U.S. patent application number 12/675620 was filed with the patent office on 2010-11-11 for composition of asymmetric rna duplex as microrna mimetic or inhibitor.
This patent application is currently assigned to Boston Biomedical, Inc.. Invention is credited to Chiang Jia Li, Harry Rogoff.
Application Number | 20100286378 12/675620 |
Document ID | / |
Family ID | 40387778 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286378 |
Kind Code |
A1 |
Li; Chiang Jia ; et
al. |
November 11, 2010 |
Composition of Asymmetric RNA Duplex As MicroRNA Mimetic or
Inhibitor
Abstract
The present invention provides double-stranded RNA molecules
that are asymmetrical in strand length. The RNA molecule of the
invention, the asymmetric RNA duplex, has one or two overhangs at
the end. In one aspect, these novel RNA duplex molecules serve as
effective mimetics of miRNA. In another aspect, they are designed
to function as effective inhibitors of miRNA. Accordingly, the RNA
molecules of the present invention can be used to modulate miRNA
pathway activities, with tremendous implications for research, drug
discovery and development, and treatment of human diseases.
Inventors: |
Li; Chiang Jia; (Cambridge,
MA) ; Rogoff; Harry; (Wrentham, MA) |
Correspondence
Address: |
Milstein Zhang & Wu LLC
49 Lexington Street, Suite 6
Newton
MA
02465-1062
US
|
Assignee: |
Boston Biomedical, Inc.
Norwood
MA
|
Family ID: |
40387778 |
Appl. No.: |
12/675620 |
Filed: |
August 27, 2008 |
PCT Filed: |
August 27, 2008 |
PCT NO: |
PCT/US08/74531 |
371 Date: |
July 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61029753 |
Feb 19, 2008 |
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12675620 |
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61038954 |
Mar 24, 2008 |
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61029753 |
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60968257 |
Aug 27, 2007 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
1/04 20180101; A61P 37/08 20180101; A61P 37/02 20180101; C12N
15/111 20130101; C12N 2310/14 20130101; A61P 31/04 20180101; C12N
2310/321 20130101; C12N 2330/51 20130101; C12N 2310/315 20130101;
A61P 29/00 20180101; C12N 2320/53 20130101; A61P 1/00 20180101;
A61P 27/02 20180101; A61P 17/00 20180101; A61P 3/00 20180101; A61P
25/00 20180101; A61P 31/12 20180101; C12N 2310/141 20130101; A61P
11/00 20180101; A61P 31/00 20180101; A61P 37/00 20180101; A61P
13/12 20180101; A61P 43/00 20180101; C12N 2310/531 20130101; C12N
2320/30 20130101; A61P 37/06 20180101; A61P 5/00 20180101; C12N
15/113 20130101; A61P 35/00 20180101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 21/02 20060101
C07H021/02 |
Claims
1. A mimetic of a microRNA (miRNA), comprising: a double stranded
RNA molecule comprising a first strand of a first length and a
second strand of a second and shorter length, said first strand
having a sequence substantially the same as at least a portion of
said miRNA, said first and second strands being substantially
complementary to each other such that they form at least one double
stranded region, wherein said RNA molecule further comprises a
terminal overhang of 1-10 nucleotides; and wherein said mimetic is
adapted to mimic said miRNA in modulating expression of at least
one gene.
2. The mimetic of claim 1 wherein said miRNA is a guide strand.
3. The mimetic of claim 1 wherein said miRNA is a mature miRNA.
4. The mimetic of claim 1 wherein said miRNA is an endogenous miRNA
duplex comprising a mature miRNA and a substantially complementary
passenger strand, and wherein said second strand of said mimetic
has a sequence substantially the same as at least a portion of said
passenger strand.
5. The mimetic of claim 1 further comprising at least one
mismatched or unmatched nucleotide in sequence between said first
and second strands.
6-13. (canceled)
14. The mimetic of claim 1 further comprising both a 3' overhang
and a 5' overhang on said first strand.
15. The mimetic of claim 14 wherein both said 3' and 5' overhangs
are of 1-3 nucleotides.
16-17. (canceled)
18. The mimetic of claim 1 wherein said first strand has a length
of 15-30 nucleotides, and said second strand has a length of 12-29
nucleotides.
19. The mimetic of claim 1 wherein said first strand has a length
of 15-28 nucleotides and said second strand has a length from 12-26
nucleotides.
20. The mimetic of claim 1 wherein said first strand has a length
of 19-25 nucleotides and said second strand has a length of 12-24
nucleotides.
21. The mimetic of claim 1 wherein said first strand has a length
of 19-23 nucleotides and said second strand has a length of 14-20
nucleotides.
22. (canceled)
23. The mimetic of claim 1 wherein said terminal overhang is
stabilized against degradation.
24. The mimetic of claim 1 further comprising a nick in at least
one of said first and second strands.
25. The mimetic of claim 1 wherein the double-stranded region
comprises a gap of one or more unpaired nucleotides.
26. The mimetic of claim 1 further comprising a modified nucleotide
or a nucleotide analogue.
27. The mimetic of claim 26 wherein said modified nucleotide or
analogue is a sugar-, backbone-, and/or base-modified
ribonucleotide.
28-30. (canceled)
31. The mimetic of claim 27, wherein the at least one modified
nucleotide or analogue is inosine, or a tritylated base.
32. The mimetic of claim 26, wherein the nucleotide analogue is a
sugar-modified ribonucleotide in which the 2'-OH group is replaced
by a group selected from the group consisting of H, OR, R, halo,
SH, SR, NH.sub.2, NHR, NR.sub.2, and CN, wherein each R is
independently selected from the group consisting of C1-C6 alkyl,
alkenyl and alkynyl, and halo is selected from the group consisting
of F, Cl, Br and I.
33. The mimetic of claim 26, wherein the nucleotide analogue is a
backbone-modified ribonucleotide containing a phosphothioate
group.
34. The mimetic of claim 1 further comprising a
deoxynucleotide.
35-36. (canceled)
37. The mimetic of claim 1, wherein said first strand shares the
same seed region as said miRNA.
38. The mimetic of claim 1 wherein the GC content of the double
stranded region is about 20-60%.
39. The mimetic of claim 1 wherein said first strand comprises a 5'
overhang with at least one nucleotide selected from the group
consisting of A, U, and dT.
40. The mimetic of claim 1, further being conjugated to an entity
selected from the group consisting of peptide, antibody, polymer,
lipid, oligonucleotide, cholesterol and aptamer.
41. The mimetic of claim 1 wherein said double stranded RNA
molecule is synthetic or isolated.
42. The mimetic of claim 1 wherein said double stranded RNA
molecule is transcribed from a recombinant vector or its
progeny.
43. The mimetic of claim 1 adapted to modulate at least 20% the
expression of said at least one gene.
44. The mimetic of claim 1 where said miRNA is of the Let7
family.
45. The mimetic of claim 1 comprising one of the following duplex
sequence: TABLE-US-00007 Sense: 5'-AUACAAUCUACUGUC Antisense:
5'-UGAGGUAGUAGGUUGUAUAGU, and Sense: 5'-ACAACCUACUACCUC Antisense:
5'-AAUGAGGUAGUAGGUUGUAUG.
46. A mimetic of a mature microRNA (miRNA) comprising: a double
stranded RNA molecule comprising a first strand of 15-28
nucleotides and a second shorter strand of 12-26 nucleotides, said
first strand having a sequence substantially the same as at least a
portion of said mature miRNA, said first and second strands being
substantially complementary to each other such that they form at
least one double stranded region, wherein said first strand further
comprises both a 3' overhang of 1-8 nucleotides and a 5' overhang
of 1-8 nucleotides, wherein said mimetic is adapted to mimic said
mature miRNA in modulating expression of at least one gene.
47. The mimetic of claim 46 wherein said second strand has a
sequence substantially the same as at least a portion of a
passenger RNA strand that forms an endogenous duplex with the
mature miRNA.
48. The mimetic of claim 46 further comprising at least one
mismatched or unmatched nucleotide in sequence between said first
and second strands.
49. The mimetic of claim 48 wherein a loop is formed by said at
least one mismatched or unmatched nucleotide.
50. The mimetic of claim 46 further comprising a
deoxynucleotide.
51. The mimetic of claim 46 wherein at least one of said 3' and 5'
overhang is stabilized against degradation.
52. The mimetic of claim 46 further comprising a nick in at least
one of said first and second strands.
53. The mimetic of claim 46 wherein the double-stranded region
comprises a gap of one or more unpaired nucleotides.
54. The mimetic of claim 46 further comprising a modified
nucleotide or a nucleotide analogue.
55-146. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
provisional patent applications Ser. Nos. 60/968,257 filed on Aug.
27, 2007, 61/029,753 filed on Feb. 19, 2008, and 61/038,954 filed
on Mar. 24, 2008, the entire contents of which applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] MicroRNA (miRNA) is a class of endogenous, small RNA
molecules that was first discovered to regulate gene expression at
the translation level, and is part of the cell's RNA interference
(RNAi) mechanism. First discovered in Caenorhabditis elegans,
miRNAs have been found in both plants and animals including humans.
The sequences of many miRNAs are homologous among different
organisms, suggesting that miRNAs represent a relativley old and
important regulatory pathway (Grosshans et al. J Cell Biol 156:
17-21 (2002)). Encoded by genes transcribed from DNA but not
translated into protein (non-protein-coding RNA), miRNAs regulate
as much as 30% of mamalian genes. (Czech, NEJM 354:1194-1195
(2006); Mack, Nature Biotech. 25:631-638 (2007); Eulalio, et al.,
Cell 132:9-14 (2008)). Recent researches have found that miRNA
represses protein production by blocking translation or causing
transcript degradation, thereby regulating gene expression. A
single miRNA may target 250-500 different mRNAs, proving this class
of RNA to be an extremely important mediator of a wide range of
cellular functions. Many of the genes regulated by miRNAs are
disease-causing genes, therefore, any mechanism that modulates
miRNA functionality has great thearapeutic potentials.
[0003] In animals, miRNAs are first expressed from the genome as
RNA transcripts called primary miRNAs (pri-miRNAs). They are
transcribed by RNA Polymerase II, and likely form hairpin
structures. In the nucleaus, the dsRNA-specific ribonuclease Drosha
processes the pri-miRNAs into shorter, about 70- to 100-nucleotide
long stem-loop structures known as pre-miRNAs, which are then
exported out into the cytoplasm, likely by Exportin-5 (Exp5). (Yi,
et al. Genes Dev. 17: 3011-3016 (2003)). In the cytoplasm, Dicer, a
member of the RNase III ribonuclease family, cleaves the pre-miRNA
into a double-stranded guide/passenger (miRNA/miRNA*) duplex with
3' overhangs at both ends. The two strands of the miRNA duplex
often have mismatches from imperfect complementarity in their
sequences and when they separate, a mature miRNA, in many cases,
between about 19 and 23 nucelotides long, is bound by the
RNA-induced silencing complex (RISC) or a similar protein complex.
RISC is also the protein complex that effects target-specific mRNA
degradation mediated by small or short interfereing RNAs
(siRNAs).
[0004] While it is not entirely clear at this point how the miRNA
duplex or the single-stranded mature miRNA interacts with RISC, it
is believed that once it is selected by a catalytic component of
RISC, argonaute, as the guide strand, the mature miRNA is
integrated into the complex, and binds to a messenger RNA (mRNA)
molecule that has a significantly, though often not perfectly,
complementary sequence. The passenger strand, miRNA*, is likely
degraded. Translation of the mRNA bound by the miRNA-RISC complex
is then repressed, resulting in reduced expression of the
corresponding gene. In some cases, the bound mRNA is cleaved or
deadenylated and degraded.
[0005] References cited herein are not admitted to be prior art to
the claimed invention.
SUMMARY OF THE INVENTION
[0006] The present invention is about the discovery that a novel
class of duplex RNAs can effectively modulate miRNA activities in
mammalian cells, which is termed here "asymmetrical interfering
RNAs" (aiRNAs). The hallmark of this novel class of RNAs is the
length asymmetry between the two RNA strands. The present invention
provides evidence that double-stranded RNAs asymmetric in
strand-length can be constructed to either mimic or inhibit miRNAs
in cells, and modulate miRNA pathway activities in both directions,
i.e., up and down.
[0007] In one aspect, the present invention provides a mimetic of a
microRNA (miRNA), comprising: a double stranded RNA molecule
comprising a first strand of a first length and a second strand of
a second and shorter length, said first strand having a sequence
substantially the same as at least a portion of said miRNA, said
first and second strands being substantially complementary to each
other such that they form at least one double stranded region,
wherein said RNA molecule further comprises a terminal overhang of
1-10 nucleotides; and wherein said mimetic is adapted to mimic said
miRNA in modulating expression of at least one gene.
[0008] In a preferred aspect, the present invention provides a
mimetic of a mature microRNA (miRNA), and the mimetic comprises: a
double stranded RNA molecule comprising a first strand of 15-28
nucleotides and a second shorter strand of 12-26 nucleotides, said
first strand having a sequence substantially the same as at least a
portion of said mature miRNA, said first and second strands being
substantially complementary to each other such that they form at
least one double stranded region, wherein said first strand further
comprises both a 3' overhang of 1-8 nucleotides and a 5' overhang
of 1-8 nucleotides, wherein said mimetic is adapted to mimic said
mature miRNA in modulating expression of at least one gene.
[0009] In one feature, the miRNA being mimicked by the mimetics of
the invention is a guide strand or a mature miRNA. In an
embodiment, the miRNA is an endogenous miRNA duplex comprising a
mature miRNA and a substantially complementary passenger strand;
said second strand of said mimetic has a sequence substantially the
same as at least a portion of said passenger strand.
[0010] In a further aspect, the present invention provides an
inhibitor of a microRNA (miRNA), comprising: a double stranded RNA
molecule comprising a first strand of a first length and a second
strand of a second and shorter length, said first strand having a
sequence substantially complementary to at least a portion of a
target miRNA, said first and second strands being substantially
complementary to each other such that they form at least one double
stranded region, wherein said RNA molecule further comprises a
terminal overhang of 1-10 nucleotides, wherein said inhibitor is
adapted to inhibit said target miRNA.
[0011] And in a preferred aspect, the present invention provides an
inhibitor of a mature microRNA (miRNA), comprising: a double
stranded RNA molecule comprising a first strand of 15-28
nucleotides and a second, shorter strand of 12-26 nucleotides, said
first strand having a sequence substantially complementary to at
least a portion of said mature miRNA, said first and second strands
being substantially complementary to each other such that they form
at least one double stranded region, wherein said first strand
further comprises both a 3' overhang of 1-8 nucleotides and a 5'
terminal overhang of 1-8 nucleotides, wherein said inhibitor is
adapted to inhibit said target miRNA.
[0012] In one feature, the miRNA targeted by the inhibitors of the
invention is a guide strand, or a mature strand. In some
embodiments, the inhibitors of the invention is capable of
decreasing the amount of mature miRNA by about at least 30%, 50%,
70%, 80% or 90%.
[0013] In one feature, the mimetics and inhibitors of the invention
further includes at least one mismatched or unmatched nucleotide in
sequence between said first and second strands. In one embodiment,
the at least one mismatched or unmatched nucleotide forms a loop.
In an alternative embodiment, the first and second strands in the
mimetics and inhibitors are perfectly complementary to each other
in said double stranded region.
[0014] The terminal overhang of the mimetics and inhibitors of the
invention, in some embodiments, is of 1-8 nucleotides, and 1-3
nucleotides, in some other embodiments. The terminal overhang can
be a 3' overhang, and in a preferred embodiment, on the first
strand, which is the longer strand. In another embodiment, the
terminal overhang is a 5' overhang, and in a preferred embodiment,
on the first strand. In some embodiments, the mimetics and
inhibitors have both a 3' overhang and a 5' overhang on said first
strand, and preferably, both said 3' and 5' overhangs are of 1-3
nucleotides. In another embodiment, the mimetics and inhibitors of
the invention has one terminal overhang on one end and a blunt end
on the other end.
[0015] In various embodiments of the mimetics: said first strand
(the longer strand) has a length of 13-100 nucleotides, and said
second strand has a length of 5-30 nucleotides; said first strand
has a length of 15-30 nucleotides, and said second strand has a
length of 12-29 nucleotides; said first strand has a length of
15-28 nucleotides and said second strand has a length from 12-26
nucleotides; said first strand has a length of 19-25 nucleotides
and said second strand has a length of 12-24 nucleotides; said
first strand has a length of 19-23 nucleotides and said second
strand has a length of 14-20 nucleotides.
[0016] In various embodiments of the inhibitors: said first strand
(the longer strand) has a length of 10-100 nucleotides, and said
second strand has a length of 5-30 nucleotides; said first strand
has a length of 15-60 nucleotides, and said second strand has a
length of 5-28 nucleotides; said first strand has a length of 15-28
nucleotides and said second strand has a length from 12-26
nucleotides; said first strand has a length of 19-25 nucleotides
and said second strand has a length of 12-20 nucleotides.
[0017] In one feature, in the mimetics and inhibitors of the
invention, said first strand is longer than said second strand by a
length selected from the group consisting of 1, 2, 3, 4, 5, 6, 7,
8, 9, and 10 nucleotides.
[0018] In one feature, in the mimetics and inhibitors of the
invention, said terminal overhang is stabilized against
degradation.
[0019] In one feature, the mimetics and inhibitors further have at
least one nick in at least one of said first and second strands. In
another feature, the double-stranded region of the mimetics or
inhibitors further comprises a gap of one or more unpaired
nucleotides.
[0020] In one feature, the mimetics and inhibitors include a
modified nucleotide or a nucleotide analogue. In another feature,
they include at least one deoxynucleotide, which can be in one or
more regions selected from the group consisting of 3'-overhang,
5'-overhang, and double-stranded region.
[0021] In one feature, in the mimetics of the invention, said first
strand has a sequence that is at least 60 percent, or in some
embodiments, at least 70 percent, the same as at least said portion
of said miRNA. In one embodiment, said first strand of the mimetics
shares the same seed region as said miRNA.
[0022] In one feature, in the inhibitors of the invention, said
first strand has a sequence that is at least 60 percent, or in some
embodiments, at least 70 percent, complementary to at least said
portion of its target miRNA.
[0023] In an embodiment, the GC content of the double stranded
region in the mimetics and inhibitors of the invention is about
20-60%, or preferably, about 30-50%.
[0024] In an embodiment of the mimetics and inhibitors, said first
strand comprises a sequence motif with at least one nucleotide
selected from the group consisting of A, U, and dT. In one further
embodiment, the 5' overhang has a sequence motif "AA," "UU," or
"dTd." In one feature, a mimetic and inhibitor of the invention is
further conjugated to an entity selected from the group consisting
of peptide, antibody, polymer, lipid, oligonucleotide, cholesterol
and aptamer.
[0025] In one feature of the mimetics and inhibitors of the
invention, the double stranded RNA molecule is synthetic or
isolated. In an embodiment, the double stranded RNA molecule of the
invention, whether a mimetic or inhibitor, is transcribed from a
recombinant vector or its progeny.
[0026] In one feature, the mimetics of the invention are adapted to
modulate at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the
expression of said at least one gene. In one embodiment, the
mimetics of the invention mimic an miRNA of the Let? family.
[0027] In an embodiment, the mimetic of the invention comprises one
of the following duplex sequence:
TABLE-US-00001 Sense: 5'-AUACAAUCUACUGUC Antisense:
5'-UGAGGUAGUAGGUUGUAUAGU, and Sense: 5'-ACAACCUACUACCUC Antisense:
5'-AAUGAGGUAGUAGGUUGUAUG.
[0028] In another embodiment, the inhibitor of the invention
comprises one of the following duplex sequence:
TABLE-US-00002 Sense: 5'-GGUAGUAGGUUGUAU Antisense:
5'-AACAUACAACCUACUACCUCA, Sense: 5'-AUCAGACUGAUGUUG Antisense:
5'-AAUCAACAUCAGUCUGAUAAG, and Sense: 5'-AUGCUAAUCGUGAUA Antisense:
5'-AACUAUCACGAUUAGCAUUAA.
[0029] In one aspect, the present invention provides an expression
vector comprising a DNA sequence encoding at least the first strand
of the double stranded RNA molecule of the mimetics and inhibitors
of the invention, said sequence operably linked to an expression
control sequence, e.g., a promoter.
[0030] In an embodiment, the expression vector further comprises a
second DNA sequence encoding at least the second strand of the
double stranded RNA molecule of the mimetics and inhibitors of the
invention, said sequence operably linked to a second promoter. The
vector may be selected from a group consisting of viral, eukaryotic
and bacterial expression vectors.
[0031] In one aspect, the present invention provides a cell
comprising the above expression vector. In another aspect, the
present invention provides a cell that comprises the double-strand
RNA molecule of the invention.
[0032] In a further aspect, the present invention provides a method
of making a mimetic of a microRNA (miRNA), said method comprising
the steps of: selecting a miRNA sequence; synthesizing a first RNA
strand having a region substantially the same as at least a portion
of contiguous nucleotides in said miRNA; synthesizing a second and
shorter RNA strand, and combining the synthesized strands under
suitable conditions to form a double stranded RNA molecule with at
least one terminal overhang such that said RNA molecule is capable
of mimicking said miRNA in modulating expression of at least one
gene.
[0033] In another aspect, the present invention provides a method
of making an inhibitor of a target microRNA (miRNA), said method
comprising the steps of: selecting a target miRNA sequence;
synthesizing a first RNA strand having a region substantially
complementary to at least a portion of contiguous nucleotides in
said target mRNA; synthesizing a second and shorter RNA strand, and
combining the synthesized strands under suitable conditions to form
a double stranded RNA molecule with at least one terminal overhang
such that said RNA molecule is capable of inhibiting said target
miRNA.
[0034] In one feature, the methods of making a mimetic or inhibitor
according to the invention further includes one or more of the
following steps: chemically modifying said at least one terminal
overhang against degradation; introducing at least one
deoxynucleotide into said double stranded RNA molecule; introducing
at least one modified nucleotide or a nucleotide analogue into said
double stranded RNA molecule; introducing at least one mismatch,
nick or gap in said double stranded region; conjugating at least
one of said first and second strands with an entity selected from
the group consisting of peptide, antibody, polymer, lipid,
oligonucleotide, cholesterol, and aptamer; modifying at least one
base in one of the strands. In an embodiment, the methods of making
a mimetic or inhibitor further includes a step of introducing at
least one modified nucleotide analogue into the duplex RNA molecule
during the synthesizing step, after the synthesizing and before the
combining step, or after the combining step.
[0035] In one feature, in the methods of making a mimetic or
inhibitor, at least one of the RNA strands is enzymatically or
biologically synthesized. In some embodiments, the first strand and
the second strand are synthesized separately or simultaneously.
[0036] In another aspect, the present invention provides a method
of modulating an miRNA pathway in a cell or organism, said method
comprising the steps of contacting said cell or organism with a
mimetic of the invention, under conditions where said mimicking of
said miRNA can occur; and modulating the expression of at least one
gene using said mimetic, thereby modulating an endogenous miRNA
pathway. In an embodiment, the first strand in the double stranded
RNA molecule of the mimetic mimics said endogenous miRNA in its
interaction with RISC.
[0037] In another aspect, the present invention provides a method
of modulating an miRNA pathway in a cell, said method comprising
the steps of: contacting said cell or organism with an inhibitor of
the invention, under conditions where said inhibition of said
target miRNA can occur; and reducing the amount of target miRNA
available with said inhibitor, thereby modulating an endogenous
miRNA pathway.
[0038] In one feature, the modulation methods of the invention,
whether using a mimetic or an inhibitor, further include the step
of introducing said duplex RNA molecule into a target cell in
culture or in an organism in which the modulation of gene
expression can occur.
[0039] In an embodiment, the introducing step is selected from the
group consisting of transfection, lipofection, electroporation,
infection, injection, oral administration, inhalation, topic and
regional administration. Further, the introducing step may use a
pharmaceutically acceptable excipient, carrier, or diluent selected
from the group consisting of a pharmaceutical carrier, a
positive-charge carrier, a liposome, a protein carrier, a polymer,
a nanoparticle, a nanoemulsion, a lipid, and a lipoid,
[0040] In another aspect, the present invention provides use of the
modulation methods of the invention for various purposes including:
determining the function or utility of a gene in a cell or an
organism, for modulating the expression of at least one gene in a
cell or an organism. In an embodiment, the gene is associated with
a disease, a pathological condition, or an undesirable condition.
In anther embodiment, the gene is associated with a human or animal
diseases. The gene may be a gene of a pathogenic microorganism, a
viral gene, a tumor-associated gene, and so on. In an embodiment,
the gene is associated with a disease selected from the group
consisting of autoimmune disease, inflammatory diseases,
degenerative diseases, infectious diseases, proliferative diseases,
metabolic diseases, immune-mediated disorders, allergic diseases,
dermatological diseases, malignant diseases, gastrointestinal
disorders, respiratory disorders, cardiovascular disorders, renal
disorders, rheumatoid disorders, neurological disorders, endocrine
disorders, and aging. In one feature, the methods of the invention
are used for studying drug target in vitro or in vivo. In a further
feature, the methods of the invention are used for treating or
preventing a disease or an undesirable condition.
[0041] In another aspect, the present invention provides a
pharmaceutical composition comprising as an active agent at least
one mimetic or inhibitor of the invention, and a pharmaceutically
acceptable excipient, carrier, or diluent. The carrier may be
selected from the group consisting of a pharmaceutical carrier, a
positive-charge carrier, a liposome, a protein carrier, a polymer,
a nanoparticle, a nanoemulsion, a lipid, and a lipoid.
[0042] In another aspect, the present invention provides a
treatment method comprising administering an effective amount of
the pharmaceutical composition of the invention to a subject in
need. In various embodiments, the pharmaceutical composition is
administered via a route selected from the group consisting of
intravascular (iv), subcutaneous (se), topic, po, inhalation,
intramuscular, intra-peritoneal (ip) and regional routes. In
various embodiments, the effective amount of the pharmaceutical
composition is about 1 ng to 1 g per day, 100 ng to 1 g per day, or
1 .mu.g to 500 mg per day. In an embodiment, the method is used in
treating cancer.
[0043] In a further aspect, the present invention provides a
research reagent comprising the mimetic of the invention, or the
inhibitor of the invention. The present invention also provides a
kit comprising said research reagent.
[0044] In yet another aspect, the present invention provides a
method of diagnosing a patient of a disease or condition,
comprising contacting cells of the patient with the mimetic or
inhibitor of the invention; and looking for at least one change
indicating said disease or condition.
[0045] Other features and advantages of the present invention are
apparent from the additional descriptions provided herein including
the different examples. The provided examples illustrate different
components and methodology useful in practicing the present
invention. The examples do not limit the claimed invention. Based
on the present disclosure the skilled artisan can identify and
employ other components and methodology useful for practicing the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1A shows the structure of a duplex RNA molecule that
has both a 3'-overhang and a 5'-overhang.
[0047] FIG. 1B shows the duplex RNA molecule of FIG. 1A with a nick
in one of the strands.
[0048] FIG. 1C shows the duplex RNA molecule of FIG. 1A with a gap
in one of the strands.
[0049] FIG. 2A shows the structure of a duplex RNA molecule that
has a blunt end, and a 5'-overhang.
[0050] FIG. 2B shows the structure of a duplex RNA molecule that
has a blunt end, and a 3'-overhang.
[0051] FIG. 2C shows the structure of a duplex RNA molecule that
has a 3'-overhang on both ends.
[0052] FIG. 2D shows the structure of a duplex RNA molecule that
has a 5'-overhang on both ends.
[0053] FIG. 2E shows an alternative structure of a duplex RNA
molecule that has both a 3% overhang and a 5'-overhang.
[0054] FIG. 2F shows an alternative structure of a duplex RNA
molecule that has a 3'-overhang on both ends.
[0055] FIG. 3 shows the induction of gene silencing of
.beta.-catenin by aiRNA (asymmetric interfering RNAs). FIG. 3A
shows the confirmation of the oligos. After annealing, the oligos
were confirmed by 20% polyacrylamide gel. Lane 1, 21 nt/21 nt; lane
2, 12 nt (a)/21 nt; lane 3, 12 nt (b)/21 nt; lane 4, 13 nt/13 nt;
lane 5, 13 nt/21 nt; lane 6, 14 nt/14 nt; lane 7, 14 nt(a)/21nt;
lane 8, 14 nt(b)/21 nt; lane 9, 15 nt/15 nt; lane 10, 15 nt/21
nt.
[0056] FIG. 3B shows the effects of the oligos in gene silencing.
HeLa cells were plated at 200,000 cells/well into a 6 well culture
plate. 24 hours later they were transfected with scramble siRNA
(lane 1), 21-bp siRNA targeted E2F1 (lane 2, as a control for
specificity) or 21-bp siRNA targeted beta-catenin (lane 3, as a
positive control), or the same concentration of aiRNA of different
length mix: 12 nt(a)/21 nt (lane 4); 12 nt (b)/21 nt (lane 5); 13
nt/21 nt (lane 6); 14nt (a)/21 nt (lane 7); 14 nt (b)/21 nt (lane
8); 15 nt/21 nt (lane 9). Cells were harvested 48 hours after
transfection. Expression of .beta.-catenin was determined by
Western blot. E2F1 and actin were used as controls.
[0057] FIGS. 4 and 5 show the structure-activity relationship of
aiRNA oligos, with or without base substitutions, in mediating gene
silencing. Hela cells were transfected with the indicated aiRNA.
Cells were harvested and lysates generated at 48 hours post
transfection. Western blots were performed to detect levels of
.beta.-catenin and actin. si stands for .beta.-catenin siRNA
oligonucleotide. The numerical labeling above each lane corresponds
to the aiRNA oligos in Table 3.
[0058] FIG. 6 shows the analysis of the mechanism of gene silencing
triggered by aiRNA.
[0059] FIG. 6a shows the northern blot analysis of .beta.-catenin
mRNA levels in cells transfected with aiRNA or siRNA for the
indicated number of days.
[0060] FIG. 6b shows the schematic of 5'-RACE-PCR for
.beta.-catenin showing cleavage of mRNA and expected PCR
product.
[0061] FIG. 6c shows .beta.-catenin cleavage products mediated by
aiRNA were amplified by 5'-RACE-PCR from cells transfected with
aiRNA for 4 or 8 hours.
[0062] FIG. 6d shows the schematic of .beta.-catenin mRNA cleavage
site confirmed by sequencing the 5'-RACE-PCR fragment.
[0063] FIG. 6e shows differential RISC loading efficiency of aiRNA
and siRNA. aiRNA or siRNA duplexes were transfected into Hela cells
48 hours after transfection with pCMV-Ago2. Ago2 was
immunoprecipitated at the indicated time points following aiRNA or
siRNA transfection, and northern blot analysis was performed to
determine levels of Ago2/RISC associated small RNAs. Levels of Ago2
(shown below) were determined by western blot following IP.
[0064] FIG. 6f shows the effects of knocking down Ago2 or Dicer on
gene siliencing activity of aiRNA and siRNA. Cells were transfected
with scramble siRNA (siCon), or siRNA targeting Ago2 (siAgo2), or
Dicer (siDicer) 24 hours prior to transfection with scramble aiRNA
(Con) or aiRNA targeting Stat3 (ai). Cells were harvested and
western blot analysis was performed at 48 hours following aiStat3
transfection.
[0065] FIG. 7 shows the advantages of incorporation of aiRNA into
RISC compared to siRNA.
[0066] FIG. 7A shows that aiRNA enters RISC with better efficiency
than siRNA. Cells transfected with Ago2 expression plasmid were
transfected with aiRNA or siRNA for the indicated times. Following
cell lysis, Ago2 was immunoprecipitaed, RNA extracted from the
immunoprecipitate, and separated on a 15% acrylamide gel. Following
transfer, the membrane was hybridized to a probe to detect the 21
mer antisense strand of the aiRNA or siRNA. IgG control lane shows
lack of signal compared to Ago2 immunoprecipitate.
[0067] FIG. 7B shows that the sense strand of aiRNA does not stay
in RISC. Membrane from (A) was stripped and re-probed to detect the
sense strand of the transfected oligo. Cartoons in (A) and (B)
illustrate the position of the sense strand (upper strand), the
antisense strand (lower strand), or the duplex on the membrane.
[0068] FIG. 8 shows that the mechanism of RISC loading by
aiRNA.
[0069] FIG. 8A shows the immunoprecipitation analysis of the
interaction between different strands of aiRNA or siRNA and Ago2.
Hela S-10 lysate containing overexpressed Ago2 was incubated with
the indicated aiRNA or siRNA duplex containing .sup.32P end labeled
sense or antisense strands. The (*) marks the location of the
label. Following Ago2 immunoprecipitation, the RNA was isolated and
separated on a 15% acrylamide gel and exposed to film. The
Ago2-associated RNAs are shown in the pellet fraction, while the
non-Ago2 bound RNAs remain in the supernatant (Sup).
[0070] FIG. 8B shows the role of sense strand cleavage in aiRNA
activity. Cells were transfected with aiRNA or aiRNA with sense
strand 2'-O-methyl at position 8 (predicted Ago2 cleavage site) or
position 9 as a control. RNA was collected at 4 hours post
transfection and qRT-PCR performed to determine relative levels of
.beta.-catenin mRNA remaining.
[0071] FIG. 9 shows the aiRNA and siRNA competition analysis.
[0072] FIG. 9A illustrates the siRNA and aiRNA duplex containing
.sup.32P end labeled antisense strands. The (*) marks the location
of the label.
[0073] FIG. 9B shows that the cold aiRNA does not compete with
labeled siRNA for Ago2. Hela S-10 lysate containing overexpressed
Ago2 was incubated with the .sup.32P end labeled siRNA and cold
aiRNA or siRNA duplex prior to Ago2 immunoprecipitation. RNA was
then isolated and analyzed on 15% acrylamide gel.
[0074] FIG. 9C shows that the cold siRNA does not compete with
labeled aiRNA for Ago2. The same S-10 lysate used in B was
incubated with the .sup.32P end labeled aiRNA and cold aiRNA or
siRNA duplex prior to Ago2 immunoprecipitation. RNA was then
isolated and analyzed on 15% acrylamide gel.
[0075] FIG. 10 illustrates the model of aiRNA and siRNA showing
observed differences in RISC loading and generation of mature
RISC.
[0076] FIG. 11 shows asymmetric RNA duplexes of 14-15 by with
antisense overhangs (aiRNA) induced potent, efficacious, rapid, and
durable gene silencing.
[0077] FIG. 11A shows the Diagram showing sequence and design of
siRNA and aiRNA targeting .beta.-catenin.
[0078] FIG. 11B shows the induction of gene silencing by aiRNA of
various lengths. .beta.-catenin protein levels were analysed by
western blot in cells transfected with indicated aiRNA for 48
hours.
[0079] FIG. 11C shows that aiRNA is more potent and efficacious
than siRNA in inducing .beta.-catenin protein depletion. Hela cells
were transfected with aiRNA or siRNA targeting .beta.-catenin at
the indicated concentrations. At 48 hours post-transfection, cell
lysates were made and western blot analysis was done.
[0080] FIG. 11D shows that the aiRNA is more efficacious, rapid,
and durable than siRNA in reducing .beta.-catenin RNA levels. Cells
were transfected with 10 nM 15 by aiRNA or 21-mer siRNA for the
indicated number of days before northern blot analysis.
[0081] FIG. 12 shows that aiRNA mediates rapid and potent
silencing.
[0082] FIG. 12A shows the sequence and structure of aiRNA and siRNA
used to target .beta.-catenin.
[0083] FIG. 12B shows RT-PCR of .beta.-catenin mRNA levels from
cells transfected with control aiRNA or aiRNA targeting
.beta.-catenin. RNA was collected at the indicated times post
transfection.
[0084] FIG. 12C shows the quantitative real-time RT-PCR of
.beta.-catenin mRNA levels in cells transfected with control,
aiRNA, or siRNA for the indicated number of hours.
[0085] FIG. 12D shows the western blot analysis of .beta.-catenin
protein levels in cells transfected with control, aiRNA, or siRNA
for the indicated times.
[0086] FIG. 13 shows the comparison of aiRNA with siRNA in gene
silencing efficacy and durability against multiple targets. Hela
cells were transfected with scramble siRNA (c), aiRNA (ai), or
siRNA (si) targeting (a) .beta.-catenin at 10 nM, (b) Stat3, (c)
EF2, or (d) NQO1 at 20 nM. RNA and protein was purified at the
indicated time points and analyzed for mRNA levels by quantitative
real time polymerase chain reaction (qRT-PCR) and protein levels by
western blot. qRT-PCR data is normalized to siCon transfected
cells.
[0087] FIG. 14 shows aiRNA mediated gene silencing is effective
against various genes in multiple cell lines.
[0088] FIG. 14a shows aiRNA duplex is more efficacious than siRNA
in targeting .beta.-catenin in different mammalian cell lines.
[0089] FIG. 14b shows the western blot analysis of Nbs1, Survivin,
Parp1, p21 from cells transfected with 20 nM of the indicated aiRNA
or siRNA for 48 hours.
[0090] FIG. 14c shows the western blot analysis of Rsk1, PCNA,
p70S6K, mTOR, and PTEN from cells transfected with 20 nM of the
indicated aiRNA or siRNA for 48 hours.
[0091] FIG. 14d shows the allele-specific gene silencing of k-Ras
by aiRNA. aiRNA targeting wildtype k-Ras was tested for silencing
of k-Ras in both k-Ras wildtype (DLD1) and k-Ras mutant (SW480)
cell lines by western blot analysis.
[0092] FIG. 15 show the lack of off-target gene silencing by
sense-strand, immuno stimulation, and serum stability of
aiRNAs.
[0093] FIG. 15a shows RT-PCR analysis of the expression of
interferon inducible genes in PBMC mock treated or incubated with
.beta.-catenin siRNA or aiRNA duplex for 16 hours.
[0094] FIG. 15b shows RT-PCR analysis of the expression of
interferon inducible genes in Hela cells mock transfected or
transfected with EF2 or Survivin aiRNA or siRNA for 24 hours.
[0095] FIG. 15c shows the microarray analysis for changes in the
expression of known interferon response related genes. Total RNA
isolated from aiRNA and siRNA transfected Hela cells was analyzed
by microarray.
[0096] FIG. 15d shows that no sense-strand mediated off-target gene
silencing is detected for aiRNA. Cells were co-transfected with
aiRNA or siRNA and either a plasmid expressing Stat3 (sense RNA) or
a plasmid expressing antisense Stat3 (antisense RNA). Cells were
harvested and RNA collected at 24 hours post transfection and
relative levels of Stat3 sense or antisense RNA were determined by
quantitative real time PCR or RT-PCR (inserts).
[0097] FIG. 15e shows the Stability of aiRNA and siRNA duplexes in
human serum. aiRNA and siRNA duplexes were incubated in 10% human
serum at 37.degree. C. for the indicated amount of time prior to
gel electrophoresis. Duplex remaining (% of control) is
indicated.
[0098] FIG. 15f illustrates the proposed model for gene specific
silencing mediated by the aiRNA duplex.
[0099] FIG. 16 shows the potent Anti-Tumor Activity of aiRNA
against .beta.-catenin in SW480 human colon xenografted mouse
model. Immunosurpressed mice with established subcutaneous SW480
human colon cancer were given intravenously (iv) with 0.6 nmol
PEI-complexed .beta.-catenin siRNAs, PEI-complexed .beta.-catenin
aiRNAs or a PEI-complexed unrelated siRNA as negative control
daily. Tumor size was evaluated periodically during treatment. Each
point represents the mean.+-.SEM of six tumors.
[0100] FIG. 17 shows the potent Anti-Tumor Activity of aiRNA
against .beta.-catenin in HT29 human colon xenografted mouse model.
Immunosurpressed mice with established subcutaneous HT29 human
colon cancer were given intravenously (iv) with 0.6 nmol
PEI-complexed .beta.-catenin siRNAs, PEI-complexed .beta.-catenin
aiRNAs or a PEI-complexed unrelated siRNA as negative control every
other day. Tumor size was evaluated periodically during treatment.
Each point represents the mean.+-.SEM of five tumors.
[0101] FIG. 18 shows that Let-7a mimetic aiRNA can function as
Let-7a with equal or better efficiency.
[0102] FIG. 18A shows the sequence of the Let-7a and Let-7a mimetic
aiRNA.
[0103] FIG. 18B shows that Let-7a mimetic aiRNA can down regulate
the mRNA level of Let-7a target k-Ras.
[0104] FIG. 19A shows the sequences and structure of the indicated
aiRNA mimetic and inhibitors.
[0105] FIG. 19B shows the sequences of the indicated mature
miRNAs.
[0106] FIG. 20A shows the effect of Let-7c mimetic aiRNA and aiRNA
Let-7c inhibitor on mRNA level of k-Ras.
[0107] FIG. 20B shows the effect of Let-7c mimetic aiRNA and aiRNA
Let-7c inhibitor on protein level of k-Ras.
[0108] FIG. 21 shows that designed aiRNAs can inhibit miRNAs.
[0109] FIG. 21A shows that anti-Let7c aiRNA potently inhibit the
expression of Let-7c.
[0110] FIG. 21B shows that anti-miR21 aiRNA potently inhibit the
expression of miR21.
[0111] FIG. 21C shows that anti-miR155 aiRNA potently inhibit the
expression of miR155.
[0112] FIG. 22A shows that MCF7 cells express high level of
miR-21.
[0113] FIG. 22B shows that FaDu cells express high level of
miR-155.
[0114] FIG. 23 shows that the anti-Let7c aiRNA is more efficacious
than the commercially available miRNA inhibitor.
[0115] FIG. 24 shows that the anti-miR21 aiRNA is more efficacious
than the commercially available miRNA inhibitor.
[0116] FIG. 25 shows that the anti-miR155 aiRNA is more efficacious
than the commercially available miRNA inhibitor.
[0117] FIG. 26 compares the potency of the anti-Let7c aiRNA and the
commercially available miRNA inhibitor.
[0118] FIG. 27 compares the potency of the anti-miR21 aiRNA and the
commercially available miRNA inhibitor.
[0119] FIG. 28 compares the potency of the anti-miR155 aiRNA and
the commercially available miRNA inhibitor.
[0120] FIG. 29 theorizes one possible mechanism for inhibition of
miRNA by the aiRNA.
[0121] FIGS. 30 and 31 list the known human miRNAs.
DETAILED DESCRIPTION OF THE INVENTION
[0122] As used in the specification and claims, the singular form
"a", "an", and "the" include plural references unless the context
clearly dictate otherwise. For example, the term "a cell" includes
a plurality of cells including mixtures thereof
[0123] As used herein, a "double stranded RNA," a "duplex RNA," or
a "RNA duplex" refers to an RNA of two strands and with at least
one double-stranded region, and includes RNA molecules that have at
least one gap, nick, bulge, loop, and/or bubble either within a
double-stranded region or between two neighboring double-stranded
regions. If one strand has a gap or a single-stranded region of
unmatched nucleotides between two double-stranded regions, that
strand is considered as having multiple fragments. A
double-stranded RNA as used here can have terminal overhangs on
either end or both ends. In some embodiments, the two strands of
the duplex RNA can be linked through certain chemical linker.
[0124] As used herein, an "antisense strand" refers to an RNA
strand that has substantial sequence complementarity against a
target messenger RNA. An antisense strand can be part of an siRNA
molecule, part of a miRNA/miRNA* duplex, or a single-strand mature
miRNA.
[0125] The term "isolated" or "purified" as used herein refer to a
material that is substantially or essentially free from components
that normally accompany it in its native state. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography.
[0126] As used herein, "modulating" and its grammatical equivalents
refer to either increasing or decreasing (e.g., silencing), in
other words, either up-regulating or down-regulating.
[0127] As used herein, the term "subject" refers to any animal
(e.g., a mammal), including, but not limited to humans, non-human
primates, rodents, and the like, which is to be the recipient of a
particular treatment. Typically, the terms "subject" and "patient"
are used interchangeably herein in reference to a human
subject.
[0128] Terms such as "treating," "treatment," "to treat,"
"alleviating" and "to alleviate" as used herein refer to both (1)
therapeutic measures that cure, slow down, lessen symptoms of
and/or halt progression of a diagnosed pathologic condition or
disorder and (2) prophylactic or preventative measures that prevent
or slow the development of a targeted pathologic condition or
disorder. Thus those in need of treatment include those already
with the disorder; those prone to have the disorder; and those in
whom the disorder is to be prevented. A subject is successfully
"treated" according to the methods of the present invention if the
patient shows one or more of the following: a reduction in the
number of or complete absence of cancer cells; a reduction in the
tumor size; inhibition of or an absence of cancer cell infiltration
into peripheral organs including the spread of cancer into soft
tissue and bone; inhibition of or an absence of tumor metastasis;
inhibition or an absence of tumor growth; relief of one or more
symptoms associated with the specific cancer; reduced morbidity and
mortality; and improvement in quality of life.
[0129] As used herein, the terms "inhibiting", "to inhibit" and
their grammatical equivalents, when used in the context of a
bioactivity, refer to a down-regulation of the bioactivity, which
may reduce or eliminate the targeted function, such as the
production of a protein or the phosphorylation of a molecule. In
particular embodiments, inhibition may refers to a reduction of
about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the targeted
activity. When used in the context of a disorder or disease, the
terms refer to success at preventing the onset of symptoms,
alleviating symptoms, or eliminating the disease, condition or
disorder.
[0130] As used herein, the term "substantially complementary"
refers to complementarity in a base-paired, double-stranded region
between two nucleic acids and not any single-stranded region such
as a terminal overhang or a gap region between two double-stranded
regions. The complementarity does not need to be perfect; there may
be any number of base pair mismatches, for example, between the two
nucleic acids. However, if the number of mismatches is so great
that no hybridization can occur under even the least stringent of
hybridization conditions, the sequence is not a substantially
complementary sequence. When two sequences are referred to as
"substantially complementary" herein, it is meant that the
sequences are sufficiently complementary to each other to hybridize
under the selected reaction conditions. The relationship of nucleic
acid complementarity and stringency of hybridization sufficient to
achieve specificity is well known in the art. Two substantially
complementary strands can be, for example, perfectly complementary
or can contain from 1 to many mismatches so long as the
hybridization conditions are sufficient to allow, for example
discrimination between a pairing sequence and a non-pairing
sequence. Accordingly, substantially complementary sequences can
refer to sequences with base-pair complementarity of 100, 95, 90,
80, 75, 70, 60, 50 percent or less, or any number in between, in a
double-stranded region.
[0131] As used herein, antagomirs are miRNA inhibitors, and can be
used in the silencing of endogenous miRNAs.
[0132] As used herein, mimetics or mimics are miRNA agonists, and
can be used to replace endogenous miRNAs as functional equivalents
and thereby up-regulating pathways affected by such endogenous
miRNAs.
[0133] As a natural form of post-transcriptional gene silencing and
each with a wide range of targets, miRNAs present great
opportunities in disease treatment and prevention. As one can
imagine, depending on the regulatory targets of a particular miRNA,
it might be desirable to either up-regulate or down-regulate that
miRNA's level in cells. For example, if its mRNA targets correspond
to one or more tumor suppressor genes, one might want to
down-regulate the miRNA, e.g., miR-21. On the other hand, one might
want to up-regulate an miRNA if its targets include the mRNA of an
oncogene, e.g., let-7 miRNAs which target the mRNA of the RAS
family of oncogenes. In situations where multiple miRNAs modulate a
single gene target or a network of related gene targets, it may be
desirable to up-regulate certain miRNA while simultaneously
down-regulate others.
[0134] Currently, effective tools devised to regulate RNAs include
single-strand RNAs (e.g., antisense) and double-stranded RNAs where
the two strands are very much the same length (e.g., siRNA
consisting of 21 nucleotide dsRNA with symmetric 2-nt 3'
overhangs). Among these, siRNA evokes RNAi in eukaryotes via an
exogenous mechanism (typically through viral or artificial
introduction) separate from miRNA, although in the end it utilizes
the same RISC as miRNA does to effect more specific gene silencing.
Single-strand antisense oligonucleotides are not stable in cell,
and although various chemical modifications have been attempted on
them, they remain largely ineffective as miRNA inhibitors
(Vermeulen A. et al. RNA 13: 726-730(2007)). In sum, while miRNA
has been discovered for over a decade, little progress has been
made in devising effective modulators, whether mimetics or
inhibitors, of miRNA (Mack G. Nat Biotech 25(6): 631-638
(2007)).
[0135] The present invention provides a novel structural scaffold
called asymmetric interfering RNA (aiRNA) that can be used to
effect siRNA-like results (described in detail in co-owned PCT and
U.S. applications filed on the same day as the present application
under the title "Composition of asymmetric interfering RNA and uses
thereof," the entire content of which is incorporated herein by
reference) and also to modulate miRNA pathway activities.
[0136] The novel structural design of aiRNA is not only
functionally potent in effecting gene regulation, but also offers
several advantages over the current state-of-art, RNAi regulators
(mainly antisense, siRNA). Among the advantages, aiRNA can have RNA
duplex structure of much shorter length than the current siRNA
constructs, which should reduce the cost of synthesis and abrogate
or reduce length-dependent triggering of nonspecific
interferon-like immune responses from host cells. The shorter
length of the passenger strand in aiRNA should also eliminate or
reduce the passenger strand's unintended incorporation in RISC, and
in turn, reduce off-target effect observed in miRNA-mediated gene
silencing. aiRNA can be used in all areas that current miRNA-based
technologies are being applied or contemplated to be applied,
including biology research, R&D in biotechnology and
pharmaceutical industries, and miRNA-based diagnostics and
therapies.
1.0. The aiRNA Structural Scaffold
[0137] The present invention is pertinent to asymmetrical double
stranded RNA molecules that are capable of modulating
miRNA-mediated gene silencing. In an embodiment, a RNA molecule of
the present invention comprises a first strand and a second strand,
wherein the second strand is substantially complementary to the
first strand, and the first strand and the second strand form at
least one double-stranded region, wherein the first strand is
longer than the second strand (length asymmetry). The RNA molecule
of the present invention has at least one double-stranded region,
and two ends independently selected from the group consisting of
5'-overhang, 3'-overhang, and blunt end (e.g., see FIGS. 1A,
2A-2D). In another embodiment, the first strand is shorter than the
second strand. The two form at least one double-stranded region,
and can have two ends independently selected from the group
consisting of 5'-overhang, 3'-overhang, and blunt end (e.g., see
FIGS. 2E-2F).
[0138] In the field of making small RNA regulators where changes,
addition and deletion of a single nucleotide can critically affect
the functionality of the molecule (Elbashir, et al, The EMBO
Journal 20:6877-6888 (2001)), the aiRNA scaffold provides a
structural platform distinct from the classic siRNA structure of
21-nt double-strand RNA which is symmetric in each strand and their
respective 3' overhangs. Further, the aiRNA of the present
invention provides a much-needed new approach in designing a new
class of small molecule regulators that, as shown by data included
in the examples below, can overcome obstacles currently encountered
in RNAi-based researches and drug development. For example, data
from aiRNAs that structurally mimic siRNAs show that aiRNAs are
more efficacious, potent, rapid-onset, durable, and specific than
siRNAs in inducing gene silencing. Further evidence is provided
below that aiRNAs can be designed to regulate miRNA pathways,
either as a mimic or inhibitor.
[0139] Any single-stranded region of the RNA molecule of the
invention, including any terminal overhangs and gaps in between two
double-stranded regions, can be stabilized against degradation,
either through chemical modification or secondary structure. The
RNA strands can have unmatched or imperfectly matched nucleotides.
Each strand may have one or more nicks (a cut in the nucleic acid
backbone, e.g., see FIG. 1B), gaps (a fragmented strand with one or
more missing nucleotides, e.g, see FIG. 1C), and modified
nucleotides or nucleotide analogues. Not only can any and all of
the nucleotides in the RNA molecule chemically modified, each
strand may be conjugated with one or more moieties to enhance its
functionality, for example, with moieties such as one or more
peptides, antibodies, antibody fragments, aptamers, polymers,
lipids, oligonucleotides and so on. In some embodiments, the
moieties are added to enhance delivery. In some other embodiments,
the moieties are added to enhance one or more pharmacological
properties, e.g., drug absorption.
[0140] In an embodiment, the first strand is at least 1 nt longer
than the second strand. In a further embodiment, the first strand
is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nt longer than the second strand. In another
embodiment, the first strand is 20-100 nt longer than the second
strand. In a further embodiment, the first strand is 2-12 nt longer
than the second strand. In an even further embodiment, the first
strand is 3-10 nt longer than the second strand.
[0141] In an embodiment, the first strand, or the long strand, has
a length of 5-100 nt, or preferably 10-30 or 12-30 nt, or more
preferably 15-28 nt. In one embodiment, the first strand is 21
nucleotides in length. In an embodiment, the second strand, or the
short strand, has a length of 3-30 nt, or preferably 3-29 nt or
10-26 nt, or more preferably 12-26 nt. In an embodiment, the second
strand has a length of 15 nucelotides.
[0142] In an embodiment, the double-stranded region has a length of
3-98 bp. In a further embodiment, the double-stranded region has a
length of 5-28 bp. In an even further embodiment, the
double-stranded region has a length of 10-19 bp. The length of the
double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 bp.
[0143] In an embodiment, the double-stranded region of the RNA
molecule does not contain any mismatch or bulge, and the two
strands are perfectly complementary to each other in the
double-stranded region. In another embodiment, the double-stranded
region of the RNA molecule contains mismatch and/or bulge.
[0144] In an embodiment, the terminal overhang is 1-10 nucleotides.
In a further embodiment, the terminal overhang is 1-8 nucleotides.
In another embodiment, the terminal overhang is 3 nt.
1.1. The Duplex RNA Molecule with Both a 5'-Overhang and a
3'-Overhang
[0145] Referring to FIG. 1A, in one embodiment of the prsent
invention, the double stranded RNA molecule has both a 5'-overhang
and a 3'-overhang on the first strand. The RNA molecule comprises a
first strand and a second strand; the first strand and the second
strand form at least one double-stranded region with substantially
complementary sequences, wherein the first strand is longer than
the second strand. On the first strand, flanking the
double-stranded region, there is an unmatched overhang on both the
5' and 3' termini.
[0146] In an embodiment, the first strand is at least 2 nt longer
than the second strand. In a further embodiment, the first strand
is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20 nt longer than the second strand. In another
embodiment, the first strand is 20-100 nt longer than the second
strand. In a further embodiment, the first strand is 2-12 nt longer
than the second strand. In an even further embodiment, the first
strand is 3-10 nt longer than the second strand.
[0147] In an embodiment, the first strand has a length of 5-100 nt.
In a further embodiment, the first strand has a length of 5-100 nt,
and the second strand has a length from 3-30 nucleotides. In an
even further embodiment, the first strand has a length of 5-100 nt,
and the second strand has a length from 3-18 nucleotides.
[0148] In an embodiment, the first strand has a length from 10-30
nucleotides. In a further embodiment, the first strand has a length
from 10-30 nucleotides, and the second strand has a length from
3-28 nucleotides. In an even further embodiment, the first strand
has a length from 10-30 nucleotides, and the second strand has a
length from 3-19 nucleotides.
[0149] In an embodiment, the first strand has a length from 12-26
nucleotides. In a further embodiment, the first strand has a length
from 12-26 nucleotides, and the second strand has a length from
10-24 nucleotides. In an even further embodiment, the first strand
has a length from 12-26 nucleotides, and the second strand has a
length from 10-19 nucleotides.
[0150] In an embodiment, the first strand has a length of 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, or 30 nt. In another embodiment, the second
strand has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nt.
[0151] In an embodiment, the first strand has a length of 21 nt,
and the second strand has a length of 15 nt. This particular
embodiment is sometimes referred to as the "15/21" configuration
herein below. In some of the "15/21" configurations, the longer
strand has a 3-nt overhang in both the 3' end and the the 5'
end.
[0152] In an embodiment, the 3'-overhang has a length of 1-10 nt.
In a further embodiment, the 3'-overhang has a length of 1-8 nt. In
an even further embodiment, the 3'-overhang has a length of 2-6 nt.
In one embodiment, the 3'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
[0153] In an embodiment, the 5'-overhang has a length of 1-10 nt.
In a further embodiment, the 5'-overhang has a length of 1-6 nt. In
an even further embodiment, the 5'-overhang has a length of 2-4 nt.
In one embodiment, the 5'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
[0154] In an embodiment, the length of the 3'-overhang is equal to
that of the 5'-overhang. In another embodiment, the 3'-overhang is
longer than the 5'-overhang. In an alternative embodiment, the
3'-overhang is shorter than the 5'-overhang.
[0155] In an embodiment, the duplex RNA molecule comprises a
double-stranded region of substantially complementary sequences of
about 15 nt, a 3-nt 3'-overhang, and a 3-nt 5'-overhang. The first
strand is 21 nt and the second strand is 15 nt. In one feature, the
double-stranded region of various embodiments consists of perfectly
complementary sequences. In an alternative feature, the double
strand region includes at least one nick (FIG. 1B), gap (FIG. 1C),
and/or mismatch (bulge or loop).
[0156] In an embodiment, the double-stranded region has a length of
3-98 bp. In a further embodiment, the double-stranded region has a
length of 5-28 bp. In an even further embodiment, the
double-stranded region has a length of 10-19 bp. The length of the
double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.
There can be more than one double-stranded region.
[0157] In an embodiment, the first strand is the guide strand,
which is capable of targeting a substantially complementary gene
transcript such as a messenger RNA (mRNA) for gene silencing either
by cleavage or by translation repression.
[0158] The same principles and features discussed above also apply
to the embodiment where the second strand is longer than the first
strand (FIG. 2E).
1.2. The Duplex RNA Molecule with a Blunt End and a 5'-Overhang or
a 3'-Overhang
[0159] In one embodiment, the duplex RNA molecule comprises a
double-stranded region, a blunt end, and a 5'-overhang or a
3'-overhang (see, e.g., FIGS. 2A and 2B). The RNA molecule
comprises a first strand and a second strand, wherein the first
strand and the second strand form a double-stranded region, wherein
the first strand is longer than the second strand.
[0160] In an embodiment, the double-stranded region has a length of
3-98 bp. In a further embodiment, the double-stranded region has a
length of 5-28 bp. In an even further embodiment, the
double-stranded region has a length of 10-18 bp. The length of the
double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 bp.
The double-stranded region can have features similar to those
described with regard to other embodiments and are not necessarily
repeated here. For example, the double-stranded region can consist
of perfectly complementary sequences or include at least one nick,
gap, and/or mismatch (bulge or loop).
[0161] In an embodiment, the first strand is at least 1 nt longer
than the second strand. In a further embodiment, the first strand
is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 nt longer than the second strand. In another
embodiment, the first strand is 20-100 nt longer than the second
strand. In a further embodiment, the first strand is 2-12 nt longer
than the second strand. In an even further embodiment, the first
strand is 4-10 nt longer than the second strand.
[0162] In an embodiment, the first strand has a length of 5-100 nt.
In a further embodiment, the first strand has a length of 5-100 nt,
and the second strand has a length from 3-30 nucleotides. In an
even further embodiment, the first strand has a length of 10-30 nt,
and the second strand has a length from 3-19 nucleotides. In
another embodiment, the first strand has a length from 12-26
nucleotides, and the second strand has a length from 10-19
nucleotides.
[0163] In an embodiment, the duplex RNA molecule comprises a
double-stranded region, a blunt end, and a 3'-overhang (see, e.g.,
FIG. 2B).
[0164] In an embodiment, the 3'-overhang has a length of 1-10 nt.
In a further embodiment, the 3'-overhang has a length of 1-8 nt. In
an even further embodiment, the 3'-overhang has a length of 2-6 nt.
In one embodiment, the 3'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
[0165] In an alternative embodiment, the duplex RNA molecule
comprises a double-stranded region, a blunt end, and a 5'-overhang
(see, e.g., FIG. 2A).
[0166] In an embodiment, the 5'-overhang has a length of 1-10 nt.
In a further embodiment, the 5'-overhang has a length of 1-6 nt. In
an even further embodiment, the 5'-overhang has a length of 2-4 nt.
In one embodiment, the 5'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
1.3. The Duplex RNA Molecule with Two 5'-Overhangs or Two
3'-Overhangs
[0167] In one embodiment, the duplex RNA molecule comprises a
double-stranded region, and two 3'-overhangs or two 5'-overhangs
(see, e.g., FIGS. 2C and 2D). The RNA molecule comprises a first
strand and a second strand, wherein the first strand and the second
strand form a double-stranded region, wherein the first strand is
longer than the second strand.
[0168] In an embodiment, the double-stranded region has a length of
3-98 bp. In a further embodiment, the double-stranded region has a
length of 5-28 bp. In an even further embodiment, the
double-stranded region has a length of 10-18 bp. The length of the
double-stranded region can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
bp.
[0169] In an embodiment, the first strand is at least 1 nt longer
than the second strand. In a further embodiment, the first strand
is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, or 20 nt longer than the second strand. In another
embodiment, the first strand is 20-100 nt longer than the second
strand. In a further embodiment, the first strand is 2-12 nt longer
than the second strand. In an even further embodiment, the first
strand is 4-10 nt longer than the second strand.
[0170] In an embodiment, the first strand has a length of 5-100 nt.
In a further embodiment, the first strand has a length of 5-100 nt,
and the second strand has a length from 3-30 nucleotides. In an
even further embodiment, the first strand has a length of 10-30 nt,
and the second strand has a length from 3-18 nucleotides. In
another embodiment, the first strand has a length from 12-26
nucleotides, and the second strand has a length from 10-16
nucleotides.
[0171] In an alternative embodiment, the duplex RNA molecule
comprises a double-stranded region, and two 3'-overhangs (see,
e.g., FIG. 2C). The double-stranded region shares similar features
as described with regard to other embodiments.
[0172] In an embodiment, the 3'-overhang has a length of 1-10 nt.
In a further embodiment, the 3'-overhang has a length of 1-6 nt. In
an even further embodiment, the 3'-overhang has a length of 2-4 nt.
In one embodiment, the 3'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
[0173] In an embodiment, the duplex RNA molecule comprises a
double-stranded region, and two 5'-overhangs (see, e.g., FIG.
2D).
[0174] In an embodiment, the 5'-overhang has a length of 1-10 nt.
In a further embodiment, the 5'-overhang has a length of 1-6 nt. In
an even further embodiment, the 5'-overhang has a length of 2-4 nt.
In one embodiment, the 3'-overhang has a length of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 nt.
[0175] The same principles and features discussed above also apply
to the embodiment where the second strand is longer than the first
strand (FIG. 2F).
2.0. The Design of aiRNAs
[0176] For ease of understanding, the principles of the invention
are sometimes explained and illustrated below using examples where,
(a) in siRNA applications, the antisense strand is the longer
strand of the aiRNA molecule; (b) in miRNA mimetic applications,
the antisense strand with sequence similar to a mature miRNA is the
longer strand of the aiRNA molecule; and (c) in miRNA inhibitor
applications, the sense strand with sequence substantially
complementary to the mature miRNA is the longer strand of the aiRNA
molecule. However, the opposite situations can be true and are
contemplated to be part of the invention, i.e., the antisense
strand can be the shorter strand of the aiRNA in both the siRNA
applications and miRNA mimetic applications, and the sense strand
can be the shorter strand in miRNA inhibitor applications--the same
features and principles described in other examples apply in those
situations and are not necessarily repeated.
[0177] siRNAs and miRNAs are widely used as research tools, and
developed as drug candidates. (see, e.g., Dykxhoorn, Novina &
Sharp. Nat. Rev. Mol. Cell Biol. 4:457-467 (2003); Kim & Rossi,
Nature Rev. Genet. 8:173-184 (2007); de Fougerolles, et al. Nature
Rev. Drug Discov. 6:443-453 (2007); Czech, NEJM 354:1194-1195
(2006); and Mack, Nature Biotech. 25:631-638 (2007)). The duplex
RNA molecules of the present invention, i.e., aiRNAs, can be
derived from siRNAs and miRNAs known in the field.
[0178] The present invention provides a method of converting an
siRNA or an miRNA into an aiRNA. The conversion results in a new
duplex RNA molecule that has at least one property improved in
comparison to the original molecule. The property can be size,
efficacy, potency, the speed of onset, durability, synthesis cost,
off-target effects, interferon response, or delivery.
[0179] In an embodiment, the original molecule is a duplex RNA
molecule, such as an siRNA or a miRNA/miRNA* (guide/passenger)
duplex. The duplex RNA molecule comprises an antisense strand
(e.g., a guide strand) and a sense strand (e.g. a passenger strand)
that form at least one double-stranded region. The method comprises
changing the length of one or both strands so that the antisense
strand is longer than the sense strand. In an embodiment, sense
passenger strand is shortened. In another embodiment, the antisense
guide strand is elongated. In an even further embodiment, the sense
strand is shortened and the antisense strand is elongated. The
antisense and sense RNA strands, intact or with changed size, can
be synthesized, and then combined under conditions, wherein an
aiRNA molecule is formed.
[0180] In a further embodiment, the method comprises changing the
length of the antisene and/or sense strand so that the duplex RNA
molecule is formed having at least one of a 3'-overhang of 1-6
nucleotides and a 5'-overhang of 1-6 nucleotides.
[0181] In an embodiment, the original molecule is a single-strand
RNA molecule, such as a mature guide miRNA or a passenger miRNA.
The method comprises synthesizing a shorter RNA strand that is
substantially complementary to the single-strand template or a
portion of it, and combining the synthesized strand with the
original single-strand RNA molecule or a shortened version of it
under hybridizing conditions, wherein an aiRNA molecule of
assymetric strand lengths is formed. In an embodiment, the template
RNA is the mature guide miRNA and the resulting aiRNA is a mimetic
of it. In another embodiment, the template RNA is the passenger
strand (miRNA*) in a miRNA/miRNA* duplex, and the resulting aiRNA
is an inhibitor of the guide miRNA.
[0182] Alternatively, the duplex RNA molecules of the present
invention can be designed de novo. A duplex RNA molecule of the
present invention can be designed taking advantage of the design
methods for siRNAs and miRNAs, such as the method of gene walk.
[0183] An RNA molecule of the present invention can be designed
with bioinformatics approaches, and then tested in vitro and in
vivo to determine its modulating efficacy against the target gene
and the existence of any off-target effects. Based on these
studies, the sequences of the RNA molecules can then be selected
and modified to improve modulating efficacy against the target
gene, and to minimize off-target effects. (see e.g., Patzel, Drug
Discovery Today 12:139-148 (2007)).
2.1. Unmatched or Mismatched Region in the Duplex RNA Molecule
[0184] The two single strands of the aiRNA duplex can have at least
one unmatched or imperfectly matched region containing, e.g., one
or more mismatches. In one embodiment, the unmatched or imperfectly
matched region is at least one end region of the RNA molecule,
including an end region with a blunt end, an end region with a
3'-recess or a 5' overhang, and an end region with a 5' recess or a
3' overhang. As used herein, the end region is a region of the RNA
molecule including one end and the neighboring area.
[0185] In an embodiment, the unmatched or imperfectly matched
region is in a double-stranded region of the aiRNA molecule. In a
further embodiment, the asymmetric RNA duplex has an unmatched
bulge or loop structure.
2.2. Sequence Motifs in the Duplex RNA Molecule
[0186] In the design of an aiRNA molecule of the invention, the
overall GC content may vary. In an embodiment, the GC content of
the double-stranded region is 20-70%. In a further embodiment, the
GC content of the double-stranded region is less than 50%, or
preferably 30-50%, to make it easier for strand separation as the
G-C pairing is stronger than the A-U pairing.
[0187] The nucleotide sequence at a terminal overhang, in some
embodiments, e.g., the 5' terminal, can be designed independently
from any template sequence (e.g., a target mRNA sequence), i.e.,
does not have to be substantially complementary to a target mRNA
(in the case of an siRNA or miRNA mimetic) or a target miRNA (in
the case of miRNA inhibitor). In one embodiment, the overhang,
e.g., at the 5' or the 3', of the longer or antisense strand, has
at least one A, U, or dT. In an embodiment, the overhang has at
least one "AA", "UU" or "dTdT" motif, which have exhibited
increased efficacy in comparison to some other motifs. In an
embodiment, the 5' overhang of the longer or antisense strand has
an "AA" motif. In another embodiment, the 3' overhang of the longer
or antisense strand has a "UU" motif.
2.3. Nucleotide Substitution
[0188] One or more of the nucleotides in the RNA molecule of the
invention can be substituted with deoxynucleotides or modified
nucleotides or nucleotide analogues. The substitution can take
place anywhere in the RNA molecule, e.g., one or both of the
overhang regions, and/or a double-stranded region. In some cases,
the substitution enhances a physical property of the RNA molecule
such as strand affinity, solubility and resistance to RNase
degradation or enhanced stability otherwise.
[0189] In one embodiment, the modified nucleotide or analogue is a
sugar-, backbone-, and/or base-modified ribonucleotide. The
backbone-modified ribonucleotide may have a modification in a
phosphodiester linkage with another ribonucleotide. In an
embodiment, the phosphodiester linkage in the RNA molecule is
modified to include at least a nitrogen and/or sulphur heteroatom.
In an embodiment, the modified nucleotide or analogue is an
unnatural base or a modified base. In an embodiment, the modified
nucleotide or analogue is inosine, or a tritylated base.
[0190] In a further embodiment, the nucleotide analogue is a
sugar-modified ribonucleotide in which the 2'-OH group is replaced
by a group selected from the group consisting of H, OR, R, halo,
SH, SR, NH.sub.2, NHR, NR.sub.2, and CN, wherein each R is
independently selected from the group consisting of C1-C6 alkyl,
alkenyl and alkynyl, and halo is selected from the group consisting
of F, Cl, Br and I.
[0191] In one embodiment, the nucleotide analogue is a
backbone-modified ribonucleotide containing a phosphothioate
group.
2.4. aiRNA as miRNA Mimetic
[0192] Novel constructs of double-stranded aiRNAs can be provided
to compensate for the lack of or insufficient level of functioning,
endogenous miRNAs in a subject. In a first approach, an endogenous
double-stranded region involving a mature miRNA is selected as a
template for the design of an aiRNA mimetic. Such a template can be
any suitable endogenous precursor to the single stranded mature
miRNA including the pri-miRNA, pre-miRNA and the guide/passenger
(miRNA/miRNA*) duplex that results from the ribonuclease Dicer
digestion of pre-miRNA. Referring to FIG. 18A, these potential
templates preserve secondary structures such as bulges from
nucleotide mismatches within the double-stranded region that can be
copied into the aiRNA design. Because such secondary structures may
play a role in the gene silencing function of the mature miRNA,
e.g., facilitation of strand separation, this approach
advantageously preserves this possibility for the aiRNA.
[0193] In the specific embodiment shown in FIG. 18A, the stem-loop
structure shown at the bottom represents the pre-miRNA of human
Let7a gene (pre-Let-7a) where the 22 nucleotides that eventually
become the mature miRNA are underlined. In designing an aiRNA
mimetic of the hsa-Let7a miRNA (the "Let-7a aiRNA oligo"), that
underlined portion of the double-strand region is used as the basis
for a 21-nt, longer strand of the aiRNA. A 15-nt stretch from the
substantially but imperfectly complementary sequence in the
double-strand region of the pre-Let-7a, which overall is a
single-stranded stem-loop hairpin molecule, is used as the shorter
strand to complete the aiRNA design. As shown in the figure, at
least the bubble structure resulting from the "GU/UG" mismatch in
the original pre-miRNA is conserved in the aiRNA design. In this
particular example, the longer strand of the aiRNA has a 3-nt
overhang on both the 3' and 5' ends.
[0194] Also, as described above in Section 2.0, a guide/passenger
miRNA duplex can be converted into an aiRNA by changing strand
length
[0195] In a second approach, a portion of the mature miRNA sequence
is copied into the longer strand of the aiRNA, and a substantially
complementary, preferably a perfectly complementary, shorter strand
is generated without regard to any secondary structure that might
have existed in the mature miRNA's natural precursors. FIG. 4A
shows, among other things, a mimetic of hsa-Let-7c miRNA ("aiLet-7c
mimic"). The sequence for the mature human Let7c miRNA (miRBase
(http://microma.sanger.ac.uk) Accession No. MIMAT0000064) is:
TABLE-US-00003 hsa-Let-7c: 5'-UGAGGUAGUAGGUUGUAUGGUU
The first 19 nucleotides (underlined in FIG. 19A) are copied into
the longer strand of the aiLet-7c mimic following an "AA" motif at
the 5' end. And a perfectly complementary sequence consisting of 15
nucleotides are provided as the shorter strand, leaving a 3-nt
overhang on both the 3' and 5' termini of the longer strand. The
"AA" motif is added at the 5' end of the longer strand because,
among the motifs tried, it appears to provide some advantage in
enhancing aiRNA functionality. As shown in examples below, both
approaches work. In an embodiment, the aiRNA mimetic adopts the
"15/21" configuration. However, other aiRNA configurations can also
be used here of course.
[0196] The present invention can be used to make mimetics of any
given miRNA of any species including plants (e.g., Arabidopsis),
worms (e.g., C. elegans), insects (e.g. Drosophila), and animals
including mammalian species like mouse and human. A list of human
miRNAs currently known and can be used to generate the RNA
molecules of the present invention is provided in FIGS. 30 and
31.
2.5. aiRNA as miRNA Inhibitor
[0197] Double-stranded aiRNAs can also be provided to inhibit
endogenous miRNA, e.g., by reducing the level of active miRNAs in
cells. Current antagomirs are based on single-stranded antisense
oligonucleotides, which are notoriously unstable in eukaryotic
cells. In contrast, the present aiRNA structural scaffold is
double-stranded, therefore much more stable in cells. Its novel
asymmetric design apparently works in effecting potent inhibition
of target miRNAs inside mammalian cells as shown in Examples 7
below.
[0198] In an embodiment, a first strand of the aiRNA, preferably
the longer strand, consists of sequence substantially complementary
to at least a portion of the target miRNA. The other aiRNA strand,
preferably the shorter strand, is then made to substantially
complement, e.g., perfectly complement, the first strand in
sequence. In an embodiment, the shorter strand is imperfectly,
i.e., only partly complementary, to the longer strand in the
double-strand region.
[0199] In an embodiment, the longer strand of the aiRNA construct
is designed to be perfectly complementary to at least a portion of
the target miRNA. For example, FIG. 19A shows, among other things,
three inhibitor constructs of hsa-Let-7c, miR-21, and miR-155,
respectively. The corresponding target miRNA sequences for each
inhibitor are shown in FIG. 19B. Each inhibitor includes, following
an "AA" motif at the 5' end, a sequence perfectly complementary to
the first 19 nucleotides of the mature target miRNA. In the
examples shown, the aiRNA constructs adopt the "15/21"
configuration with the longer 21-nt strand having an overhang on
both the 3' and 5' ends. Other aiRNA configurations can also be
used here of course.
[0200] While not to be bound by theory, one possible mechanism for
inhibition of miRNAs by the aiRNAs of the present invention is
illustrated through FIG. 29 and as follows: inside the cell,
strands of both the endogenous miRNA/miRNA* duplex and the
introduced aiRNA duplex are constantly in a dynamic equilibrium
under which condition some duplexes are constantly base-pairing
together while others are constantly separating. The ratio, at any
given time, between duplexes formed and unraveled, or, in other
words, between an on-rate and off-rate, is largely dependent upon
the affinity between the two strands forming the duplex. While not
clear at this time, the longer strand of the aiRNA, possibly by
virtue of its designed complementarity or the lack of structures
that facilitate strand separation, once base-pairs with the target
miRNA strand, exhibits stronger affinity for the target miRNA
(i.e., a lower off-rate) than the endogenous passenger miRNA
(miRNA*) strand. This can be especially true when there are
mismatches or unmatched regions in the endogenous miRNA/miRNA*
duplex that favors dissociation. As a result, over time, the longer
strand of the aiRNA is able to compete against the endogenous
passenger miRNA in forming longer-lasting duplexes with the mature
guide miRNA, thus reducing the amount of unbound guide miRNAs at
any given time that are available to effect intended gene
silencing. The length asymmetry of the aiRNA construct, in
comparison to a symmetric construct, favors dissociation between
the longer strand and the shorter strand, making more of the longer
strands available to compete for the target miRNA strand.
[0201] In a preferred embodiment, the longer strand of the aiRNA
has perfect base-pair complementarity with at least a portion of
the targeted guide miRNA, but this does not have to be true--as
long as the complementarity is sufficiently high for the longer
strand to compete against the endogenous passenger miRNA in terms
of forming a duplex with the guide miRNA.
[0202] The present invention can be used to make inhibitors of any
given miRNA of any species including plants (e.g., Arabidopsis),
worms (e.g., C. elegans), insects (e.g. Drosophila), and animals
including mammalian species like mouse and human. A list of human
miRNAs currently known and can be used to generate the RNA
molecules of the present invention is provided in FIGS. 30 and
31.
3. The Utilities
3.1. Research and Drug Discovery Tools
[0203] MicroRNAs serve gene-regulatory functions in cells; some
have been found to associate with various types of human diseases
including cancers, viral infections, etc. Therefore, miRNA mimetics
and inhibitors can be used to study gene targets modulated by
miRNAs, and the mechanisms and components of various miRNA pathways
and their interactions.
[0204] Methods and constructs of the invention can be used to study
miRNA pathways and related gene function in vitro and in vivo. RNA
molecules of the invention can also be used to transfect cultured
animal cells as a research tool in drug target/pathway
identification and validation. For example, after transfecting or
otherwise delivering the RNA molecules of the invention into host
cells, these cells can be monitored for phenotypical or morphology
changes that suggest some pathway of interest has been affected.
Such phenotypical changes can involve numbers of nuclei, nuclei
morphology, cell death, cell proliferation, DNA fragmentation, cell
surface marker, and mitotic index, etc. In another example,
interaction between a molecule/substrate in the host cell and the
miRNA modulator (mimetic or inhibitor) transfected into the cell
can be isolated or identified to discover potential therapeutic
target. That target can be upstream and modulates the miRNA
activity, or, the target can be downstream and its activity is
modulated by the miRNA. Furthermore, the RNA molecules of the
invention can be used to conduct drug target discovery and
validation in vivo, e.g., in animal models with xenograft of
diseased tissue or cell populations.
[0205] With evidence that a network or a number of miRNAs may
target the same gene product or pathway (Sethupathy P. et al. RNA
12: 192-197 (2006)), multiple RNA molecules of the invention,
including mimetics of different miRNAs, inhibitors of different
miRNAs, and mixture of mimetics and inhibitors of different miRNAs
can be used to study the interrelationship between various miRNA
pathways including coregulation of the same gene product.
[0206] In terms of additional in vivo applications, since the
present invention provides functional miRNA mimetics and
inhibitors, after they are respectively introduced into a host
body, a systems biology approach can be adopted in studying the
effect of certain miRNA, which is being mimicked or inhibited, on
different cell types and different tissues.
[0207] Further, with their ability to modulate post-transcriptional
gene silencing mediated by miRNA, the RNA molecules of the present
invention can be used to create gene "knockdown" in animal models
as opposed to genetically engineered knockout models in order to
study and validate gene functions.
[0208] The RNA molecule of the invention can be supplied as
research reagents either in double-stranded duplex, or separated as
single strands to be used in double-stranded form. Accordingly, the
present invention also provides a kit that contains an RNA molecule
of the invention in any of suitable forms including those described
above.
3.2. Therapeutic Uses
[0209] The RNA molecules of the present invention can be used for
the treatment and or prevention of various diseases, including the
diseases summarized in Dykxhoorn, Novina & Sharp. Nat. Rev.
Mol. Cell Biol. 4:457-467 (2003); Kim & Rossi, Nature Rev.
Genet. 8:173-184 (2007); de Fougerolles, et al. Nature Rev. Drug
Discov. 6:443-453 (2007); Czech, NEJM 354:1194-1195 (2006); and
Mack, Nature Biotech. 25:631-638 (2007); Tong and Nemunaitis,
Cancer Gene Therapy 15: 341-355 (2008).
[0210] In one embodiment, a mimetic of the present invention is
used to reconstitute desired miRNA function to the normal level,
thereby treating the disease or alleviating the symptom. In another
embodiment, an inhibitor of the present invention is used to reduce
or eliminate undesired miRNA activity, thereby treating diseases
caused by overexpression or misexpression of the miRNA.
Combinatorial use of multiple mimetics, multiple inhibitors, and a
mixture of mimetic and inhibitor can also be used.
[0211] In an embodiment, the present invention is used as a cancer
therapy or to prevent cancer by targeting one or more
cancer-related genes. This method is effected by using miRNA
mimetics and/or inhibitors of the invention to up-regulate
tumor-suppressing genes, and/or by using miRNA inhibitors and/or
mimetics of the invention to silence genes involved with cell
proliferation or other cancer phenotypes.
[0212] In an embodiment, the therapeutic mimetics according to the
invention mimic miRNAs that target oncogenes. Examples of such
tumor-suppressing miRNAs may include: let-7 family, miR-15a,
miR-16-1, miR-34a, miR-143, miR-145 and so on. Examples of various
oncogenes targeted by these miRNAs include k-Ras and bcl-2. For
example, k-Ras has been shown to be regulated by miRNA Let-7. These
oncogenes are active and relevant in the majority of clinical
cases. For example, k-Ras is aberrantly active in the majority of
human colon cancer, pancreatic cancer, and non small cell lung
cancers. Further, k-Ras mutation confers resistance to chemotherapy
and current targeted therapy.
[0213] In an embodiment, the therapeutic inhibitors according to
the invention inhibit miRNAs that target tumor-suppressor genes.
Examples of such miRNAs may include miR-17/92 cluster, miR-21,
miR-106, miR-155, miR-221, miR-222 and so on. Examples of tumor
suppressing genes targeted by these miRNAs include ERK5 and p27
(kipi) (Tong and Nemunaitis, Cancer Gene Therapy 15: 341-355
(2008)).
[0214] These RNA molecules can also be used to modulate non-cancer
genes targeted by the miRNAs being mimicked or inhibited. The RNA
molecules of the invention can also be used to treat or prevent
ocular diseases, (e.g., age-related macular degeneration (AMD) and
diabetic retinopathy (DR)); infectious diseases (e.g. HIV/AIDS,
hepatitis B virus (HBV), hepatitis C virus (HCV), human
papillomavirus (HPV), herpes simplex virus (HSV), RCV,
cytomegalovirus (CMV), dengue fever, west Nile virus); respiratory
diseases (e.g., respiratory syncytial virus (RSV), asthma, cystic
fibrosis); neurological diseases (e.g., Huntingdon's disease (HD),
amyotrophic lateral sclerosis (ALS), spinal cord injury,
Parkinson's disease, Alzheimer's disease, pain); cardiovascular
diseases; metabolic disorders (e.g., diabetes); genetic disorders;
and inflammatory conditions (e.g., inflammatory bowel disease
(1BD), arthritis, rheumatoid disease, autoimmune disorders),
dermatological diseases, psychological disorders (e.g., bipolar
disorder).
4. Manufacture and Use
[0215] 4.1. Making the aiRNA Molecules
[0216] The RNA molecules of the present invention can be made via
any suitable means, including through chemical reactions and
synthesis, through biological processes, and/or an enzyme-effected
processes.
[0217] Chemical synthesis of RNA molecules of a given sequence is
well known in the art. Biological processes for making RNA
molecules are also well known. For example, a DNA expression
vector, viral, eukaryotic, or bacterial, can be constructed with an
appropriate promoter to transcribe a corresponding DNA sequence
into the designed RNA sequence once transfected into host cells
(e.g., bacteria). In an embodiment, the transcript may be a
precursor to the final RNA duplex such that enzyme actions such as
ribonuclease-effected site-specific cleavage is needed. For
example, both strands of the aiRNA molecule of the invention may be
transcribed into a single strand that needs to be cut into two
separate strands to form the double-stranded RNA molecule of the
invention.
[0218] One aspect of the invention is directed to an expression
vector that includes a DNA sequence encoding part or all of the
double-stranded RNA molecule of the present invention (e.g., one of
the asymmetric strands), the DNA sequence being operably linked to
an expression control sequence, e.g., a promoter. In an embodiment,
the vector is single-stranded. In a further embodiment, two
different vectors each encoding for a different strand of the RNA
molecule of the invention are provided to co-transfect a cell,
inside which the two expressed strands form a duplex. In another
embodiment, the vector is double stranded and each strand contains
the DNA sequence for a different strand of the RNA molecule
operably linked to an expression control sequence. Further, the
present invention provides a cell that includes such an expression
vector. The cell can be a mammalian, avian or bacterial cell.
[0219] The two strands of the aiRNA molecule can be manufactured
separately or at the same time, in any of the above processes. For
example, in a biological process, two vectors can be constructed to
express the shorter strand and the longer strand of aiRNA
separately, or a single vector with two strands can be constructed
to express the two strands of the aiRNA molecule
simultaneously.
4.2. Modifications of the RNA Molecules
[0220] Naked RNA molecules are relatively unstable and can be
degraded in vivo relatively quickly. Chemical modifications can be
introduced to the RNA molecules of the present invention, including
mimetics of siRNAs, of miRNAs and inhibitors of miRNAs, to improve
their half-life and to further reduce the risk of non-specific
effects of gene targeting, without reducing their biological
activities.
[0221] The modifications of RNA molecules have been investigated to
improve the stability of various RNA molecules, including antisense
RNA, ribozyme, aptamer, and RNAi. (see, e.g., Chiu & Rana, RNA
9:1034-1048 (2003); Czaudema, et al, Nucleic Acids Research
31:2705-2716 (2003); Zhang H Y, et al, Curr Top Med Chem. 6:893-900
(2006); Kim & Rossi, Nature Rev. Genet. 8:173-184 (2007); de
Fougerolles, et al. Nature Rev. Drug Discov. 6:443-453 (2007); and
Schmit, Nature Biotech. 25:273-275 (2007); and Mack, Nature
Biotech. 25:631-638 (2007)).
[0222] Any stabilizing modification known to one skill in the art
can be used to improve the stability of the RNA molecules of the
present invention. Within the RNA molecules of the present
invention, chemical modifications can be introduced to the
phosphate backbone (e.g., phosphorothioate linkages), the ribose
(e.g., locked nucleic acids, 2'-deoxy-2'-fluorouridine,
2'-O-mthyl), and/or the base (e.g., 2'-fluoropyrimidines). Several
examples of such chemical modifications are summarized below.
[0223] Chemical modifications at the 2' position of the ribose,
such as 2'-O-mthylpurines and 2'-fluoropyrimidines, which increase
resistance to endonuclease activity in serum, can be adopted to
stabilize the RNA molecules of the present invention. The position
for the introduction of the modification should be carefully
selected to avoid significantly reducing the silencing potency of
the RNA molecule. For example, the modifications on 5' end of the
guide strand can reduce the silencing activity. On the other hand,
2'-O-methyl modifications can be staggered between the two RNA
strands at the double-stranded region to improve the stability
while reserving the gene silencing potency. The 2'-O-methyl
modifications can also eliminate or reduce the interferon
induction.
[0224] Another stabilizing modification is phosphorothioate (P=S)
linkage. The introduction of phosphorothioate (P=S) linkage into
the RNA molecules, e.g., at the 3'-overhang, can provide protection
against exonuclease.
[0225] The introduction of deoxyribonucleotides into the RNA
molecules can also reduce the manufacture cost, and increase
stability.
[0226] In an embodiment, the 3'-overhang, 5'-overhang, or both are
stabilized against degradation. In an embodiment, the RNA molecule
contains at least one modified nucleotide or its analogue. In a
further embodiment, the modified ribonucleotide is modified at its
sugar, backbone, base, or any combination of the three.
[0227] In an embodiment, the nucleotide analogue is a
sugar-modified ribonucleotide. In a further embodiment, the 2'-OH
group of the nucleotide analogue is replaced by a group selected
from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein each R
is independently C1-C6 alkyl, alkenyl or alkynyl, and halo is F,
Cl, Br or I.
[0228] In an alternative embodiment, the nucleotide analogue is a
backbone-modified ribonucleotide containing a phosphothioate
group.
[0229] In an embodiment, the duplex RNA molecule contains at least
one deoxynucleotide. In a further embodiment, the first strand
comprises 1-6 deoxynucleotides. In an even further embodiment, the
first strand comprises 1-3 deoxynucleotides. In another embodiment,
the 3'-overhang comprises 1-3 deoxynucleotides. In a further
embodiment, the 5'-overhang comprises 1-3 deoxynucleotides. In an
alternative embodiment, the second strand comprises 1-5
deoxynucleotides.
[0230] In an embodiment, the duplex RNA molecule comprises a
3'-overhang or 5'-overhang that contains at least one
deoxynucleotide. In another embodiment, the 3'-overhang and/or
5'-overhang of the RNA consists of deoxynucleotides.
[0231] In an embodiment, the duplex RNA molecule is conjugated to
an entity. In a further embodiment, the entity is selected from the
group consisting of peptide, antibody, polymer, lipid,
oligonucleotide, and aptamer.
[0232] In another embodiment, the first strand and the second
strand are joined by a chemical linker.
4.3. In Vivo Delivery of the RNA Molecules
[0233] One major obstacle for the therapeutic use of RNAi is the
delivery of siRNA to the target cell (Zamore P D, Aronin N. Nature
Medicine 9:266-8 (2003)). Various approaches have been developed
for the delivery of RNA molecules, especially siRNA molecules (see,
e.g., Dykxhoom, Novina & Sharp. Nat. Rev. Mol. Cell Biol.
4:457-467 (2003); Kim & Rossi, Nature Rev. Genet. 8:173-184
(2007); and de Fougerolles, et al. Nature Rev. Drug Discov.
6:443-453 (2007)). Any delivery approach known to one skilled in
the art can be used for the delivery of the RNA molecules of the
present invention.
[0234] Major issues in delivery include instability in serum,
non-specific distribution, tissue barriers, and non-specific
interferon response (Lu & Woodle, Methods in Mol Biology 437:
93-107 (2008)). Compared to their siRNA and miRNA counterparts,
aiRNA molecules possess several advantages that should make a wider
ranger of methods available for delivery purpose. First, aiRNAs can
be designed to be smaller than their siRNA and miRNA counterparts,
therefore, reducing or eliminating any interferon responses.
Second, aiRNAs are more potent, faster-onsetting, more efficacious
and last longer, therefore, less amount/dosage of aiRNAs is
required to achieve a therapeutic goal. Third, aiRNA are double
stranded and more stable than single-stranded antisense oligos and
miRNAs, and they can be further modified chemically to enhance
stability. Therefore, the RNA molecules of the invention can be
delivered into a subject via a variety of systemic or local
delivery routes. In some embodiments, molecules of the invention
are delivered through systemic delivery routes include intra-venous
(I.V.) and intra-peritoneal (ip). In other embodiments, molecules
of the invention are delivered through local delivery routes, e.g.,
intra-nasal, intra-vitreous, intra-tracheal, intra-cerebral,
intra-muscle, intra-articular, and intra-tumor.
[0235] Examples of the delivery technologies include direct
injection of naked RNA molecules, conjugation of the RNA molecules
to a natural ligand such as cholesterol, or an aptamer,
liposome-formulated delivery, and non-covalently binding to
antibody-protamine fusion proteins. Other carrier choices include
positive charged carriers (e.g., cationic lipids and polymers) and
various protein carriers. In one embodiment, the delivery of the
molecules of the invention uses a ligand-targeted delivery system
based on the cationic liposome complex or polymer complex systems
(Woodle, et al. J Control Release 74: 309-311; Song, et al. Nat
Biotechnol. 23(6): 709-717 (2005); Morrissey et al. Nat Biotechnol.
23(8): 1002-1007 (2005)).
[0236] In one embodiment, molecules of the invention are formulated
with a collagen carrier, e.g., atelocollagen, for in vivo delivery.
Atelocollagen has been reported to protect siRNA from being
digested by RNase and to enable sustained release (Minakuchi, et
al. Nucleic Acids Res. 32: e109 (2004); Takei et al. Cancer Res.
64: 3365-3370 (2004)). In another embodiment, molecules of the
invention are formulated with nanoparticles or form a nanoemulsion,
e.g., RGD peptide ligand targeted nanoparticles. It has been shown
that different siRNA oligos can be combined in the same RGD ligand
targeted nanoparticle to target several genes at the same time
(Woodle et al. Materials Today 8 (suppl 1): 34-41 (2005)).
[0237] Viral vectors can also be used for the delivery of the RNA
molecules of the present invention. In an embodiment, lentiviral
vectors are used to deliver the RNA molecule transgenes that
integrate into the genome for stable expression. In another
embodiment, adenoviral and adeno-associated virus (AAV) are used to
deliver the RNA molecule transgenes that do not integrate into the
genome and have episomal expression.
[0238] Moreover, bacteria can be used for the delivery of the RNA
molecules of the present invention. (see Xiang, Fruehauf, & Li,
Nature Biotechnology 24:697-702 (2006)).
4.4. The Pharmaceutical Compositions and Formulations
[0239] The pharmaceutical compositions and formulations of the
present invention can be the same or similar to the pharmaceutical
compositions and formulations developed for siRNA, miRNA, and
antisense RNA (see, e.g., Kim & Rossi, Nature Rev. Genet.
8:173-184 (2007); and de Fougerolles, et al. Nature Rev. Drug
Discov. 6:443-453 (2007)), except for the RNA ingredient. The
siRNA, miRNA, and antisense RNA in the pharmaceutical compositions
and formulations can be replaced by the duplex RNA molecules of the
present information. The pharmaceutical compositions and
formulations can also be further modified to accommodate the duplex
RNA molecules of the present information.
[0240] A "pharmaceutically acceptable salt" or "salt" of the
disclosed duplex RNA molecule is a product of the disclosed duplex
RNA molecule that contains an ionic bond, and is typically produced
by reacting the disclosed duplex RNA molecule with either an acid
or a base, suitable for administering to a subject.
Pharmaceutically acceptable salt can include, but is not limited
to, acid addition salts including hydrochlorides, hydrobromides,
phosphates, sulphates, hydrogen sulphates, alkylsulphonates,
arylsulphonates, acetates, benzoates, citrates, maleates,
fumarates, succinates, lactates, and tartrates; alkali metal
cations such as Na, K, Li, alkali earth metal salts such as Mg or
Ca, or organic amine salts.
[0241] A "pharmaceutical composition" is a formulation containing
the disclosed duplex RNA molecules in a form suitable for
administration to a subject. In one embodiment, the pharmaceutical
composition is in bulk or in unit dosage form. The unit dosage form
is any of a variety of forms, including, for example, a capsule, an
IV bag, a tablet, a single pump on an aerosol inhaler, or a vial.
The quantity of active ingredient (e.g., a formulation of the
disclosed duplex RNA molecule or salts thereof) in a unit dose of
composition is an effective amount and is varied according to the
particular treatment involved. One skilled in the art will
appreciate that it is sometimes necessary to make routine
variations to the dosage depending on the age and condition of the
patient. The dosage will also depend on the route of
administration. A variety of routes are contemplated, including
oral, pulmonary, rectal, parenteral, transdermal, subcutaneous,
intravenous, intramuscular, intraperitoneal, intranasal, and the
like. Dosage forms for the topical or transdermal administration of
a duplex RNA molecule of this invention include powders, sprays,
ointments, pastes, creams, lotions, gels, solutions, patches and
inhalants. In one embodiment, the active duplex RNA molecule is
mixed under sterile conditions with a pharmaceutically acceptable
carrier, and with any preservatives, buffers, or propellants that
are required.
[0242] The present invention also provides pharmaceutical
formulations comprising a duplex RNA molecule of the present
invention in combination with at least one pharmaceutically
acceptable excipient or carrier. As used herein, "pharmaceutically
acceptable excipient" or "pharmaceutically acceptable carrier" is
intended to include any and all solvents, dispersion media,
coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Suitable carriers are described in
"Remington: The Science and Practice of Pharmacy, Twentieth
Edition," Lippincott Williams & Wilkins, Philadelphia, Pa.,
which is incorporated herein by reference. Examples of such
carriers or diluents include, but are not limited to, water,
saline, Ringer's solutions, dextrose solution, and 5% human serum
albumin. Liposomes and non-aqueous vehicles such as fixed oils may
also be used. The use of such media and agents for pharmaceutically
active substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active duplex
RNA molecule, use thereof in the compositions is contemplated.
Supplementary active duplex RNA molecules can also be incorporated
into the compositions.
[0243] In an embodiment, the pharmaceutically acceptable excipient,
carrier, or diluent comprises a lipid for intravenous delivery. The
lipid can be: phospholipids, synthetic phophatidylcholines, natural
phophatidylcholines, sphingomyelin, ceramides,
phophatidylethanolamines, phosphatidylglycerols, phosphatidic
acids, cholesterol, cholesterol sulfate, and hapten and PEG
conjugated lipids. The lipid may be in the form of nanoemulsion,
micelles, emulsions, suspension, nanosuspension, niosomes, or
liposomes. In an embodiment, the pharmaceutically acceptable
excipient, carrier, or diluent is in a form of micellar emulsion,
suspension, or nanoparticle suspension, and it further comprises an
intravenously acceptable protein, e.g., human albumin or a
derivative thereof, for intravenous delivery.
[0244] In an embodiment, the pharmaceutically acceptable excipient,
carrier, or diluent comprises a waxy material for oral delivery.
The waxy material may be mono-, di-, or tri-glycerides, mono-,
di-fatty acid esters of PEG, PEG conjugated vitamin E (vitamin E
TPGs), Gelucire and/or Gelucire 44/14. In an embodiment, the
pharmaceutically acceptable excipient, e.g., Gelucire 44/14, is
mixed with a surfactant, which can be Tween 80 or Tween 20. These
embodiments of pharmaceutical compositions can be further
formulated for oral administration. Methods for formulation are
disclosed in PCT International Application PCT/US02/24262
(WO03/011224), U.S. Patent Application Publication No. 2003/0091639
and U.S. Patent Application Publication No. 2004/0071775, each of
which is incorporated by reference herein.
[0245] A duplex RNA molecule of the present invention is
administered in a suitable dosage faun prepared by combining a
therapeutically effective amount (e.g., an efficacious level
sufficient to achieve the desired therapeutic effect through
inhibition of tumor growth, killing of tumor cells, treatment or
prevention of cell proliferative disorders, etc.) of a duplex RNA
molecule of the present invention (as an active ingredient) with
standard pharmaceutical carriers or diluents according to
conventional procedures (i.e., by producing a pharmaceutical
composition of the invention). These procedures may involve mixing,
granulating, and compressing or dissolving the ingredients as
appropriate to attain the desired preparation. In another
embodiment, a therapeutically effective amount of a duplex RNA
molecule of the present invention is administered in a suitable
dosage form without standard pharmaceutical carriers or
diluents.
[0246] Pharmaceutically acceptable carriers include solid carriers
such as lactose, terra alba, sucrose, talc, gelatin, agar, pectin,
acacia, magnesium stearate, stearic acid and the like. Exemplary
liquid carriers include syrup, peanut oil, olive oil, water and the
like. Similarly, the carrier or diluent may include time-delay
material known in the art, such as glyceryl monostearate or
glyceryl distearate, alone or with a wax, ethylcellulose,
hydroxypropylmethylcellulose, methylmethacrylate or the like. Other
fillers, excipients, flavorants, and other additives such as are
known in the art may also be included in a pharmaceutical
composition according to this invention.
[0247] The pharmaceutical compositions containing active duplex RNA
molecules of the present invention may be manufactured in a manner
that is generally known, e.g., by means of conventional mixing,
dissolving, granulating, dragee-making, levigating, emulsifying,
encapsulating, entrapping, or lyophilizing processes.
Pharmaceutical compositions may be formulated in a conventional
manner using one or more physiologically acceptable carriers
comprising excipients and/or auxiliaries which facilitate
processing of the active duplex RNA molecules into preparations
that can be used pharmaceutically. Of course, the appropriate
formulation is dependent upon the route of administration
chosen.
[0248] A duplex RNA molecule or pharmaceutical composition of the
invention can be administered to a subject in many of the
well-known methods currently used for chemotherapeutic treatment.
For example, for treatment of cancers, a duplex RNA molecule of the
invention may be injected directly into tumors, injected into the
blood stream or body cavities or taken orally or applied through
the skin with patches. For treatment of psoriatic conditions,
systemic administration (e.g., oral administration), or topical
administration to affected areas of the skin, are preferred routes
of administration. The dose chosen should be sufficient to
constitute effective treatment but not so high as to cause
unacceptable side effects. The state of the disease condition
(e.g., cancer, psoriasis, and the like) and the health of the
patient should be closely monitored during and for a reasonable
period after treatment.
[0249] The duplex RNA molecule or pharmaceutical composition of the
invention can be administered to a subject in any suitable dosing
range, dosing frequencies and plasma concentration. In an
embodiment, the subject is being treated with a pharmaceutical
composition of the invention with an effective dosage amount of 1
ng to 1 g per day, 100 ng to 1 g per day, or 1 .mu.g to 500 mg per
day, and so on.
Examples
[0250] Examples are provided below to further illustrate different
features of the present invention. The examples also illustrate
useful methodology for practicing the invention. These examples do
not limit the claimed invention.
Methods and Materials
Cell Culture and Reagents
[0251] Hela, SW480, DLD1, HT29, and H1299 cells were obtained from
ATCC, and cultured in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS), 100 units/ml penicillin,
100 .mu.g/ml streptomycin and 2 mM L-glutamine (Invitrogen). Fresh
peripheral blood mononuclear cells (PBMC) were obtained from
AllCells LLC and maintained in RPMI-1640 medium containing 10% FBS
and pen/strep (Invitrogen). Small RNAs described in this study were
synthesized by Dharmacon, Qiagen, or Integrated DNA technologies
(Table 2) and annealed following the manufacturer's instructions
(FIG. 3a). siRNAs targeting human Ago2, and Dicer (Ambion) were
used at 100 nM. Transfections of the RNAs were performed using
DharmaFECT1 (Dharmacon) at the indicated concentrations. Human
Argonaute2 (Ago2) expression vector (OriGene) was transfected using
Lipofectamine 2000 (Invitrogen). Serum stability was determined by
incubation of aiRNA or siRNA duplex with 10% human serum (Sigma)
for the indicated amount of time followed by non-denaturing
TBE-acrylamide gel electrophoresis and ethidium bromide
staining.
Northern Blot Analysis.
[0252] To determine levels of .beta.-catenin, total RNA was
extracted with TRIZOL (Invitrogen) from siRNA or aiRNA transfected
Hela cells at various time points. 20 .mu.g of total cellular RNA
was loaded to each lane of a denaturing agarose gel. After
electrophoresis, RNA was transferred to Hybond-XL Nylon membrane
(Amersham Biosciences), UV crosslinked, and baked at 80.degree. C.
for 30 min. Probes detecting .beta.-catenin and actin mRNA was
prepared using Prime-It II Random Primer Labeling Kit (Stratagene)
from .beta.-catenin cDNA fragment (1-568 nt) and actin cDNA
fragment (1-500 nt). To analyze small RNA RISC loading, siRNA or
aiRNA were transfected into Hela cells 48 hours after transfection
with pCMV-Ago2. Cells were lysed at the indicated timepoints and
immunoprecipitated with Ago2 antibody. Immunoprecipitates were
washed, RNA isolated from the complex by TRIZOL extraction, and
loaded on a 15% TBE-Urea PAGE gel (Bio-Rad). Following
electrophoreses, RNA was transferred to Hybond-XL Nylon membrane.
mirVana miRNA Probe Kit (Ambion) was used to generate 5' .sup.32P
labeled RNA probes. Antisense probe (5'-GUAGCUGAUAUUGAUGGACUU-3').
Sense probe (5'-UCCAUCAAUAUCAGC-3')
In Vitro Ago2-RISC Loading.
[0253] aiRNA or siRNA sense and anti-sense strands were .sup.32P
end labeled using T4 kinase (Promega). End labeled RNAs were
purified by phenol/chloroform/isoamyl alcohol, precipitated with
EtOH, and resuspended in water. Labeled RNAs were then annealed to
siRNA or aiRNA anti-sense strands as described. For in vitro
lysates, Hela cells were transfected for 24 hours with human Ago2
expression vector, and S10 lysates generated essentially as
described (Dignam et al., 1983). 5' sense strand or anti-sense
strand labeled duplex aiRNA or siRNA was then added to the Ago2-S10
lysate. Following a 5 min incubation at 37.degree. C., Ago2 was
immunoprecipited as described, and Ago2-associated (pellet) and
non-Ago2 associated (supernatant) fractions were separated on a 20%
TBE-acrylamide gel and gel exposed to film to detect sense
strand-Ago2 association. For aiRNA and siRNA competition
experiments, up to 100 folds cold aiRNA and siRNA were used to
compete with .sup.32P labeled aiRNA or siRNA to load to RISC.
Briefly, S10 lysates were generated from Hela cells transfected
with Ago2 expression vector as described. Labeled aiRNA or siRNA
was then added to the S10 lysates followed immediately by addition
of unlabeled aiRNA or siRNA. Reaction was incubated for 5 min at
37.degree. C. and processed as described above.
qRT-PCR.
[0254] Cells transfected with siRNA, the indicated aiRNA, the
indicated aiRNA anti-miRNA, or the commercially available miRNA
inhibitor (Ambion) were harvested at the indicated time points
following transfection. RNA was isolated with TRIZOL. For mRNAs,
qRT-PCR performed using TaqMan one-step RT-PCR reagents on a
StepOne real-time PCR and primer probe sets for the indicated mRNA
(Applied Biosystems). Data is presented relative to control
transfected cells and each sample is normalized to actin mRNA
levels. For miRNAs, reverse transcription of miRNAs was performed
using TaqMan microRNA reverse transcription kits (Applied
Biosystems) and cDNA was subjected to real-time PCR using TaqMan
microRNA assays (Applied Biosystems) on a StepOne real-time PCR
machine (Applied Biosciences), each sample is normalized to
U6snRNA. For the experiment in FIG. 14d, Stat3 constructs were
created by cloning Stat3 cDNA (Origene) into either pcDNA3.1.sup.+
or pcDNA3.1.sup.- at the HindIII-Xho1 sites. Stat3 forward or
reverse expression vectors were then co-transfected into Hela cells
with aiStat3 or siStat3 for 24 hours. Cells were then harvested,
RNA isolated by TRIZOL, and qRT-PCR performed using TaqMan one-step
RT-PCR reagents and primer probe sets for Stat3 or actin (Applied
Biosystems). RT-PCR was performed on the same RNA samples using
Superscript One-Step RT-PCR kit (Invitrogen) and Stat3 forward
(5'-GGATCTAGAATCAGCTACAGCAGC-3') and Stat3 reverse
(5'-TCCTCTAGAGGGCAATCTCCATTG-3') primers and actin forward
(5'-CCATGGATGATGATATCGCC-3') and actin reverse
(5'-TAGAAGCATTTGCGGTGGAC-3') primers.
RT-PCR.
[0255] Total RNA was prepared using the TRIZOL, and cDNA was
synthesized using random primers with Thermoscript RT-PCR System
(Invitrogen). PCR was run for 20 cycles using Pfx polymerase.
Primers: ACTIN-1, 5' CCATGGATGATGATATCGCC-3'; ACTIN-2,
5'-TAGAAGCATTTGCGGTGGAC-3'; .beta.-catenin-1,
5'-GACAATGGCTACTCAAGCTG-3'; .beta.-catenin-2,
5'-CAGGTCAGTATCAAACCAGG-3'.
Western Blot
[0256] Cells were washed twice with ice-cold phosphate-buffered
saline and lysed in lysis buffer (50 mM HEPES, pH 7.5, 0.5% Nonidet
P-40, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate,
1 mM dithiothreitol, 1 mM NaF, 2 mM phenylmethylsulfonyl fluoride,
and 10 .mu.g/ml each of pepstatin, leupeptin, and aprotinin). 20
.mu.g of soluble protein was separated by SDS-PAGE and transferred
to PVDF membranes. Primary Antibodies against .beta.-catenin, Nbs1,
Survivin, p21, Rsk1, k-Ras, Stat3, PCNA, NQO1, Actin (Santa Cruz),
EF2, p70S6K, mTOR, PTEN (Cell Signaling Technology), Ago2 (Wako),
Dicer (Novus), and Parp1 (EMD Biosciences) were used in this study.
The antigen-antibody complexes were visualized by enhanced
chemiluminescence (GE Biosciences).
RT-PCR and Western Blot Analysis on miRNAs
[0257] Hela cells were transfected with 100 nM of the indicated
aiRNA or microRNA inhibitor. 24 hours after transfection, cells
harvested for RNA using TRIZOL (Invitrogen) or for protein using
whole cell extract buffer (50 mM HEPES, 2 mM magnesium chloride,
250 mM sodium chloride, 0.1 mM EDTA, 1 mM EGTA, 0.1% Nonidet P-40,
1 mM dithiothreitol, 1.times. mammalian protease inhibitor cocktail
[Sigma], 1.times. phosphatase inhibitor cocktails I and II [Sigma])
by incubation for 30 min on ice. Soluble proteins were separated by
centrifugation at 13,000.times.g in a microcentrifuge, and
supernatants were stored at -70.degree. C. Proteins were separated
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
analysis and transferred to a polyvinylidene difluoride membrane by
electroblotting. Primary antibodies used to detect k-Ras and actin
(Santa Cruz Biotechnology) were incubated with the membrane
followed by HRP-linked secondary antibody (GE Biosciences) and
visualized by chemiluminesence (GE Biosciences). RT-PCR was
performed on the RNA samples using Superscript One-Step RT-PCR kit
(Invitrogen) and k-Ras forward (5'-AGTACAGTGCAATGAGGGACCAGT), k-Ras
reverse (5'-AGCATCCTCCACTCTCTGTCTTGT), Actin forward
(5'-CCATGGATGATGATATCGCC) and actin reverse
(5'-TAGAAGCATTTGCGGTGGAC) primers.
5'-RACE Analysis
[0258] Total RNA (5 .mu.g) from Hela cells treated with
non-silencing aiRNA or aiRNA was ligated to GeneRacer.TM. RNA
adaptor (Invitrogen,
5'-CGACUGGAGCACGAGGACACUGACAUGGACUGAAGGAGUAGAAA-3') without any
prior processing. Ligated RNA was reverse transcribed into cDNA
using a random primer. To detect cleavage product, PCR was
performed using primers complementary to the RNA adaptor
(GeneRacer.TM. 5' Nested Primer: 5'-GGACACTGACATGGACTGAAGGAGTA-3')
and .beta.-catenin specific primer (GSP:
5'-CGCATGATAGCGTGTCTGGAAGCTT-3'). Amplification fragments were
resolved on 1.4% agarose gel and sized using a 1-kb Plus DNA Ladder
(Invitrogen). Specific cleavage site was further confirmed by DNA
sequencing.
Interferon-Response Detection.
[0259] For the experiment in FIG. 15a, PBMC were incubated directly
with 100 nM .beta.-catenin siRNA or aiRNA. Total RNA was purified
at 16 hours using TRIZOL, and levels of interferon responsive gene
expression were determined by RT-PCR as described by the
manufacturer (System Biosciences). For the experiment in FIG. 15b,
Hela cells were mock transfected or transfected with 100 nM of the
indicated aiRNA or siRNA for 24 hours. Total RNA was purified using
TRIZOL and levels of interferon responsive gene expression were
determined by RT-PCR. For microarray analysis, Hela cells were
transfected with 100 nM aiRNA or siRNA. Total RNA was purified at
24 hours using TRIZOL, and RNA was used for hybridization to Human
Genome U133 Plus 2.0 GeneChip (Affymetrix) according to the
manufacturer's protocol (ExpressionAnalysis, Inc.). RNA from
DharmaFECT 1 treated cells was used as control. To calculate
transcript expression values, Microarray Suite 5.0 was used with
quantile normalization, and transcripts with sufficient
hybridization signals to be called present (P) were used in this
study.
aiRNA and siRNA Sequences
[0260] Sequence and structure of aiRNA and siRNA duplexes were
listed in Table 2. Location of point mutation is framed in the
k-Ras aiRNA.
TABLE-US-00004 TABLE 2 ##STR00001## ##STR00002##
In Life Evaluations
[0261] Daily examinations into the health status of each animal
were also conducted. Body weights were checked every three days.
Food and water was supplied daily according to the animal husbandry
procedures of the facility. Treatment producing >20% lethality
and or >20% net body weight loss were considered toxic. Results
are expressed as mean tumor volume (mm.sup.3) .+-.SE. P
Values<0.05 are considered to be statistically relevant.
Animal Husbandry
[0262] Male or female athymic nude mice 4-5 weeks (Charles River
Laboratories, Wilmington, Mass.), were acclimated to the animal
housing facility for at least 1 week before study initiation. All
of the experimental procedures utilized were consistent with the
guidelines outlined by the American Physiology Society and the
Guide for the Care and Use of Laboratory Animals and were also
approved by the Institutional Animal Care and Use Committee of
Boston Biomedical Inc. The animals were housed in groups of four in
wood chip bedded cages in a room having controlled temperature
(68.degree. F.-72.degree. F.), light (12-h light-dark cycle), and
humidity (45-55%). The animals were allowed free access to water
and food during the experiment.
Example 1
Asymmetric Interfering RNA (aiRNA) Causes Gene-Specific Silencing
in Mammalian Cells
[0263] The siRNA structural scaffold is considered the essential
configuration for incorporating into RISC and mediating
RNAi(Elbashir et al., 2001a; Elbashir et al., 2001b; Elbashir et
al., 2001c; Rana, 2007; Zamore et al., 2000). However, very little
is known about RNA duplex scaffold requirements for RISC
incorporation and gene silencing. To investigate the structural
scaffold requirements for an efficient RNAi mediator and RISC
substrate, we first determined if RNA duplexes shorter than siRNAs
could mediate gene silencing. The length of double stranded (ds)
RNA is an important determinant of its propensity in activating
protein kinase R (PKR)-mediated non-specific interferon responses,
increased synthesis cost, and delivery challenges (Elbashir et al.,
2001b; Sledz et al., 2003). We designed a series of short dsRNAs
ranging from 12 to 21 by with 2 nucleotide 3' overhangs or blunt
ends targeting different mammalian genes. No gene silencing was
detected after the length was reduced below 19 by (data not shown),
which is consistent with previous reports in Drosophila
Melanogaster cell lysate(Elbashir et al., 2001b) and the notion
that 19-21 bp is the shortest siRNA duplex that mediates RNAi
(Elbashir et al., 2001a; Elbashir et al., 2001b; Elbashir et al.,
2001c; Rana, 2007; Zamore et al., 2000).
[0264] We next tested if RNA duplexes of non-siRNA scaffold with an
asymmetric configuration of overhangs can mediate gene silencing.
The siRNA duplex contains a symmetrical sense strand and an
antisense strand. While the duplex siRNA structure containing a 3'
overhang is required for incorporation into the RISC complex,
following Argonaute (Ago) mediated cleavage of the sense strand,
the antisense strand directs cleavage of the target mRNA (Hammond
et al., 2001; Matranga et al., 2005; Tabara et al., 1999). We
sought to make asymmetric RNA duplexes of various lengths with
overhangs at the 3' and 5' ends of the antisense strand.
[0265] Oligos with sequences shown in Table 3 were confirmed by 20%
polyacrylamide gel after annealing. As shown in FIG. 3A, each lane
was loaded as follows: lane 1, 21nt/21nt; lane 2, 12nt (a)/21nt;
lane 3, 12nt (b)/21nt; lane 4, 13nt/13nt; lane 5, 13nt/21nt; lane
6, 14nt/14nt; lane 7, 14nt(a)/21nt; lane 8, 14nt(b)/21nt; lane 9,
15nt/15nt; lane 10, 15nt/21nt.
TABLE-US-00005 TABLE 3 SEQ ID Oligos Sequences NO: 21nt/21nt
5'-GUAGCUGAUAUUGAUGGACTT-3' 1 3'-TTCAUCGACUAUAACUACCUG-5' 2
12nt/21nt (a) 5'-UGAUAUUGAUGG-3' 3 3'-CAUCGACUAUAACUACCUGAA-5' 4
12nt/21nt (b) 5'-CUGAUAUUGAUG-3' 5 3'-CAUCGACUAUAACUACCUGAA-5' 4
13nt/21nt 5'-CUGAUAUUGAUGG-3' 6 3'-CAUCGACUAUAACUACCUGAA-5' 4
14nt/21nt (a) 5'-GCUGAUAUUGAUGG-3' 7 3'-CAUCGACUAUAACUACCUGAA-5' 4
14nt/21nt (b) 5'-CUGAUAUUGAUGGA-3' 8 3'-CAUCGACUAUAACUACCUGAA-5' 4
15nt/21nt 5'-GCUGAUAUUGAUGGA-3' 9 3'-CAUCGACUAUAACUACCUGAA-5' 4
[0266] HeLa cells were plated at 200,000 cells/well into a 6 well
culture plate. As shown in FIG. 3B, 24 hours later they were
transfected with scramble siRNA (lane 1), 21-bp siRNA targeted E2F1
(lane 2, as a control for specificity) or 21-bp siRNA targeted
beta-catenin (lane 3, as a positive control), or the same
concentration of aiRNA of different length mix: 12nt(a)/21nt (lane
4); 12nt (b)/21nt (lane 5); 13nt/21nt (lane 6); 14nt (a)/21nt (lane
7); 14nt (b)/21nt (lane 8); 15nt/21nt (lane 9). Cells were
harvested 48 hours after transfection. Expression of .beta.-catenin
was determined by Western blot. E2F1 and actin are used as
controls. The results demonstrate that asymmetric interfering RNA
(aiRNA) causes gene-specific silencing in mammalian cells.
[0267] In order to determine the structural features of aiRNA
important in aiRNA function, we generated multiple aiRNA
oligonucleotides based on modification of the core 15/21 dual
anti-sense overhang structure (Table 4). The aiRNAs, summarized in
Table 4, contained modifications including, but not limited to,
length of the sense and anti-sense strands, degree of sense and
anti-sense overhangs, and RNA-DNA hybrid oligonucleotides.
[0268] Modification to the parental 15/21 aiRNA structure was done
by altering the sense strand, anti-sense strand, or both (Table 4).
Modified aiRNA duplexes were transfected into Hela cells at 50 nM
for 48 hours. Western blots for .beta.-catenin and actin were used
to examine the degree of gene silencing compared to the parental
15/21 aiRNA and to the traditional siRNA structure. aiRNA
modifications were also tested which contained dual sense strand
overhangs. These oligonucleotides contain a 21 base sense strand
paired to differing length anti-sense strands. In addition, we also
examined the activity of aiRNA oligonucleotides that have been
modified with DNA bases. DNA substitutions were done on both the
anti-sense and sense strands (Table 3). RNA-DNA hybrid
oligonucleotides tested contained 1 or more DNA substitutions in
either the sense or anti-sense strand, or contained 21 base
anti-sense RNA paired with indicated length of DNA sense strand.
The gene silencing results of these various aiRNAs were shown in
FIGS. 4 and 5.
[0269] Taken together, these data provide structural clues to aiRNA
function.
[0270] Regarding the sense strand, our data indicate that the
length of 15 bases works well, while lengths between 14 and 19
bases remain functional. The sense strand can match any part of the
anti-sense strand, provided that the anti-sense overhang rules are
met. Replacement of a single RNA base with DNA at either the 5' or
3' end of the sense strand is tolerated and may even provide
increased activity.
[0271] With respect to the anti-sense strand length, the length of
21 bases works well, 19-22 bases retains activity, and activity is
decreased when the length falls below 19 bases or increases above
22 bases. The 3' end of the anti-sense strand requires an overhang
of 1-5 bases with a 2-3 base overhang being preferred, blunt end
shows a decrease in activity. Base pairing with the target RNA
sequence is preferred, and DNA base replacement up to 3 bases is
tolerated without concurrent 5' DNA base replacement. The 5' end of
the anti-sense strand prefers a 0-4 base overhang, and does not
require an overhang to remain active. The 5' end of the anti-sense
strand can tolerate 2 bases not matching the target RNA sequence,
and can tolerate DNA base replacement up to 3 bases without
concurrent 3' DNA base replacement.
[0272] With respect to mismatched or chemically modified bases, we
find that both mismatches and one or more chemically modified bases
in either the sense or anti-sense strand is tolerated by the aiRNA
structure.
TABLE-US-00006 TABLE 4 aiRNA sequences used for FIG. 4-5 aiRNA #
Generic Structure Sequence 1 15-21 (NNN---NNN) 5'-GCUGAUAUUGAUGGA
CAUCGACUAUAACUACCUGAA-5' 2 15-21a (NNNNNN---blunt)
5'-GAUAUUGAUGGACUU CAUCGACUAUAACUACCUGAA-5' 3 15-21b
(blunt---NNNNNN) 5'-GUAGCUGAUAUUGAU CAUCGACUAUAACUACCUGAA-5' 4
15-21c (NNNN---NN) 5'-CUGAUAUUGAUGGAC CAUCGACUAUAACUACCUGAA-5' 5
15-21d (NN---NNNN) 5'-AGCUGAUAUUGAUGG CAUCGACUAUAACUACCUGAA-5' 7
15-18b (blunt cut 3'---NNN) 5'-GCUGAUAUUGAUGGA
CGACUAUAACUACCUGAA-5' 8 15-21d (N---NNNNN) 5'-UAGCUGAUAUUGAUG
CAUCGACUAUAACUACCUGAA-5' 9 15-21e (NNNNN---N) 5'-UGAUAUUGAUGGACU
CAUCGACUAUAACUACCUGAA-5' 10 15-22a (NNNN---NNN) 5'-GCUGAUAUUGAUGGA
UCAUCGACUAUAACUACCUGAA-5' 11 15-22b (NNN---NNNN) 5'-GCUGAUAUUGAUGGA
CAUCGACUAUAACUACCUGAAA-5' 13 15-24a (NNNNN---NNNN)
5'-GCUGAUAUUGAUGGA UUCAUCGACUAUAACUACCUGUAA-5' 14 15-24b
(NNNN---NNNNN) 5'-GCUGAUAUUGAUGGA UCAUCGACUAUAACUACCUGUCAA-5' 15
15-27 (NNNNNN---NNNNNN) 5'-GCUGAUAUUGAUGGA
GUUCAUCGACUAUAACUACCUGUCAUA-5' 16 15-20a (NNN---NN)
5'-GCUGAUAUUGAUGGA CAUCGACUAUAACUACCUGA-5' 17 15-20b (NNNN---N)
5'-GCUGAUAUUGAUGGA UCAUCGACUAUAACUACCUG-5' 18 15-20c (NN---NNN)
5'-GCUGAUAUUGAUGGA AUCGACUAUAACUACCUGAA-5' 21 15-19c (NNNN-blunt)
5'-GCUGAUAUUGAUGGA UCAUCGACUAUAACUACCU-5' 22 15-18a (NN---N)
5'-GCUGAUAUUGAUGGA AUCGACUAUAACUACCUG-5' 23 15-18b (NNN-blunt)
5'-GCUGAUAUUGAUGGA CAUCGACUAUAACUACCU-5' 24 15-18c (blunt---NNN)
5'-GCUGAUAUUGAUGGA CGACUAUAACUACCUGAA-5' 25 15-17a (NN---blunt)
5'-GCUGAUAUUGAUGGA AUCGACUAUAACUACCU-5' 26 15-17b (blunt---NN)
5'-GCUGAUAUUGAUGGA CGACUAUAACUACCUGA-5' 29 14-20 (NNN---NNN)
5'-GCUGAUAUUGAUGG CAUCGACUAUAACUACCUGA-5' 30 14-19a (NNN---NN)
5'-GCUGAUAUUGAUGG CAUCGACUAUAACUACCUG-5' 31 14-19b (NN---NNN)
5'-GCUGAUAUUGAUGG AUCGACUAUAACUACCUGA-5' 33 14-18b (NNN---N)
5'-GCUGAUAUUGAUGG CAUCGACUAUAACUACCU-5' 34 16-21a (NNN---NN)
5'-GCUGAUAUUGAUGGAC CAUCGACUAUAACUACCUGAA-5' 35 16-21b (NN---NNN)
5'-AGCUGAUAUUGAUGGA CAUCGACUAUAACUACCUGAA-5' 36 17-21 (NN---NN)
5'-AGCUGAUAUUGAUGGAC CAUCGACUAUAACUACCUGAA-5' 37 18-21a (NN---N)
5'-AGCUGAUAUUGAUGGACU CAUCGACUAUAACUACCUGAA-5' 38 18-21b (N---NN)
5'-UAGCUGAUAUUGAUGGAC CAUCGACUAUAACUACCUGAA-5' 39 18-21c
(NNN---blunt) 5'-GCUGAUAUUGAUGGACUU CAUCGACUAUAACUACCUGAA-5' 40
19-21a (NN---blunt) 5'-AGCUGAUAUUGAUGGACUU CAUCGACUAUAACUACCUGAA-5'
41 18-21b (blunt---NNN) 5'-GUAGCUGAUAUUGAUGGA
CAUCGUCUAUAACUACCUGAA-5' 42 19-21c (N---N) 5'-UAGCUGAUAUUGAUGGACU
CAUCGACUAUAACUACCUGAA-5' 43 20-21a (N---blunt)
5'-UAGCUGAUAUUGAUGGACUU CAUCGACUAUAACUACCUGAA-5' 44 20-21b
(blunt---N) 5'-GUAGCUGAUAUUGAUGGACU CAUCGACUAUAACUACCUGAA-5' 45
Mismatch and miRNA 5'-GCUGAUAUUGAAGGA CAUCGACUAUAACUACCUGAA-5' 46
5' end homologous to target 5'-GCUGAUAUUGAUGGA
CAUCGACUAUAACUACCUGUC-5' 47 NNNNNNNNNNNNNNN 5'-GCUGAUAUUGAUGGA
3'NNNNNNNNNNNNNNNNNNDDD-5' CAUCGACUAUAACUACCUgaa-5' 48
NNNNNNNNNNNNNNN 5'-GCUGAUAUUGAUGGA 3'DDDNNNNNNNNNNNNNNNNNN-5'
catCGACUAUAACUACCUGAA-5' 49 NNNNNNNNNNNNNNN 5'-GCUGAUAUUGAUGGA
3'DDDNNNNNNNNNNNNNNNDDD-5' catCGACUAUAACUACCUgaa-5' 51
DNDNNNNNNNDNDND 5'-gCtGAUAUUGaUgGa 3'NNNNNNNNNNNNNNNNNNNNN-5'
CAUCGACUAUAACUACCUGAA-5' 52 DNNNNNNNNNNNNNN 5'-gCUGAUAUUGAUGGA
3'NNNNNNNNNNNNNNNNNNNNN-5' CAUCGACUAUAACUACCUGAA-5' 53
NNNNNNNNNNNNNND 5'-GCUGAUAUUGAUGGa 3'NNNNNNNNNNNNNNNNNNNNN-5'
CAUCGACUAUAACUACCUGAA-5' 54 5'-UAGCUGAUAUUGAUG
UUCAUCGACUAUAACUACCUG-5' 55 5'-GUAGCUGAUAUUGAUGGA
UUCAUCGACUAUAACUACCUG-5' 56 5'-AGCUGAUAUUGAUGGA
UUCAUCGACUAUAACUACCUG-5' 57 DNNNNNNNNNNNNNN 5'-gCUGAUAUUGAUGGA
3'DDDNNNNNNNNNNNNNNNNNN-5' catCGACUAUAACUACCUGAA-5' 58
DNNNNNNNNNNNNNN 5'-gCUGAUAUUGAUGGA 3'NNNNNNNNNNNNNNNNNNDDD-5'
CAUCGACUAUAACUACCUgaa-5' 59 NNNNNNNNNNNNNND 5'-GCUGAUAUUGAUGGa
3'DDDNNNNNNNNNNNNNNNNNN-5' catCGACUAUAACUACCUGAA-5' 60
NNNNNNNNNNNNNND 5'-GCUGAUAUUGAUGGa 3'NNNNNNNNNNNNNNNNNNDDD-5'
CAUCGACUAUAACUACCUgaa-5' 61 NNNNNNNNNNNNNNN 5'-UAGCUGAUAUUGAUGGACU
3'DDDNNNNNNNNNNNNNNNNNN-5 catCGACUAUAACUACCUGAA-5' 62
NNNNNNNNNNNNNNN 5'-UAGCUGAUAUUGAUGGACU 3'NNNNNNNNNNNNNNNNNNDDD-5'
CAUCGACUAUAACUACCUgaa-5' In table 4, A, U, G, C represent
nucleotides, while a, t, g, c represent deoxynucleotides.
Example 2
Mechanism of Gene Silencing Triggered by aiRNA
[0273] To investigated the mechanism of gene knockdown induced by
aiRNA, we first determined if the gene silencing by aiRNA occurs at
translational or mRNA level. Northern blot analysis of
.beta.-catenin in cells transfected with 10 nM of the 15 by aiRNA
showed that the aiRNA reduced mRNA levels by over 95% within 24
hours and the decrease lasted more than 4 days (FIG. 6a),
suggesting that aiRNA mediates gene silencing at the mRNA level.
The reduction of .beta.-catenin mRNA induced by aiRNA was
substantially more rapid, efficacious and durable than by siRNA
(FIG. 6a). We further determined if the 15 by aiRNA catalyzed the
site-specific cleavage of .beta.-catenin mRNA. Total RNA isolated
from cells transfected with the 15 by aiRNA was examined by rapid
amplification of cDNA ends (5'-RACE) and PCR for the presence of
the .beta.-catenin mRNA cleavage fragments (FIG. 6b). We detected
.beta.-catenin cleavage fragments at 4 and 8 hours following aiRNA
transfection (FIG. 6c). Sequence analysis showed that cleavage was
taking place within the aiRNA target sequence between bases 10 and
11 relative to the 5' end of the aiRNA antisense strand (FIG. 6d).
No such cleavage fragments were observed following transfection
with a scrambled aiRNA (FIG. 6c). These results demonstrate that
aiRNA induced potent and efficacious gene silencing through
sequence-specific cleavage of the target mRNA.
[0274] We next determined whether the novel asymmetric scaffold of
aiRNA can be incorporated into the RISC. RNAi is catalyzed by RISC
enzyme complex with an Argonaute protein (Ago) as the catalytic
unit of the complex (Liu et al., 2004; Matranga et al., 2005). To
determine if aiRNA is incorporated into the Ago/RISC complex, we
immunoprecipitated myc-tagged human Ago1 from cells expressing
myc-tagged Ago1 (Siolas et al., 2005) after cells were transfected
with aiRNA. Small RNAs associated with the RISC complex were
detected by northern blotting of Ago immunoprecipitates. Northern
blot analysis revealed that the aiRNA entered the RISC complex with
high efficiency (FIG. 6e). These data suggest the asymmetric
scaffold of aiRNA can be efficiently incorporated into RISC.
[0275] Since aiRNA induced more efficient gene silencing than
siRNA, we tested if aiRNA can give rise to RISC complex more
efficiently than siRNA. As shown in FIG. 6e, aiRNA-Ago2/RISC
complexes formed faster and more efficient than the siRNA-Ago2/RISC
complexes, with more aiRNA contained in the RISC complex than the
corresponding siRNA (FIG. 6e and FIG. 7A). Of note, siRNA displayed
a typical pattern (21) that is consistent with formation of
secondary structures by siRNA (FIG. 6e and FIG. 7). In contrast,
aiRNA displayed a single band, suggesting that the shorter length
of aiRNA may reduce or eliminate the secondary structure formation
as occurred with siRNA.
[0276] Further, the asymmetric configuration of aiRNA may
facilitate the formation of active RISC with antisense strand and
reduce the ineffective RISC formed with the sense strand (Ref. 16).
Our data proved this is true as shown in FIG. 7B, no sense strand
can be detected in the RISC complex. FIG. 8A also demonstrates that
while the anti-sense strand of the aiRNA strongly associates with
Ago 2, the sense-strand does not. In contrast, both the anti-sense
and sense strand of the siRNA associate with Ago 2. These data
suggest that aiRNA has higher efficiency in forming RISC than siRNA
in cells, which may underlie the superior gene silencing efficiency
of aiRNA.
[0277] In addition, it has been shown that the sense strand of
siRNA is required to be cleaved in order to be functional.
Therefore, we tested if the same requirement is true for aiRNA. To
do that, the nucleotide at position 8 or 9 of the aiRNA sense
strand was modified with 2'-O-methyl to make it uncleavable. Our
results show that the aiRNAs with the uncleavable sense strand are
still functional (FIG. 8B), demonstrating aiRNA is quite different
than siRNA in terms of their mechanism.
[0278] Further we asked if there is any different loading pocket
for aiRNA and siRNA. We used cold aiRNA or siRNA to compete with
the radioactively labelled siRNA or aiRNA for the RISC complex
(FIG. 9). Surprisingly, the results show that cold aiRNA does not
compete with the siRNA for RISC complex (FIG. 9B) and cold siRNA
does not compete with aiRNA for the RISC complex either (FIG. 9C).
These data indicate that aiRNA and siRNA may load to different
pockets of RISC complex.
[0279] Together, the data above suggest that aiRNA represents the
first non-siRNA scaffold that is incorporated into RISC, providing
a novel structural scaffold that interacts with RISC. The
difference of the RISC loading of aiRNA and siRNA is illustrated in
our model shown in FIG. 10. Briefly, because of the asymmetric
property, only the anti-sense strand is selected to stay in the
RISC complex and results in a 100% efficiency in strand selection.
In contrast, siRNA is structurally symmetric. Both anti-sense
strand and sense strand of the siRNA has a chance to be selected to
stay in the RISC complex and therefore siRNA has an inefficient
strand selection and at the same time may cause non-specific gene
silencing due to the sense strand RISC complex.
Example 3
aiRNA Mediates a More Rapid, Potent, Efficacious, and Durable Gene
Silencing than siRNA
[0280] To compare aiRNA with siRNA in gene silencing properties, we
first determined the optimal aiRNA structure for gene
silencing.
[0281] The siRNA duplex contains a symmetrical sense strand and an
antisense strand. While the duplex siRNA structure containing a 3'
overhang is required for incorporation into the RISC complex,
following Argonaute (Ago) mediated cleavage of the sense strand,
the antisense strand directs cleavage of the target mRNA(Hammond et
al., 2001; Matranga et al., 2005; Tabara et al., 1999). We sought
to make asymmetric RNA duplexes of various lengths with overhangs
at the 3' and 5' ends of the antisense strand. We designed one set
of such asymmetrical RNA duplexes of 12 to15 by with 3' and 5'
antisense overhangs to target .beta.-catenin (FIG. 11A), an
endogenous gene implicated in cancer and stem cells (Clevers,
2006). An optimized siRNA of the standard configuration has been
designed to target .beta.-catenin for triggering RNAi (Xiang et
al., 2006). All aiRNAs against .beta.-catenin were designed within
the same sequence targeted by the siRNA (FIG. 11A). The results
showed that the optimal gene silencing achieved was with the 15 by
aiRNA (FIG. 11B). Therefore, we used 15 by aiRNA to be compared
with 21-mer siRNA duplex in the subsequent experiments.
[0282] To our surprise, we found that aiRNA induced potent and
highly efficacious reduction of .beta.-catenin protein while
sparing the non-targeted control genes actin (FIG. 11C).
[0283] We next examined the onset of gene silencing by aiRNA and
siRNA targeting .beta.-catenin. The sequence of the aiRNA and siRNA
used is shown in FIG. 11A. As shown in FIG. 12, aiRNA has a more
rapid onset (FIGS. 12C and D) and also a better efficacy (FIGS. 12B
and D).
[0284] We also compared the gene silencing effects of aiRNA and
siRNA on various targets and multiple human cell lines. The aiRNAs
were designed to target genes of different functional categories
including Stat3 (FIG. 13b), NQO1 (FIG. 12d), elongation factor 2
(EF2) (FIG. 13c), Nbs1 (FIG. 14b), Survivin (FIG. 14b), Parp1 (FIG.
14b), p21 (FIG. 14b), Rsk1 (FIG. 14c), PCNA (FIG. 14c), p70S6K
(FIG. 14c), mTOR (FIG. 14c), and PTEN (FIG. 14c), besides
.beta.-catenin (FIG. 13a) at the same sequences that have been
targeted with siRNA with low efficiency (Rogoff et al., 2004). As
shown in FIGS. 13 and 14, aiRNA is more efficacious than siRNA in
silencing Stat3, .beta.-catenin, Rsk1, p70S6K, Nbs1, mTOR, and EF2,
and is as efficacious as siRNA in silencing NQO1, PCNA, Survivin,
PTEN, Parp1, and p21. Since the target sequences were chosen based
on the optimization for siRNA, it is possible that the efficacy and
potency of aiRNA can be further increased by targeting sites that
are optimized for aiRNA. In addition, our data also shows that
aiRNA is more efficacious than siRNA against b-catenin in multiple
cell lines including Hela (FIG. 13a), H1299 (FIG. 14a, left panel)
and Dld1 (FIG. 14a, right panel).
[0285] Taken together, these data demonstrate that aiRNA is more
efficacious, potent, rapid-onset, and durable than siRNA in
mediating gene silencing in mammalian cells.
Example 4
Specificity of Gene Silencing Medicated by aiRNA
[0286] We next investigated the specificity of gene silencing
mediated by aiRNAs. We first analyzed aiRNAs that target the
wildtype k-Ras allele. DLD1 cells contain wild-type k-Ras while
SW480 cells contain mutant k-Ras that has a single base pair
substitution (FIG. 14d). Transfection of DLD1 cells with aiRNA
targeting wildtype k-Ras showed effective silencing, but no
silencing of mutant k-Ras was observed in the SW480 cells. These
data demonstrate that aiRNA mediates allele specific gene
silencing.
[0287] The activation of an interferon-like response is a major
non-specific mechanism of gene silencing. A primary reason that
siRNAs are used for gene silencing is that the dsRNA of shorter
than 30 by has reduced ability to activate the interferon-like
response in mammalian cells (Bernstein et al., 2001; Martinez and
Tuschl, 2004; Sledz et al., 2003). We tested if aiRNA showed any
signs of activating the interferon-like response in mammalian
cells. RNA collected from PBMC cells transfected with aiRNA against
.beta.-catenin and Hela cells transfected with aiRNA against EF2 or
Survivin was analyzed by RT-PCR for interferon inducible genes. We
found that aiRNA transfection showed no increase by RT-PCR of any
of the interferon inducible genes tested, while levels of targeted
mRNAs were reduced relative to control transfected cells (FIGS. 15a
and b). Microarray analysis was also performed to compare the
changes in the expression of known interferon response related
genes induced by aiRNA and miRNA. As shown in FIG. 15c, much less
changes were observed for aiRNA compared to siRNA.
[0288] In addition, as mentioned above, sense strand-RISC complex
may cause non-specific gene silencing. To compare aiRNA and siRNA
on the non-specific gene silencing mediated by sense-strand-RISC
complex, cells were co-transfected with aiRNA or siRNA and either a
plasmid expressing Stat3 (sense RNA) or a plasmid expressing
antisense Stat3 (antisense RNA). Cells were harvested and RNA
collected at 24 hours post transfection and relative levels of
Stat3 sense or antisense RNA were determined by quantitative real
time PCR or RT-PCR (inserts). The results show that aiRNA has no
effect on the antisense Stat3 mRNAs while siRNA does (FIG. 15d).
This result demonstrate aiRNA completely abolish the undesired
non-specific gene-silencing mediated by the sense strand-RISC
complex.
[0289] In summary, we have shown that aiRNA is a novel class of
gene-silencing inducers, the non-siRNA type and the smallest
structural scaffold for RISC substrates and RNAi mediators (FIG.
15f). Our data suggest that aiRNA works through RISC, the cellular
RNAi machinery. After incorporation into RISC, aiRNA mediates
sequence-specific cleavage of the mRNA between base 10 and 11
relative to the 5' end of the aiRNA antisense strand. The
asymmetrical configuration of aiRNA can interact more efficiently
with RISC than siRNA. Consistent with high RISC binding efficiency,
aiRNA is more potent, efficacious, rapid-onset, and durable than
siRNA in mediating gene-specific silencing against genes tested in
our study. While previous studies have proposed a role of Dicer in
facilitating efficient RISC formation, our data suggest aiRNA can
give rise to active RISC complexes with high efficiency independent
of Dicer-mediated processing.
[0290] The key feature of this novel RNA duplex scaffold is
antisense overhangs at the 3' and 5' ends. The 12-15 by aiRNA are
the shortest RNA duplex known to induce RNAi. While long dsRNAs
triggered potent gene silencing in C. elegans and Drosophila
Melanogaster, gene-specific silencing in mammalian cells was not
possible until siRNA duplexes were used. The siRNA scaffold, as
defined by Dicer digestion, is characterized by symmetry in strand
lengths of 19-21 by and 3' overhangs (Bernstein et al., 2001),
which has been considered the essential structure for incorporating
into RISC to mediate RNAi. Therefore, optimization efforts for RNAi
inducers have been focused on siRNA precursors, which are
invariably larger than siRNA (Soutschek et al., 2004; Zhang and
Farwell, 2007). Our data suggest that siRNA is not the essential
scaffold for incorporating into RISC to mediate RNAi. The aiRNAs of
different lengths displayed a spectrum of gene silencing efficacy
and RISC incorporation efficiency, offering unique opportunity for
understanding the mechanism of RISC incorporation and activation.
Research is needed to further understand the structure-activity
relationship of aiRNAs in RISC incorporation and RNAi induction,
which should help establish a rational basis for optimizing aiRNAs
with regards to target sequence selection, length, structure,
chemical composition and modifications for various RNAi
applications.
Example 5
aiRNA is More Efficacious than siRNA In Vivo
[0291] To investigate if aiRNA is efficacious in vivo and to
compare it with siRNA, we tested the effects of aiRNA and siRNA in
human colon cancer xenograft models.
[0292] Human Colon Cancer is the second leading cause of cancer
death in the U.S. The Wnt .beta.-catenin signaling pathway is
tightly regulated and has important functions in development,
tissue homeostasis, and regeneration. Deregulation of
Wnt/.beta.-catenin signaling is frequently found in various human
cancers. Eighty percent of colorectal cancers alone reveal
activation of this pathway by either inactivation of the
tumor-suppressor gene adenomatous polyposis coli or mutation of the
proto-oncogene .beta.-catenin.
[0293] Activation of Wnt/.beta.-catenin signaling has been found to
be important for both initiation and progression of cancers of
different tissues. Therefore, targeted inhibition of
Wnt/.beta.-catenin signaling is a rational and promising new
approach for the therapy of cancers of various origins.
[0294] In vitro, by ribozyme-targeting we have demonstrated the
reduction of .beta.-catenin expression in human colon cancer SW480
cells and associated induction of cell death, indicating that
.beta.-catenin expression is rate-limiting for tumor growth in
vitro.
[0295] SW480 human colon cancer cells were inoculated
subcutaneously into female athymic nude mice (8.times.10.sup.6
cells/mouse) and allowed to form palpable tumors. In this study,
dosing began when the tumors reached approximately 120 mm.sup.3.
Animals were treated intravenously (iv) with 0.6 nmol PET-complexed
.beta.-catenin siRNAs, PEI-complexed .beta.-catenin aiRNAs or a
PEI-complexed unrelated siRNA as a negative control daily. The
animals received a total of 10 doses of siRNA, aiRNA or control.
Tumors were measured throughout treatment. As shown in FIG. 16,
intravenous treatment with siRNA and aiRNA as a monotherapy at 0.6
nmol mg/kg significantly inhibited tumor growth. The % T/C value of
siRNA was calculated to be 48.8% with a p value of 0.0286. The
treatment with the .beta.-catenin-specific aiRNAs, however,
resulted in a much more potent reduction in tumor growth. The % T/C
value was calculated to be 9.9% with a p value of 0.0024. There was
no significant change in body weight due to iv administration of
the siRNA, aiRNA or control. These data suggest that the systemic
in vivo application of aiRNAs through PEI complexation upon
targeting of the .beta.-catenin offers an avenue for the
development of highly efficient, specific and safe agents for
therapeutic applications for patients with colon cancer.
[0296] In addition, we also tested the effects of aiRNA and siRNA
in HT29 human colon cancer xenograft model. HT29 human colon cancer
cells were inoculated subcutaneously into female athymic nude mice
(6.times.10.sup.6 cells/mouse) and allowed to form palpable tumors.
In this study, dosing began when the tumors reached approximately
200 mm.sup.3. Animals were treated intravenously (iv) with 0.6 nmol
PEI-complexed .beta.-catenin siRNAs, PEI-complexed .beta.-catenin
aiRNAs or a PEI-complexed unrelated siRNA as a negative control
every other day. The animals received a total of 8 doses of siRNA,
aiRNA or control. Tumors were measured throughout treatment. As
shown in FIG. 17, intravenous treatment with siRNA and aiRNA as a
monotherapy at 0.6 nmol mg/kg significantly inhibited tumor growth.
The % T/C value of siRNA was calculated to be 78% with a p value of
0.21. Again, the treatment with the .beta.-catenin-specific aiRNAs
resulted in an even more potent reduction in tumor growth. The %
T/C value was calculated to be 41% with a p value of 0.016. There
was no significant change in body weight due to iv administration
of the siRNA, aiRNA or control. These data second that the systemic
in vivo application of aiRNAs through PEI complexation upon
targeting of the .beta.-catenin offers an avenue for the
development of highly efficient, specific and safe agents for
therapeutic applications for patients with colon cancer.
[0297] Together, the aiRNA may significantly improve broad RNAi
applications. The siRNA-based therapeutics have met with
challenges, including limited efficacy, delivery difficulty,
interferon-like responses and manufacture cost (de Fougerolles et
al., 2007; loins et al., 2007; Rana, 2007). The improved efficacy,
potency, durability, and smaller size of aiRNAs may help or
overcome these challenges since aiRNA is smaller and may need less
material for its delivery. Therefore, aiRNA represents new and
smallest RNA duplexes that enter RISC and mediates gene silencing
of better efficacy, potency, onset of action, and durability than
siRNA in mammalian cells, holding significant potential for broad
RNAi applications in gene function study and RNAi-based
therapies.
Example 6
aiRNAs Function as Mimetic miRNAs
[0298] Micro-RNAs (miRNA) are an additional regulator of gene
expression with some similarity to siRNAs. However, miRNA primarily
regulates gene expression through a mechanism(s) distinct from
siRNA-mediated target cleavage. Silencing by miRNAs occurs through
interaction of the miRNA with the 3' untranslated region (UTR) of
the target RNA that leads to translation inhibition and/or target
RNA degradation. Unlike single target specificity of siRNA, a
single miRNA can regulate expression of multiple targets.
[0299] The aiRNA structure was tested for its ability to function
as a miRNA (amiRNA). Specifically in this example, an aiRNA (Let-7a
aiRNA) was constructed to have a 21-nt longer strand and a 15-nt
shorter strand forming a double stranded region of 15 nt flanked by
a 3-nt 3' overhang and a 3-nt 5' overhang, both on the longer
strand. The aiRNA was constrcuted as a mimetic of the Let-7a miRNA.
As shown in FIG. 18A, the entire aiRNA construct (boxed area) was
derived directly from a portion of the stem-loop structure of the
pre-miRNA of Let-7a (pre-Let-7a) and preserves the secondary
structure, a bulge resulting from a 2-bp mismatch, in the
endogenous pre-miRNA structure. The boxed area was selected such
that the longer strand of the aiRNA contains most of the mature
guide miRNA of Let-7a, the sequence of which is underlined in the
pre-Let-7a structure.
[0300] The sequence structure of a positive control Let7a miRNA
oligo, a double-stranded RNA molecule of symmetrical strand length
(23 nt) and including the same portion of the Let-7a miRNA sequence
(underlined) as Let-7a aiRNA is also shown in FIG. 18A. Hela and
Dld1 cells were transfected with the miRNA duplex or the aiRNA
duplex at 100 nM for 24 hours at which time RNA was isolated and
RT-PCR was performed to detect levels of k-Ras mRNA, a known
silencing target of Let-7a miRNA. As shown in FIG. 18B, both the
aiRNA duplex (labeled as aiLet7) and the miRNA duplex (labeled as
miLet7) led to a decrease in k-Ras mRNA in both cell lines. These
data suggest that aiRNA can function as a mimetic in the miRNA
pathway to alter gene expression.
[0301] We next determined whether a miRNA mimetic aiRNA could alter
protein levels of known miRNA targets. The k-Ras gene can be
regulated by members of the Let7 miRNA family. We therefore
determined if k-Ras mRNA and/or protein was modulated following
transfection with the miRNA mimetic aiRNA. The k-Ras mRNA levels
were determined by RT-PCR and the protein levels of k-Ras were
determined by western blot analysis (FIG. 19). The k-Ras mRNA
levels were down regulated by the Let7 mimetic aiRNA (FIG. 20A),
and the protein levels were also down regulated by the Let7 mimetic
aiRNA (FIG. 20B).
Example 7
aiRNAs Function as miRNA Inhibitors
[0302] To see if aiRNA structure could be used to create inhibitors
of miRNAs, we designed aiRNAs against hsa-Let-7c, hsa-miR-21, and
hsa-miR-155. Their sequences are shown in FIG. 19A. The full length
of mature hsa-Let-7c, hsa-miR-21, and hsa-miR-155 are shown in FIG.
19B. RT-PCR and western blot analyses were performed to analyze the
effect of the aiRNA-Let7c inhibitor on the expression of k-Ras, one
of the targets of Let-7c. As shown in FIG. 20A, k-Ras mRNA level
was down regulated by aiRNA-Let7c mimic and was up regulated by
aiRNA-Let7c inhibitor. Similar results were observed at the protein
level as shown in FIG. 20B.
[0303] Relative levels of Let-7c in Hela cells treated with
aiRNA-Let7c inhibitor (FIG. 21A), miR-21 in MCF-7 cells treated
with aiRNA-miR21 inhibitor (FIG. 21B), or miR-155 in FaDu cells
treated with aiRNA-miR-155 inhibitor (FIG. 21C) were also
determined by qRT-PCR. As shown in FIG. 21, these aiRNAs can
potently inhibit their specific target--the endogenous
corresponding miRNAs. Note that MCF7 cells contain relatively high
levels of miR-21 (FIG. 22A) and FaDu cells contain relatively high
levels of miR-155 (FIG. 22B). These cell lines were therefore used
to test whether transfection of the aiRNA miRNA inhibitor could
reduce the levels of miR-21 or miR-155 in MCF7 and FaDu cells,
respectively.
[0304] We next compared the efficacy of aiRNA miRNA inhibitors to
the commercially available miRNA inhibitors (Ambion). Transfection
of 100 nM of either the aiRNA miRNA inhibitor or the Ambion
inhibitor resulted in a decrease in the levels of Let-7c at 24
hours (FIG. 23), miR-21 at 72 hours (FIG. 24), and miR-155 at 72
hours (FIG. 25) following transfection. In all cases, the aiRNA
miRNA showed comparable or enhanced efficacy.
[0305] We next compared the potency of the aiRNA miRNA inhibitors
to the commercially available inhibitors from Ambion. Cells were
transfected with aiRNA or miRNA inhibitor targeting Let-7c (FIG.
26), miR-21 (FIG. 27), or miR-155 (FIG. 28) at 1, 10, and 100 nM
for 24 hours. Quantitative RT-PCR (qRT-PCR) analyses were then
performed to determine levels of miRNA remaining, relative to
non-targeting control aiRNA transfected cells. The aiRNA miRNA
showed similar potency to the commercial inhibitor at the 10 and
100 nM doses. At the 1 nM dose, aiRNA showed enhanced potency, in
the case of miR-21, similar potency, in the case of miR-155, or
reduced potency, in the case of Let-7c.
[0306] Together, these data demonstrate that the aiRNA structure
can function as both an inhibitor of endogenous miRNA that may show
enhanced efficacy and/or duration of gene silencing compared to the
antagomir, and as a mimic of miRNA function, causing the repression
of endogenous miRNA target gene expression.
[0307] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
is specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supersede and/or take precedence
over any such contradictory material.
[0308] All numbers expressing quantities of ingredients, reaction
conditions, analytical results and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should be construed in
light of the number of significant digits and ordinary rounding
approaches.
[0309] Modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to
those skilled in the art. The specific embodiments described herein
are offered by way of example only and are not meant to be limiting
in any way. It is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the
invention being indicated by the following claims.
Sequence CWU 1
1
154115RNAArtificial SequenceStrand of duplex RNA 1auacaaucua cuguc
15221RNAArtificial SequenceStrand of duplex RNA 2ugagguagua
gguuguauag u 21315RNAArtificial SequenceStrand of duplex RNA
3acaaccuacu accuc 15421RNAArtificial SequenceStrand of duplex RNA
4aaugagguag uagguuguau g 21515RNAArtificial SequenceStrand of
duplex RNA 5gguaguaggu uguau 15621RNAArtificial SequenceStrand of
duplex RNA 6aacauacaac cuacuaccuc a 21715RNAArtificial
SequenceStrand of duplex RNA 7aucagacuga uguug 15821RNAArtificial
SequenceStrand of duplex RNA 8aaucaacauc agucugauaa g
21915RNAArtificial SequenceStrand of duplex RNA 9augcuaaucg ugaua
151021RNAArtificial SequenceStrand of duplex RNA 10aacuaucacg
auuagcauua a 211122RNAArtificial SequenceStrand of duplex RNA
11ugagguagua gguuguaugg uu 221221RNAArtificial SequenceStrand of
duplex RNA 12guagcugaua uugauggacu u 211315RNAArtificial
SequenceStrand of duplex RNA 13uccaucaaua ucagc 151424DNAHomo
sapiens 14ggatctagaa tcagctacag cagc 241524DNAHomo sapiens
15tcctctagag ggcaatctcc attg 241620DNAHomo sapiens 16ccatggatga
tgatatcgcc 201720DNAHomo sapiens 17tagaagcatt tgcggtggac
201820DNAHomo sapiens 18gacaatggct actcaagctg 201920DNAHomo sapiens
19caggtcagta tcaaaccagg 202024DNAHomo sapiens 20agtacagtgc
aatgagggac cagt 242124DNAHomo sapiens 21agcatcctcc actctctgtc ttgt
242244RNAArtificial SequenceGeneRacer RNA adaptor 22cgacuggagc
acgaggacac ugacauggac ugaaggagua gaaa 442326DNAHomo sapiens
23ggacactgac atggactgaa ggagta 262425DNAHomo sapiens 24cgcatgatag
cgtgtctgga agctt 252521RNAArtificial SequenceStrand of duplex RNA
25guagcugaua uugauggacu u 212621RNAArtificial SequenceStrand of RNA
duplex 26guccaucaau aucagcuacu u 212715RNAArtificial SequenceStrand
of duplex RNA 27gcugauauug augga 152821RNAArtificial SequenceStrand
of duplex RNA 28aaguccauca auaucagcua c 212921RNAArtificial
SequenceStrand of duplex RNA 29aucaugcugu guuaacugcu u
213021RNAArtificial SequenceStrand of duplex RNA 30gcaguuaaca
cagcaugauu u 213115RNAArtificial SequenceStrand of duplex RNA
31augcuguguu aacug 153221RNAArtificial SequenceStrand of duplex RNA
32aagcaguuaa cacagcauga u 213321RNAArtificial SequenceStrand of
duplex RNA 33ggcccucuua ugauguauau u 213421RNAArtificial
SequenceStrand of duplex RNA 34uauacaucau aagagggccu u
213515RNAArtificial SequenceStrand of duplex RNA 35ccucuuauga uguau
153621RNAArtificial SequenceStrand of duplex RNA 36aauauacauc
auaagagggc c 213721RNAArtificial SequenceStrand of duplex RNA
37gccagcaaag aaucacaugu u 213821RNAArtificial SequenceStrand of
duplex RNA 38caugugauuc uuugcuggcu u 213915RNAArtificial
SequenceStrand of duplex RNA 39agcaaagaau cacau 154021RNAArtificial
SequenceStrand of duplex RNA 40aacaugugau ucuuugcugg c
214121RNAArtificial SequenceStrand of duplex RNA 41agcuaaaggu
gaagauauau u 214221RNAArtificial SequenceStrand of duplex RNA
42uauaucuuca ccuuuagcuu u 214315RNAArtificial SequenceStrand of
duplex RNA 43uaaaggugaa gauau 154421RNAArtificial SequenceStrand of
duplex RNA 44aauauaucuu caccuuuagc u 214521RNAArtificial
SequenceStrand of duplex RNA 45ccguguuuga uuuggauuuu u
214621RNAArtificial SequenceStrand of duplex RNA 46aaauccaaau
caaacacggu u 214715RNAArtificial SequenceStrand of duplex RNA
47uguuugauuu ggauu 154821RNAArtificial SequenceStrand of duplex RNA
48aaaaauccaa aucaaacacg g 214921RNAArtificial SequenceStrand of
duplex RNA 49gcagaauugu caagggauau u 215021RNAArtificial
SequenceStrand of duplex RNA 50uaucccuuga caauucugcu u
215115RNAArtificial SequenceStrand of duplex RNA 51gaauugucaa gggau
155221RNAArtificial SequenceStrand of duplex RNA 52aauaucccuu
gacaauucug c 215321RNAArtificial SequenceStrand of duplex RNA
53ggaaauugga acacaguuuu u 215421RNAArtificial SequenceStrand of
duplex RNA 54aaacuguguu ccaauuuccu u 215515RNAArtificial
SequenceStrand of duplex RNA 55aauuggaaca caguu 155621RNAArtificial
SequenceStrand of duplex RNA 56aaaaacugug uuccaauuuc c
215721RNAArtificial SequenceStrand of duplex RNA 57uggagaugcu
guuguaauuu u 215821RNAArtificial SequenceStrand of duplex RNA
58aauuacaaca gcaucuccau u 215915RNAArtificial SequenceStrand of
duplex RNA 59agaugcuguu guaau 156021RNAArtificial SequenceStrand of
duplex RNA 60aaaauuacaa cagcaucucc a 216121RNAArtificial
SequenceStrand of duplex RNA 61guggcgaaga agaaaucuau u
216221RNAArtificial SequenceStrand of duplex RNA 62uagauuucuu
cuucgccacu u 216315RNAArtificial SequenceStrand of duplex RNA
63gcgaagaaga aaucu 156421RNAArtificial SequenceStrand of duplex RNA
64aauagauuuc uucuucgcca c 216521RNAArtificial SequenceStrand of
duplex RNA 65aaggagauca acauuuucan n 216621RNAArtificial
SequenceStrand of duplex RNA 66ugaaaauguu gaucuccuun n
216715RNAArtificial SequenceStrand of duplex RNA 67aggagaucaa cauuu
156821RNAArtificial SequenceStrand of duplex RNA 68ugaaaauguu
gaucuccuun n 216921RNAArtificial SequenceStrand of duplex RNA
69gccgcagacc uugugauaun n 217021RNAArtificial SequenceStrand of
duplex RNA 70auaucacaag gucugcggcn n 217115RNAArtificial
SequenceStrand of duplex RNA 71gcagaccuug ugaua 157221RNAArtificial
SequenceStrand of duplex RNA 72aaauaucaca aggucugcgg c
217321RNAArtificial SequenceStrand of duplex RNA 73aggcccgcuc
uacaucuucu u 217421RNAArtificial SequenceStrand of duplex RNA
74gaagauguag agcgggccuu u 217515RNAArtificial SequenceStrand of
duplex RNA 75cccgcucuac aucuu 157621RNAArtificial SequenceStrand of
duplex RNA 76aagaagaugu agagcgggcc u 217715RNAArtificial
SequenceStrand of duplex RNA 77ggagcuguug gcgua 157815RNAArtificial
SequenceStrand of duplex RNA 78ggagcuguug gcgua 157921RNAArtificial
SequenceStrand of duplex RNA 79guagcugaua uugauggacn n
218021RNAArtificial SequenceStrand of duplex RNA 80guccaucaau
aucagcuacn n 218112RNAArtificial SequenceStrand of duplex RNA
81ugauauugau gg 128221RNAArtificial SequenceStrand of duplex RNA
82aaguccauca auaucagcua c 218312RNAArtificial SequenceStrand of
duplex RNA 83cugauauuga ug 128413RNAArtificial SequenceStrand of
duplex RNA 84cugauauuga ugg 138514RNAArtificial SequenceStrand of
duplex RNA 85gcugauauug augg 148614RNAArtificial SequenceStrand of
duplex RNA 86cugauauuga ugga 148715RNAArtificial SequenceStrand of
duplex RNA 87gcugauauug augga 158815RNAArtificial SequenceStrand of
duplex RNA 88gauauugaug gacuu 158915RNAArtificial SequenceStrand of
duplex RNA 89guagcugaua uugau 159015RNAArtificial SequenceStrand of
duplex RNA 90cugauauuga uggac 159115RNAArtificial SequenceStrand of
duplex RNA 91agcugauauu gaugg 159215RNAArtificial SequenceStrand of
duplex RNA 92uagcugauau ugaug 159315RNAArtificial SequenceStrand of
duplex RNA 93ugauauugau ggacu 159422RNAArtificial SequenceStrand of
duplex RNA 94aaguccauca auaucagcua cu 229522RNAArtificial
SequenceStrand of duplex RNA 95aaaguccauc aauaucagcu ac
229624RNAArtificial SequenceStrand of duplex RNA 96aauguccauc
aauaucagcu acuu 249724RNAArtificial SequenceStrand of duplex RNA
97aacuguccau caauaucagc uacu 249827RNAArtificial SequenceStrand of
duplex RNA 98auacugucca ucaauaucag cuacuug 279920RNAArtificial
SequenceStrand of duplex RNA 99aguccaucaa uaucagcuac
2010020RNAArtificial SequenceStrand of duplex RNA 100guccaucaau
aucagcuacu 2010120RNAArtificial SequenceStrand of duplex RNA
101aaguccauca auaucagcua 2010219RNAArtificial SequenceStrand of
duplex RNA 102uccaucaaua ucagcuacu 1910318RNAArtificial
SequenceStrand of duplex RNA 103guccaucaau aucagcua
1810418RNAArtificial SequenceStrand of duplex RNA 104uccaucaaua
ucagcuac 1810518RNAArtificial SequenceStrand of duplex RNA
105aaguccauca auaucagc 1810617RNAArtificial SequenceStrand of
duplex RNA 106uccaucaaua ucagcua 1710717RNAArtificial
SequenceStrand of duplex RNA 107aguccaucaa uaucagc
1710814RNAArtificial SequenceStrand of duplex RNA 108gcugauauug
augg 1410916RNAArtificial SequenceStrand of duplex RNA
109gcugauauug auggac 1611017RNAArtificial SequenceStrand of duplex
RNA 110agcugauauu gauggac 1711118RNAArtificial SequenceStrand of
duplex RNA 111agcugauauu gauggacu 1811218RNAArtificial
SequenceStrand of duplex RNA 112uagcugauau ugauggac
1811318RNAArtificial SequenceStrand of duplex RNA 113gcugauauug
auggacuu 1811419RNAArtificial SequenceStrand of duplex RNA
114agcugauauu gauggacuu 1911518RNAArtificial SequenceStrand of
duplex RNA 115guagcugaua uugaugga 1811621RNAArtificial
SequenceStrand of duplex RNA 116aaguccauca auaucugcua c
2111719RNAArtificial SequenceStrand of duplex RNA 117uagcugauau
ugauggacu 1911820RNAArtificial SequenceStrand of duplex RNA
118uagcugauau ugauggacuu 2011920RNAArtificial SequenceStrand of
duplex RNA 119guagcugaua uugauggacu 2012015RNAArtificial
SequenceStrand of duplex RNA 120gcugauauug aagga
1512121RNAArtificial SequenceStrand of duplex RNA 121cuguccauca
auaucagcua c 2112221RNAArtificial SequenceStrand of duplex RNA
122nnnuccauca auaucagcua c 2112321RNAArtificial SequenceStrand of
duplex RNA 123aaguccauca auaucagcnn n 2112421RNAArtificial
SequenceStrand of duplex RNA 124nnnuccauca auaucagcnn n
2112515RNAArtificial SequenceStrand of duplex RNA 125ncngauauug
nungn 1512615RNAArtificial SequenceStrand of duplex RNA
126ncugauauug augga 1512715RNAArtificial SequenceStrand of duplex
RNA 127gcugauauug auggn 1512821RNAArtificial SequenceStrand of
duplex RNA 128guccaucaau aucagcuacu u 2112915RNAArtificial
SequenceStrand of duplex RNA 129nnnnnnnnnn nnnnn
1513021RNAArtificial SequenceStrand of duplex RNA 130nnnnnnnnnn
nnnnnnnnnn n 2113121RNAArtificial SequenceStrand of duplex RNA
131nnnnnnnnnn nnnnnnnnnn n 2113221RNAArtificial SequenceStrand of
duplex RNA 132dddnnnnnnn nnnnnnnndd d 2113315RNAArtificial
SequenceStrand of duplex RNA 133nnnnnnnnnn nnnnn
1513421RNAArtificial SequenceStrand of duplex RNA 134nnnnnnnnnn
nnnnnnnnnn n 2113515RNAArtificial SequenceStrand of duplex RNA
135nnnnnnnnnn nnnnn 1513615RNAArtificial SequenceStrand of duplex
RNA 136nnnnnnnnnn nnnnn 1513719RNAArtificial SequenceStrand of
duplex RNA 137nnnnnnnnnn nnnnnnnnn 1913822RNAArtificial
SequenceStrand of duplex RNA 138ugagguagua gguuguauag uu
2213922RNAArtificial SequenceStrand of duplex RNA 139ugagguagua
gguuguaugg uu 2214022RNAArtificial SequenceStrand of duplex RNA
140uagcuuauca gacugauguu ga 2214123RNAArtificial SequenceStrand of
duplex RNA 141uuaaugcuaa ucgugauagg ggu 2314223RNAArtificial
SequenceStrand of duplex RNA 142acuauacaau cuacugucuu ucc
2314323RNAArtificial SequenceStrand of duplex RNA 143ugagguagua
gguuguauag uuu 2314415RNAArtificial SequenceStrand of duplex RNA
144auacaaucua cuguc 1514521RNAArtificial SequenceStrand of duplex
RNA 145ugagguagua gguuguauag u 2114615RNAArtificial SequenceStrand
of duplex RNA 146acaaccuacu accuc 1514721RNAArtificial
SequenceStrand of duplex RNA 147aaugagguag uagguuguau g
2114815RNAArtificial SequenceStrand of duplex RNA 148gguaguaggu
uguau 1514921RNAArtificial SequenceStrand of duplex RNA
149aacauacaac cuacuaccuc a 2115015RNAArtificial SequenceStrand of
duplex RNA 150aucagacuga uguug 1515121RNAArtificial SequenceStrand
of duplex RNA 151aaucaacauc agucugauaa g 2115215RNAArtificial
SequenceStrand of duplex RNA 152augcuaaucg ugaua
1515321RNAArtificial SequenceStrand of duplex RNA 153aacuaucacg
auuagcauua a 2115479RNAArtificial SequenceStrand of duplex RNA
154ugggaugagg uaguagguug uauaguuuua gggucacacc accacuggga
gauaacuaua 60caaucuacug ucuuuccua 79
* * * * *
References