U.S. patent application number 10/357529 was filed with the patent office on 2004-01-22 for double-stranded oligonucleotides.
This patent application is currently assigned to Sequitur, Inc.. Invention is credited to Wiederholt, Kristin A., Woolf, Tod M..
Application Number | 20040014956 10/357529 |
Document ID | / |
Family ID | 41112152 |
Filed Date | 2004-01-22 |
United States Patent
Application |
20040014956 |
Kind Code |
A1 |
Woolf, Tod M. ; et
al. |
January 22, 2004 |
Double-stranded oligonucleotides
Abstract
Antisense sequences, including duplex RNAi compositions, which
possess improved properties over those taught in the prior art are
disclosed. The invention provides optimized antisense oligomer
compositions and method for making and using the both in in vitro
systems and therapeutically. The invention also provides methods of
making and using the improved antisense oligomer compositions.
Inventors: |
Woolf, Tod M.; (Sudbury,
MA) ; Wiederholt, Kristin A.; (Hudson, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Sequitur, Inc.
Natick
MA
|
Family ID: |
41112152 |
Appl. No.: |
10/357529 |
Filed: |
February 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60353203 |
Feb 1, 2002 |
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60436238 |
Dec 23, 2002 |
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60438608 |
Jan 7, 2003 |
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60353381 |
Feb 1, 2002 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
A61P 27/02 20180101;
A61K 31/713 20130101; C12N 2320/11 20130101; C12N 15/111 20130101;
C12N 2320/30 20130101; A61P 31/12 20180101; C12Y 301/03048
20130101; A61P 1/04 20180101; C12N 15/1135 20130101; C12N 15/113
20130101; A61P 43/00 20180101; C12N 2310/315 20130101; A61P 29/00
20180101; C12N 2310/14 20130101; C12N 2330/30 20130101; C12N
15/1137 20130101; A61P 35/00 20180101; A61P 17/06 20180101; A61P
31/18 20180101; C12N 2320/31 20130101; A61P 9/00 20180101; C12N
2310/111 20130101; C12Y 207/11022 20130101; A61P 37/02 20180101;
C12N 2310/11 20130101; C12N 2320/50 20130101; A61P 31/20
20180101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 021/02; C07H
021/04 |
Claims
1. A double-stranded oligonucleotide composition having the
structure: 1wherein (1) n is a nucleomonomer in complementary
oligonucleotide strands of equal length and where the sequence of
Ns corresponds to a target gene sequence and (2) X and Y are each
independently selected from a group consisting of nothing; from
about 1 to about 20 nucleotides of 5' overhang; from about 1 to
about 20 nucleotides of 3' overhang; and a loop structure
consisting from about 4 to about 20 nucleomonomers, where the
nucleomonomers are selected from the group consisting of G and
A.
2. A double-stranded oligonucleotide composition having the
structure: 2wherein (1) oligoA is an oligonucleotide of a number of
nucleomonomers; (2) oligoB is an oligonucleotide that has the same
number of nucleomonomers as oligoA and that is complementary to
oligoA; (3) either oligoA or oligoB corresponds to a target gene
sequence; (4) X is selected from a group consisting of (a) nothing;
(b) an oligonucleotide of about 1 to about 20 nucleotides
covalently bonded to the 5' end of oligoA and constituting a 5'
overhang; (c) an oligonucleotide of about 1 to about 20 nucleotides
covalently bonded to the 3' end of oligoB and constituting a 3'
overhang; (d) and an oligonucleotide of about 4 to about 20
nucleomonomers covalently bonded to the 3' end of oligoB and the 5'
end of oligoA and constituting a loop structure, where the
nucleomonomers are selected from the group consisting of G and A
and (5) Y is selected from a group consisting of (a) nothing; (b)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 5' end of oligoB and constituting a 5' overhang; (c)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 3' end of oligoA and constituting a 3' overhang; (d)
and an oligonucleotide of about 4 to about 20 nucleomonomers
covalently bonded to the 3' end of oligoA and the 5' end of oligoB
and constituting a loop structure, where the nucleomonomers are
selected from the group consisting of G and A.
3. The composition of claim 1, wherein the number of nucleomonomers
in each strand of the duplex is between about 12 and about 40.
4. The composition of claim 2, wherein the number of nucleomonomers
in each strand oligoA and oligoB is between about 12 and about
40.
5. The composition of claim 1, wherein the number of nucleomonomers
in each strand of the duplex is about 27.
6. The composition of claim 2, wherein the number of nucleomonomers
in each strand of oligoA and oligoB is about 27.
7. The composition of claim 1, wherein X is a sequence of about 4
to about 20 nucleomonomers which form a loop, wherein the
nucleomonomers are selected from the group consisting of G and
A.
8. The composition of claim 2, wherein X or Y is a sequence of
about 4 to about 20 nucleomonomers that forms a loop, wherein the
nucleomonomers are selected from the group consisting of G and
A.
9. The composition of claim 8, wherein two of the adjacent Ns are
unlinked.
10. The composition of claim 8, wherein the nucleotide sequence of
the loop is GAAA.
11. A double-stranded oligonucleotide composition having the
structure:
10 5'-(Z).sub.2-8-(N).sub.15-40-(M).sub.2-8-3'
3'-(Z).sub.2-8-(N).sub.15-40-(M).sub.2-8-5'
wherein (1) each of N, Z, and M is independently a nucleomonomer;
(2) both of the sequences of Ns are complementary oligonucleotide
strands of equal length having between about 15 and about 40
nucleomonomers; (3) at least one of the sequences of Ns, optionally
with some or all of the flanking Ms or Zs, corresponds to a target
gene sequence; (4) both of the sequences of Zs are complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length; and (5) both of the sequences of Ms are
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length.
12. The composition of claim 11, wherein each Z and M nucleomonomer
is selected from the group consisting of C and G.
13. The composition of claim 12 wherein, the sequence of Zs or Ms
is CC, GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC,
GGGG, GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
14. A double-stranded oligonucleotide composition having the
structure: 3wherein (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length and where the sequence of
Ns corresponds to a target gene sequence and (2) X is selected from
the group consisting of nothing; 1-20 nucleotides of 5' overhang;
1-20 nucleotides of 3' overhang; a loop structure consisting of
from about 4 to about 20 nucleomonomers, where the nucleomonomers
are selected from the group consisting of G and A, and (3) where M
is a nucleomonomer in complementary oligonucleotide strands of
between about 2 and about 8 nucleomonomers in length which
optionally correspond to the target sequence.
15. A double-stranded oligonucleotide composition having the
structure: 4wherein (1) oligoA is 5'-(N).sub.15-40-(M).sub.2-8-3'
and oligoB is 5'-(N).sub.15-40-(M).sub.2-8-3', wherein each of N
and M is independently a nucleomonomer; (2) both of the sequences
of Ns are complementary oligonucleotide strands of equal length
having between about 15 and 40 nucleomonomers; (3) at least one of
the sequences of Ns, optionally with some or all of the flanking
Ms, corresponds to a target gene sequence; (4) X is selected from a
group consisting of (a) nothing; (b) an oligonucleotide of about 1
to about 20 nucleotides covalently bonded to the 5' end of oligoA
and constituting a 5' overhang; (c) an oligonucleotide of about 1
to about 20 nucleotides covalently bonded to the 3' end of oligoB
and constituting a 3' overhang; (d) and an oligonucleotide of about
4 to about 20 nucleomonomers covalently bonded to the 3' end of
oligoB and the 5' end of oligoA and constituting a loop structure,
where the nucleomonomers are selected from the group consisting of
G and A; and (5) both of the sequences of Ms are complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length.
16. The composition of claim 15, wherein M nucleomonomer is
selected from the group consisting of contain C and G.
17. The composition of claim 16, wherein the sequence of M is CC,
GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG,
GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
18. A double-stranded oligonucleotide composition having the
structure: 5wherein (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length and which correspond to a
target gene sequence and (2) Y is selected from the group
consisting of nothing; 1-20 nucleotides of 5' overhang; 1-20
nucleotides of 3' overhang; a loop consisting of a sequence of from
about 4 to about 20 nucleomonomers, where the nucleomonomers are
all either Gs or A's and (3) where Z is a are nucleomonomer in
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length and which comprise a sequence which can
optionally correspond to the target sequence.
19. A double-stranded oligonucleotide composition having the
structure: 6wherein (1) oligoA is 5'-(Z).sub.2-8-(N).sub.12-40-3'
and oligoB is 5'-(Z).sub.2-8-(N).sub.12-40-3', wherein each of N
and Z is independently a nucleomonomer; (2) both of the sequences
of Ns are complementary oligonucleotide strands of equal length
having between about 12 and 40 nucleomonomers; (3) at least one of
the sequences of Ns, optionally with some or all of the flanking
Zs, corresponds to a target gene sequence; (4) Y is selected from a
group consisting of (a) nothing; (b) an oligonucleotide of about 1
to about 20 nucleotides covalently bonded to the 5' end of oligoB
and constituting a 5' overhang; (c) an oligonucleotide of about 1
to about 20 nucleotides covalently bonded to the 3' end of oligoA
and constituting a 3' overhang; (d) and an oligonucleotide of about
4 to about 20 nucleomonomers covalently bonded to the 3' end of
oligoA and the 5' end of oligoB and constituting a loop structure,
where the nucleomonomers are selected from the group consisting of
G and A; and (5) both of the sequences of Zs are complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length.
20. The composition of claim 19, wherein the Z nucleomonomers are
selected from the group consisting of C and G.
21. The composition of claim 20, wherein the sequence of Z is CC,
GG, CG, GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG,
GCGC, CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
22. A method of regulating gene expression in a cell, comprising
contacting a cell with the double-stranded duplex oligonucleotide
composition of claim 1, to thereby regulate gene expression in a
cell.
23. A method of increasing the nuclease resistance of an antisense
sequence, comprising forming a double-stranded oligonucleotide
composition of claim 1, such that a double-stranded duplex is
formed, wherein the nuclease resistance of the antisense sequence
is increased compared to a control composition.
Description
RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
patent application No. 60/353,203, filed on Feb. 1, 2002,
application No. 60/436,238, filed Dec. 23, 2002, and application
No. 60/438,608, filed Jan. 7, 2003. This application also claims
the priority of 60/353,381, filed Feb. 1, 2002. The entire contents
of the aforementioned applications are hereby expressly
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Complementary oligonucleotide sequences are promising
therapeutic agents and useful research tools in elucidating gene
function. However, oligonucleotide molecules of the prior art are
often subject to nuclease degradation when applied to biological
systems. Therefore, it is often difficult to achieve efficient
inhibition of gene expression (including protein synthesis) using
such compositions.
[0003] In order to maximize the usefulness, such as the potential
therapeutic activity and in vitro utility, of oligonucleotides that
are complementary to other sequences of interest, it would be of
great benefit to improve upon the prior art oligonucleotides by
designing improved oligonucleotides having increased stability both
against serum nucleases and cellular nucleases and nucleases found
in other bodily fluids.
SUMMARY OF THE INVENTION
[0004] The instant invention is based, at least in part, on the
discovery that double-stranded oligonucleotides comprising an
antisense oligonucleotide and a protector oligonucleotide, are
capable of inhibiting gene function. Thus, the invention improves
the prior art antisense sequences, inter alia, by providing
oligonucleotides which are resistant to degradation by cellular
nucleases.
[0005] Accordingly, the invention provides optimized
oligonucleotide compositions and methods for making and using both
in in vitro, and in vivo systems, e.g., therapeutically.
[0006] In one aspect, the invention pertains to a double-stranded
oligonucleotide composition having the structure:
[0007] where (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length and where the sequence of
Ns corresponds to a target gene sequence and (2) X and Y are each
independently selected from a group consisting of nothing; from
about 1 to about 20 nucleotides of 5' overhang; from about 1 to
about 20 nucleotides of 3' overhang; and a loop structure
consisting from about 4 to about 20 nucleomonomers, where the
nucleomonomers are selected from the group consisting of G and
A.
[0008] An "overhang" is a relatively short single-stranded
nucleotide sequence on the 5'- or 3'-hydroxyl end of a
double-stranded oligonucleotide molecule (also referred to as an
"extension," "protruding end," or "sticky end").
[0009] In one embodiment, the number of Ns in each strand of the
duplex is between about 12 and about 50 (i.e., in the figure above,
oligo(N) has between about 12 and about 50 nucleomonomers). In
other embodiments, the number of Ns in each strand of the duplex is
between about 12 and about 40; or between about 15 and about 35; or
more particularly between about 20 and about 30; or even between
about 21 and about 25.
[0010] In one embodiment, X is a sequence of about 4 to about 20
nucleomonomers which form a loop, wherein the nucleomonomers are
selected from the group consisting of G and A.
[0011] In one embodiment, two of the Ns are unlinked, i.e., there
is no phosphodiester bond between the two nucleomonomers. In one
embodiment, the unlinked Ns are not in the antisense sequence.
[0012] In one embodiment, the nucleotide sequence of the loop is
GAAA.
[0013] In another aspect, the invention pertains to a
double-stranded oligonucleotide composition having the
structure:
[0014] where (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length where the sequence of Ns
corresponds to a target gene sequence; and (2) Z is a nucleomonomer
in complementary oligonucleotide strands of between about 2 and
about 8 nucleomonomers in length and where the sequence of Zs
optionally corresponds to the target sequence; and (3) where M is a
nucleomonomer in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and where the sequence
of Ms optionally corresponds to the target sequence. Although the
sequences of N nucleomonomers should be of the same length, the
sequences of Z and M nucleomonomers may optionally be of the same
length.
[0015] In one embodiment, Z and M are nucleomonomers selected from
the group consisting of C and G.
[0016] In one embodiment, the sequence of Zs or Ms is CC, GG, CG,
GC, CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC,
CGCG, GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
[0017] In another aspect, the invention pertains to a
double-stranded oligonucleotide composition having the
structure:
[0018] where (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length and where the sequence of
Ns corresponds to a target gene sequence and (2) X is selected from
the group consisting of nothing; 1-20 nucleotides of 5' overhang;
1-20 nucleotides of 3' overhang.
[0019] In some embodiments, X is a loop structure consisting of
from about 4 to about 20 nucleomonomers, where the nucleomonomers
are selected from the group consisting of G and A.
[0020] In the structure above, M is a nucleomonomer in
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length which optionally correspond to the
target sequence. In one embodiment, M is a nucleomonomer selected
from the group consisting of contain C and G.
[0021] In one embodiment, the sequence of M is CC, GG, CG, GC, CCC,
GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC,
CCCG, CGGG, GCCC, GGCC, or CCGG.
[0022] In another aspect, the invention pertains to a
double-stranded oligonucleotide composition having the
structure:
[0023] where (1) N is a nucleomonomer in complementary
oligonucleotide strands of equal length and which correspond to a
target gene sequence and (2) Y is selected from the group
consisting of nothing; 1-20 nucleotides of 5' overhang; 1-20
nucleotides of 3' overhang; a loop consisting of a sequence of from
about 4 to about 20 nucleomonomers, where the nucleomonomers are
all either Gs or A's and (3) where Z is a nucleomonomer in
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length and which comprise a sequence which can
optionally correspond to the target sequence.
[0024] In one embodiment, Zs are nucleomonomers selected from the
group consisting of C and G.
[0025] In one embodiment, the sequence of Zs is CC, GG, CG, GC,
CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG,
GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG.
[0026] In another aspect, the invention pertains to a method of
regulating gene expression in a cell, comprising forming a
double-stranded oligonucleotide composition as described herein and
contacting a cell with the double-stranded duplex, to thereby
regulate gene expression in a cell.
[0027] In one embodiment, the invention pertains to a method of
increasing the nuclease resistance of an antisense sequence,
comprising forming a double-stranded oligonucleotide composition as
described herein, such that a double-stranded duplex is formed,
wherein the nuclease resistance of the antisense sequence is
increased compared to a double-stranded, unmodified RNA
molecule.
[0028] Methods of stabilizing oligonucleotides, particularly
antisense oligonucleotides, by formation of a oligonucleotide
compositions comprising at least 3 different oligonucleotides, are
disclosed in co-pending application no. U.S. Ser. No. ______, filed
on the same day as the present application, bearing attorney docket
number "SRI-013," and entitled "Oligonucleotide Compositions with
Enhanced Efficiency." This application and all of its teachings is
hereby expressly incorporated herein by reference in its
entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows that the length of double-stranded
oligonucleotides and the presence or absence of overhangs has no
effect on function.
[0030] FIG. 1B shows the effect of structural changes on the
efficacy of siRNAs targeting .beta.-3-Integrin.
[0031] FIG. 2 shows that there is no correlation was observed
between the length of the double-stranded oligonucleotide and the
level of PKR induction for the given sequences.
[0032] FIG. 2B shows effect of .beta.-3-integrin targeted 21-mer
and 27-mers on PKR expression in HMVEC Cells.
[0033] FIG. 3 shows the effect of 5' or 3' modification on activity
of double-stranded RNA duplexes.
[0034] FIG. 4 shows the effect of the size of the modifying group
on activity of the double-stranded RNA duplex.
[0035] FIG. 5 shows the results of 2'-O-Me modifications on the
activity of double-stranded RNA duplexes.
[0036] FIG. 6 shows the inhibition of p53 by 32- and 37-mer
blunt-end siRNAs.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The instant invention advances the prior art by providing
double-stranded oligonucleotide compositions for use, both in vitro
and in vivo, e.g., therapeutically, and by providing methods of
making and using the double-stranded antisense oligomer
compositions.
[0038] Double-Stranded Oligonucleotide Compositions
[0039] Double-stranded oligonucleotides of the invention are
capable of inhibiting the synthesis of a target protein, which is
encoded by a target gene. The target gene can be endogenous or
exogenous (e.g., introduced into a cell by a virus or using
recombinant DNA technology) to a cell. As used herein, the term
"target gene" includes polynucleotides comprising a region that
encodes a polypeptide or polynucleotide region that regulates
replication, transcription, translation, or other process important
in expression of the target protein; or a polynucleotide comprising
a region that encodes the target polypeptide and a region that
regulates expression of the target polypeptide; or non-coding
regions such as the 5' or 3' UTR or introns. Accordingly, the term
"target gene" as used herein may refer to, for example, an mRNA
molecule produced by transcription a gene of interest. Furthermore,
the term "correspond," as in "an oligomer corresponds to a target
gene sequence," means that the two sequences are complementary or
homologous or bear such other biologically rational relationship to
each other (e.g., based on the sequence of nucleomonomers and their
base-pairing properties).
[0040] The "target gene" to which an RNA molecule of the invention
is directed may be associated with a pathological condition. For
example, the gene may be a pathogen-associated gene, e.g., a viral
gene, a tumor-associated gene, or an autoimmune disease-associated
gene. The target gene may also be a heterologous gene expressed in
a recombinant cell or a genetically altered organism. By
determining or modulating (e.g., inhibiting) the function of such a
gene, valuable information and therapeutic benefits in medicine,
veterinary medicine, and biology may be obtained.
[0041] The term "oligonucleotide" includes two or more
nucleomonomers covalently coupled to each other by linkages (e.g.,
phosphodiesters) or substitute linkages. In one embodiment, it may
be desirable to use a single-stranded nucleic acid molecule which
forms a duplex structure (e.g., as described in more detail below).
For example, in one embodiment, the oligonucleotide can include a
nick in either the sense of the antisense sequence.
[0042] The term "antisense" refers to a nucleotide sequence that is
inverted relative to its normal orientation for transcription and
so expresses an RNA transcript that is complementary to a target
gene mRNA molecule expressed within the host cell (e.g., it can
hybridize to the target gene mRNA molecule through Watson-Crick
base pairing). An antisense strand may be constructed in a number
of different ways, provided that it is capable of interfering with
the expression of a target gene. For example, the antisense strand
can be constructed by inverting the coding region (or a portion
thereof) of the target gene relative to its normal orientation for
transcription to allow the transcription of its complement, (e.g.,
RNAs encoded by the antisense and sense gene may be complementary).
Furthermore, the antisense oligonucleotide strand need not have the
same intron or exon pattern as the target gene, and noncoding
segments of the target gene may be equally effective in achieving
antisense suppression of target gene expression as coding
segments.
[0043] Accordingly, one aspect of the invention is a method of
inhibiting the activity of a target gene by introducing an RNAi
agent into a cell, such that the dsRNA component of the RNAi agent
is targeted to the gene. In one embodiment, an RNA oligonucleotide
molecule may contain at least one nucleomonomer that is a modified
nucleotide analogue. The nucleotide analogues may be located at
positions where the target-specific activity, e.g., the RNAi
mediating activity is not substantially effected, e.g., in a region
at the 5'-end or the 3'-end of the double-stranded molecule, where
the overhangs may be stabilized by incorporating modified
nucleotide analogues.
[0044] In another aspect, double-stranded RNA molecules known in
the art can be used in the methods of the present invention.
Double-stranded RNA molecules known in the art may also be modified
according to the teachings herein in conjunction with such methods,
e.g., by using modified nucleomonomers. For example, see U.S. Pat.
No. 6,506,559; U.S. Pat. No. 2002/0,173,478 A1; U.S. Pat. No.
2002/0,086,356 A1; Shuey, et al., "RNAi: gene-silencing in
therapeutic intervention." Drug Discov. Today 2002 Oct
15;7(20):1040-6; Aoki, et al., "Clin. Exp. Pharmacol. Physiol. 2003
Jan;30(1-2):96-102; Cioca, et al., "RNA interference is a
functional pathway with therapeutic potential in human myeloid
leukemia cell lines. Cancer Gene Ther. 2003 Feb;10(2):125-33.
[0045] Further examples of double-stranded RNA molecules include
those disclosed in the following references: Kawasaki, et al.,
"Short hairpin type of dsRNAs that are controlled by tRNA(Val)
promoter significantly induce RNAi-mediated gene silencing in the
cytoplasm of human cells." Nucleic Acids Res. 2003 Jan
15;31(2):700-7; Cottrell, et al., "Silence of the strands: RNA
interference in eukaryotic pathogens." Trends Microbiol. 2003 Jan;
11(1):37-43; Links, "Mammalian RNAi for the masses." Trends Genet.
2003 Jan;19(1):9-12; Hamada, et al., "Effects on RNA interference
in gene expression (RNAi) in cultured mammalian cells of mismatches
and the introduction of chemical modifications at the 3'-ends of
siRNAs." Antisense Nucleic Acid Drug Dev. 2002 Oct;12(5):301-9;
Links, "RNAi and related mechanisms and their potential use for
therapy." Curr. Opin. Chem. Biol. 2002 Dec;6(6):829-34; Kawasaki,
et al., "Short hairpin type of dsRNAs that are controlled by
tRNA(Val) promoter significantly induce RNAi-mediated gene
silencing in the cytoplasm of human cells." Nucleic Acids Res. 2003
Jan 15;31(2):700-7.)
[0046] A nick is two non-linked nucleomonomers in an
oligonucleotide. A nick can be included at any point along the
sense or antisense nucleotide sequence. In a preferred embodiment,
a nick is in the sense sequence. In another preferred embodiment,
the nick is at least about four nucleomonomers in from an end of
the duplexed region of the oligonucleotide (e.g., is at least about
four nucleomonomers away from the 5' or 3' end of the
oligonucleotide or away from a loop structure. For example, in one
embodiment, the nick is present in the middle of the sense strand
of the duplex molecule (e.g., if the sense sequence of the duplex
is 30 nucleomonomers in length, nucleomonomers 14 and 15 or 15 and
16 are unlinked). In an embodiment, a nick may optionally be
ligated to form a circular nucleic acid molecule.
[0047] For example, in the structure below, the indicated U
nucleomonomer is not bonded to the neighboring nucleomonomer, e.g.,
by a phosphodiester bond. The 5' OH of the nick may optionally be
phosphorylated to allow enzymatic ligation of the oligonucleotide
into a circle.
[0048] As used herein, the term "nucleotide" includes any monomeric
unit of DNA or RNA containing a sugar moiety (pentose), a
phosphate, and a nitrogenous heterocyclic base. The base is usually
linked to the sugar moiety via the glycosidic carbon (at the 1'
carbon of pentose) and that combination of base and sugar is called
a "nucleoside." The base characterizes the nucleotide with the four
customary bases of DNA being adenine (A), guanine (G), cytosine (C)
and thymine (T). Inosine (I) is an example of a synthetic base that
can be used to substitute for any of the four, naturally-occurring
bases (A, C, G, or T). The four RNA bases are A, G, C, and uracil
(U). Accordingly, an oligonucleotide may be a nucleotide sequence
comprising a linear array of nucleotides connected by
phosphodiester bonds between the 3' and 5' carbons of adjacent
pentoses. Other modified nucleosides/nucleotides are described
herein and may also be used in the oligonucleotides of the
invention.
[0049] Oligonucleotides may comprise, for example,
oligonucleotides, oligonucleosides, polydeoxyribonucleotides
(containing 2'-deoxy-D-ribose) or modified forms thereof, e.g.,
DNA, polyribonucleotides (containing D-ribose or modified forms
thereof), RNA, or any other type of polynucleotide which is an
N-glycoside or C-glycoside of a purine or pyrimidine base, or
modified purine or pyrimidine base. The term oligonucleotide
includes compositions in which adjacent nucleomonomers are linked
via phosphorothioate, amide or other linkages (e.g., Neilsen, P.
E., et al. 1991. Science. 254:1497). Generally, the term "linkage"
refers to any physical connection, preferably covalent coupling,
between two or more nucleic acid components, e.g., catalyzed by an
enzyme such as a ligase.
[0050] In addition to its art-recognized meaning (e.g., a
relatively short length single or double-stranded sequences of
deoxyribonucleotides or ribonucleotides linked via phosphodiester
bonds), the term "oligonucleotide" includes any structure that
serves as a scaffold or support for the bases of the
oligonucleotide, where the scaffold permits binding to the target
nucleic acid molecule in a sequence-dependent manner.
[0051] Oligonucleotides of the invention are isolated. The term
"isolated" includes nucleic acid molecules which are synthesized
(e.g., chemically, enzymatically, or recombinantly) or are
naturally occurring but separated from other nucleic acid molecules
which are present in a natural source of the nucleic acid.
Preferably, a naturally occurring "isolated" nucleic acid molecule
is free of sequences which naturally flank the nucleic acid
molecule (i.e., sequences located at the 5' and 3' ends of the
nucleic acid molecule) in a nucleic acid molecule in an organism
from which the nucleic acid molecule is derived.
[0052] The term "nucleomonomer" includes a single base covalently
linked to a second moiety. Nucleomonomers include, for example,
nucleosides and nucleotides. Nucleomonomers can be linked to form
oligonucleotides that bind to target nucleic acid sequences in a
sequence specific manner.
[0053] In one embodiment, modified (non-naturally occurring)
nucleomonomers can be used in the oligonucleotides described
herein. For example, nucleomonomers which are based on bases
(purines, pyrimidines, and derivatives and analogs thereof) bound
to substituted and unsubstituted cycloalkyl moieties, e.g.,
cyclohexyl or cyclopentyl moieties, and substituted and
unsubstituted heterocyclic moieties, e.g., 6-member morpholino
moieties or, preferably, sugar moieties.
[0054] Sugar moieties include natural, unmodified sugars, e.g.,
monosaccharides (such as pentoses, e.g., ribose, deoxyribose),
modified sugars and sugar analogs. Possible modifications of
nucleomonomers, particularly of a sugar moiety, include, for
example, replacement of one or more of the hydroxyl groups with a
halogen, a heteroatom, an aliphatic group, or the functionalization
of the hydroxyl group as an ether, an amine, a thiol, or the like.
One particularly useful group of modified nucleomonomers are
2'-O-methyl nucleotides, especially when the 2'-O-methyl
nucleotides are used as nucleomonomers in the ends of the
oligomers. Such 2'O-methyl nucleotides may be referred to as
"methylated," and the corresponding nucleotides may be made from
unmethylated nucleotides followed by alkylation or directly from
methylated nucleotide reagents. Modified nucleomonomers may be used
in combination with unmodified nucleomonomers. For example, an
oligonucleotide of the invention may contain both methylated and
unmethylated nucleomonomers.
[0055] Some exemplary modified nucleomonomers include sugar-or
backbone-modified ribonucleotides. Modified ribonucleotides may
contain a nonnaturally occurring base (instead of a naturally
occurring base) such as uridines or cytidines modified at the
5-position, e.g., 5-(2-amino)propyl uridine and 5-bromo uridine;
adenosines and guanosines modified at the 8-position, e.g., 8-bromo
guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and
N-alkylated nucleotides, e.g., N6-methyl adenosine. Also,
sugar-modified ribonucleotides may have the 2'-OH group replaced by
a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as
NH.sub.2, NHR, NR.sub.2,), or CN group, wherein R is lower alkyl,
alkenyl, or alkynyl.
[0056] Modified ribonucleotides may also have the phosphoester
group connecting to adjacent ribonucleotides replaced by a modified
group, e.g., of phosphothioate group. More generally, the various
nucleotide modifications may be combined.
[0057] In one embodiment, sense oligomers may have 2' modifications
on the ends (1 on each end, 2 on each end, 3 on each end, and 4 on
each end, and so on; as well as 1 on one end, 2 on one end, 3 on
one end, and 4 on one end, and so on; and even unbalanced
combinations such as 1 on one end and 2 on the other end, and so
on). Likewise, the antisense strand may have 2' modifications on
the ends (1 on each end, 2 on each end, 3 on each end, and 4 on
each end, and so on; as well as 1 on one end, 2 on one end, 3 on
one end, and 4 on one end, and so on; and even unbalanced
combinations such as 1 on one end and 2 on the other end, and so
on). In preferred aspects, such 2'-modifications are in the sense
RNA strand or the sequences other than the antisense strand.
[0058] To further maximize endo- and exonuclease resistance, in
addition to the use of 2' modified nucleomonomers in the ends,
inter-nucleomonomer linkages other than phosphodiesters may be
used. For example, such end blocks may be used alone or in
conjunction with phosphothorothioate linkages between the
2'-O-methly linkages. Preferred 2'-modified nucleomonomers are
2'-modified C and U bases.
[0059] Although the antisense strand may be substantially identical
to at least a portion of the target gene (or genes), at least with
respect to the base pairing properties, the sequence need not be
perfectly identical to be useful, e.g., to inhibit expression of a
target gene's phenotype. Generally, higher homology can be used to
compensate for the use of a shorter antisense gene. In some cases,
the antisense strand generally will be substantially identical
(although in antisense orientation) to the target gene.
[0060] One particular example of a composition of the invention has
end-blocks on both ends of a sense oligonucleotide and only the 3'
end of an antisense oligonucleotide. Without wishing to be bound by
theory, the inventors believe that a 2'-O-modified sense strand
works less well than unmodified because it is not efficiently
unwound. Accordingly, another embodiment of the invention includes
duplexes in which nucleomonomer-nucleomonomer mismatches are
present in a sense 2'-O-methly strand (and are thought to be easier
to unwind).
[0061] Accordingly, for a given first oligonucleotide strand, a
number of complementary second oligonucleotide strands are
permitted according to the invention. For example, in the following
Tables, a targeted and a non-targeted oligonucleotide are
illustrated with several possible complementary oligonucleotides.
The individual nucleotides may be 2'-OH RNA nucleotides (R) or the
corresponding 2'-OMe nucleotides (M), and the oligonucleotides
themselves may contain mismatched nucleotides (lower case
letters).
[0062] Targeted Oligonucleotide:
1 First CCCUUCUGUCUUGAACAUGAG (SEQ ID NO: ##) Strand: Second
CTgATGTTCAAGACAGAAcGG (SEQ ID NO: ##) Strand: (methyl
MMMMMMMMMMMMMMMMMMMMM groups .fwdarw.) CTgATGTTCAAGACAGAAcGG (SEQ
ID NO: ##) RRRRRRRRRRRRRRRRRRRDD CTCAUGUUCAAGACAGAAGGG (SEQ ID NO:
##) RRRRRRMMMMMMMMMRRRRRR CTCAUGUUCAAGACAGAAGGG (SEQ ID NO: ##)
MMMMMMRRRRRRRRRMMMMMM CTCAUGUUCAAGACAGAAGGG (SEQ ID NO: ##)
RMRMRMRMRMRMRMRMRMRMR
[0063] Non-Targeted Oligonucleotide:
2 First GAGTACAAGTTCTGTCTTCCC (SEQ ID NO: ##) Strand: Second
GGcAAGACAGAACTTGTAgTC (SEQ ID NO: ##) Strand: (methyl
MMMMMMMMMMMMMMMMMMMMM groups .fwdarw.) GGGAAGACAGAACTTGTACTC (SEQ
ID NO: ##) RRRRRRMMMMMMMMMRRRRRR GGGAAGACAGAACTTGTACTC (SEQ ID NO:
##) MMMMMMRRRRRRRRRMMMMMM GGGAAGACAGAACTTGTACTC (SEQ ID NO: ##)
RMRMRMRMRMRMRMRMRMRMR
[0064] Another example of further modifications that may be used in
conjunction with 2'-O-methyl nucleomonomers are modification of the
sugar residues themselves, for example alternating modified and
unmodified sugars, particularly in the sense strand.
[0065] In some embodiments, the length of the sense strand can be
29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides, with
a complementary duplexed RNA strand, optionally having
overhangs.
[0066] As a further example, the use of 2'-O-methyl RNA may
beneficially be used in circumstances in which it is desirable to
minimize cellular stress responses. RNA having 2'-O-methyl
nucleomonomers may not be recognized by cellular machinery that is
thought to recognize unmodified RNA. The use of 2'-O-methylated or
partially 2'-O-methylated RNA may avoid the interferon response to
double-stranded nucleic acids, while maintaining target RNA
inhibition. This RNAi ("stealth RNAi") is useful for avoiding the
interferon or other cellular stress responses, both in short RNAi
(e.g., siRNA) sequences that induce the interferon response, and in
longer RNAi sequences that may induce the interferon response.
[0067] An especially advantageous use of the present invention is
in gene function studies in which multiple RNAi sequences are used.
According to present methods known in the art, frequently there is
no way of predicting which sequences might induce a stress
response, including the interferon response, and in this regard the
present invention significantly advances the state of the art. For
example, if all of the multiple sequences are partially
2-O-methylated, the stress response, including interferon response,
may be avoided, and thus avoid confounding results in which some
sequences affect cellular phenotype independent of the target gene
inhibition. Other chemical modifications in addition to
2'-O-methylation may also achieve this effect.
[0068] For example, modified sugars include D-ribose, 2'-O-alkyl
(including 2'-O-methyl and 2'-O-ethyl), i.e., 2'-alkoxy, 2'-amino,
2'-S-alkyl, 2'-halo (including 2'-fluoro), 2'-methoxyethoxy,
2'-allyloxy (--OCH.sub.2CH.dbd.CH.sub.2), 2'-propargyl, 2'-propyl,
ethynyl, ethenyl, propenyl, and cyano and the like. In one
embodiment, the sugar moiety can be a hexose and incorporated into
an oligonucleotide as described (Augustyns, K., et al., Nucl.
Acids. Res. 1992. 18:4711). Exemplary nucleomonomers can be found,
e.g., in U.S. Pat. No. 5,849,902, incorporated by reference
herein.
[0069] The term "alkyl" includes saturated aliphatic groups,
including straight-chain alkyl groups (e.g., methyl, ethyl, propyl,
butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.),
branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl,
etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl,
cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl
groups, and cycloalkyl substituted alkyl groups. In certain
embodiments, a straight chain or branched chain alkyl has 6 or
fewer carbon atoms in its backbone (e.g., C.sub.1-C.sub.6 for
straight chain, C.sub.3-C.sub.6 for branched chain), and more
preferably 4 or fewer. Likewise, preferred cycloalkyls have from
3-8 carbon atoms in their ring structure, and more preferably have
5 or 6 carbons in the ring structure. The term C.sub.1-C.sub.6
includes alkyl groups containing 1 to 6 carbon atoms.
[0070] Moreover, unless otherwise specified, the term alkyl
includes both "unsubstituted alkyls" and "substituted alkyls," the
latter of which refers to alkyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
Cycloalkyls can be further substituted, e.g., with the substituents
described above. An "alkylaryl" or an "arylalkyl" moiety is an
alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The
term "alkyl" also includes the side chains of natural and unnatural
amino acids. The term "n-alkyl" means a straight chain (i.e.,
unbranched) unsubstituted alkyl group.
[0071] The term "alkenyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but that contain at least one double bond. For
example, the term "alkenyl" includes straight-chain alkenyl groups
(e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl,
octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups,
cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl,
cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl
substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl
substituted alkenyl groups. In certain embodiments, a straight
chain or branched chain alkenyl group has 6 or fewer carbon atoms
in its backbone (e.g., C.sub.2-C.sub.6 for straight chain,
C.sub.3-C.sub.6 for branched chain). Likewise, cycloalkenyl groups
may have from 3-8 carbon atoms in their ring structure, and more
preferably have 5 or 6 carbons in the ring structure. The term
C.sub.2-C.sub.6 includes alkenyl groups containing 2 to 6 carbon
atoms.
[0072] Moreover, unless otherwise specified, the term alkenyl
includes both "unsubstituted alkenyls" and "substituted alkenyls,"
the latter of which refers to alkenyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, alkyl groups,
alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0073] The term "alkynyl" includes unsaturated aliphatic groups
analogous in length and possible substitution to the alkyls
described above, but which contain at least one triple bond. For
example, the term "alkynyl" includes straight-chain alkynyl groups
(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl,
octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups,
and cycloalkyl or cycloalkenyl substituted alkynyl groups. In
certain embodiments, a straight chain or branched chain alkynyl
group has 6 or fewer carbon atoms in its backbone (e.g.,
C.sub.2-C.sub.6 for straight chain, C.sub.3-C.sub.6 for branched
chain). The term C.sub.2-C.sub.6 includes alkynyl groups containing
2 to 6 carbon atoms.
[0074] Moreover, unless otherwise specified, the term alkynyl
includes both "unsubstituted alkynyls" and "substituted alkynyls,"
the latter of which refers to alkynyl moieties having substituents
replacing a hydrogen on one or more carbons of the hydrocarbon
backbone. Such substituents can include, for example, alkyl groups,
alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy,
arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy,
carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato,
cyano, amino (including alkyl amino, dialkylamino, arylamino,
diarylamino, and alkylarylamino), acylamino (including
alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),
amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,
trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an
aromatic or heteroaromatic moiety.
[0075] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to five carbon atoms in its backbone structure.
"Lower alkenyl" and "lower alkynyl" have chain lengths of, for
example, 2-5 carbon atoms.
[0076] The term "alkoxy" includes substituted and unsubstituted
alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen
atom. Examples of alkoxy groups include methoxy, ethoxy,
isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of
substituted alkoxy groups include halogenated alkoxy groups. The
alkoxy groups can be substituted with groups such as alkenyl,
alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,
alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,
arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,
dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,
phosphonato, phosphinato, cyano, amino (including alkyl amino,
dialkylamino, arylamino, diarylamino, and alkylarylamino),
acylamino (including alkylcarbonylamino, arylcarbonylamino,
carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio,
arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato,
sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,
heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moieties.
Examples of halogen substituted alkoxy groups include, but are not
limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy,
chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
[0077] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0078] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.- (with an appropriate counterion).
[0079] The term "halogen" includes fluorine, bromine, chlorine,
iodine, etc. The term "perhalogenated" generally refers to a moiety
wherein all hydrogens are replaced by halogen atoms.
[0080] The term "substituted" includes substituents which can be
placed on the moiety and which allow the molecule to perform its
intended function. Examples of substituents include alkyl, alkenyl,
alkynyl, aryl, (CR'R").sub.0-3NR'R", (CR'R").sub.0-3CN, NO.sub.2,
halogen, (CR'R").sub.O-3C(halogen).sub.3,
(CR'R").sub.0-3CH(halogen).sub.2, (CR'R").sub.0-3CH.sub.2(halogen),
(CR'R").sub.0-3CONR'R", (CR'R").sub.0-3S(O).sub.1-2NR'R",
(CR'R").sub.0-3CHO, (CR'R").sub.0-3O(CR'R").sub.0-3H,
(CR'R").sub.0-3S(O).sub.0-2R', (CR'R").sub.0-3O(CR'R").sub.0-3H,
(CR'R").sub.0-3COR', (CR'R").sub.0-3CO.sub.2R', or
(CR'R").sub.0-3OR' groups; wherein each R' and R" are each
independently hydrogen, a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5
alkenyl, C.sub.2-C.sub.5 alkynyl, or aryl group, or R' and R" taken
together are a benzylidene group or a --(CH.sub.2).sub.2O
(CH.sub.2).sub.2- group.
[0081] The term "amine" or "amino" includes compounds or moieties
in which a nitrogen atom is covalently bonded to at least one
carbon or heteroatom. The term "alkyl amino" includes groups and
compounds wherein the nitrogen is bound to at least one additional
alkyl group. The term "dialkyl amino" includes groups wherein the
nitrogen atom is bound to at least two additional alkyl groups.
[0082] The term "ether" includes compounds or moieties which
contain an oxygen bonded to two different carbon atoms or
heteroatoms. For example, the term includes "alkoxyalkyl," which
refers to an alkyl, alkenyl, or alkynyl group covalently bonded to
an oxygen atom which is covalently bonded to another alkyl
group.
[0083] The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including
heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine),
derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and
1-alkynyl derivatives) and tautomers thereof. Examples of purines
include adenine, guanine, inosine, diaminopurine, and xanthine and
analogs (e.g., 8-oxo-N.sup.6-methyladenin- e or 7-diazaxanthine)
and derivatives thereof. Pyrimidines include, for example, thymine,
uracil, and cytosine, and their analogs (e.g., 5-methylcytosine,
5-methyluracil, 5-(1-propynyl)uracil, 5-(1-propynyl)cytosine and
4,4-ethanocytosine). Other examples of suitable bases include
non-purinyl and non-pyrimidinyl bases such as 2-aminopyridine and
triazines.
[0084] In a preferred embodiment, the nucleomonomers of an
oligonucleotide of the invention are RNA nucleotides. In another
preferred embodiment, the nucleomonomers of an oligonucleotide of
the invention are modified RNA nucleotides.
[0085] The term "nucleoside" includes bases which are covalently
attached to a sugar moiety, preferably ribose or deoxyribose.
Examples of preferred nucleosides include ribonucleosides and
deoxyribonucleosides. Nucleosides also include bases linked to
amino acids or amino acid analogs which may comprise free carboxyl
groups, free amino groups, or protecting groups. Suitable
protecting groups are well known in the art (see P. G. M. Wuts and
T. W. Greene, "Protective Groups in Organic Synthesis", 2.sup.nd
Ed., Wiley-Interscience, New York, 1999).
[0086] The term "nucleotide" includes nucleosides which further
comprise a phosphate group or a phosphate analog.
[0087] As used herein, the term "linkage" includes a naturally
occurring, unmodified phosphodiester moiety
(--O--(PO.sub.2.sup.-)--O--) that covalently couples adjacent
nucleomonomers. As used herein, the term "substitute linkage"
includes any analog or derivative of the native phosphodiester
group that covalently couples adjacent nucleomonomers. Substitute
linkages include phosphodiester analogs, e.g., phosphorothioate,
phosphorodithioate, and P-ethyoxyphosphodiester,
P-ethoxyphosphodiester, P-alkyloxyphosphotriester,
methylphosphonate, and nonphosphorus containing linkages, e.g.,
acetals and amides. Such substitute linkages are known in the art
(e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers
et al. 1991. Nucleosides Nucleotides. 10:47).
[0088] In certain embodiments, oligonucleotides of the invention
comprise 3' and 5' termini (except for circular oligonucleotides).
In one embodiment, the 3' and 5' termini of an oligonucleotide can
be substantially protected from nucleases e.g., by modifying the 3'
or 5' linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For
example, oligonucleotides can be made resistant by the inclusion of
a "blocking group." The term "blocking group" as used herein refers
to substituents (e.g., other than OH groups) that can be attached
to oligonucleotides or nucleomonomers, either as protecting groups
or coupling groups for synthesis (e.g., FITC, propyl
(CH.sub.2-CH.sub.2-CH.sub.3), phosphate (PO.sub.3.sup.2-), hydrogen
phosphonate, or phosphoramidite). "Blocking groups" also include
"end blocking groups" or "exonuclease blocking groups" which
protect the 5' and 3' termini of the oligonucleotide, including
modified nucleotides and non-nucleotide exonuclease resistant
structures.
[0089] Exemplary end-blocking groups include cap structures (e.g.,
a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3'-3'
or 5'-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense
Res. Dev. 2:129), methylphosphonate, phosphoramidite,
non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers,
conjugates) and the like. The 3' terminal nucleomonomer can
comprise a modified sugar moiety. The 3' terminal nucleomonomer
comprises a 3'-O that can optionally be substituted by a blocking
group that prevents 3'-exonuclease degradation of the
oligonucleotide. For example, the 3'-hydroxyl can be esterified to
a nucleotide through a 3'.fwdarw.3' internucleotide linkage. For
example, the alkyloxy radical can be methoxy, ethoxy, or
isopropoxy, and preferably, ethoxy. Optionally, the 3'.fwdarw.3'
linked nucleotide at the 3' terminus can be linked by a substitute
linkage. To reduce nuclease degradation, the 5' most 3'.fwdarw.5'
linkage can be a modified linkage, e.g., a phosphorothioate or a
P-alkyloxyphosphotriester linkage. Preferably, the two 5' most
3'.fwdarw.5' linkages are modified linkages. Optionally, the 5'
terminal hydroxy moiety can be esterified with a phosphorus
containing moiety, e.g., phosphate, phosphorothioate, or
P-ethoxyphosphate.
[0090] In one embodiment, the sense strand of an oligonucleotide
comprises a 5' group that allows for RNAi activity but which
renders the sense strand inactive in terms of gene targeting.
Preferably, such a 5' modifying group is a phosphate group or a
group larger than a phosphate group
[0091] In another embodiment, the antisense strand of an
oligonucleotide comprises a 5' phosphate group.
[0092] In one embodiment, the oligonucleotides included in the
composition are high affinity oligonucleotides. The term "high
affinity" as used herein includes oligonucleotides that have a Tm
(melting temperature) of or greater than about 60.degree. C.,
greater than about 65.degree. C., greater than about 70.degree. C.,
greater than about 75.degree. C., greater than about 80.degree. C.
or greater than about 85.degree. C. The Tm is the midpoint of the
temperature range over which the oligonucleotide separates from the
target nucleotide sequence. At this temperature, 50% helical
(hybridized) versus coil (unhybridized) forms are present. Tm is
measured by using the UV spectrum to determine the formation and
breakdown (melting) of hybridization. Base stacking occurs during
hybridization, which leads to a reduction in UV absorption. Tm
depends both on GC content of the two nucleic acid molecules and on
the degree of sequence complementarity. Tm can be determined using
techniques that are known in the art (see for example, Monia et al.
1993. J Biol. Chem. 268:145; Chiang et al. 1991. J Biol. Chem.
266:18162; Gagnor et al. 1987. Nucleic Acids Res. 15:10419; Monia
et al. 1996. Proc. Natl. Acad. Sci. 93:15481; Publisis and Tinoco.
1989. Methods in Enzymology 180:304; Thuong et al. 1987. Proc.
Natl. Acad. Sci. USA 84:5129).
[0093] In one embodiment, an oligonucleotide can include an agent
which increases the affinity of the oligonucleotide for its target
sequence. The term "affinity enhancing agent" includes agents that
increase the affinity of an oligonucleotide for its target. Such
agents include, e.g., intercalating agents and high affinity
nucleomonomers. Intercalating agents interact strongly and
nonspecifically with nucleic acids. Intercalating agents serve to
stabilize RNA-DNA duplexes and thus increase the affinity of the
oligonucleotides for their targets. Intercalating agents are most
commonly linked to the 3' or 5' end of oligonucleotides. Examples
of intercalating agents include acridine, chlorambucil,
benzopyridoquinoxaline, benzopyridoindole, benzophenanthridine, and
phenazinium. The agents may also impart other characteristics to
the oligonucleotide, for example, increasing resistance to
endonucleases and exonucleases.
[0094] In one embodiment, a high affinity nucleomonomer is
incorporated into an oligonucleotide. The language "high affinity
nucleomonomer" as used herein includes modified bases or base
analogs that bind to a complementary base in a target nucleic acid
molecule with higher affinity than an unmodified base, for example,
by having more energetically favorable interactions with the
complementary base, e.g., by forming more hydrogen bonds with the
complementary base. For example, high affinity nucleomonomer
analogs such as aminoethyoxy phenoxazine (also referred to as a G
clamp), which forms four hydrogen bonds with guanine are included
in the term "high affinity nucleomonomer." A high affinity
nucleomonomer is illustrated below (see, e.g., Flanagan, et al.,
1999. Proc. Natl. Acad. Sci. 96:3513).
[0095] Other exemplary high affinity nucleomonomers are known in
the art and include 7-alkenyl, 7-alkynyl, 7-heteroaromatic-, or
7-alkynyl-heteroaromatic-substituted bases or the like which can be
substituted for adenosine or guanosine in oligonucleotides (see,
e.g., U.S. Pat. No. 5,594,121). Also, 7-substituted deazapurines
have been found to impart enhanced binding properties to
oligonucleotides, i.e., by allowing them to bind with higher
affinity to complementary target nucleic acid molecules as compared
to unmodified oligonucleotides. High affinity nucleomonomers can be
incorporated into the oligonucleotides of the instant invention
using standard techniques.
[0096] In another embodiment, an agent that increases the affinity
of an oligonucleotide for its target comprises an intercalating
agent. As used herein, the language "intercalating agent" includes
agents which can bind to a DNA double helix. When covalently
attached to an oligonucleotide of the invention, an intercalating
agent enhances the binding of the oligonucleotide to its
complementary genomic DNA target sequence. The intercalating agent
may also increase resistance to endonucleases and exonucleases.
[0097] Exemplary intercalating agents are taught by Helene and
Thuong (1989. Genome 31:413), and include e.g., acridine
derivatives (Lacoste et al. 1997. Nucleic Acids Research. 25:1991;
Kukreti et al. 1997. Nucleic Acids Research. 25:4264); quinoline
derivatives (Wilson et al. 1993. Biochemistry 32:10614);
benzo[f]quino[3,4-b]quioxaline derivatives (Marchand et al. 1996.
Biochemistry. 35:5022; Escude et al. 1998. Proc. Natl. Acad. Sci.
95:3591).
[0098] Intercalating agents can be incorporated into an
oligonucleotide using any convenient linkage. For example, acridine
or psoralen can be linked to the oligonucleotide through any
available --OH or --SH group, e.g., at the terminal 5' position of
the oligonucleotide, the 2' positions of sugar moieties, or an OH,
NH.sub.2, COOH, or SH incorporated into the 5-position of
pyrimidines using standard methods.
[0099] In one embodiment, when included in an RNase H activating
antisense nucleotide sequence, an agent that increases the affinity
of an oligonucleotide for its target is not positioned adjacent to
an RNase activating region of the oligonucleotide, e.g., is
positioned adjacent to a non-RNase activating region. Preferably,
the agent that increases the affinity of an oligonucleotide for its
target is placed at a distance as far as possible from the RNase
activating domain of the chimeric antisense sequence such that the
specificity of the chimeric antisense sequence is not altered when
compared with the specificity of a chimeric antisense sequence
which lacks the intercalating compound. In one embodiment, this can
be accomplished by positioning the agent adjacent to a non-RNase
activating region. The specificity of the oligonucleotide can be
tested by demonstrating that transcription of a non-target
sequence, preferably a non-target sequence which is structurally
similar to the target (e.g., has some sequence homology or identity
with the target sequence but which is not identical in sequence to
the target), is not inhibited to a greater degree by an
oligonucleotide comprising an affinity enhancing agent than by an
oligonucleotide directed against the same target that does not
comprise an affinity enhancing agent.
[0100] The double-stranded oligonucleotides of the invention may be
formed by a single, self-complementary nucleic acid strand or two
separate complementary nucleic acid strands. Duplex formation can
occur either inside or outside the cell containing the target
gene.
[0101] As used herein, the term "double-stranded" includes one or
more nucleic acid molecules comprising a region of the molecule in
which at least a portion of the nucleomonomers are complementary
and hydrogen bond to form a duplex.
[0102] As used herein, the term "duplex" includes the region of the
double-stranded nucleic acid molecule(s) that is (are) hydrogen
bonded to a complementary sequence.
[0103] The double-stranded oligonucleotides of the invention
comprise a nucleotide sequence that is sense to a target gene and a
complementary sequence that is antisense to the target gene. The
sense and antisense nucleotide sequences correspond to the target
gene sequence, e.g., are identical or are sufficiently identical to
effect target gene inhibition (e.g., are about at least about 98%,
96% identical, 94%, 90% identical, 85% identical, or 80% identical)
to the target gene sequence.
[0104] When comprised of two separate complementary nucleic acid
molecules, the individual nucleic acid molecules can be of
different lengths.
[0105] In one embodiment, a double-stranded oligonucleotide of the
invention is double-stranded over its entire length, i.e., with no
overhanging single-stranded sequence at either end of the molecule,
i.e., is blunt-ended. In another embodiment, a double-stranded
oligonucleotide of the invention is not double-stranded over its
entire length. For instance, when two separate nucleic acid
molecules are used, one of the molecules, e.g., the first molecule
comprising an antisense sequence can be longer than the second
molecule hybridizing thereto (leaving a portion of the molecule
single-stranded). Likewise, when a single nucleic acid molecule is
used a portion of the molecule at either end can remain
single-stranded.
[0106] In one embodiment, a double-stranded oligonucleotide of the
invention is double-stranded over at least about 70% of the length
of the oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 80% of the length of the oligonucleotide. In another
embodiment, a double-stranded oligonucleotide of the invention is
double-stranded over at least about 90%-95% of the length of the
oligonucleotide. In another embodiment, a double-stranded
oligonucleotide of the invention is double-stranded over at least
about 96%-98% of the length of the oligonucleotide.
[0107] In one embodiment, the double-stranded duplex constructs of
the invention can be further stabilized against nucleases by
forming loop structures at the 5' or 3' end of the sense or
antisense strand of the construct. For example, the construct can
take the form:
[0108] where the Ns are nucleomonomers in complementary
oligonucleotide strands (i.e., the top N strand is complementary to
the bottom N strand) of equal length (e.g., between about 12 and
about 40 nucleotides in length) and X and Y are each independently
selected from a group consisting of nothing (i.e., the construct is
a blunt ended construct with no loops and no overhang); from about
1 to about 20 nucleotides of 5' overhang; from about 1 to about 20
nucleotides of 3' overhang; a GAAA loop (tetra-loop); and a loop
consisting from about 4 to about 20 nucleomonomers (where the
nucleomonomers are all either Gs or A's).
[0109] The sequence of Ns corresponds to the target gene sequence
(e.g., is homologous or identical to a nucleotide sequence that is
sense or antisense to the target gene sequence), while the
nucleotide sequence of the loop structure does not correspond to
the target gene sequence.
[0110] For example, such loops can comprise all Gs and A's and be
from about 4 to about 20 nucleotides in length. In one embodiment,
such a loop can be a tetra-loop having a sequence GAAA:
[0111] In one embodiment, the number of Ns is about 27.
[0112] In embodiments in which loops are at one or both ends of the
construct, the oligonucleotide can be divided by having a "nick"
which is two non-linked nucleomonomers at any point along the sense
or antisense strand, but preferably along the sense strand.
Preferably, the nick is at least four bases from the nearest end of
the duplexed region (to provide enough thermodynamic
stability).
[0113] In another embodiment, a construct of the invention can take
the form:
[0114] where the Ns are complementary nucleomonomers in
oligonucleotide strands of equal length (e.g., between 12-40
nucleomonomers in length); Zs are nucleomonomers in complementary
oligonucleotide strands of between about 2 and about 8
nucleomonomers in length and which comprise a sequence which can
optionally correspond to the target sequence; and where Ms are
nucleomonomers in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and which can
optionally correspond to the target sequence.
[0115] Preferably, the Zs and Ms are nucleomonomers selected from
the group consisting of Cs and Gs to make the end of the duplex
more thermodynamically stable. Ends of duplexes can become single
stranded transiently, and since duplex RNA is more stable than
single-stranded RNA, the enhanced stability of the duplex on the
ends will result in higher nuclease stability.
[0116] A preferred sequence for Z or M in the antisense strand is
from 2-8 nucleomonomers in length or preferably from 3-4
nucleomonomers in length, e.g., (from 5' to 3') CC, GG, CG, GC,
CCC, GGG, CGG, GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG,
GGGC, CCCG, CGGG, GCCC, GGCC, or CCGG. The complementary strand
would have the corresponding complementary sequence.
[0117] In still another embodiment, a construct of the invention
has the form:
[0118] where Ns are nucleomonomers in complementary oligonucleotide
strands (i.e., the top N strand is complementary to the bottom N
strand) of equal length (e.g., from between about 12 to about 40
nucleomonomers in length) and X is selected from the group
consisting of nothing (i.e., leaving blunt ends with no loop or
overhang); 1-20 nucleotides of 5' overhang; 1-20 nucleotides of 3'
overhang; a GAAA loop (tetra-loop); and a loop consisting of from
about 4 to about 20 nucleomonomers (where the nucleomonomers are
all either Gs or A's) and where Ms are nucleomonomers in
complementary oligonucleotide strands of between about 2 and about
8 nucleomonomers in length (which can optionally correspond to the
target sequence). Preferably, Ms are nucleomonomers selected from
the group consisting of contain Cs and Gs.
[0119] A preferred sequence for M in the antisense strand is from
2-8 nucleomonomers in length or preferably from 3-4 nucleomonomers
in length, e.g., (from 5' to 3') CC, GG, CG, GC, CCC, GGG, CGG,
GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,
CGGG, GCCC, GGCC, or CCGG and the corresponding complement on the
opposite strand.
[0120] In another embodiment, the construct can take the form:
[0121] where Ns are nucleomonomers in complementary oligonucleotide
strands of equal length (e.g., from between about 12 to about 40
nucleomonomers in length) and Y is selected from the group
consisting of nothing (i.e., leaving blunt ends with no loop or
overhang; 1-20 nucleotides of 5' overhang; 1-20 nucleotides of 3'
overhang; a GAAA loop (tetra-loop); and a loop consisting of a
sequence of from about 4 to about 20 nucleomonomers (where the
nucleomonomers are all either Gs or A's) and where Zs are
nucleomonomers in complementary oligonucleotide strands of between
about 2 and about 8 nucleomonomers in length and which comprise a
sequence which can optionally correspond to the target sequence.
Preferably, the Zs are nucleomonomers selected from the group
consisting of Cs and Gs to make the end of the duplex more
stable.
[0122] A preferred sequence for Z in the antisense strand is from
2-8 nucleomonomers in length or preferably from 3-4 nucleomonomers
in length, e.g., (from 5' to 3') CC, GG, CG, GC, CCC, GGG, CGG,
GCC, GCG, CGC, CGGG, GCCC, CCCC, GGGG, GCGC, CGCG, GGGC, CCCG,
CGGG, GCCC, GGCC or CCGG (and the corresponding complement on the
opposite strand). For example, in the following structure, GGCC on
the end (and its complement) confers additional stability:
[0123] The invention also relates to a double-stranded
oligonucleotide composition having the following structure:
[0124] wherein (1) oligoA is an oligonucleotide of a number of
nucleomonomers; (2) oligoB is an oligonucleotide that has the same
number of nucleomonomers as oligoA and that is complementary to
oligoA; (3)either oligoA or oligoB corresponds to a target gene
sequence.
[0125] In this structure, X may be selected from (a) nothing; (b)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 5' end of oligoA and constituting a 5' overhang; (c)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 3' end of oligoB and constituting a 3' overhang; (d)
and an oligonucleotide of about 4 to about 20 nucleomonomers
covalently bonded to the 3' end of oligoB and the 5' end of oligoA
and constituting a loop structure, where the nucleomonomers are
selected from the group consisting of G and A.
[0126] Similarly, Y may be selected from (a) nothing; (b) an
oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 5' end of oligoB and constituting a 5' overhang; (c)
an oligonucleotide of about 1 to about 20 nucleotides covalently
bonded to the 3' end of oligoA and constituting a 3' overhang; (d)
and an oligonucleotide of about 4 to about 20 nucleomonomers
covalently bonded to the 3' end of oligoA and the 5' end of oligoB
and constituting a loop structure, where the nucleomonomers are
selected from the group consisting of G and A.
[0127] Similarly, the invention includes a double-stranded
oligonucleotide composition having the structure:
[0128] wherein (1) oligoA is 5'-(N).sub.15-40-(M).sub.2-8-3' and
oligoB is 5'-(N).sub.15-40-(M).sub.2-8-3', wherein each of N and M
is independently a nucleomonomer; (2) both of the sequences of Ns
are complementary oligonucleotide strands of equal length having
between about 15 and 40 nucleomonomers; (3) at least one of the
sequences of Ns, optionally with some or all of the flanking Ms,
corresponds to a target gene sequence. Both of the sequences of Ms
are complementary oligonucleotide strands of between about 2 and
about 8 nucleomonomers in length. The two M strands are optionally
of the same length.
[0129] The group X indicated by the curved line is selected from
(a) nothing; (b) an oligonucleotide of about 1 to about 20
nucleotides covalently bonded to the 5' end of oligoA and
constituting a 5' overhang; (c) an oligonucleotide of about 1 to
about 20 nucleotides covalently bonded to the 3' end of oligoB and
constituting a 3' overhang; (d) and an oligonucleotide of about 4
to about 20 nucleomonomers covalently bonded to the 3' end of
oligoB and the 5' end of oligoA and constituting a loop structure,
where the nucleomonomers are selected from the group consisting of
G and A.
[0130] Likewise, the invention pertains to a double-stranded
oligonucleotide composition having the structure:
[0131] wherein (1) oligoA is 5'-(Z).sub.2-8-(N).sub.12-40-3' and
oligoB is 5'-(Z).sub.2-8-(N).sub.12-40-3', wherein each of N and Z
is independently a nucleomonomer; (2) both of the sequences of Ns
are complementary oligonucleotide strands of equal length having
between about 12 and 40 nucleomonomers; (3) at least one of the
sequences of Ns, optionally with some or all of the flanking Zs,
corresponds to a target gene sequence. Both of the sequences of Zs
are complementary oligonucleotide strands of between about 2 and
about 8 nucleomonomers in length. The two Z strands are optionally
of the same length.
[0132] Here, Y is selected from (a) nothing; (b) an oligonucleotide
of about 1 to about 20 nucleotides covalently bonded to the 5' end
of oligoB and constituting a 5' overhang; (c) an oligonucleotide of
about 1 to about 20 nucleotides covalently bonded to the 3' end of
oligoA and constituting a 3' overhang; (d) and an oligonucleotide
of about 4 to about 20 nucleomonomers covalently bonded to the 3'
end of oligoA and the 5' end of oligoB and constituting a loop
structure, where the nucleomonomers are selected from the group
consisting of G and A.
[0133] In one embodiment, the double-stranded duplex of an
oligonucleotide of the invention is from between about 12 to about
50 nucleomonomers in length, i.e., the number of nucleotides of the
double-stranded oligonucleotide which hybridize to the
complementary sequence of the double-stranded oligonucleotide to
form the double-stranded duplex structure is from about 12 to about
50 nuclemonomers in length. In another embodiment, the
double-stranded duplex of an oligonucleotide of the invention is
from between about 12 to about 40 nucleomonomers in length.
[0134] In one embodiment, the double-stranded duplex of an
oligonucleotide of the invention is at least about 25
nucleomonomers in length. In one embodiment, the double-stranded
duplex is greater than about 25 nucleomonomers in length. In one
embodiment, a double-stranded duplex is at least about 26, 27, 28,
29, 30, at least about 40, at least about 50, or at least about 60,
at least about 70, at least about 80, or at least about 90
nucleomonomers in length. In another embodiment, the
double-stranded duplex is less than about 25 nucleomonomers in
length. In one embodiment, a double-stranded duplex is at least
about 10, at least about 15, at least about 20, at least about 22,
at least about 23 or at least about 24 nucleomonomers in
length.
[0135] In one embodiment, the number of Ns in each strand of the
duplex is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, or 27. In another embodiment, the number of Ns in each
strand of the duplex is about 30, 35, 40, 45, or 50. In one
embodiment, the number of Ns in each strand of the duplex is about
19. In a preferred embodiment, the number of Ns in each strand of
the duplex is about 27. In another embodiment, the number of Ns in
each strand of the duplex is about 27 (e.g., is 26, 27, or 28). In
another embodiment, the number of Ns in each strand of the duplex
is 27.
[0136] In one embodiment, an individual nucleic acid molecule of a
double-stranded oligonucleotide of the invention is at least about
25 nucleomonomers in length. For example, when the double-stranded
oligonucleotide of the invention is comprised of one nucleic acid
molecule, that individual molecule is at least about 25
nucleomonomers in length or when the double-stranded
oligonucleotide of the invention is comprised of two separate
nucleic acid molecules, the length of at least one of the
individual nucleic acid molecules is at least about 25
nucleomonomers in length.
[0137] A variety of nucleotides of different lengths may be used.
In one embodiment, an individual nucleic acid molecule comprising a
double-stranded oligonucleotide of the invention is greater than
about 25 nucleomonomers in length. In one embodiment, an individual
nucleic acid molecule comprising a double-stranded oligonucleotide
of the invention is at least about 26, 27, 28, 29, 30, at least
about 40, at least about 50, or at least about 60, at least about
70, at least about 80, or at least about 90 nucleomonomers in
length. In another embodiment, an individual nucleic acid molecule
comprising a double-stranded oligonucleotide of the invention is
less than about 25 nucleomonomers in length. In one embodiment, an
individual nucleic acid molecule comprising a double-stranded
oligonucleotide of the invention is at least about 10, at least
about 15, at least about 20, at least about 22, at least about 23
or at least about 24 nucleomonomers in length.
[0138] The double-stranded molecules of the invention comprise a
first nucleotide sequence which is antisense to at least part of
the target gene and a second nucleotide sequence which is
complementary to the first nucleotide sequence; i. e., is sense to
at least part of the target gene. In one embodiment, the second
nucleotide sequence of the double-stranded molecule comprises a
nucleotide sequence which is at least about 100% complementary to
the antisense molecule.
[0139] In another embodiment, the second nucleotide sequence of the
double-stranded molecule comprises a nucleotide sequence which is
at least about 95% complementary to the antisense molecule. In
another embodiment, the second nucleotide sequence of the
double-stranded molecule comprises a nucleotide sequence which is
at least about 90% complementary to the antisense molecule. In
another embodiment, the second nucleotide sequence of the
double-stranded molecule comprises a nucleotide sequence which is
at least about 80% complementary to the antisense molecule. In
another embodiment, the second nucleotide sequence of the
double-stranded molecule comprises a nucleotide sequence which is
at least about 60% complementary to the antisense molecule. In
another embodiment, the second nucleotide sequence of the
double-stranded molecule comprises a nucleotide sequence which is
at least about 100% complementary to the antisense molecule.
[0140] To determine the percent identity of two nucleic acid
sequences, the sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-identical sequences can be disregarded for
comparison purposes). When a position in the first sequence is
occupied by the same nucleotide as the corresponding position in
the second sequence, then the molecules are identical at that
position. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences. The percent complementarity can be determined
analogously; when a position in one sequence occupied by a
nucleotide that is complementary to the nucleotide in the other
sequence, then the molecules are complementary at that
position.
[0141] The comparison of sequences and determination of percent
identity between two sequences can be accomplished using a
mathematical algorithm. In a preferred embodiment, the percent
identity between two nucleotide sequences is determined using e.g.,
the GAP program in the GCG software package, using a NWSgapdna. CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length
weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent
identity between two nucleotide sequences is determined using the
algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17
(1988)) which has been incorporated into the ALIGN program (version
2.0), using a PAM120 weight residue table, a gap length penalty of
12 and a gap penalty of 4.
[0142] The nucleic acid sequences of the present invention can
further be used as a "query sequence" to perform alignments against
sequences in public databases. Such searches can be performed using
the NBLAST and XBLAST programs (version 2.0) of Altschul et al.
(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be
performed with the NBLAST program, score=100, wordlength=12. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al. (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. See, e.g., the NIH website.
[0143] In yet another embodiment, a first antisense sequence of the
double-stranded molecule hybridizes to its complementary second
sequence of the double-stranded molecule under stringent
hybridization conditions. As used herein, the term "hybridizes
under stringent conditions" is intended to describe conditions for
hybridization and washing under which nucleotide sequences at least
60% complementary to each other typically remain hybridized to each
other. Preferably, the conditions are such that sequences at least
about 70%, more preferably at least about 80%, even more preferably
at least about 85% or 90% complementary to each other typically
remain hybridized to each other.
[0144] Such stringent conditions are known to those skilled in the
art and can be found in Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred,
non-limiting example of stringent hybridization conditions are
hybridization in 6.times.sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 50.degree. C., preferably at 55.degree.
C., more preferably at 60.degree. C., and even more preferably at
65.degree. C. Ranges intermediate to the above-recited values,
e.g., at 60-65.degree. C. or at 55-60.degree. C. are also intended
to be encompassed by the present invention. Alternatively,
formamide can be included in the hybridization solution, using
methods and conditions also known in the art.
[0145] One of the sequences (or molecules) of the double-stranded
oligonucleotide of the invention is antisense to the target gene.
As used herein, the term "antisense sequence" includes nucleotide
sequences which bind to the "sense" strand of the nucleotide
sequence of the target gene (e.g., polynucleotides such as DNA,
mRNA (including pre-mRNA)) molecules. When the antisense sequences
of the invention bind to nucleic acid molecules, they can bind to
any region of a nucleic acid molecule, including e.g., introns,
exons, 5', or 3' untranslated regions. Antisense sequences that
work by binding to a target and activating RNase H preferably bind
within an intron, an exon, the 5' untranslated region, or the 3'
untranslated region of a nucleic acid target molecule.
[0146] Preferably, the oligonucleotide compositions of the
invention do not activate the interferon pathway, e.g., as
evidenced by the lack of induction of the double-stranded RNA,
interferon-inducible protein kinase, PKR.
[0147] In one embodiment, modifications are made to a
double-stranded RNA molecule which would normally activate the
interferon pathway such that the interferon pathway is not
activated. For example, the interferon pathway is activated by
double-stranded unmodified RNA. The cellular recognition of
double-stranded RNA is highly specific and modifying one or woth of
the strands of a double-stranded duplex enable the double-stranded
RNA molecule to evade the double-stranded RNA recognition machinery
of the cell but would still allow for the activation of the RNAi
pathway.
[0148] The ability of a double-stranded oligonucleotide to activate
interferon could be assessed by testing for expression of the
double-stranded RNA, Interferon-Inducible Protein Kinase, PKR using
techniques known in the art and also testing for the ability of the
double-stranded molecule to effect target gene inhibition.
Accordingly, in one embodiment, the invention provides a method of
testing for the ability of a double-stranded RNA molecule to induce
interferon by testing for the ability of the oligonucleotide to
activate PKR. Compositions that do not activate PKR (i.e., do not
activate the interferon pathway) are then selected for use to
inhibit gene transcription in cells, e.g., in therapeutics or
functional genomics.
[0149] Without being limited to any particular mechanism of action,
an antisense sequence used in a double-stranded oligonucleotide
composition of the invention that can specifically hybridize with a
nucleotide sequence within the target gene (i.e., can be
complementary to a nucleotide sequence within the target gene) may
achieve its affects based on, e.g.,: (1) binding to target mRNA and
stericly blocking the ribosome complex from translating the mRNA;
(2) binding to target mRNA and triggering mRNA cleavage by RNase H;
(3) binding to double-stranded DNA in the nucleus and forming a
triple helix; (4) hybridizing to open DNA loops created by RNA
polymerase; (5) interfering with mRNA splicing; (6) interfering
with transport of mRNA from the nucleus to the cytoplasm; or (7)
interfering with translation through inhibition of the binding of
initiation factors or assembly of ribosomal subunits (i.e., at the
start codon).
[0150] In one embodiment, an antisense sequence of the
double-stranded oligonucleotides of the invention is complementary
to a target nucleic acid sequence over at least about 80% of the
length of the antisense sequence. In another embodiment, the
antisense sequence of the double-stranded oligonucleotide of the
invention is complementary to a target nucleic acid sequence over
at least about 90-95% of the length of the antisense sequence. In
another embodiment, the antisense sequence of the double-stranded
oligonucleotide of the invention is complementary to a target
nucleic acid sequence over the entire length of the antisense
sequence.
[0151] In yet another embodiment, an antisense sequence of the
double-stranded oligonucleotide hybridizes to at least a portion of
the target gene under stringent hybridization conditions.
[0152] In one embodiment, antisense sequences of the invention are
substantially complementary to a target nucleic acid sequence. In
one embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 100% complementary to a portion of
the target gene. In another embodiment, an antisense RNA molecule
comprises a nucleotide sequence which is at least about 90%
complementary to a portion of the target gene. In another
embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 80% complementary to a portion of
the target gene. In another embodiment, an antisense RNA molecule
comprises a nucleotide sequence which is at least about 60%
complementary to a portion of the target gene. In another
embodiment, an antisense RNA molecule comprises a nucleotide
sequence which is at least about 100% complementary to a portion of
the target gene. Preferably, no loops greater than about 8
nucleotides are formed by areas of non-complementarity between the
oligonucleotide and the target.
[0153] In one embodiment, an antisense nucleotide sequence of the
invention is complementary to a target nucleic acid sequence over
at least about 80% of the length of the antisense sequence. In
another embodiment, an antisense sequence of the invention is
complementary to a target nucleic acid sequence over at least about
90-95% of the length of the antisense sequence. In another
embodiment, an antisense sequence of the invention is complementary
to a target nucleic acid sequence over the entire length of the
antisense sequence.
[0154] The antisense sequences used in an oligonucleotide
composition of the invention may be of any type, e.g., including
morpholino oligonucleotides, RNase H activating oligonucleotides,
or ribozymes.
[0155] In one embodiment, a double-stranded oligonucleotide of the
invention can comprise (i.e., be a duplex of) one nucleic acid
molecule which is DNA and one nucleic acid molecule which is
RNA.
[0156] Antisense sequences of the invention can be "chimeric
oligonucleotides" which comprise an RNA-like and a DNA-like region.
The language "RNase H activating region" includes a region of an
oligonucleotide, e.g., a chimeric oligonucleotide, that is capable
of recruiting RNase H to cleave the target RNA strand to which the
oligonucleotide binds. Typically, the RNase activating region
contains a minimal core (of at least about 3-5, typically between
about 3-12, more typically, between about 5-12, and more preferably
between about 5-10 contiguous nucleomonomers) of DNA or DNA-like
nucleomonomers. (See, e.g., U.S. Pat. No. 5,849,902). Preferably,
the RNase H activating region comprises about nine contiguous
deoxyribose containing nucleomonomers.
[0157] In one embodiment, the contiguous nucleomonomers are linked
by a substitute linkage, e.g., a phosphorothioate linkage. In one
embodiment, an antisense sequence of the invention is unstable,
i.e., is degraded in a cell, in the absence of the second strand
(or self complementary sequence) which forms a double-stranded
oligonucleotide of the invention. For example, in one embodiment, a
chimeric antisense sequence comprises unmodified DNA nucleomonomers
in the gap rather than phosphorothioate DNA.
[0158] The language "non-activating region" includes a region of an
antisense sequence, e.g., a chimeric oligonucleotide, that does not
recruit or activate RNase H. Preferably, a non-activating region
does not comprise phosphorothioate DNA. The oligonucleotides of the
invention comprise at least one non-activating region. In one
embodiment, the non-activating region can be stabilized against
nucleases or can provide specificity for the target by being
complementary to the target and forming hydrogen bonds with the
target nucleic acid molecule, which is to be bound by the
oligonucleotide.
[0159] Antisense sequences of the present invention may include
"morpholino oligonucleotides." Morpholino oligonucleotides are
non-ionic and function by an RNase H-independent mechanism. Each of
the 4 genetic bases (Adenine, Cytosine, Guanine, and
Thymine/Uracil) of the morpholino oligonucleotides is linked to a
6-membered morpholine ring. Morpholino oligonucleotides are made by
joining the 4 different subunit types by, e.g., non-ionic
phosphorodiamidate inter-subunit linkages. An example of a 2
subunit morpholino oligonucleotide is shown below.
[0160] Morpholino oligonucleotides have many advantages including:
complete resistance to nucleases (Antisense & Nuc. Acid Drug
Dev. 1996. 6:267); predictable targeting (Biochemica Biophysica
Acta. 1999. 1489:141); reliable activity in cells (Antisense &
Nuc. Acid Drug Dev. 1997. 7:63); excellent sequence specificity
(Antisense & Nuc. Acid Drug Dev. 1997. 7:151); minimal
non-antisense activity (Biochemica Biophysica Acta. 1999.
1489:141); and simple osmotic or scrape delivery (Antisense &
Nuc. Acid Drug Dev. 1997. 7:291). Morpholino oligonucleotides are
also preferred because of their non-toxicity at high doses. A
discussion of the preparation of morpholino oligonucleotides can be
found in Antisense & Nuc. Acid Drug Dev. 1997. 7:187.
[0161] Uptake Of Oligonucleotides By Cells
[0162] Oligonucleotides and oligonucleotide compositions are
contacted with (i.e., brought into contact with, also referred to
herein as administered or delivered to) and taken up by one or more
cells or a cell lysate. The term "cells" includes prokaryotic and
eukaryotic cells, preferably vertebrate cells, and, more
preferably, mammalian cells. In a preferred embodiment, the
oligonucleotide compositions of the invention are contacted with
human cells.
[0163] Oligonucleotide compositions of the invention can be
contacted with cells in vitro, e.g., in a test tube or culture
dish, (and may or may not be introduced into a subject) or in vivo,
e.g., in a subject such as a mamalian subject. Oligonucleotides are
taken up by cells at a slow rate by endocytosis, but endocytosed
oligonucleotides are generally sequestered and not available, e.g.,
for hybridization to a target nucleic acid molecule. In one
embodiment, cellular uptake can be facilitated by electroporation
or calcium phosphate precipitation. However, these procedures are
only useful for in vitro or ex vivo embodiments, are not convenient
and, in some cases, are associated with cell toxicity.
[0164] In another embodiment, delivery of oligonucleotides into
cells can be enhanced by suitable art recognized methods including
calcium phosphate, DMSO, glycerol or dextran, electroporation, or
by transfection, e.g., using cationic, anionic, or neutral lipid
compositions or liposomes using methods known in the art (see e.g.,
WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355;
Bergan et al. 1993. Nucleic Acids Research. 21:3567). Enhanced
delivery of oligonucleotides can also be mediated by the use of
vectors (See e.g., Shi, Y. 2003. Trends Genet 2003 Jan 19:9;
Reichhart J M et al. Genesis. 2002. 34(1-2):160-4, Yu et al. 2002.
Proc Natl Acad Sci U S A 99:6047; Sui et al. 2002. Proc Natl Acad
Sci U S A 99:5515) viruses, polyamine or polycation conjugates
using compounds such as polylysine, protamine, or N1, N12-bis
(ethyl) spermine (see, e.g., Bartzatt, R. et al. 1989. Biotechnol.
Appl. Biochem. 11:133; Wagner E. et al. 1992. Proc. Natl. Acad.
Sci. 88:4255)
[0165] Conjugating Agents
[0166] Conjugating agents bind to the oligonucleotide in a covalent
manner. In one embodiment, oligonucleotides can be derivitized or
chemically modified by binding to a conjugating agent to facilitate
cellular uptake. For example, covalent linkage of a cholesterol
moiety to an oligonucleotide can improve cellular uptake by 5- to
10-fold which in turn improves DNA binding by about 10-fold
(Boutorin et al., 1989, FEBS Letters 254:129-132). Conjugation of
octyl, dodecyl, and octadecyl residues enhances cellular uptake by
3-, 4-, and 10-fold as compared to unmodified oligonucleotides
(Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108).
Similarly, derivatization of oligonucleotides with poly-L-lysine
can aid oligonucleotide uptake by cells (Schell, 1974, Biochem.
Biophys. Acta 340:323, and Lemaitre et al., 1987, Proc. Natl. Acad.
Sci. USA 84:648).
[0167] Certain protein carriers can also facilitate cellular uptake
of oligonucleotides, including, for example, serum albumin, nuclear
proteins possessing signals for transport to the nucleus, and viral
or bacterial proteins capable of cell membrane penetration.
Therefore, protein carriers are useful when associated with or
linked to the oligonucleotides. Accordingly, the present invention
provides for derivatization of oligonucleotides with groups capable
of facilitating cellular uptake, including hydrocarbons and
non-polar groups, cholesterol, long chain alcohols (i.e., hexanol),
poly-L-lysine and proteins, as well as other aryl or steroid groups
and polycations having analogous beneficial effects, such as phenyl
or naphthyl groups, quinoline, anthracene or phenanthracene groups,
fatty acids, fatty alcohols and sesquiterpenes, diterpenes, and
steroids. A major advantage of using conjugating agents is to
increase the initial membrane interaction that leads to a greater
cellular accumulation of oligonucleotides.
[0168] Encapsulating Agents
[0169] Encapsulating agents entrap oligonucleotides within
vesicles. In another embodiment of the invention, an
oligonucleotide may be associated with a carrier or vehicle, e.g.,
liposomes or micelles, although other carriers could be used, as
would be appreciated by one skilled in the art. Liposomes are
vesicles made of a lipid bilayer having a structure similar to
biological membranes. Such carriers are used to facilitate the
cellular uptake or targeting of the oligonucleotide, or improve the
oligonucleotide's pharmacokinetic or toxicologic properties.
[0170] For example, the oligonucleotides of the present invention
may also be administered encapsulated in liposomes, pharmaceutical
compositions wherein the active ingredient is contained either
dispersed or variously present in corpuscles consisting of aqueous
concentric layers adherent to lipidic layers. The oligonucleotides,
depending upon solubility, may be present both in the aqueous layer
and in the lipidic layer, or in what is generally termed a
liposomic suspension. The hydrophobic layer, generally but not
exclusively, comprises phopholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, more or less ionic
surfactants such as diacetylphosphate, stearylamine, or
phosphatidic acid, or other materials of a hydrophobic nature. The
diameters of the liposomes generally range from about 15 nm to
about 5 microns.
[0171] The use of liposomes as drug delivery vehicles offers
several advantages. Liposomes increase intracellular stability,
increase uptake efficiency and improve biological activity.
Liposomes are hollow spherical vesicles composed of lipids arranged
in a similar fashion as those lipids which make up the cell
membrane. They have an internal aqueous space for entrapping water
soluble compounds and range in size from 0.05 to several microns in
diameter. Several studies have shown that liposomes can deliver
nucleic acids to cells and that the nucleic acids remain
biologically active. For example, a liposome delivery vehicle
originally designed as a research tool, such as Lipofectin, can
deliver intact nucleic acid molecules to cells.
[0172] Specific advantages of using liposomes include the
following: they are non-toxic and biodegradable in composition;
they display long circulation half-lives; and recognition molecules
can be readily attached to their surface for targeting to tissues.
Finally, cost-effective manufacture of liposome-based
pharmaceuticals, either in a liquid suspension or lyophilized
product, has demonstrated the viability of this technology as an
acceptable drug delivery system.
[0173] Complexing Agents
[0174] Complexing agents bind to the oligonucleotides of the
invention by a strong but non-covalent attraction (e.g., an
electrostatic, van der Waals, pi-stacking, etc. interaction). In
one embodiment, oligonucleotides of the invention can be complexed
with a complexing agent to increase cellular uptake of
oligonucleotides. An example of a complexing agent includes
cationic lipids. Cationic lipids can be used to deliver
oligonucleotides to cells.
[0175] The term "cationic lipid" includes lipids and synthetic
lipids having both polar and non-polar domains and which are
capable of being positively charged at or around physiological pH
and which bind to polyanions, such as nucleic acids, and facilitate
the delivery of nucleic acids into cells. In general cationic
lipids include saturated and unsaturated alkyl and alicyclic ethers
and esters of amines, amides, or derivatives thereof.
Straight-chain and branched alkyl and alkenyl groups of cationic
lipids can contain, e.g., from 1 to about 25 carbon atoms.
Preferred straight chain or branched alkyl or alkene groups have
six or more carbon atoms. Alicyclic groups include cholesterol and
other steroid groups. Cationic lipids can be prepared with a
variety of counterions (anions) including, e.g., Cl.sup.-,
Br.sup.-, I.sup.-, F.sup.-, acetate, trifluoroacetate, sulfate,
nitrite, and nitrate.
[0176] Examples of cationic lipids include polyethylenimine,
polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a
combination of DOTMA and DOPE), Lipofectase, Lipofectamine, DOPE,
Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (J
B L, San Luis Obispo, Calif.). Exemplary cationic liposomes can be
made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
chloride (DOTMA),
N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP),
3.beta.-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol
(DC-Chol),
2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-
-propanaminium trifluoroacetate (DOSPA),
1,2-dimyristyloxypropyl-3-dimethy- l-hydroxyethyl ammonium bromide;
and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylam- monium chloride
(DOTMA), for example, was found to increase 1000-fold the antisense
effect of a phosophorothioate oligonucleotide. (Vlassov et al.,
1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides
can also be complexed with, e.g., poly (L-lysine) or avidin and
lipids may, or may not, be included in this mixture, e.g.,
steryl-poly (L-lysine).
[0177] Cationic lipids have been used in the art to deliver
oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910;
5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996.
Proc. Natl. Acad. Sci. USA 93:3176; Hope et al 1998. Molecular
Membrane Biology 15:1). Other lipid compositions which can be used
to facilitate uptake of the instant oligonucleotides can be used in
connection with the claimed methods. In addition to those listed
supra, other lipid compositions are also known in the art and
include, e.g, those taught in U.S. Pat. No. 4,235,871, U.S. Pat.
Nos. 4,501,728; 4,837,028; 4,737,323.
[0178] In one embodiment lipid compositions can further comprise
agents, e.g., viral proteins to enhance lipid-mediated
transfections of oligonucleotides (Kamata, et al., 1994. Nucl.
Acids. Res. 22:536). In another embodiment, oligonucleotides are
contacted with cells as part of a composition comprising an
oligonucleotide, a peptide, and a lipid as taught, e.g., in U.S.
Pat. No. 5,736,392. Improved lipids have also been described which
are serum resistant (Lewis, et al., 1996. Proc. Natl. Acad. Sci.
93:3176). Cationic lipids and other complexing agents act to
increase the number of oligonucleotides carried into the cell
through endocytosis.
[0179] In another embodiment N-substituted glycine oligonucleotides
(peptoids) can be used to optimize uptake of oligonucleotides.
Peptoids have been used to create cationic lipid-like compounds for
transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci.
95:1517). Peptoids can be synthesized using standard methods (e.g.,
Zuckermann, R. N., et al. 1992. J. Am. Chem. Soc. 114:10646;
Zuckermann, R. N., et al. 1992. Int. J Peptide Protein Res.
40:497). Combinations of cationic lipids and peptoids, liptoids,
can also be used to optimize uptake of the subject oligonucleotides
(Hunag, et al., 1998. Chemistry and Biology. 5:345). Liptoids can
be synthesized by elaborating peptoid oligonucleotides and coupling
the amino terminal submonomer to a lipid via its amino group
(Hunag, et al., 1998. Chemistry and Biology. 5:345).
[0180] It is known in the art that positively charged amino acids
can be used for creating highly active cation lipids (Lewis et al.
1996. Proc. Natl. Acad. Sci. U.S.A. 93:3176). In one embodiment, a
composition for delivering oligonucleotides of the invention
comprises a number of arginine, lysine, histadine or ornithine
residues linked to a lipophilic moiety (see e.g., U.S. Pat. No.
5,777,153).
[0181] In another, a composition for delivering oligonucleotides of
the invention comprises a peptide having from between about one to
about four basic residues. These basic residues can be located,
e.g., on the amino terminal, C-terminal, or internal region of the
peptide. Families of amino acid residues having similar side chains
have been defined in the art. These families include amino acids
with basic side chains (e.g., lysine, arginine, histidine), acidic
side chains (e.g., aspartic acid, glutamic acid), uncharged polar
side chains (e.g., glycine (can also be considered non-polar),
asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan), beta-branched side
chains (e.g., threonine, valine, isoleucine) and aromatic side
chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
Apart from the basic amino acids, a majority or all of the other
residues of the peptide can be selected from the non-basic amino
acids, e.g., amino acids other than lysine, arginine, or histidine.
Preferably a preponderance of neutral amino acids with long neutral
side chains are used. For example, a peptide such as (N-term)
His-Ile-Trp-Leu-Ile-Tyr-Leu-Trp-Ile-Val-(C-term) (SEQ ID NO: ##)
could be used. In one embodiment such a composition can be mixed
with the fusogenic lipid DOPE as is well known in the art.
[0182] In one embodiment, the cells to be contacted with an
oligonucleotide composition of the invention are contacted with a
mixture comprising the oligonucleotide and a mixture comprising a
lipid, e.g., one of the lipids or lipid compositions described
supra for between about 12 h to about 24 h. In another embodiment,
the cells to be contacted with an oligonucleotide composition are
contacted with a mixture comprising the oligonucleotide and a
mixture comprising a lipid, e.g., one of the lipids or lipid
compositions described supra for between about 1 and about five
days. In one embodiment, the cells are contacted with a mixture
comprising a lipid and the oligonucleotide for between about three
days to as long as about 30 days. In another embodiment, a mixture
comprising a lipid is left in contact with the cells for at least
about five to about 20 days. In another embodiment, a mixture
comprising a lipid is left in contact with the cells for at least
about seven to about 15 days.
[0183] For example, in one embodiment, an oligonucleotide
composition can be contacted with cells in the presence of a lipid
such as cytofectin CS or GSV(available from Glen Research;
Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as
described herein.
[0184] In one embodiment the incubation of the cells with the
mixture comprising a lipid and an oligonucleotide composition does
not reduce the viability of the cells. Preferably, after the
transfection period the cells are substantially viable. In one
embodiment, after transfection, the cells are between at least
about 70 and at least about 100 percent viable. In another
embodiment, the cells are between at least about 80 and at least
about 95% viable. In yet another embodiment, the cells are between
at least about 85% and at least about 90% viable.
[0185] In one embodiment, oligonucleotides are modified by
attaching a peptide sequence that transports the oligonucleotide
into a cell, referred to herein as a "transporting peptide." In one
embodiment, the composition includes an oligonucleotide which is
complementary to a target nucleic acid molecule encoding the
protein, and a covalently attached transporting peptide.
[0186] The language "transporting peptide" includes an amino acid
sequence that facilitates the transport of an oligonucleotide into
a cell. Exemplary peptides which facilitate the transport of the
moieties to which they are linked into cells are known in the art,
and include, e.g., HIV TAT transcription factor, lactoferrin,
Herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al.
1998. Nature Biotechnology. 16:857; and Derossi et al. 1998. Trends
in Cell Biology. 8:84; Elliott and O'Hare. 1997. Cell 88:223).
[0187] For example, in one embodiment, the transporting peptide
comprises an amino acid sequence derived from the antennapedia
protein. Preferably, the peptide comprises amino acids 43-58 of the
antennapedia protein
(Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys)
(SEQ ID NO: ##) or a portion or variant thereof that facilitates
transport of an oligonucleotide into a cell (see, e.g., WO 91/1898;
Derossi et al. 1998. Trends Cell Biol. 8:84). Exemplary variants
are shown in Derossi et al., supra.
[0188] In one embodiment, the transporting peptide comprises an
amino acid sequence derived from the transportan, galanin
(1-12)-Lys-mastoparan (1-14) amide, protein. (Pooga et al. 1998.
Nature Biotechnology 16:857). Preferably, the peptide comprises the
amino acids of the transportan protein shown in the sequence
GWTLNSAGYLLGKFNLKALAALAKKIL (SEQ ID NO: ##) or a portion or variant
thereof that facilitates transport of an oligonucleotide into a
cell.
[0189] In one embodiment, the transporting peptide comprises an
amino acid sequence derived from the HIV TAT protein. Preferably,
the peptide comprises amino acids 37-72 of the HIV TAT protein,
e.g., shown in the sequence C(Acm)FITKALGISYGRKKRRQRRRPPQC (SEQ ID
NO: ##) (TAT 37-60; where C(Acm) is Cys-acetamidomethyl) or a
portion or variant thereof, e.g., C(Acm)GRKKRRQRRRPPQC (SEQ ID NO:
##)(TAT 48-40) or C(Acm)LGISYGRKKRRQRRPPQC (SEQ ID NO: ##) (TAT
43-60) that facilitates transport of an oligonucleotide into a cell
(Vives et al. 1997. J. Biol. Chem. 272:16010). In another
embodiment the peptide (G)CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ (SEQ
ID NO: ##) can be used.
[0190] Portions or variants of transporting peptides can be readily
tested to determine whether they are equivalent to these peptide
portions by comparing their activity to the activity of the native
peptide, e.g., their ability to transport fluorescently-labeled
oligonucleotides to cells. Fragments or variants that retain the
ability of the native transporting peptide to transport an
oligonucleotide into a cell are functionally equivalent and can be
substituted for the native peptides.
[0191] Oligonucleotides can be attached to the transporting peptide
using known techniques, e.g., ( Prochiantz, A. 1996. Curr. Opin.
Neurobiol. 6:629; Derossi et al. 1998. Trends Cell Biol. 8:84; Troy
et al. 1996. J. Neurosci. 16:253), Vives et al. 1997. J. Biol.
Chem. 272:16010). For example, in one embodiment, oligonucleotides
bearing an activated thiol group are linked via that thiol group to
a cysteine present in a transport peptide (e.g., to the cysteine
present in the .beta. turn between the second and the third helix
of the antennapedia homeodomain as taught, e.g., in Derossi et al.
1998. Trends Cell Biol. 8:84; Prochiantz. 1996. Current Opinion in
Neurobiol. 6:629; Allinquant et al. 1995. J Cell Biol. 128:919). In
another embodiment, a Boc-Cys-(Npys)OH group can be coupled to the
transport peptide as the last (N-terminal) amino acid and an
oligonucleotide bearing an SH group can be coupled to the peptide
(Troy et al. 1996. J. Neurosci. 16:253).
[0192] In one embodiment, a linking group can be attached to a
nucleomonomer and the transporting peptide can be covalently
attached to the linker. In one embodiment, a linker can function as
both an attachment site for a transporting peptide and can provide
stability against nucleases. Examples of suitable linkers include
substituted or unsubstituted C.sub.1-C.sub.20 alkyl chains,
C.sub.2-C.sub.20 alkenyl chains, C.sub.2-C.sub.20 alkynyl chains,
peptides, and heteroatoms (e.g., S, O, NH, etc.). Other exemplary
linkers include bifunctional crosslinking agents such as
sulfosuccinimidyl-4-(maleimidophenyl)-butyrat- e (SMPB) (see, e.g.,
Smith et al. Biochem J 1991. 276: 417-2).
[0193] In one embodiment, oligonucleotides of the invention are
synthesized as molecular conjugates which utilize receptor-mediated
endocytotic mechanisms for delivering genes into cells (see, e.g.,
Bunnell et al. 1992. Somatic Cell and Molecular Genetics. 18:559
and the references cited therein).
[0194] Targeting Agents
[0195] The delivery of oligonucleotides can also be improved by
targeting the oligonucleotides to a cellular receptor. The
targeting moieties can be conjugated to the oligonucleotides or
attached to a carrier group (i.e., poly(L-lysine) or liposomes)
linked to the oligonucleotides. This method is well suited to cells
that display specific receptor-mediated endocytosis.
[0196] For instance, oligonucleotide conjugates to
6-phosphomannosylated proteins are internalized 20-fold more
efficiently by cells expressing mannose 6-phosphate specific
receptors than free oligonucleotides. The oligonucleotides may also
be coupled to a ligand for a cellular receptor using a
biodegradable linker. In another example, the delivery construct is
mannosylated streptavidin which forms a tight complex with
biotinylated oligonucleotides. Mannosylated streptavidin was found
to increase 20-fold the internalization of biotinylated
oligonucleotides. (Vlassov et al. 1994. Biochimica et Biophysica
Acta 1197:95-108).
[0197] In addition specific ligands can be conjugated to the
polylysine component of polylysine-based delivery systems. For
example, transferrin-polylysine, adenovirus-polylysine, and
influenza virus hemagglutinin HA-2 N-terminal fusogenic
peptides-polylysine conjugates greatly enhance receptor-mediated
DNA delivery in eucaryotic cells. Mannosylated glycoprotein
conjugated to poly(L-lysine) in aveolar macrophages has been
employed to enhance the cellular uptake of oligonucleotides. Liang
et al. 1999. Pharmazie 54:559-566.
[0198] Because malignant cells have an increased need for essential
nutrients such as folic acid and transferrin, these nutrients can
be used to target oligonucleotides to cancerous cells. For example,
when folic acid is linked to poly(L-lysine) enhanced
oligonucleotide uptake is seen in promyelocytic leukaemia (HL-60)
cells and human melanoma (M-14) cells. Ginobbi et al. 1997.
Anticancer Res. 17:29. In another example, liposomes coated with
maleylated bovine serum albumin, folic acid, or ferric
protoporphyrin IX, show enhanced cellular uptake of
oligonucleotides in murine macrophages, KB cells, and 2.2.15 human
hepatoma cells. Liang et al. 1999. Pharmazie 54:559-566.
[0199] Liposomes naturally accumulate in the liver, spleen, and
reticuloendothelial system (so-called, passive targeting). By
coupling liposomes to various ligands such as antibodies are
protein A, they can be actively targeted to specific cell
populations. For example, protein A-bearing liposomes may be
pretreated with H-2K specific antibodies which are targeted to the
mouse major histocompatibility complex-encoded H-2K protein
expressed on L cells. (Vlassov et al. 1994. Biochimica et
Biophysica Acta 1197:95-108).
[0200] Assays of Oligonucleotide Stability
[0201] Preferably, the double-stranded oligonucleotides of the
invention are stabilized, i.e., substantially resistant to
endonuclease and exonuclease degradation. An oligonucleotide is
defined as being substantially resistant to nucleases when it is at
least about 3-fold more resistant to attack by an endogenous
cellular nuclease, and is highly nuclease resistant when it is at
least about 6-fold more resistant than a corresponding,
single-stranded oligonucleotide. This can be demonstrated by
showing that the oligonucleotides of the invention are
substantially resist nucleases using techniques which are known in
the art.
[0202] One way in which substantial stability can be demonstrated
is by showing that the oligonucleotides of the invention function
when delivered to a cell, e.g., that they reduce transcription or
translation of target nucleic acid molecules, e.g., by measuring
protein levels or by measuring cleavage of mRNA. Assays which
measure the stability of target RNA can be performed at about 24
hours post-transfection (e.g., using Northern blot techniques,
RNase Protection Assays, or QC-PCR assays as known in the art).
Alternatively, levels of the target protein can be measured.
Preferably, in addition to testing the RNA or protein levels of
interest, the RNA or protein levels of a control, non-targeted gene
will be measured (e.g., actin, or preferably a control with
sequence similarity to the target) as a specificity control. RNA or
protein measurements can be made using any art-recognized
technique. Preferably, measurements will be made beginning at about
16-24 hours post transfection. (M. Y. Chiang, et al. 1991. J. Biol
Chem. 266:18162-71; T. Fisher, et al. 1993. Nucleic Acids Research.
21 3857).
[0203] The ability of an oligonucleotide composition of the
invention to inhibit protein synthesis can be measured using
techniques which are known in the art, for example, by detecting an
inhibition in gene transcription or protein synthesis. For example,
Nuclease S1 mapping can be performed. In another example, Northern
blot analysis can be used to measure the presence of RNA encoding a
particular protein. For example, total RNA can be prepared over a
cesium chloride cushion (see, e.g., Ausebel et al., 1987. Current
Protocols in Molecular Biology (Greene & Wiley, New York)).
Northern blots can then be made using the RNA and probed (see,
e.g., Id.). In another example, the level of the specific mRNA
produced by the target protein can be measured, e.g., using PCR. In
yet another example, Western blots can be used to measure the
amount of target protein present. In still another embodiment, a
phenotype influenced by the amount of the protein can be detected.
Techniques for performing Western blots are well known in the art,
see, e.g., Chen et al. J. Biol. Chem. 271:28259.
[0204] In another example, the promoter sequence of a target gene
can be linked to a reporter gene and reporter gene transcription
(e.g., as described in more detail below) can be monitored.
Alternatively, oligonucleotide compositions that do not target a
promoter can be identified by fusing a portion of the target
nucleic acid molecule with a reporter gene so that the reporter
gene is transcribed. By monitoring a change in the expression of
the reporter gene in the presence of the oligonucleotide
composition, it is possible to determine the effectiveness of the
oligonucleotide composition in inhibiting the expression of the
reporter gene. For example, in one embodiment, an effective
oligonucleotide composition will reduce the expression of the
reporter gene.
[0205] A "reporter gene" is a nucleic acid that expresses a
detectable gene product, which may be RNA or protein. Detection of
mRNA expression may be accomplished by Northern blotting and
detection of protein may be accomplished by staining with
antibodies specific to the protein. Preferred reporter genes
produce a readily detectable product. A reporter gene may be
operably linked with a regulatory DNA sequence such that detection
of the reporter gene product provides a measure of the
transcriptional activity of the regulatory sequence. In preferred
embodiments, the gene product of the reporter gene is detected by
an intrinsic activity associated with that product. For instance,
the reporter gene may encode a gene product that, by enzymatic
activity, gives rise to a detectable signal based on color,
fluorescence, or luminescence. Examples of reporter genes include,
but are not limited to, those coding for chloramphenicol acetyl
transferase (CAT), luciferase, .beta.-galactosidase, and alkaline
phosphatase.
[0206] One skilled in the art would readily recognize numerous
reporter genes suitable for use in the present invention. These
include, but are not limited to, chloramphenicol acetyltransferase
(CAT), luciferase, human growth hormone (hGH), and
beta-galactosidase. Examples of such reporter genes can be found in
F. A. Ausubel et al., Eds., Current Protocols in Molecular Biology,
John Wiley & Sons, New York, (1989). Any gene that encodes a
detectable product, e.g., any product having detectable enzymatic
activity or against which a specific antibody can be raised, can be
used as a reporter gene in the present methods.
[0207] One reporter gene system is the firefly luciferase reporter
system. (Gould, S. J., and Subramani, S. 1988. Anal. Biochem.,
7:404-408 incorporated herein by reference). The luciferase assay
is fast and sensitive. In this assay, a lysate of the test cell is
prepared and combined with ATP and the substrate luciferin. The
encoded enzyme luciferase catalyzes a rapid, ATP dependent
oxidation of the substrate to generate a light-emitting product.
The total light output is measured and is proportional to the
amount of luciferase present over a wide range of enzyme
concentrations.
[0208] CAT is another frequently used reporter gene system; a major
advantage of this system is that it has been an extensively
validated and is widely accepted as a measure of promoter activity.
(Gorman C. M., Moffat, L. F., and Howard, B. H. 1982. Mol. Cell.
Biol., 2:1044-1051). In this system, test cells are transfected
with CAT expression vectors and incubated with the candidate
substance within 2-3 days of the initial transfection. Thereafter,
cell extracts are prepared. The extracts are incubated with acetyl
CoA and radioactive chloramphenicol. Following the incubation,
acetylated chloramphenicol is separated from nonacetylated form by
thin layer chromatography. In this assay, the degree of acetylation
reflects the CAT gene activity with the particular promoter.
[0209] Another suitable reporter gene system is based on
immunologic detection of hGH. This system is also quick and easy to
use. (Selden, R., Burke-Howie, K. Rowe, M. E., Goodman, H. M., and
Moore, D. D. (1986), Mol. Cell, Biol., 6:3173-3179 incorporated
herein by reference). The hGH system is advantageous in that the
expressed hGH polypeptide is assayed in the media, rather than in a
cell extract. Thus, this system does not require the destruction of
the test cells. It will be appreciated that the principle of this
reporter gene system is not limited to hGH but rather adapted for
use with any polypeptide for which an antibody of acceptable
specificity is available or can be prepared.
[0210] In one embodiment, nuclease stability of a double-stranded
oligonucleotide of the invention is measured and compared to a
control, e.g., an RNAi molecule typically used in the art (e.g., a
duplex oligonucleotide of less than 25 nucleotides in length and
comprising 2 nucleotide base overhangs) or an unmodified RNA duplex
with blunt ends.
[0211] Oligonucleotide Synthesis
[0212] Oligonucleotides of the invention can be synthesized by any
method known in the art, e.g., using enzymatic synthesis and
chemical synthesis. The oligonucleotides can be synthesized in
vitro (e.g., using enzymatic synthesis and chemical synthesis) or
in vivo (using recombinant DNA technology well known in the
art).
[0213] In a preferred embodiment, chemical synthesis is used.
Chemical synthesis of linear oligonucleotides is well known in the
art and can be achieved by solution or solid phase techniques.
Preferably, synthesis is by solid phase methods. Oligonucleotides
can be made by any of several different synthetic procedures
including the phosphoramidite, phosphite triester, H-phosphonate,
and phosphotriester methods, typically by automated synthesis
methods.
[0214] Oligonucleotide synthesis protocols are well known in the
art and can be found, e.g., in U.S. Pat. No. 5,830,653; WO
98/13526; Stec et al. 1984. J. Am. Chem. Soc. 106:6077; Stec et al.
1985. J. Org. Chem. 50:3908; Stec et al. J. Chromatog. 1985.
326:263; LaPlanche et al. 1986. Nuc. Acid. Res. 1986. 14:9081;
Fasman G. D., 1989. Practical Handbook of Biochemistry and
Molecular Biology. 1989. CRC Press, Boca Raton, Fla.; Lamone. 1993.
Biochem. Soc. Trans. 21:1; U.S. Pat. No. 5,013,830; U.S. Pat. No.
5,214,135; U.S. Pat. No. 5,525,719; Kawasaki et al. 1993. J. Med.
Chem. 36:831; WO 92/03568; U.S. Pat. No. 5,276,019; U.S. Pat. No.
5,264,423.
[0215] The synthesis method selected can depend on the length of
the desired oligonucleotide and such choice is within the skill of
the ordinary artisan. For example, the phosphoramidite and
phosphite triester method can produce oligonucleotides having 175
or more nucleotides while the H-phosphonate method works well for
oligonucleotides of less than 100 nucleotides. If modified bases
are incorporated into the oligonucleotide, and particularly if
modified phosphodiester linkages are used, then the synthetic
procedures are altered as needed according to known procedures. In
this regard, Uhlmann et al. (1990, Chemical Reviews 90:543-584)
provide references and outline procedures for making
oligonucleotides with modified bases and modified phosphodiester
linkages. Other exemplary methods for making oligonucleotides are
taught in Sonveaux. 1994. "Protecting Groups in Oligonucleotide
Synthesis"; Agrawal. Methods in Molecular Biology 26:1. Exemplary
synthesis methods are also taught in "Oligonucleotide Synthesis--A
Practical Approach" (Gait, M. J. IRL Press at Oxford University
Press. 1984). Moreover, linear oligonucleotides of defined
sequence, including some sequences with modified nucleotides, are
readily available from several commercial sources.
[0216] The oligonucleotides may be purified by polyacrylamide gel
electrophoresis, or by any of a number of chromatographic methods,
including gel chromatography and high pressure liquid
chromatography. To confirm a nucleotide sequence, oligonucleotides
may be subjected to DNA sequencing by any of the known procedures,
including Maxam and Gilbert sequencing, Sanger sequencing,
capillary electrophoresis sequencing the wandering spot sequencing
procedure or by using selective chemical degradation of
oligonucleotides bound to Hybond paper. Sequences of short
oligonucleotides can also be analyzed by laser desorption mass
spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J.
Am. Chem. Soc. 104:976; Viari, et al., 1987, Biomed. Environ. Mass
Spectrom. 14:83; Grotjahn et al., 1982, Nuc. Acid Res. 10:4671).
Sequencing methods are also available for RNA oligonucleotides.
[0217] The quality of oligonucleotides synthesized can be verified
by testing the oligonucleotide by capillary electrophoresis and
denaturing strong anion HPLC (SAX-HPLC) using, e.g., the method of
Bergot and Egan. 1992. J. Chrom. 599:35.
[0218] Other exemplary synthesis techniques are well known in the
art (see, e.g., Sambrook et al., Molecular Cloning: a Laboratory
Manual, Second Edition (1989); DNA Cloning, Volumes I and II (D N
Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984;
Nucleic Acid Hybridisation (B D Hames and S J Higgins eds. 1984); A
Practical Guide to Molecular Cloning (1984); or the series, Methods
in Enzymology (Academic Press, Inc.)).
[0219] Uses of Oligonucleotides
[0220] This invention also features methods of inhibiting
expression of a protein in a cell including contacting the cell
with one of the above-described oligonucleotide compositions.
[0221] The oligonucleotides of the invention can be used in a
variety of in vitro and in vivo situations to specifically inhibit
protein expression. The instant methods and compositions are
suitable for both in vitro and in vivo use.
[0222] The methods of the invention may be used for determining the
function of a gene in a cell or an organism or for modulating the
function of a gene in a cell or an organism, being capable of
responding to or mediating RNA interference. The cell is preferably
a eukaryotic cell or a cell line, e.g., an animal cell such as a
mammalian cell, e.g., an embryonic cell, a pluripotent stem cell, a
tumor cell, e.g., a teratocarcinoma cell, or a virus-infected cell.
The organism is preferably a eukaryotic organism, e.g., an animal
such as a mammal, particularly a human.
[0223] The invention includes methods to inhibit expression of a
target gene in a cell in vitro. For example, such methods may
include introduction of RNA into a cell in an amount sufficient to
inhibit expression of the target gene, where the RNA is a
double-stranded molecule of the invention. By way of a further
example, such an RNA molecule may have a first strand consisting
essentially of a ribonucleotide sequence that corresponds to a
nucleotide sequence of the target gene, and a second strand
consisting essentially of a ribonucleotide sequence that is
complementary to the nucleotide sequence of the target gene, in
which the first and the second strands are separate complementary
strands or are joined by a loop, and they hybridize to each other
to form said double-stranded molecule, such that the duplex
composition inhibits expression of the target gene. The duplex
composition may include modified nucleomonomers as discussed
above.
[0224] The invention also relates to a method to inhibit expression
of a target gene in an invertebrate organism. Such methods include
providing an invertebrate organism containing a target cell that
contains the target gene, in which the target cell is susceptible
to RNA interference and the target gene is expressed in the target
cell. Such methods further include contacting the invertebrate
organism with an RNA composition of the invention. For example, the
RNA may be a double-stranded molecule with a first strand
consisting essentially of a ribonucleotide sequence that
corresponds to a nucleotide sequence of the target gene and a
second strand consisting essentially of a ribonucleotide sequence
that is complementary to the nucleotide sequence of the target
gene. In such cases, the first and the second ribonucleotide
sequences may be separate complementary strands or joined by a
loop, and they hybridize to each other to form the double-stranded
molecule. Finally, such methods include a step of introducing the
duplex RNA composition into the target cell to thereby inhibiting
expression of the target gene.
[0225] In one embodiment, the oligonucleotides of the invention can
be used to inhibit gene function in vitro in a method for
identifying the functions of genes. In this manner, the
transcription of genes that are identified, but for which no
function has yet been shown, can be inhibited to thereby determine
how the phenotype of a cell is changed when the gene is not
transcribed. Such methods are useful for the validation of genes as
targets for clinical treatment, e.g., with oligonucleotides or with
other therapies.
[0226] To determine the effect of a composition of the invention, a
variety of end points can be used. In addition to the assays
described previously herein, for example, nucleic acid probes
(e.g., in the form of arrays) can be used to evaluate transcription
patterns produced by cells. Probes can also be used detect
peptides, proteins, or protein domains, e.g., antibodies can be
used to detect the expression of a particular protein. In yet
another embodiment, the function of a protein (e.g., enzymatic
activity) can be measured. In yet another embodiment, the phenotype
of a cell can be evaluated to determine whether or not a target
protein is expressed. For example, the ability of a composition to
affect a phenotype of a cell that is associated with cancer can be
tested.
[0227] In one embodiment, one or more additional agents (e.g.,
activating agents, inducing agents, proliferation enhancing agents,
tumor promoters) can be added to the cells.
[0228] In another embodiment, the compositions of the invention can
be used to monitor biochemical reactions such as, e.g.,
interactions of proteins, nucleic acids, small molecules, or the
like, for example the efficiency or specificity of interactions
between antigens and antibodies; or of receptors (such as purified
receptors or receptors bound to cell membranes) and their ligands,
agonists or antagonists; or of enzymes (such as proteases or
kinases) and their substrates, or increases or decreases in the
amount of substrate converted to a product; as well as many others.
Such biochemical assays can be used to characterize properties of
the probe or target, or as the basis of a screening assay. For
example, to screen samples for the presence of particular proteases
(e.g., proteases involved in blood clotting such as proteases Xa
and VIIa), the samples can be assayed, for example using probes
which are fluorogenic substrates specific for each protease of
interest. If a target protease binds to and cleaves a substrate,
the substrate will fluoresce, usually as a result, e.g., of
cleavage and separation between two energy transfer pairs, and the
signal can be detected. In another example, to screen samples for
the presence of a particular kinase(s) (e.g., a tyrosine kinase),
samples containing one or more kinases of interest can be assayed,
e.g., using probes are peptides which can be selectively
phosphorylated by one of the kinases of interest. Using
art-recognized, routinely determinable conditions, samples can be
incubated with an array of substrates, in an appropriate buffer and
with the necessary cofactors, for an empirically determined period
of time. If necessary, reactions can be stopped, e.g., by washing
and the phosphorylated substrates can be detected by, for example,
incubating them with detectable reagents such as, e.g.,
fluorescein-labeled anti-phosphotyrosine or anti-phosphoserine
antibodies and the signal can be detected.
[0229] In another embodiment, the compositions of the invention can
be used to screen for agents which modulate a pattern of gene
expression. Arrays of oligonucleotides can be used, for example, to
identify mRNA species whose pattern of expression from a set of
genes is correlated with a particular physiological state or
developmental stage, or with a disease condition ("correlative"
genes, RNAs, or expression patterns). By the terms "correlate" or
"correlative," it is meant that the synthesis pattern of RNA is
associated with the physiological condition of a cell, but not
necessarily that the expression of a given RNA is responsible for
or is causative of a particular physiological state. For example, a
small subset of mRNAs can be identified which are modulated (e.g.,
upregulated or downregulated) in cells which serve as a model for a
particular disease state. This altered pattern of expression as
compared to that in a normal cell, which does not exhibit a
pathological phenotype, can serve as a indicator of the disease
state ("indicator" or "correlatvie" genes, RNAs, or expression
patterns).
[0230] Compositions which modulate the chosen indicator expression
pattern (e.g., compared to control compositions comprising, for
example oligonucleotides which comprise a nucleotide sequence which
is the reverse of the oligonucleotide, or which contains mismatch
bases) can indicate that a particular target gene is a potential
target for therapeutic intervention. Moreover, such compositions
may be useful as therapeutic agents to modulate expression patters
of cells in an in vitro expression system or in in vivo therapy. As
used herein, "modulate" means to cause to increase or decrease the
amount or activity of a molecule or the like which is involved in a
measurable reaction. In one embodiment, a series of cells (e.g.,
from a disease model) can be contacted with a series of agents
(e.g., for a period of time ranging from about 10 minutes to about
48 hours or more) and, using routine, art-recognized methods (e.g.,
commercially available kits), total RNA or mRNA extracts can be
made. If it is desired to amplify the amount of RNA, standard
procedures such as RT-PCR amplification can be used (see, e.g.,
Innis et al eds., (1996) PCR Protocols: A Guide to Methods in
Amplification, Academic Press, New York). The extracts (or
amplified products from them) can be allowed to contact (e.g.,
incubate with) probes for appropriate indicator RNAs, and those
agents which are associated with a change in the indicator
expression pattern can be identified.
[0231] Similarly, agents can be identified which modulate
expression patterns associated with particular physiological states
or developmental stages. Such agents can be man-made or
naturally-occurring substances, including environmental factors
such as substances involved in embryonic development or in
regulating physiological reactions.
[0232] In one embodiment, the methods described herein can be
performed in a "high throughput" manner, in which a large number of
target genes (e.g., as many as about 1000 or more, depending on the
particular format used) are assayed rapidly and concurrently.
Further, many assay formats (e.g., plates or surfaces) can be
processed at one time. For example, because the oligonucleotides of
the invention do not need to be tested individually before
incorporating them into a composition, they can be readily
synthesized and large numbers of target genes can be tested at one
time. For example, a large number of samples, each comprising a
biological sample containing a target nucleic acid molecule (e.g.,
a cell) and a composition of the invention can be added to separate
regions of an assay format and assays can be performed on each of
the samples.
[0233] Administration of Oligonucleotide Compositions
[0234] The optimal course of administration or delivery of the
oligonucleotides may vary depending upon the desired result and/or
on the subject to be treated. As used herein "administration"
refers to contacting cells with oligonucleotides and can be
performed in vitro or in vivo. The dosage of oligonucleotides may
be adjusted to optimally reduce expression of a protein translated
from a target nucleic acid molecule, e.g., as measured by a readout
of RNA stability or by a therapeutic response, without undue
experimentation.
[0235] For example, expression of the protein encoded by the
nucleic acid target can be measured to determine whether or not the
dosage regimen needs to be adjusted accordingly. In addition, an
increase or decrease in RNA or protein levels in a cell or produced
by a cell can be measured using any art recognized technique. By
determining whether transcription has been decreased, the
effectiveness of the oligonucleotide in inducing the cleavage of a
target RNA can be determined.
[0236] Any of the above-described oligonucleotide compositions can
be used alone or in conjunction with a pharmaceutically acceptable
carrier. As used herein, "pharmaceutically acceptable carrier"
includes appropriate solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, it can be used in the therapeutic compositions.
Supplementary active ingredients can also be incorporated into the
compositions.
[0237] Oligonucleotides may be incorporated into liposomes or
liposomes modified with polyethylene glycol or admixed with
cationic lipids for parenteral administration. Incorporation of
additional substances into the liposome, for example, antibodies
reactive against membrane proteins found on specific target cells,
can help target the oligonucleotides to specific cell types.
[0238] Moreover, the present invention provides for administering
the subject oligonucleotides with an osmotic pump providing
continuous infusion of such oligonucleotides, for example, as
described in Rataiczak et al. (1992 Proc. Natl. Acad. Sci. USA
89:11823-11827). Such osmotic pumps are commercially available,
e.g., from Alzet Inc. (Palo Alto, Calif.). Topical administration
and parenteral administration in a cationic lipid carrier are
preferred.
[0239] With respect to in vivo applications, the formulations of
the present invention can be administered to a patient in a variety
of forms adapted to the chosen route of administration, e.g.,
parenterally, orally, or intraperitoneally. Parenteral
administration, which is preferred, includes administration by the
following routes: intravenous; intramuscular; interstitially;
intraarterially; subcutaneous; intra ocular; intrasynovial; trans
epithelial, including transdermal; pulmonary via inhalation;
ophthalmic; sublingual and buccal; topically, including ophthalmic;
dermal; ocular; rectal; and nasal inhalation via insufflation.
[0240] Pharmaceutical preparations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
or water-dispersible form. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension include, for example, sodium
carboxymethyl cellulose, sorbitol, or dextran, optionally, the
suspension may also contain stabilizers. The oligonucleotides of
the invention can be formulated in liquid solutions, preferably in
physiologically compatible buffers such as Hank's solution or
Ringer's solution. In addition, the oligonucleotides may be
formulated in solid form and redissolved or suspended immediately
prior to use. Lyophilized forms are also included in the
invention.
[0241] Pharmaceutical preparations for topical administration
include transdermal patches, ointments, lotions, creams, gels,
drops, sprays, suppositories, liquids and powders. In addition,
conventional pharmaceutical carriers, aqueous, powder or oily
bases, or thickeners may be used in pharmaceutical preparations for
topical administration.
[0242] Pharmaceutical preparations for oral administration include
powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. In addition,
thickeners, flavoring agents, diluents, emulsifiers, dispersing
aids, or binders may be used in pharmaceutical preparations for
oral administration.
[0243] For transmucosal or transdermal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are known in the art, and include, for
example, for transmucosal administration bile salts and fusidic
acid derivatives, and detergents. Transmucosal administration may
be through nasal sprays or using suppositories. For oral
administration, the oligonucleotides are formulated into
conventional oral administration forms such as capsules, tablets,
and tonics. For topical administration, the oligonucleotides of the
invention are formulated into ointments, salves, gels, or creams as
known in the art.
[0244] Drug delivery vehicles can be chosen e.g., for in vitro, for
systemic, or for topical administration. These vehicles can be
designed to serve as a slow release reservoir or to deliver their
contents directly to the target cell. An advantage of using some
direct delivery drug vehicles is that multiple molecules are
delivered per uptake. Such vehicles have been shown to increase the
circulation half-life of drugs that would otherwise be rapidly
cleared from the blood stream. Some examples of such specialized
drug delivery vehicles which fall into this category are liposomes,
hydrogels, cyclodextrins, biodegradable nanocapsules, and
bioadhesive microspheres.
[0245] The described oligonucleotides may be administered
systemically to a subject. Systemic absorption refers to the entry
of drugs into the blood stream followed by distribution throughout
the entire body. Administration routes which lead to systemic
absorption include: intravenous, subcutaneous, intraperitoneal, and
intranasal. Each of these administration routes delivers the
oligonucleotide to accessible diseased cells. Following
subcutaneous administration, the therapeutic agent drains into
local lymph nodes and proceeds through the lymphatic network into
the circulation. The rate of entry into the circulation has been
shown to be a function of molecular weight or size. The use of a
liposome or other drug carrier localizes the oligonucleotide at the
lymph node. The oligonucleotide can be modified to diffuse into the
cell, or the liposome can directly participate in the delivery of
either the unmodified or modified oligonucleotide into the
cell.
[0246] The chosen method of delivery will result in entry into
cells. Preferred delivery methods include liposomes (10-400 nm),
hydrogels, controlled-release polymers, and other pharmaceutically
applicable vehicles, and microinjection or electroporation (for ex
vivo treatments).
[0247] The pharmaceutical preparations of the present invention may
be prepared and formulated as emulsions. Emulsions are usually
heterogenous systems of one liquid dispersed in another in the form
of droplets usually exceeding 0.1 .mu.m in diameter.
[0248] The emulsions of the present invention may contain
excipients such as emulsifiers, stabilizers, dyes, fats, oils,
waxes, fatty acids, fatty alcohols, fatty esters, humectants,
hydrophilic colloids, preservatives, and anti-oxidants may also be
present in emulsions as needed. These excipients may be present as
a solution in either the aqueous phase, oily phase or itself as a
separate phase.
[0249] Examples of naturally occurring emulsifiers that may be used
in emulsion formulations of the present invention include lanolin,
beeswax, phosphatides, lecithin and acacia. Finely divided solids
have also been used as good emulsifiers especially in combination
with surfactants and in viscous preparations. Examples of finely
divided solids that may be used as emulsifiers include polar
inorganic solids, such as heavy metal hydroxides, nonswelling clays
such as bentonite, attapulgite, hectorite, kaolin, montmorillonite,
colloidal aluminum silicate and colloidal magnesium aluminum
silicate, pigments and nonpolar solids such as carbon or glyceryl
tristearate.
[0250] Examples of preservatives that may be included in the
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Examples of antioxidants
that may be included in the emulsion formulations include free
radical scavengers such as tocopherols, alkyl gallates, butylated
hydroxyanisole, butylated hydroxytoluene, or reducing agents such
as ascorbic acid and sodium metabisulfite, and antioxidant
synergists such as citric acid, tartaric acid, and lecithin.
[0251] In one embodiment, the compositions of oligonucleotides are
formulated as microemulsions. A microemulsion is a system of water,
oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution. Typically microemulsions
are prepared by first dispersing an oil in an aqueous surfactant
solution and then adding a sufficient amount of a 4th component,
generally an intermediate chain-length alcohol to form a
transparent system.
[0252] Surfactants that may be used in the preparation of
microemulsions include, but are not limited to, ionic surfactants,
non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers,
polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310),
tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310),
hexaglycerol pentaoleate (PO500), decaglycerol monocaprate
(MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate
(SO750), decaglycerol decaoleate (DA0750), alone or in combination
with cosurfactants. The cosurfactant, usually a short-chain alcohol
such as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules.
[0253] Microemulsions may, however, be prepared without the use of
cosurfactants and alcohol-free self-emulsifying microemulsion
systems are known in the art. The aqueous phase may typically be,
but is not limited to, water, an aqueous solution of the drug,
glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and
derivatives of ethylene glycol. The oil phase may include, but is
not limited to, materials such as Captex 300, Captex 355, Capmul
MCM, fatty acid esters, medium chain (C.sub.8-C.sub.12) mono, di,
and tri-glycerides, polyoxyethylated glyceryl fatty acid esters,
fatty alcohols, polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0254] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both oil/water and water/oil)
have been proposed to enhance the oral bioavailability of
drugs.
[0255] Microemulsions offer improved drug solubilization,
protection of drug from enzymatic hydrolysis, possible enhancement
of drug absorption due to surfactant-induced alterations in
membrane fluidity and permeability, ease of preparation, ease of
oral administration over solid dosage forms, improved clinical
potency, and decreased toxicity (Constantinides et al.,
Pharmaceutical Research, 1994, 11:1385; Ho et al., J. Pharm. Sci.,
1996, 85:138-143). Microemulsions have also been effective in the
transdermal delivery of active components in both cosmetic and
pharmaceutical applications. It is expected that the microemulsion
compositions and formulations of the present invention will
facilitate the increased systemic absorption of oligonucleotides
from the gastrointestinal tract, as well as improve the local
cellular uptake of oligonucleotides within the gastrointestinal
tract, vagina, buccal cavity and other areas of administration.
[0256] In an embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides, to the skin of animals. Even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
increasing the diffusion of non-lipophilic drugs across cell
membranes, penetration enhancers also act to enhance the
permeability of lipophilic drugs.
[0257] Five categories of penetration enhancers that may be used in
the present invention include: surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants. Other
agents may be utilized to enhance the penetration of the
administered oligonucleotides include: glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-15 pyrrol, azones,
and terpenes such as limonene, and menthone.
[0258] The oligonucleotides, especially in lipid formulations, can
also be administered by coating a medical device, for example, a
catheter, such as an angioplasty balloon catheter, with a cationic
lipid formulation. Coating may be achieved, for example, by dipping
the medical device into a lipid formulation or a mixture of a lipid
formulation and a suitable solvent, for example, an aqueous-based
buffer, an aqueous solvent, ethanol, methylene chloride, chloroform
and the like. An amount of the formulation will naturally adhere to
the surface of the device which is subsequently administered to a
patient, as appropriate. Alternatively, a lyophilized mixture of a
lipid formulation may be specifically bound to the surface of the
device. Such binding techniques are described, for example, in K.
Ishihara et al., Journal of Biomedical Materials Research, Vol. 27,
pp. 1309-1314 (1993), the disclosures of which are incorporated
herein by reference in their entirety.
[0259] The useful dosage to be administered and the particular mode
of administration will vary depending upon such factors as the cell
type, or for in vivo use, the age, weight and the particular animal
and region thereof to be treated, the particular oligonucleotide
and delivery method used, the therapeutic or diagnostic use
contemplated, and the form of the formulation, for example,
suspension, emulsion, micelle or liposome, as will be readily
apparent to those skilled in the art. Typically, dosage is
administered at lower levels and increased until the desired effect
is achieved. When lipids are used to deliver the oligonucleotides,
the amount of lipid compound that is administered can vary and
generally depends upon the amount of oligonucleotide agent being
administered. For example, the weight ratio of lipid compound to
oligonucleotide agent is preferably from about 1:1 to about 15:1,
with a weight ratio of about 5:1 to about 10:1 being more
preferred. Generally, the amount of cationic lipid compound which
is administered will vary from between about 0.1 milligram (mg) to
about 1 gram (g). By way of general guidance, typically between
about 0.1 mg and about 10 mg of the particular oligonucleotide
agent, and about 1 mg to about 100 mg of the lipid compositions,
each per kilogram of patient body weight, is administered, although
higher and lower amounts can be used.
[0260] The agents of the invention are administered to subjects or
contacted with cells in a biologically compatible form suitable for
pharmaceutical administration. By "biologically compatible form
suitable for administration" is meant that the oligonucleotide is
administered in a form in which any toxic effects are outweighed by
the therapeutic effects of the oligonucleotide. In one embodiment,
oligonucleotides can be administered to subjects. Examples of
subjects include mammals, e.g., humans and other primates; cows,
pigs, horses, and farming (agricultural) animals; dogs, cats, and
other domesticated pets; mice, rats, and transgenic non-human
animals.
[0261] Administration of an active amount of an oligonucleotide of
the present invention is defined as an amount effective, at dosages
and for periods of time necessary to achieve the desired result.
For example, an active amount of an oligonucleotide may vary
according to factors such as the type of cell, the oligonucleotide
used, and for in vivo uses the disease state, age, sex, and weight
of the individual, and the ability of the oligonucleotide to elicit
a desired response in the individual. Establishment of therapeutic
levels of oligonucleotides within the cell is dependent upon the
rates of uptake and efflux or degradation. Decreasing the degree of
degradation prolongs the intracellular half-life of the
oligonucleotide. Thus, chemically-modified oligonucleotides, e.g.,
with modification of the phosphate backbone, may require different
dosing.
[0262] The exact dosage of an oligonucleotide and number of doses
administered will depend upon the data generated experimentally and
in clinical trials. Several factors such as the desired effect, the
delivery vehicle, disease indication, and the route of
administration, will affect the dosage. Dosages can be readily
determined by one of ordinary skill in the art and formulated into
the subject pharmaceutical compositions. Preferably, the duration
of treatment will extend at least through the course of the disease
symptoms.
[0263] Dosage regima may be adjusted to provide the optimum
therapeutic response. For example, the oligonucleotide may be
repeatedly administered, e.g., several doses may be administered
daily or the dose may be proportionally reduced as indicated by the
exigencies of the therapeutic situation. One of ordinary skill in
the art will readily be able to determine appropriate doses and
schedules of administration of the subject oligonucleotides,
whether the oligonucleotides are to be administered to cells or to
subjects.
[0264] Treatment of Diseases or Disorders
[0265] By inhibiting the expression of a gene, the oligonucleotide
compositions of the present invention can be used to treat any
disease involving the expression of a protein. Examples of diseases
that can be treated by oligonucleotide compositions include:
cancer, retinopathies, autoimmune diseases, inflammatory diseases
(i.e., ICAM-1 related disorders, Psoriasis, Ulcerative Colitus,
Crohn's disease), viral diseases (i.e., HIV, Hepatitis C), and
cardiovascular diseases.
[0266] In one embodiment, in vitro treatment of cells with
oligonucleotides can be used for ex vivo therapy of cells removed
from a subject (e.g., for treatment of leukemia or viral infection)
or for treatment of cells which did not originate in the subject,
but are to be administered to the subject (e.g., to eliminate
transplantation antigen expression on cells to be transplanted into
a subject). In addition, in vitro treatment of cells can be used in
non-therapeutic settings, e.g., to evaluate gene function, to study
gene regulation and protein synthesis or to evaluate improvements
made to oligonucleotides designed to modulate gene expression or
protein synthesis. In vivo treatment of cells can be useful in
certain clinical settings where it is desirable to inhibit the
expression of a protein. There are numerous medical conditions for
which antisense therapy is reported to be suitable (see, e.g., U.S.
Pat. No. 5,830,653) as well as respiratory syncytial virus
infection (WO 95/22,553) influenza virus (WO 94/23,028), and
malignancies (WO 94/08,003). Other examples of clinical uses of
antisense sequences are reviewed, e.g., in Glaser. 1996. Genetic
Engineering News 16:1. Exemplary targets for cleavage by
oligonucleotides include, e.g., protein kinase Ca, ICAM-1, c-raf
kinase, p53, c-myb, and the bcr/abl fusion gene found in chronic
myelogenous leukemia.
[0267] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, microbiology, recombinant DNA, and
immunology, which are within the skill of the art. Such techniques
are explained fully in the literature. See, for example, Molecular
Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, J. et al.
(Cold Spring Harbor Laboratory Press (1989)); Short Protocols in
Molecular Biology, 3rd Ed., ed. by Ausubel, F. et al. (Wiley, N.Y.
(1995)); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);
Oligonucleotide Synthesis (M. J. Gait ed. (1984)); Mullis et al.
U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames
& S. J. Higgins eds. (1984)); the treatise, Methods In
Enzymology (Academic Press, Inc., N.Y.); Immunochemical Methods In
Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,
London (1987)); Handbook Of Experimental Immunology, Volumes I-IV
(D. M. Weir and C. C. Blackwell, eds. (1986)); and Miller, J.
Experiments in Molecular Genetics (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. (1972)).
[0268] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Oligonucleotide Compositions Comprising Chimeric Antisense
Sequences
[0269] A gapped antisense oligonucleotide comprising 2'-O-methyl
RNA arms and an unmodified DNA gap was synthesized. A complementary
oligonucleotide was also synthesized using unmodified RNA. A
double-stranded duplex was formed and the composition was found to
inhibit expression of the target gene.
Example 2
Length of Double-Stranded ligonucleotides and the Presence or
Absence of Overhangs Has No Effect on Function
[0270] Twenty one and 27-mers were designed to target each of two
sites on the p53 molecule (89-90 site, and 93-94 site). The
double-stranded molecules were designed with or without 3'-deoxy TT
overhangs. The test oligonucleotides were 21-mers with 2 nucleotide
3' deoxy TT overhangs and without overhangs (blunt ends); and
27-mers with 2 nucleotide 3' deoxy TT overhangs and without
overhangs (blunt ends). Two positive controls were included in the
experiment (p53) and two negative controls were also included
(FITC)
[0271] A549 cells were transfected with 100 nM of the
double-stranded molecules plus 2 ug/mL Lipofectamine 2000. A549
cells were examined 24 hours post-transfection. FITC-labeled
molecules were taken up well by cells. Both 21-mers (with or
without overhangs) and 27-mers (with or without overhangs) were
non-toxic to cells. FIG. 1 shows the result of an experiment
comparing the ability of different oligonucleotide constructs to
inhibit p53 and shows that length or the presence or absence of a
3' deoxy TT overhang did not affect the activity of the
oligonucleotide. The results in FIG. 1 show the amount of p53 mRNA
normalized to the amount of an irrelevant message, GAPDH. The level
of mRNA was determined using RT-PCR analysis. The observed percent
inhibition of p53 expression is shown below:
3 21-MER 27-MER SITE overhang no overhang overhang no overhang
93-94 58% 65% 62% 62% 89-90 81% 75% 67% 70%
[0272] Similar results were observed for .beta.-3-integrin; both 21
-mer and 27-mer double-stranded molecules were found to inhibit
integrin mRNA. Two double-stranded RNA complexes designed to target
the same site of the .beta.-3-integrin gene were transfected in
HMVEC cells. Both complexes contained a two nucleotide (TT)
overhang: one complex was a 21-mer (with 19 nucleotides
complementary to the target gene) and the other was a 27-mer (with
25 nucleotides complementary to the target gene). RT-PCR analysis
showed that the two complexes inhibited the target gene to the same
extent. HMVEC cells were transfected using 100 nM oligomer
complexed with 2ug/mL of Lipofecatmine 2000 in media containing
serum for 24 hours. Twenty-four hours after transfection, the cells
were lysed and the RNA was isolated for analysis by RT-PCR. No
significant toxicity was observed. The results in FIG. 1B show the
amount of .beta.-3-integrin mRNA normalized to the amount of GAPDH,
as determined by RT-PCR analysis.
Example 3
Activation of the Double-Stranded RNA, Interferon-Inducible Protein
Kinase, PKR
[0273] PKR is activated by double-stranded RNA molecules. Active
PKR leads to the inhibition of protein synthesis, activation of
transcription, and a variety of other cellular effects, including
signal transduction, cell differentiation, cell growth inhibition,
apoptosis, and antiviral effects. The effect of p53-targetd
double-stranded RNA molecules on PKR expression was tested. The
level of mRNA was determined using RT-PCR analysis. As shown in
FIG. 2, no correlation was observed between the length of the
double-stranded oligonucleotide and the level of PKR induction.
Accordingly, long oligonucleotides can be used without activating
PKR, a marker for interferon induction.
[0274] As illustrated in FIG. 2B, analysis of relative amounts of
PKR mRNA after the 21- and 27-mer transfection in HMVEC cells
showed approximately a 2 fold increase in PKR mRNA of the siRNA
control sequences over no treatment, and approximately a 2 fold
increase of PKR mRNA of the 27-mer compared to the 21-mer targeted
double-stranded RNA complexes.
Example 4
The Effect of 5' vs. 3' Modification on the Activity of
Double-Stranded Oligonucleotides
[0275] Oligonucleotide duplexes were modified at either the 3' or
5' end with FITC groups. The modifications were made on either the
antisense strand or the sense strand. 5' or 3' modification of the
sense strand had no effect on the percent inhibition of p53 mRNA.
3' modification of the antisense strand had little affect on
activity, while 5' modification of the antisense strand reduced
activity significantly. 3' modification of both strands also had
little affect on activity, while 3' and 5' modification of both
strands reduced activity. See FIG. 3.
[0276] The effect of the size of the group used to modify the 5'
end was tested. The results of this experiment are shown in FIG. 4.
The inclusion of a 5' phosphate group had little affect on
activity, whereas the modification of the antisense strand or both
strands had a greater effect. The inclusion of a propyl group had
more of an effect, with a 5' propyl group on the antisense strand
showing a large reduction in activity; there was also an effect
when this group was added to both strands. Similarly, the inclusion
of a FITC group at the 5' end of the antisense molecule (or to both
molecules) also significantly reduced the activity of the RNA
duplex.
Example 5
Comparison of the Efficacy of 2'-O-Me Modified and Unmodified
Double-Stranded RNA Oligonucleotides
[0277] A549 cells were transfected with modified or unmodified RNA
duplexes complexed at 100 nM with 2 ug/mL Lipofectamine 2000
(Invitrogen) and were transfected for 24 hours. The A549 cells were
plated at 20,000/well in 48 well plates. After 24 hours,
FITC-labeled double-stranded oligonucleotides were visible in A549
cells; the inclusion of a 2'-O-Me group did not affect uptake. The
Table below shows the results of this experiment.
4 2'-O-Me Oligonucleotide Duplexes Anti- sense/Sense Anti- Anti-
Anti-sense/ 2'-O-Me/2'-O- sense/Sense sense/Sense Sense Me
2'-O-Me/RNA RNA/2'-O-Me RNA/RNA targeted 18639/18640 18639/16194
16193/18640 18876 non- 19039/19040 19039/19044 19043/19040 18850
& targeted 16197/16198 FITC-2'-O-Me/ FITC-2'-O-Me/
FITC-2'-O-Me/ 2'-O-Me/ FITC 2'-O-Me FITC-RNA RNA FITC-RNA non-
19209 19037/19042 19037/19044 19039/19042 targeted
[0278] The affect of 2'-O-Me modifications to one or both strands
of a double-stranded RNA molecule is shown in FIG. 5.
Example 6
Toxicity of p53-Targeted siRNAs in A549 Cells
[0279] 27-mer siRNAs targeting p53 were not toxic to cells when
compared to standard 21-mer siRNAs having 3' deoxy TT overhangs. In
this experiment, both siRNA constructs inhibited p53 to a similar
extent (83% inhibition for 27-mer vs. 90% inhibition for 21-mer).
siRNAs were designed to target p53 and were constructed as
blunt-end 27-mers or as 21-mers with 3' deoxy TT overhangs. A549
cells were plated at 20,000 cells per well in 48-well plates on the
day prior to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL Lipofectamine 2000 for 24 hours.
Following transfection, cells were stained with Dead Red stain to
visualize the extent of cell death. The siRNA sequences used were
as follows:
5 21-mer with overhangs targeted (5'-3'): ACCUCAAAGCUGUUCCGUCTT
(SEQ ID NO: ##) GACGGAACAGCUUUGAGGUTT (SEQ ID NO: ##) Blunt-end
27-mer targeted (5'-3'): ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO:
##) GGGACGGAACAGCTTTGAGGTGTGCGT (SEQ ID NO: ##)
Example 7
Toxicity of Blunt-End 27-mer siRNAs Targeting p53 in A549 Cells
[0280] The toxicity of targeted blunt-end 27-mer siRNAs targeting
p53 was observed to be not significantly different than a control
nucleic acid or no treatment. siRNAs were designed to target p53
and were constructed as blunt-end 27-mers. The corresponding
control consisted of chemistry-matched, scrambled sequences with a
similar base-pair composition. A549 cells were plated at 20,000
cells per well in 48-well plates on the day prior to transfection.
On the day of transfection, cells were approximately 60-70%
confluent. Cells were transfected with 100 nM siRNAs complexed with
2 ug/mL Lipofectamine 2000 for 24 hours. Following transfection,
the cells were stained with Dead Red stain to visualize the extent
of cell death. The siRNA sequences used were as follows:
6 Blunt-end 27-mer targeted (5'-3' on top):
ACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##)
GGGACGGAACAGCTTTGAGGTGTGCGT (SEQ ID NO: ##) Corresponding control
(5'-3' on top): CCCTGCCTTGTCGAAACTCCACACGC- A (SEQ ID NO: ##)
TGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: ##)
Example 8
Toxicity of Blunt End 32-mer siRNAs Targeting p53 in A549 Cells
[0281] Similarly, blunt-end 32-mer siRNAs targeting p53 were not
observed to be toxic to cells in comparison with a control nucleic
acid and no treatment, as determined by Dead Red staining. siRNAs
were designed to target p53 and were constructed as blunt-end
32-mers. The corresponding control consisted of chemistry-matched,
scrambled sequences with a similar base-pair composition. A549
cells were plated at 20,000 cells per well in 48-well plates on the
day prior to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL Lipofectamine 2000 for 24 hours.
Following transfection, cells were stained with Dead Red stain to
visualize the extent of cell death. The siRNA sequences used were
as follows:
7 Targeted blunt-end 32-mer (5'-3' on top:)
CCCTCACGCACACCUCAAAGCUGUUCCGUCCC (SEQ ID NO: ##)
GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG (SEQ ID NO: ##) Corresponding
control (5'-3' on top): CCCTGCCTTGTCGAAACTCCACACGCA- CTCCC (SEQ ID
NO: ##) GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: ##)
Example 9
Inhibition of p53 by 32- and 37-mer Blunt-End siRNAs
[0282] FIG. 6 depicts the results of inhibition of p53 by 32- and
37-mer blunt-end siRNAs in comparison with various control
experiments. siRNAs were designed to target each of two sites
(93-93 site) and (89-90 site) along the coding region of p53.
siRNAs were constructed as blunt-end 32-mers or blunt-end 37-mers.
Positive control siRNAs were 21-mers with 3' deoxy TT overhangs.
Corresponding controls consisted of chemistry-matched, scrambled
sequences with a similar base-pair composition. A549 cells were
plated at 20,000 cells per well in 48-well plates on the day prior
to transfection. On the day of transfection, cells were
approximately 60-70% confluent. Cells were transfected with 100 nM
siRNAs complexed with 2 ug/mL Lipofectamine 2000 for 24 hours.
Following transfection, cells were lysed and poly(A) mRNA was
harvested for RT-PCR. Inhibition of p53 expression was determined
by quantitative real-time RT-PCR (TaqMan) analysis. Expression of
p53 was standardized by quantifying GAPDH for each sample. The data
in FIG. 6 represent three separate transfections analyzed in
duplicate and normalized to the internal control (GAPDH). The siRNA
sequences used were as follows (depicted with the 5'-3' strand on
top):
8 Targeted 32-mer (89-90 site): CCCTCACGCACACCUCAAAGCUGUUC- CGUCCC
(SEQ ID NO: ##) GGGACGGAACAGCTTTGAGGTGTGCGTGAGGG (SEQ ID NO: ##)
32-mer control (89-90 site): CCCTGCCTTGTCGAAACTCCACACGCACTCCC (SEQ
ID NO: ##) GGGAGTGCGTGTGGAGTTTCGACAAGGCAGGG (SEQ ID NO: ##) 32-mer
targeted (93-94 site): CCCUUCUGUCUUGAACAUGAGTTTTTTATGGC (SEQ ID NO:
##) GCCATAAAAAACTCATGTTCAAGACAGAAGGG (SEQ ID NO: ##) 32-mer control
(93-94 site): CGGTATTTTTTGAGTACAAGTTCTGTCTTC- CC (SEQ ID NO: ##)
GGGAAGACAGAACTTGTACTCAAAAAATACCG (SEQ ID NO: ##) 37-mer targeted
(93-94 site): CCCTTCTGTCTTGAACATGAGTTTTTTATGGCGGGAG (SEQ ID NO: ##)
CTCCCGCCATAAAAAACTCATGTTCAAGACAGAAGGG (SEQ ID NO: ##) 37-mer
control (93-94 site): GAGGGCGGTATTTTTTGAGTACAAGTTCTGTCTTCCC (SEQ ID
NO: ##) GGGAAGACAGAACTTGTACTCAAAAAATACCGCCCTC (SEQ ID NO: ##)
21-mer targeted (89-90 site): ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO: ##)
GACGGAACAGCUUUGAGGUTT (SEQ ID NO: ##) 21-mer targeted (93-94 site):
CCCUUCUGUCUUGAACAUGTT (SEQ ID NO: ##) CAUGUUCAAGACAGAAGGGTT (SEQ ID
NO: ##)
Example 10
Enhanced Cellular Stability of Double-Stranded 2'-O-Methyl RNA
[0283] In this example, the single-stranded control oligomer was
transfected at 800 nM. Accumulation was observed in the nucleus at
6 hours post transfection, however by 25 hours the fluorescence of
the single-stranded oligomer had largely dissipated, indicating the
oligomer was no longer intact (Fisher, T., T. Terhorst, et al.
(1993). "Intracellular disposition and metabolism of
fluorescently-labeled unmodifieed and modified oligonucleotides
microinjected into mammalian cells." NAR 21: 3857-3865). The
relative fluorescence of fluorescently-labeled oligomers
transfected into A549 cells was observed to fit the following
pattern:
9 single-stranded (800 nM) double-stranded (100 nm) 6 h ++++ +++++
25 h + +++++
[0284] The double-stranded oligomer duplex, wherein the second
strand was 2'-O-methyl modified RNA, was transfected at 100 nM, and
was also clearly visible at 6 hours post transfection. However, in
contrast to the single-stranded oligomer, the double-stranded was
still largely intact in the nucleus at 24 hours, even though the
concentration transfected was 8-fold less, thereby demonstrating
that the 2'-O-methyl second strand stabilized the oligomer in the
cell.
[0285] The oligomers were all 2'-O-CH.sub.3 with a phosphodiester
backbone containing 6-carboxyfluorescein (6-FAM) tethered to the 5'
hydroxyl. The single-stranded control oligomer was transfected at
800 nM complexed with 4 ug/mL of Lipofectamine 2000, and the
double-stranded complex was transfected at 100 nM complexed with 1
ug/mL of Lipofectamine 2000.
[0286] Fluorescent signal was seen accumulating in the nucleus at 6
hours post transfection, however by 24 hours the single-stranded
oligomer has significantly dissipated, indicating the oligomer is
no longer intact. The double-stranded duplexes (wherein the second
strand is 2'-O-methyl modified RNA with a 5' 6-FAM) was transfected
at 100 nM, and was also clearly visible at 6 hours post
transfection. In contrast to the single-stranded oligomer, the
double-stranded was still largely intact in the nucleus at 24
hours, even though the concentration transfected was 8-fold less.
This experiment demonstrates that the 2'-O-methyl second strand
stabilizes the duplex in the cell.
Example 11
Enhanced Stability in Cells and Accumulation in Cytoplasm of RNA
Hybridized to 2'-O-Methyl RNA
[0287] The fluorescence signal, corresponding to uptake of
FITC-labeled RNA and 2'-O-methyl modified RNA duplexes, was
measured at 6 and 24 hours. RNA complexes were transfected in A549
cells with 100 nM oligomer complexed with 2 ug/mL Lipofecatmine
2000 as described below. Cells were continuously transfected for 24
hours and fluorescent uptake was assessed at 6 and 24 hours.
Oligomers were 2'-O-methyl modified RNA with 5' 6-FAM
(FITC-2'-OMe), 19-mer RNA with two deoxynucleotides on the 3' end
with 5' 6-FAM (FITC-RNA) or 19-mer RNA with two deoxynucleotides on
the 3' end (RNA) complexed. At 6 hours, the FITC-2'-O-methyl
duplexes show localization in the nucleus and the
FITC-2'-O-methyl/RNA and 2'-O-methyl/FITC-RNA complexes show a more
diffuse pattern of uptake (these RNA/2'-O-methyl complexes are a
substrate for the RISC complex and are therefore retained in the
cytoplasm where the RISC complex has been reported to be active).
At 24 hours, the FITC-2'-O-methyl/RNA and 2'-O-methyl/FITC-RNA
complexes were still visible in the cell, whereas typically not
even the single-stranded FITC-2'-O- was visible, even when
transfected at significantly higher concentrations, demonstrating
that the 2'-O-methyl RNA protects the RNA strand from degradation
in the cell.
[0288] RNA oligomers having a phosphodiester backbone with
2'-O-methyl nucleotides were synthesized using standard
phosphoramidite chemistry. Oligomers were purified by denaturing
polyacrylamide gel electrophoresis (PAGE). Purity of oligomers was
confirmed by (PAGE) and mass spectrometry. All oligomers were
greater than 90% full length, and mass data obtained was consistent
with expected values. Target-specific siRNA duplexes consisted of
21-nt sense and 21-nt antisense strands with symmetric 2-nt 3'
deoxy TT overhangs. 21-nt RNAs were chemically synthesized using
phosphoramidite chemistry. For duplex preparation, sense- and
antisense oligomers (each at 50 .mu.M) were combined in equal
volumes in annealing buffer (30 mM HEPES pH 7.0, 100 mM potassium
acetate, and 2 mM magnesium acetate), heat-denatured at 90.degree.
C. for 1 min and annealed at 37.degree. C. for one hour. Duplexes
were stored at 80.degree. C. until used.
[0289] A549 cells (ATCC #CCL-185) were cultured at 37.degree. C. in
Dulbecco's Modified Eagle Medium (DMEM, Life Technologies
#11960-044) supplemented with 2 mM L-glutamine, 100 units/mL
penicillin, 100 .mu.g/mL streptomycin, and 10% fetal bovine serum
(FBS). HeLa cells (ATCC #CCL-2) were cultured at 37.degree. C. in
Minimal Essential Medium (MEM, Life Technologies #10370-021)
supplemented with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate, 1.0
mM sodium pyruvate, 100 units/mL penicillin, 100 .mu.g/mL
streptomycin, and 10% FBS. Cells were passaged regularly to
maintain exponential growth. On the day prior to transfection,
cells were trypsinized, counted, and seeded in 48-well plates at a
density of 20.times.103 cells per well in 250 .mu.L fresh media. On
the day of transfection cells were typically 60-65% confluent.
Transfection of siRNA duplexes and oligomers was carried out using
Lipofectamine 2000 (Life Technologies). Briefly, a 10.times. stock
of Lipofectamine 2000 was prepared in Opti-Mem (Life Technologies)
and incubated at room temperature for 15 minutes. An equal volume
of a 10.times. stock of siRNA duplex or oligomers in Opti-Mem was
added and complexation carried out for 15 minutes at room
temperature. Complexes were then diluted 5-fold in full growth
media. Culture media was removed from each well prior to the
addition of 250 .mu.L complexes per well. Cells were incubated at
37.degree. C./5% CO.sub.2 for 6 or 24 hours prior to assessing the
uptake.
[0290] Equivalents
[0291] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. The entire contents of all patents, published
patent applications and other references cited herein are hereby
expressly incorporated herein in their entireties by reference.
* * * * *