stability, or activity
of the dsRNA when contacted with a biological sample. Exemplary
peptide conjugate members for use within these aspects of this
disclosure, include peptides PN27, PN28, PN29, PN58, PN61, PN73,
PN158, PN159, PN173, PN182, PN202, PN204, PN250, PN361, PN365,
PN404, PN453, and PN509 are described, for example, in U.S. Patent
Application Publication Nos. 2006/0040882 and 2006/0014289, and
U.S. Provisional Patent Application No. 60/939,578, which are all
incorporated herein by reference. In certain embodiments, when
peptide conjugate partners are used to enhance delivery of dsRNA or
analogs thereof of this disclosure, the resulting dsRNA
formulations and methods will often exhibit further reduction of an
interferon response in target cells as compared to dsRNAs delivered
in combination with alternate delivery vehicles, such as lipid
delivery vehicles (e.g., Lipofectamine.TM.).
[0178] In still another embodiment, a dsRNA or analog thereof of
this disclosure may be conjugated to the polypeptide and admixed
with one or more non-cationic lipids or a combination of a
non-cationic lipid and a cationic lipid to form a composition that
enhances intracellular delivery of the dsRNA as compared to
delivery resulting from contacting the target cells with a naked
dsRNA. In more detailed aspects of this disclosure, the mixture,
complex or conjugate comprising a dsRNA and a polypeptide can be
optionally combined with (e.g., admixed or complexed with) a
cationic lipid, such as Lipofectine.TM.. To produce these
compositions comprised of a polypeptide, dsRNA and a cationic
lipid, the dsRNA and peptide may be mixed together first in a
suitable medium such as a cell culture medium, after which the
cationic lipid is added to the mixture to form a dsRNA/delivery
peptide/cationic lipid composition. Optionally, the peptide and
cationic lipid can be mixed together first in a suitable medium
such as a cell culture medium, followed by the addition of the
dsRNA to form the dsRNA/delivery peptide/cationic lipid
composition.
[0179] This disclosure also features the use of dsRNA compositions
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 (Lasic et
al., Chem. Rev. 95:2601, 1995; Ishiwata et al., Chem. Pharm. Bull.
43:1005, 1995). Such liposomes have been shown to accumulate
selectively in tumors, presumably by extravasation and capture in
the neovascularized target tissues (Lasic et al., Science 267:1275,
1995; Oku et al., Biochim. Biophys. Acta 1238:86, 1995). The
long-circulating liposomes enhance the pharmacokinetics and
pharmacodynamics of nucleic acid molecules as compared to
conventional cationic liposomes, which are known to accumulate in
tissues of the mononuclear phagocytic system (MPS) (Liu et al., J.
Biol. Chem. 42:24864, 1995; Choi et al., PCT Publication No. WO
96/10391; Ansell et al., PCT Publication No. WO 96/10390; Holland
et al., PCT Publication No. WO 96/10392). Long-circulating
liposomes may also provide additional protection from nuclease
degradation as compared to cationic liposomes in theory due to
avoiding accumulation in metabolically aggressive MPS tissues, such
as the liver and spleen.
[0180] In one embodiment, this disclosure provides compositions
suitable for administering dsRNA molecules of this disclosure to
specific cell types, such as hepatocytes. For example, the
asialoglycoprotein receptor (ASGPr) (Wu and Wu, J. Biol. Chem.
262:4429, 1987) 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, Cell 22:
611, 1980; Connolly et al., J. Biol. Chem. 257:939, 1982). Lee and
Lee (Glycoconjugate J. 4:317, 1987) 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., J. Med. Chem. 24:1388, 1981). 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. The use of
bioconjugates can also provide a reduction in the required dose of
therapeutic compounds required for treatment. Furthermore,
therapeutic bioavailability, pharmacodynamics, and pharmacokinetic
parameters can be modulated through the use of dsRNA bioconjugates
of this disclosure.
[0181] The present disclosure also features a method for preparing
dsRNA nanoparticles. A first solution containing melamine
derivatives is dissolved in an organic solvent such as dimethyl
sulfoxide, or dimethyl formamide to which an acid such as HCl has
been added. The concentration of HCl would be about 3.3 moles of
HCl for every mole of the melamine derivative. The first solution
is then mixed with a second solution, which includes a nucleic acid
dissolved or suspended in a polar or hydrophilic solvent (e.g., an
aqueous buffer solution containing, for instance,
ethylenediaminetraacetic acid (EDTA), or tris(hydroxymethyl)
aminomethane (TRIS), or combinations thereof. The mixture forms a
first emulsion. The mixing can be done using any standard technique
such as, for example, sonication, vortexing, or in a
microfluidizer. This causes complexing of the nucleic acids with
the melamine derivative forming a trimeric nucleic acid complex.
While not being bound to theory or mechanism, it is believed that
three nucleic acids are complexed in a circular fashion about one
melamine derivative moiety, and that a number of the melamine
derivative moieties can be complexed with the three nucleic acid
molecules depending on the size of the number of nucleotides that
the nucleic acid has. The concentration should be from about 1 to
about 7 moles of the melamine derivative for every mole of a
double-stranded nucleic acid having about 20 nucleotide pairs, more
if the double-stranded nucleic acid is larger. The resultant
nucleic acid particles can be purified and the organic solvent
removed using size-exclusion chromatography or dialysis or
both.
[0182] The complexed nucleic acid nanoparticles can then be mixed
with an aqueous solution containing either polyarginine or a
Gln-Asn polymer, or both, in an aqueous solution. A preferred
molecular weight of each polymer is about 5000 to about 15,000
Daltons. This forms a solution containing nanoparticles of nucleic
acid complexed with the melamine derivative and the polyarginine
and the Gln-Asn polymers. The mixing steps are carried out in a
manner that minimizes shearing of the nucleic acid while producing
nanoparticles on average smaller than about 200 nanometers in
diameter. While not wishing to be bound by theory, it is believed
that the polyarginine complexes with the negative charge of the
phosphate groups within the minor groove of the nucleic acid, and
the polyarginine wraps around the trimeric nucleic acid complex. At
either terminus of the polyarginine other moieties, such as the TAT
polypeptide, mannose or galactose, can be covalently bound to the
polymer to direct binding of the nucleic acid complex to specific
tissues, such as to the liver when galactose is used. While not
being bound to theory, it is believed that the Gln-Asn polymer
complexes with the nucleic acid complex within the major groove of
the nucleic acid through hydrogen bonding with the bases of the
nucleic acid. The polyarginine and the Gln-Asn polymer should be
present at a concentration of 2 moles per every mole of nucleic
acid having 20 base pairs. The concentration should be increased
proportionally for a nucleic acid having more than 20 base pairs.
For example, if the nucleic acid has 25 base pairs, the
concentration of the polymers should be 2.5-3 moles per mole of
double-stranded nucleic acid. An example of is a polypeptide
operatively linked to an N-terminal protein transduction domain
from HIV TAT. The HIV TAT construct for use in such a protein is
described in detail in Vocero-Akbani et al., Nature Med. 5:23,
1999. See also, U.S. Patent Application Publication No.
2004/0132161, published on Jul. 8, 2004. The resultant
nanoparticles can be purified by standard means such as size
exclusion chromatography followed by dialysis. The purified
complexed nanoparticles can then be lyophilized using techniques
well known in the art.
[0183] One embodiment of the present disclosure provides
nanoparticles less than 100 nanometers (nm) comprising dsRNA that
decreases expression of one or more target gene by RNAi. More
specifically, the dsRNA is less than about 30 base pairs in length,
or is from about 20 to about 25 base pairs in length.
[0184] 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) a disease state. The
pharmaceutically effective dose depends on the type of disease, the
composition used, the route of administration, the type of subject
being treated, the physical characteristics of the specific subject
under consideration for treatment, concurrent medication, and other
factors that those skilled in the medical arts will recognize. For
example, an amount between 0.1 mg/kg and 100 mg/kg body weight/day
of active ingredients may be administered depending on the potency
of a dsRNA of this disclosure.
[0185] Dosage levels in the order of about 0.1 mg to about 140 mg
per kilogram of body weight per day can be useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 7 g per
patient 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.
[0186] It is understood that the specific dose level for any
particular patient 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. Following
administration of dsRNA according to the formulations and methods
of this disclosure, test subjects will exhibit about a 10% up to
about a 99% reduction in one or more symptoms associated with the
disease or disorder being treated, as compared to placebo-treated
or other suitable control subjects.
[0187] Nucleic acid molecules and polypeptides can be administered
to cells by a variety of methods known to those of skill in the
art, including administration within formulations that comprise the
dsRNA and polypeptide alone, or that further comprise one or more
additional components, such as a pharmaceutically acceptable
carrier, diluent, excipient, adjuvant, emulsifier, buffer,
stabilizer, preservative, or the like. In certain embodiments, the
dsRNA or the polypeptide can be encapsulated in liposomes,
administered by iontophoresis, or incorporated into other vehicles,
such as hydrogels, cyclodextrins, biodegradable nanocapsules,
bioadhesive microspheres, or proteinaceous vectors (see, e.g., PCT
Publication No. WO 00/53722). Alternatively, a nucleic
acid/peptide/vehicle combination can be locally delivered by direct
injection or by use of an infusion pump. Direct injection of the
nucleic acid molecules of this disclosure, whether subcutaneous,
intramuscular, or intradermal, can take place using standard needle
and syringe methodologies, or by needle-free technologies, such as
those described in Conroy et al., Clin. Cancer Res. 5:2330, 1999,
and PCT Publication No. WO 99/31262.
[0188] The dsRNAs can also be administered in the form of
suppositories, for example, 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.
[0189] 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.
[0190] Further methods for delivery of nucleic acid molecules, such
as the dsRNAs of this disclosure, are described, for example, in
Boado et al., J. Pharm. Sci. 87:1308, 1998; Tyler et al., FEBS
Lett. 421:280, 1999; Pardridge et al., Proc. Nat'l Acad. Sci. USA
92:5592, 1995; Boado, Adv. Drug Delivery Rev. 15:73, 1995;
Aldrian-Herrada et al., Nucleic Acids Res. 26:4910, 1998; Tyler et
al., Proc. Nat'l Acad. Sci. USA 96:7053, 1999; Akhtar et al.,
Trends Cell Bio. 2:139, 1992; "Delivery Strategies for Antisense
Oligonucleotide Therapeutics," ed. Akhtar, 1995, Maurer et al.,
Mol. Membr. Biol. 16:129, 1999; Hofland and Huang, Handb. Exp.
Pharmacol. 137:165, 1999; and Lee et al., ACS Symp. Ser. 752:184,
2000. Sullivan et al. (PCT Publication No. WO 94/02595) further
describe general methods for delivery of enzymatic nucleic acid
molecules, which methods can be used to supplement or complement
delivery of dsRNA contemplated within this disclosure.
[0191] In addition to in vivo gene inhibition, a skilled artisan
will appreciate that the dsRNAs of the present disclosure are
useful in a wide variety of in vitro applications, such as in
scientific and commercial research (e.g., elucidation of
physiological pathways, drug discovery and development), and
medical and veterinary diagnostics. In general, the method involves
the introduction of the dsRNA agent into a cell using known
techniques (e.g., absorption through cellular processes, or by
auxiliary agents or devices, such as electroporation, lipofection,
or through the use of peptide conjugates), then maintaining the
cell for a time sufficient to obtain degradation of one or more
target mRNA.
[0192] All U.S. patents, U.S. patent application publications, U.S.
patent applications, foreign patents, foreign patent applications,
non-patent publications, figures, and websites referred to in this
specification are expressly incorporated herein by reference, in
their entirety.
EXAMPLES
Example 1
Knockdown of .beta.-Galactosidase Activity by Gapped dsRNA Dicer
Substrate
[0193] The activity of a Dicer substrate dsRNA containing a gap in
the double-stranded structure in silencing LacZ mRNA as compared to
the normal Dicer substrate dsRNA (i.e., not having a gap) was
examined.
Nucleotide Sequences of dsRNA and mdRNA Targeting LacZ mRNA
[0194] The nucleic acid sequence of the one or more sense strands,
and the antisense strand of the dsRNA and gapped dsRNA (also
referred to herein as a meroduplex or mdRNA) are shown below and
were synthesized using standard techniques. The RISC activator LacZ
dsRNA comprises a 21 nucleotide sense strand and a 21 nucleotide
antisense strand, which can anneal to form a double-stranded region
of 19 base pairs with a two deoxythymidine overhang on each strand
(referred to as 21/21 dsRNA).
LacZ dsRNA (21/21)--RISC Activator
TABLE-US-00001 (SEQ ID NO: 1) Sense 5'-CUACACAAAUCAGCGAUUUdTdT-3'
(SEQ ID NO: 2) Antisense 3'-dTdTGAUGUGUUUAGUCGCUAAA-5'
[0195] The Dicer substrate LacZ dsRNA comprises a 25 nucleotide
sense strand and a 27 nucleotide antisense strand, which can anneal
to form a double-stranded region of 25 base pairs with one blunt
end and a cytidine and uridine overhang on the other end (referred
to as 25/27 dsRNA).
LacZ dsRNA (25/27)--Dicer Substrate
TABLE-US-00002 (SEQ ID NO: 3) Sense
5'-CUACACAAAUCAGCGAUUUCCAUdGdT-3' (SEQ ID NO: 4) Antisense
3'-CUGAUGUGUUUAGUCGCUAAAGGUA C A- 5'
[0196] The LacZ mdRNA comprises two sense strands of 13 nucleotides
(5'-portion) and 11 nucleotides (3'-portion) and a 27 nucleotide
antisense strand, which three strands can anneal to form two
double-stranded regions of 13 and 11 base pairs separated by a
single nucleotide gap (referred to as a 13, 11/27 mdRNA). The
5'-end of the 11 nucleotide sense strand fragment may be optionally
phosphorylated. The "*" indicates a gap--in this case, a single
nucleotide gap (i.e., a cytidine is missing).
LacZ mdRNA (13, 11/27)--Dicer Substrate
TABLE-US-00003 (SEQ ID NOS: 5, 6) Sense
5'-CUACACAAAUCAG*GAUUUCCAUdGdT-3' (SEQ ID NO: 4) Antisense
3'-CUGAUGUGUUUAGUCGCUAAAGGUA C A- 5'
Each of the LacZ dsRNA or mdRNA was used to transfect 9lacZ/R
cells.
Transfection
[0197] Six well collagen-coated plates were seeded with
5.times.10.sup.5 9lacZ/R cells/well in a 2 ml volume per well, and
incubated overnight at 37.degree. C./5% CO.sub.2 in DMEM/high
glucose media. Preparation for transfection: 250 .mu.l of OPTIMEM
media without serum was mixed with 5 .mu.l of 20 pmol/.mu.l dsRNA
and 5 .mu.l of HIPERFECT transfection solution (Qiagen) was mixed
with another 250 .mu.l OPTIMEM media. After both mixtures were
allowed to equilibrate for 5 minutes, the RNA and transfection
solutions were combined and left at room temperature for 20 minutes
to form transfection complexes. The final concentration of
HIPERFECT was 50 .mu.M, and the dsRNAs were tested at 0.05 nM, 0.1
nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, and 10 nM, while the mdRNA
was tested at 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, and
50 nM. Complete media was removed, the cells were washed with
incomplete OPTIMEM, and then 500 transfection mixture was applied
to the cells, which were incubated with gentle shaking at
37.degree. C. for 4 hours. After transfecting, the transfection
media was removed, cells were washed once with complete DMEM/high
glucose media, fresh media added, and the cells were then incubated
for 48 hours at 37.degree. C., 5% CO.sub.2.
.beta.-Galactosidase Assay
[0198] Transfected cells were washed with PBS, and then detached
with 0.5 ml trypsin/EDTA. The detached cells were suspended in 1 ml
complete DMEM/high glucose and transferred to a clean tube. The
cells were harvested by centrifugation at 250.times.g for 5
minutes, and then resuspended in 50 .mu.l 1.times. lysis buffer at
4.degree. C. The lysed cells were subjected to two freeze-thaw
cycles on dry ice and a 37.degree. C. water bath. The lysed samples
were centrifuged for 5 minutes at 4.degree. C. and the supernatant
was recovered. For each sample, 1.5 .mu.l and 10 .mu.l of lysate
was transferred to a clean tube and sterile water added to a final
volume of 30 .mu.l followed by the addition of 70 .mu.l
o-nitrophenyl-.beta.-D-galactopyranose (ONPG) and 200 .mu.l
1.times. cleavage buffer with .beta.-mercaptoethanol. The samples
were mixed briefly, incubated for 30 minutes at 37.degree. C., and
then 500 .mu.l stop buffer was added (final volume 800 .mu.l).
.beta.-Galactosidase activity for each sample was measured in
disposable cuvettes at 420 nm. Protein concentration was determined
by the BCA (bicinchoninic acid) method. For the purpose of the
instant example, the level of measured LacZ activity was correlated
with the quantity of LacZ transcript within 9L/LacZ cells. Thus, a
reduction in .beta.-galactosidase activity after dsRNA
transfection, absent a negative impact on cell viability, was
attributed to a reduction in the quantity of LacZ transcripts
resulting from targeted degradation mediated by the LacZ dsRNA.
Results
[0199] Knockdown activity in transfected and untransfected cells
was normalized to a Qneg control dsRNA and presented as a
normalized value of the Qneg control (i.e., Qneg represented 100%
or "normal" gene expression levels). Both the lacZ RISC activator
and Dicer substrate dsRNAs molecule showed good knockdown of
.beta.-galactosidase activity at concentration as low as 0.1 nM
(FIG. 1), while the Dicer substrate antisense strand alone (single
stranded 27mer) had no silencing effect. Surprisingly, a gapped
mdRNA showed good knockdown although somewhat lower than that of
intact RISC activator and Dicer substrate dsRNAs (FIG. 1). The
presence of the gapmer cytidine (i.e., the missing nucleotide) at
various concentrations (0.1 .mu.M to 50 .mu.M) had no effect on the
activity of the mdRNA (data not shown). None of the dsRNA or mdRNA
solutions showed any detectable toxicity in the transfected 9L/LacZ
cells. The IC.sub.50 of the lacZ mdRNA was calculated to be 3.74
nM, which is about 10 fold lower than what had been previously
measured for lacZ dsRNA 21/21 (data not shown). These results show
that a meroduplex (gapped dsRNA) is capable of inducing gene
silencing.
Example 2
Knockdown of Influenza Gene Expression by Nicked dsRNA
[0200] The activity of a nicked dsRNA (21/21) in silencing
influenza gene expression as compared to a normal dsRNA (i.e., not
having a nick) was examined.
Nucleotide Sequences of dsRNA and mdRNA Targeting Influenza
mRNA
[0201] The dsRNA and nicked dsRNA (another form of meroduplex,
referred to herein as ndsRNA) are shown below and were synthesized
using standard techniques. The RISC activator influenza G1498 dsRNA
comprises a 21 nucleotide sense strand and a 21 nucleotide
antisense strand, which can anneal to form a double-stranded region
of 19 base pairs with a two deoxythymidine overhang on each
strand.
G1498-wt dsRNA (21/21)
TABLE-US-00004 (SEQ ID NO: 7) Sense 5'-GGAUCUUAUUUCUUCGGAGdTdT-3'
(SEQ ID NO: 8) Antisense 3'-dTdTCCUAGAAUAAAGAAGCCUC-5'
[0202] The RISC activator influenza G1498 dsRNA was nicked on the
sense strand after nucleotide 11 to produce a ndsRNA having two
sense strands of 11 nucleotides (5'-portion, italic) and 10
nucleotides (3'-portion) and a 21 nucleotide antisense strand,
which three strands can anneal to form two double-stranded regions
of 11 (shown in italics) and 10 base pairs separated by a one
nucleotide gap (which may be referred to as G1498 11, 10/21
ndsRNA-wt). The 5'-end of the 10 nucleotide sense strand fragment
may be optionally phosphorylated, as depicted by a "p" preceding
the nucleotide (e.g., pC).
G1498 ndsRNA-wt (11, 10/21)
TABLE-US-00005 (SEQ ID NO: 9, 10) Sense
5'-GGAUCUUAUUUCUUCGGAGdTdT-3' (SEQ ID NO: 8) Antisense
3'-dTdTCCUAGAAUAAAGAAGCCUC-5'
G1498 ndsRNA-wt (11, 10/21)
TABLE-US-00006 (SEQ ID NOS: 9, 10) Sense
5'-GGAUCUUAUUUpCUUCGGAGdTdT-3' (SEQ ID NO: 8) Antisense
3'-dTdTCCUAGAAUAAAGAAGCCUC-5'
In addition, each of these G1498 dsRNAs were made with each U
substituted with a 5-methyluridine (ribothymidine) and are referred
to as G1498 dsRNA-rT. Each of the G1498 dsRNA or ndsRNA
(meroduplex), with or without the 5-methyluridine substitution, was
used to transfect HeLa S3 cells having an influenza target sequence
associated with a luciferase gene. Also, the G1498 antisense strand
alone or the antisense strand annealed to the 11 nucleotide sense
strand portion alone or the 10 nucleotide sense strand portion
alone were examined for activity.
Transfection and Dual Luciferase Assay
[0203] The reporter plasmid psiCHECK.TM.-2 (Promega, Madison,
Wis.), which constitutively expresses both firefly luc2 (Photinus
pyralis) and Renilla (Renilla reniformis, also known as sea pansy)
luciferases, was used to clone in a portion of the influenza NP
gene downstream of the Renilla translational stop codon that
results in a Renilla-influenza NP fusion mRNA. The firefly
luciferase in the psiCHECK.TM.-2 vector is used to normalize
Renilla luciferase expression and serves as a control for
transfection efficiency.
[0204] Multi-well plates were seeded with HeLa S3 cells/well in 100
.mu.l Ham's F12 medium and 10% fetal bovine serum, and incubated
overnight at 37.degree. C./5% CO.sub.2. Using essentially the same
transfection procedure as described in Example 1, the HeLa S3 cells
were transfected with the psiCHECK.TM.-influenza plasmid (75 ng)
and G1498 dsRNA or ndsRNA (final concentration of 10 nM or 100 nM)
formulated in Lipofectamine.TM. 2000 and OPTIMEM reduced serum
medium. The transfection mixture was incubated with the HeLa S3
cells with gentle shaking at 37.degree. C. for about 18 to 20
hours.
[0205] After transfecting, firefly luciferase reporter activity was
measured first by adding Dual-G10.TM. Luciferase Reagent (Promega,
Madison, Wis.) for 10 minutes with shaking, and then quantitating
the luminescent signal using a VICTOR.sup.3.TM. 1420 Multilabel
Counter (PerkinElmer, Waltham, Mass.). After measuring the firefly
luminescence, Stop & Glo.RTM. Reagent (Promega, Madison, Wis.)
was added for 10 minutes with shaking to simultaneously quench the
firefly reaction and initiate the Renilla luciferase reaction, and
then the Renilla luciferase luminescent signal was quantitated
VICTOR.sup.3.TM. 1420 Multilabel Counter (PerkinElmer, Waltham,
Mass.).
Results
[0206] Knockdown activity in transfected and untransfected cells
was normalized to a Qneg control dsRNA and presented as a
normalized value of the Qneg control (i.e., Qneg represented 100%
or "normal" gene expression levels). Thus, a smaller value
indicates a greater knockdown effect. The G1498 dsRNA-wt and
dsRNA-rT showed similar good knockdown at a 100 nM concentration
(FIG. 2). Surprisingly, the G1498 ndsRNA-rT, whether phosphorylated
or not, showed good knockdown although somewhat lower than the
G1498 dsRNA-wt (FIG. 2). Similar results were obtained with dsRNA
or ndsRNA at 10 nM (data not shown). None of the G1498 dsRNA or
ndsRNA solutions showed any detectable toxicity in HeLa S3 cells at
either 10 nM or 100 nM. Even the presence of only half a nicked
sense strand (an 11 nucleotide or 10 nucleotide strand alone) with
a G1498 antisense strand showed some detectable activity. These
results show that a nicked-type meroduplex dsRNA molecule is
unexpectedly capable of promoting gene silencing.
Example 3
Knockdown Activity of mdRNA Having a Nick in Different
Positions
[0207] In this example, the activity of a dicer substrate LacZ
dsRNA of Example 1 having a sense strand with a nick at various
positions was examined. In addition, a dideoxy nucleotide (i.e.,
ddG) was incorporated at the 5'-end of the 3'-most strand of a
sense sequence having a nick or a single nucleotide gap to
determine whether the in vivo ligation of the nicked sense strand
is "rescuing" activity. The ddG is not a substrate for ligation.
Also examined was the influenza dicer substrate dsRNA of Example 6
having a sense strand with a nick at one of positions 8 to 14. The
"p" designation indicates that the 5'-end of the 3'-most strand of
the nicked sense influenza sequence was phosphorylated. The "L"
designation indicates that the G at position 2 of the 5'-most
strand of the nicked sense influenza sequence was substituted for a
locked nucleic acid G. The Qneg is a negative control dsRNA.
[0208] The dual fluorescence assay of Example 2 was used to measure
knockdown activity with 5 nM of the LacZ sequences and 0.5 nM of
the influenza sequences. The lacZ dicer substrate (25/27, LacZ-DS)
and lacZ RISC activator (21/21, LacZ) are equally active, and the
LacZ-DS can be nicked in any position between 8 and 14 without
affecting activity (FIG. 3). In addition, the inclusion of a ddG on
the 5'-end of the 3'-most LacZ sense sequence having a nick
(LacZ:DSNkd13-3'dd) or a one nucleotide gap (LacZ:DSNkd13D1-3'dd)
was essentially as active as the unsubstituted sequence (FIG. 3).
The influenza dicer substrate (G1498DS) nicked at any one of
positions 8 to 14 was also highly active (FIG. 4). Phosphorylation
of the 5'-end of the 3'-most strand of the nicked sense influenza
sequence had essentially no effect on activity, but addition of a
locked nucleic acid appears to improve activity.
Example 4
Mean Inhibitory Concentration of mdRNA
[0209] In this example, a dose response assay was performed to
measure the mean inhibitory concentration (IC.sub.50) of the
influenza dicer substrate dsRNA of Example 7 having a sense strand
with a nick at position 12, 13, or 14, including or not a locked
nucleic acid. The dual luciferase assay of Example 2 was used. The
influenza dicer substrate dsRNA (G1498DS) was tested at 0.0004 nM,
0.002 nM, 0.005 nM, 0.019 nM, 0.067 nM, 0.233 nM, 0.816 nM, 2.8 nM,
and 10 nM, while the mdRNA with a nick at position 13
(G1498DS:Nkd13) was tested at 0.001 nM, 0.048 nM, 0.167 nM, 1 nM, 2
nM, 7 nM, and 25 nM (see FIG. 5). Also tested were RISC activator
molecules (21/21) with or without a nick at various positions,
G1498DS:Nkd12, and G1498DS:Nkd14, each of the nicked versions with
a locked nucleic acid as described above (data not shown). The Qneg
is a negative control dsRNA.
[0210] The IC.sub.50 of the RISC activator G1498 was calculated to
be about 22 pM, while the dicer substrate G1498DS IC.sub.50 was
calculated to be about 6 pM. The IC.sub.50 of RISC and Dicer mdRNAs
range from about 200 pM to about 15 nM. The inclusion of a single
locked nucleic acid reduced the IC.sub.50 of Dicer mdRNAs by up 4
fold (data not shown). These results show that a meroduplex dsRNA
having a nick or gap in any position is capable of inducing gene
silencing.
Example 5
Knockdown Activity of mdRNA Having a Gap of Different Sizes and
Positions
[0211] The activity of an influenza dicer substrate dsRNA having a
sense strand with a gap of differing sizes and positions was
examined. The influenza dicer substrate dsRNA of Example 7 was
generated with a sense strand having a gap of 0 to 6 nucleotides at
position 8, a gap of 4 nucleotides at position 9, a gap of 3
nucleotides at position 10, a gap of 2 nucleotides at position 11,
and a gap of 1 nucleotide at position 12 (see Table 3). The Qneg is
a negative control dsRNA. Each of the mdRNAs were tested at a
concentration of 5 nM (data not shown) and 10 nM. The mdRNAs have
the following antisense strand 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU (SEQ
ID NO:11) and nicked or gapped sense strands as shown in Table
3.
TABLE-US-00007 TABLE 3 5' Sense* 3' Sense Gap Gap % mdRNA (SEQ ID
NO.) (SEQ ID NO.) Pos Size KD G1498:DSNkd8 GGAUCUUA (12)
UUUCUUCGGAGACAAdTdG (13) 8 0 67.8 G1498:DSNkd8D1 GGAUCUUA (12)
UUCUUCGGAGACAAdTdG (14) 8 1 60.9 G1498:DSNkd8D2 GGAUCUUA (12)
UCUUCGGAGACAAdTdG (15) 8 2 48.2 G1498:DSNkd8D3 GGAUCUUA (12)
CUUCGGAGACAAdTdG (16) 8 3 44.1 G1498:DSNkd8D4 GGAUCUUA (12)
UUCGGAGACAAdTdG (17) 8 4 30.8 G1498:DSNkd8D5 GGAUCUUA (12)
UCGGAGACAAdTdG (18) 8 5 10.8 G1498:DSNkd8D6 GGAUCUUA (12)
CGGAGACAAdTdG (19) 8 6 17.9 G1498:DSNkd9D4 GGAUCUUAU (20)
UCGGAGACAAdTdG (18) 9 4 38.9 G1498:DSNkd10D3 GGAUCUUAUU (21)
UCGGAGACAAdTdG (18) 10 3 38.4 G1498:DSNkd11D2 GGAUCUUAUUU (22)
UCGGAGACAAdTdG (18) 11 2 46.2 G1498:DSNkd12D1 GGAUCUUAUUUC (23)
UCGGAGACAAdTdG (18) 12 1 49.6 Plasmid -- -- -- -- 5.3 *G indicates
a locked nucleic acid G in the 5' sense strand.
[0212] The dual fluorescence assay of Example 2 was used to measure
knockdown activity. Similar results were obtained at both the 5 nM
and 10 nM concentrations. These data show that an mdRNA having a
gap of up to 6 nucleotides still has activity, although having four
or fewer missing nucleotides shows the best activity (see, also,
FIG. 6). Thus, mdRNA having various sizes gaps that are in various
different positions have knockdown activity.
[0213] To examine the general applicability of a sequence having a
sense strand with a gap of differing sizes and positions, a
different dsRNA sequence was tested. The lacZ RISC dsRNA of Example
1 was generated with a sense strand having a gap of 0 to 6
nucleotides at position 8, a gap of 5 nucleotides at position 9, a
gap of 4 nucleotides at position 10, a gap of 3 nucleotides at
position 11, a gap of 2 nucleotides at position 12, a gap of 1
nucleotide at position 12, and a nick (gap of 0) at position 14
(see Table 4). The Qneg is a negative control dsRNA. Each of the
mdRNAs was tested at a concentration of 5 nM (data not shown) and
25 nM. The lacZ mdRNAs have the following antisense strand
5'-AAAUCGCUGAUUUGUGUAGdTdTUAAA (SEQ ID NO:2) and nicked or gapped
sense strands as shown in Table 4.
TABLE-US-00008 TABLE 4 5' Sense* 3' Sense* Gap Gap mdRNA (SEQ ID
NO.) (SEQ ID NO.) Pos Size LacZ:Nkd8 CUACACAA (24) AUCAGCGAUUUdTdT
(25) 8 0 LacZ:Nkd8D1 CUACACAA (24) UCAGCGAUUUdTdT (26) 8 1
LacZ:Nkd8D2 CUACACAA (24) CAGCGAUUUdTdT (27) 8 2 LacZ:Nkd8D3
CUACACAA (24) AGCGAUUUdTdT (28) 8 3 LacZ:Nkd8D4 CUACACAA (24)
GCGAUUUdTdT (29) 8 4 LacZ:Nkd8D5 CUACACAA (24) CGAUUUdTdT (30) 8 5
LacZ:Nkd8D6 CUACACAA (24) GAUUUdTdT (31) 8 6 LacZ:Nkd9D5 CUACACAAA
(32) GAUUUdTdT (31) 9 5 LacZ:Nkd10D4 CUACACAAAU (33) GAUUUdTdT (31)
10 4 LacZ:Nkd11D3 CUACACAAAUC (34) GAUUUdTdT (31) 11 3 LacZ:Nkd12D2
CUACACAAAUCA (35) GAUUUdTdT (31) 12 2 LacZ:Nkd13D1 CUACACAAAUCAG
(36) GAUUUdTdT (31) 13 1 LacZ:Nkd14 CUACACAAAUCAGC (37) GAUUUdTdT
(31) 14 0 *A indicates a locked nucleic acid A in each sense
strand.
[0214] The dual fluorescence assay of Example 2 was used to measure
knockdown activity. FIG. 7 shows that an mdRNA having a gap of up
to 6 nucleotides has substantial activity and the position of the
gap may affect the potency of knockdown. Thus, mdRNA having various
sizes gaps that are in various different positions and in different
mdRNA sequences have knockdown activity.
Example 6
Knockdown Activity of Substituted mdRNA
[0215] The activity of an influenza dsRNA RISC sequences having a
nicked sense strand and the sense strands having locked nucleic
acid substitutions were examined. The influenza RISC sequence G1498
of Example 2 was generated with a sense strand having a nick at
positions 8 to 14 counting from the 5'-end. Each sense strand was
substituted with one or two locked nucleic acids as shown in Table
5. The Qneg and Plasmid are negative controls. Each of the mdRNAs
was tested at a concentration of 5 nM. The antisense strand used
was 5'-CUCCGAAGAAAUAAGAUCCdTdT (SEQ ID NO:8).
TABLE-US-00009 TABLE 5 5' Sense* 3' Sense* Nick % mdRNA (SEQ ID
NO.) (SEQ ID NO.) Pos KD G1498-wt GGAUCUUAUUUCUUCGGAGdTdT (7) -- --
85.8 G1498-L GGAUCUUAUUUCUUCGGAGdTdT (61) -- -- 86.8 G1498:Nkd8-1
GGAUCUUA (12) UUUCUUCGGAGdTdT (47) 8 36.0 G1498:Nkd8-2 GGAUCUUA
(40) UUUCUUCGGAGdTdT (54) 8 66.2 G1498:Nkd9-1 GGAUCUUAU (20)
UUCUUCGGAGdTdT (48) 9 60.9 G1498:Nkd9-2 GGAUCUUAU (41)
UUCUUCGGAGdTdT (55) 9 64.4 G1498:Nkd10-1 GGAUCUUAUU (21)
UCUUCGGAGdTdT (49) 10 58.2 G1498:Nkd10-2 GGAUCUUAUU (42)
UCUUCGGAGdTdT (56) 10 68.5 G1498:Nkd11-1 GGAUCUUAUUU (22)
CUUCGGAGdTdT (50) 11 75.9 G1498:Nkd11-2 GGAUCUUAUUU (43)
CUUCGGAGdTdT (57) 11 67.1 G1498:Nkd12-1 GGAUCUUAUUUC (23)
UUCGGAGdTdT (51) 12 59.9 G1498:Nkd12-2 GGAUCUUAUUUC (44)
UUCGGAGdTdT (58) 12 72.8 G1498:Nkd13-1 GGAUCUUAUUUCU (38)
UCGGAGdTdT (52) 13 37.1 G1498:Nkd13-2 GGAUCUUAUUUCU (45) UCGGAGdTdT
(59) 13 74.3 G1498:Nkd14-1 GGAUCUUAUUUCUU (39) CGGAGdTdT (53) 14
29.0 G1498:Nkd14-2 GGAUCUUAUUUCUU (46) CGGAGdTdT (60) 14 60.2 Qneg
-- -- -- 0 Plasmid -- -- -- 3.6 *Nucleotides that are bold and
underlined are locked nucleic acids.
[0216] The dual fluorescence assay of Example 2 was used to measure
knockdown activity. These data show that increasing the number of
locked nucleic acid substitutions tends to increase activity of an
mdRNA having a nick at any of a number of positions. The single
locked nucleic acid per sense strand is most active when the nick
is at position 11 (see FIG. 8). But, multiple locked nucleic acids
on each sense strand make mdRNA having a nick at any position as
active as the most optimal nick position with a single substitution
(i.e., position 11) (FIG. 8). Thus, mdRNA having duplex stabilizing
modifications make mdRNA essentially equally active regardless of
the nick position.
[0217] Similar results were observed when locked nucleic acid
substitutions were made in the LacZ dicer substrate mdRNA of
Example 1 (SEQ ID NOS:3 and 4). The lacZ dicer was nicked at
positions 8 to 14, and a duplicate set of nicked LacZ dicer
molecules were made with the exception that the A at position 3
(from the 5'-end) of the 5' sense strand was substituted for a
locked nucleic acid A (LNA-A). As is evident from FIG. 11, most of
the nicked lacZ dicer molecules containing LNA-A were as potent in
knockdown activity as the unsubstituted lacZ dicer (see FIG.
9).
Example 7
mdRNA Knockdown of Influenza Virus Titer
[0218] The activity of a dicer substrate nicked dsRNA in reducing
influenza virus titer as compared to a wild-type dsRNA (i.e., not
having a nick) was examined. The influenza dicer substrate sequence
(25/27) is as follows:
TABLE-US-00010 (SEQ ID NO: 62) Sense 5'-GGAUCUUAUUUCUUCGGAGACAAdTdG
(SEQ ID NO: 11) Antisense 5'-CAUUGUCUCCGAAGAAAUAAGAUCCUU
These mdRNA sequences are nicked after position 12, 13, and 14,
respectively, as counted from the 5'-end, and each sense strand
also has a G at position 2 substituted with locked nucleic acid
G.
[0219] For the viral infectivity assay, Vero cells were seeded at
6.5.times.10.sup.4 cells/well the day before transfection in 500
.mu.l 10% FBS/DMEM media per well. Samples of 100, 10, 1, 0.1, and
0.01 nM stock of each dsRNA were complexed with 1.0 .mu.l (1 mg/ml
stock) of Lipofectamine.TM. 2000 (Invitrogen, Carlsbad, Calif.) and
incubated for 20 minutes at room temperature in 150 .mu.l OPTIMEM
(total volume) (Gibco, Carlsbad, Calif.). Vero cells were washed
with OPTIMEM, and 150 .mu.l of the transfection complex in OPTIMEM
was then added to each well containing 150 .mu.l of OPTIMEM media.
Triplicate wells were tested for each condition. An additional
control well with no transfection condition was prepared. Three
hours post transfection, the media was removed. Each well was
washed once with 200 .mu.l PBS containing 0.3% BSA and 10 mM
HEPES/PS. Cells in each well were infected with WSN strain of
influenza virus at an MOI 0.01 in 200 .mu.l of infection media
containing 0.3% BSA/10 mM HEPES/PS and 4 .mu.g/ml trypsin. The
plate was incubated for 1 hour at 37.degree. C. Unadsorbed virus
was washed off with the 200 .mu.l of infection media and discarded,
then 400 .mu.l DMEM containing 0.3% BSA/10 mM HEPES/PS and 4
.mu.g/ml trypsin was added to each well. The plate was incubated at
37.degree. C., 5% CO.sub.2 for 48 hours, then 50 .mu.l supernatant
from each well was tested in duplicate by TCID.sub.50 assays
(Tissue-Culture Infective Dose 50, WHO protocol) in MDCK cells and
titers were estimated using the Spearman and Karber formula.
[0220] The results show that all of the G1498 nicked mdRNAs caused
a 10-fold reduction in influenza viral titers (FIG. 10). That is,
these mdRNAs show about a 50% to 60% viral titer knockdown, even at
a concentration as low as 10 pM (FIG. 11).
[0221] An in vivo influenza mouse model was also used to examine
the activity of a dicer substrate nicked dsRNA in reducing
influenza virus titer as compared to a wild-type dsRNA (i.e., not
having a nick). Female BALB/c mice (age 8-10 weeks with 5-10 mice
per group) were dosed intranasally with 120 nmol/kg/day dsRNA
(formulated in
C12-norArg(NH.sub.3+Cl--)-C12/DSPE-PEG2000/DSPC/cholesterol at a
ratio of 30:1:20:49) for three consecutive days before intranasal
challenge with influenza strain PR8 (20 PFU/mouse). Two days after
infection, whole lungs are harvested from each mouse and placed in
a solution of PBS/0.3% BSA with antibiotics, homogenize, and
measure the viral titer (TCID.sub.50). Doses were well tolerated by
the mice, indicated by less than 2% body weight reduction in any of
the dose groups. The mdRNAs tested exhibit similar, if not slightly
greater, virus reduction in vivo as compared to unmodified and
unnicked G1498 dicer substrate (see FIG. 12). Hence, mdRNA are
active in vivo.
Example 8
Effect of mdRNA on Cytokine Induction
[0222] The effect of the mdRNA structure on cytokine induction in
vivo was examined Female BALB/c mice (age 7-9 weeks) were dosed
intranasally with about 50 .mu.M dsRNA (formulated in
C12-norArg(NH.sub.3+Cl+C12/DSPE-PEG2000/DSPC/cholesterol at a ratio
of 30:1:20:49) or with 605 nmol/kg/day naked dsRNA for three
consecutive days. About four hours after the final dose is
administered, the mice were sacrificed to collect bronchoalveolar
fluid (BALF), and collected blood is processed to serum for
evaluation of the cytokine response. Bronchial lavage was performed
with 0.5 mL ice-cold 0.3% BSA in saline two times for a total of 1
mL. BALF was spun and supernatants collected and frozen until
cytokine analysis. Blood was collected from the vena cava
immediately following euthanasia, placed into serum separator
tubes, and allowed to clot at room temperature for at least 20
minutes. The samples were processed to serum, aliquoted into
Millipore ULTRAFREE 0.22 nm filter tubes, spun at 12,000 rpm,
frozen on dry ice, and then stored at -70.degree. C. until
analysis. Cytokine analysis of BALF and plasma were performed using
the Procarta.TM. mouse 10-Plex Cytokine Assay Kit (Panomics,
Fremont, Calif.) on a Bio-Plex.TM. array reader. Toxicity
parameters were also measured, including body weights prior to the
first dose on day 0 and again on day 3, just prior to euthanasia.
Spleens were harvested, weighed, and weights were normalized to
final body weights. The results are provided in Table 6.
TABLE-US-00011 TABLE 6 In vivo Cytokine Induction by Naked mdRNA
G1498:Nkd G1498:DSNkd G1498:DSNkd G1498:DSNkd Cytokine G1498 11-1
G1498:DS 12-1 13-1 14-1 IL-6 Conc 90.68 10.07 77.35 17.17 18.21
38.59 (pg/mL) Fold decrease -- 9 -- 5 4 2 IL-12 Conc 661.48 20.32
1403.61 25.07 37.70 57.02 (p40) (pg/mL) Fold decrease -- 33 -- 56
37 25 TNF.alpha. Conc 264.49 25.59 112.95 20.52 29.00 64.93 (pg/mL)
Fold decrease -- 10 -- 6 4 2
[0223] The mdRNA (RISC or dicer sized) induced cytokines to lesser
extent than the intact (i.e., not nicked) parent molecules. The
decrease in cytokine induction was greatest when looking at
IL-12(p40), the cytokine with consistently the highest levels of
induction of the 10 cytokine multiplex assay. For the mdRNA, the
decrease in IL-12 (p40) ranges from 25- to 56-fold, while the
reduction in either IL-6 or TNF.alpha. induction was more modest
(the decrease in these two cytokines ranges from 2- to 10-fold).
Thus, the mdRNA structure appears to provide an advantage in vivo
in that cytokine induction is minimized compared to unmodified
dsRNA.
[0224] Similar results were obtained with the formulated mdRNA,
although the reduction in induction was not as prominent. In
addition, the presence or absence of a locked nucleic acid has no
effect on cytokine induction. These results are shown in Table
7.
TABLE-US-00012 TABLE 7 In vivo Cytokine Induction by Formulated
mdRNA G1498:Nkd G1498:Nkd G1498:DSNkd G1498:DSNkd Cytokine G1498:DS
12-1 13-1 14-1 13 IL-6 Conc (pg/mL) 29.04 52.95 10.28 7.79 44.29
Fold decrease -- -1.8 3 4 -1.5 IL-12 (p40) Conc (pg/mL) 298.93
604.24 136.45 126.71 551.49 Fold decrease -- 0 2 2 1 TNF.alpha.
Conc (pg/mL) 13.49 21.35 3.15 3.15 18.69 Fold decrease -- -1.6 4 4
1.4
[0225] The teachings of all of references cited herein including
patents, patent applications and journal articles are incorporated
herein in their entirety by reference. Although the foregoing
disclosure has been described in detail by way of example for
purposes of clarity of understanding, it will be apparent to the
artisan that certain changes and modifications may be practiced
within the scope of the appended claims which are presented by way
of illustration not limitation. In this context, various
publications and other references have been cited within the
foregoing disclosure for economy of description. It is noted,
however, that the various publications discussed herein are
incorporated solely for their disclosure prior to the filing date
of the present application, and the inventors reserve the right to
antedate such disclosure by virtue of prior invention.
Sequence CWU 1
1
62121DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 1cuacacaaau cagcgauuut t 21221DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 2aaaucgcuga
uuuguguagt t 21325DNAArtificial SequenceSynthetic oligonucleotide;
combined DNA/RNA 3cuacacaaau cagcgauuuc caugt 25427RNAArtificial
SequenceSynthetic oligonucleotide 4acauggaaau cgcugauuug uguaguc
27513RNAArtificial SequenceSynthetic oligonucleotide 5cuacacaaau
cag 13611DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 6gauuuccaug t 11721DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 7ggaucuuauu ucuucggagt t
21821DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 8cuccgaagaa auaagaucct t 21911RNAArtificial
SequenceSynthetic oligonucleotide 9ggaucuuauu u 111010DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 10cuucggagtt
101127RNAArtificial SequenceSynthetic oligonucleotide 11cauugucucc
gaagaaauaa gauccuu 27128RNAArtificial SequenceSynthetic
oligonucleotide 12ggaucuua 81317DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 13uuucuucgga gacaatg
171416DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 14uucuucggag acaatg 161515DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 15ucuucggaga caatg
151614DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 16cuucggagac aatg 141713DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 17uucggagaca atg
131812DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 18ucggagacaa tg 121911DNAArtificial SequenceSynthetic
oligonucleotide 19cggagacaat g 11209RNAArtificial SequenceSynthetic
oligonucleotide 20ggaucuuau 92110RNAArtificial SequenceSynthetic
oligonucleotide 21ggaucuuauu 102211RNAArtificial SequenceSynthetic
oligonucleotide 22ggaucuuauu u 112312RNAArtificial
SequenceSynthetic oligonucleotide 23ggaucuuauu uc
12248RNAArtificial SequenceSynthetic oligonucleotide 24cuacacaa
82513DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 25aucagcgauu utt 132612DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 26ucagcgauuu tt
122711DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 27cagcgauuut t 112810DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 28agcgauuutt 10299DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 29gcgauuutt
9308DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 30cgauuutt 8317DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 31gauuutt 7329RNAArtificial
SequenceSynthetic oligonucleotide 32cuacacaaa 93310RNAArtificial
SequenceSynthetic oligonucleotide 33cuacacaaau 103411RNAArtificial
SequenceSynthetic oligonucleotide 34cuacacaaau c
113512RNAArtificial SequenceSynthetic oligonucleotide 35cuacacaaau
ca 123613RNAArtificial SequenceSynthetic oligonucleotide
36cuacacaaau cag 133714RNAArtificial SequenceSynthetic
oligonucleotide 37cuacacaaau cagc 143813RNAArtificial
SequenceSynthetic oligonucleotide 38ggaucuuauu ucu
133914RNAArtificial SequenceSynthetic oligonucleotide 39ggaucuuauu
ucuu 14408RNAArtificial SequenceSynthetic oligonucleotide
40ggaucuua 8419RNAArtificial SequenceSynthetic oligonucleotide
41ggaucuuau 94210RNAArtificial SequenceSynthetic oligonucleotide
42ggaucuuauu 104311RNAArtificial SequenceSynthetic oligonucleotide
43ggaucuuauu u 114412RNAArtificial SequenceSynthetic
oligonucleotide 44ggaucuuauu uc 124513RNAArtificial
SequenceSynthetic oligonucleotide 45ggaucuuauu ucu
134614RNAArtificial SequenceSynthetic oligonucleotide 46ggaucuuauu
ucuu 144713DNAArtificial SequenceSynthetic oligonucleotide;
combined DNA/RNA 47uuucuucgga gtt 134812DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 48uucuucggag tt
124911DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 49ucuucggagt t 115010DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 50cuucggagtt 10519DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 51uucggagtt
9528DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 52ucggagtt 8537DNAArtificial SequenceSynthetic
oligonucleotide 53cggagtt 75413DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 54uuucuucgga gtt
135512DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 55uucuucggag tt 125611DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 56ucuucggagt t
115710DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 57cuucggagtt 10589DNAArtificial SequenceSynthetic
oligonucleotide; combined DNA/RNA 58uucggagtt 9598DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 59ucggagtt
8607DNAArtificial SequenceSynthetic oligonucleotide 60cggagtt
76121DNAArtificial SequenceSynthetic oligonucleotide; combined
DNA/RNA 61ggaucuuauu ucuucggagt t 216225DNAArtificial
SequenceSynthetic oligonucleotide; combined DNA/RNA 62ggaucuuauu
ucuucggaga caatg 25
* * * * *
References