U.S. patent application number 10/357826 was filed with the patent office on 2004-03-18 for oligonucleotide compositions with enhanced efficiency.
This patent application is currently assigned to Sequitur, Inc.. Invention is credited to Taylor, Margaret F., Woolf, Tod M..
Application Number | 20040054155 10/357826 |
Document ID | / |
Family ID | 41112152 |
Filed Date | 2004-03-18 |
United States Patent
Application |
20040054155 |
Kind Code |
A1 |
Woolf, Tod M. ; et
al. |
March 18, 2004 |
Oligonucleotide compositions with enhanced efficiency
Abstract
The oligonucleotide compositions of the present invention make
use of combinations of oligonucleotides. In one aspect, the
invention features an oligonucleotide composition including at
least 2 different oligonucleotides targeted to a target gene. This
invention also provides methods of inhibiting protein synthesis in
a cell and methods of identifying oligonucleotide compositions that
inhibit synthesis of a protein in a cell.
Inventors: |
Woolf, Tod M.; (Sudbury,
MA) ; Taylor, Margaret F.; (Hopkinton, MA) |
Correspondence
Address: |
FOLEY & LARDNER
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Sequitur, Inc.
Natick
MA
|
Family ID: |
41112152 |
Appl. No.: |
10/357826 |
Filed: |
October 2, 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: |
536/22.1 |
Current CPC
Class: |
A61P 29/00 20180101;
C12N 2310/111 20130101; A61P 37/02 20180101; C12N 15/111 20130101;
A61P 31/12 20180101; A61P 1/04 20180101; A61P 17/06 20180101; C12N
2320/30 20130101; A61P 31/20 20180101; A61P 9/00 20180101; A61P
31/18 20180101; C12N 2330/30 20130101; C12N 15/1135 20130101; C12N
2320/11 20130101; C12N 2320/50 20130101; A61P 43/00 20180101; C12N
2310/11 20130101; C12Y 301/03048 20130101; C12N 2320/31 20130101;
C12N 2310/315 20130101; A61K 31/713 20130101; A61P 27/02 20180101;
C12Y 207/11022 20130101; C12N 15/1137 20130101; C12N 2310/14
20130101; C12N 15/113 20130101; A61P 35/00 20180101 |
Class at
Publication: |
536/022.1 |
International
Class: |
C07H 019/00 |
Claims
1. An oligonucleotide composition comprising at least 3 different
oligonucleotides targeted to at least three different nucleotide
sequences within a target gene, wherein (i) the oligonucleotides
bind to their target nucleotide sequence with high affinity and
(ii) the oligonucleotides are GC enriched.
2. The oligonucleotide composition of claim 1, wherein the
oligonucleotides are antisense oligonucleotides.
3. The oligonucleotide composition of claim 1, wherein the
oligonucleotides are double-stranded RNA oligonucleotides.
4. The oligonucleotide composition of claim 1, wherein the
oligonucleotide compositions bind to their target nucleotide
sequence with a Tm of at least about 60.degree. C.
5. The oligonucleotide composition of claim 1, wherein the
oligonucleotides have a GC content of at least about 20%.
6. The oligonucleotide composition of claim 1, wherein the
composition comprises at least about 4 antisense oligonucleotides
targeting at least four different nucleic acid sequences.
7. The oligonucleotide composition of claim 1, wherein the
composition comprises at least about 5 oligonucleotides targeting
at least five different nucleic acid sequences.
8. The oligonucleotide composition of claim 1, wherein the
composition comprises at least about 6 oligonucleotides targeting
at least six different nucleic acid sequences.
9. The oligonucleotide composition of claim 1, wherein the
oligonucleotides are at least about 25 nucleomonomers in
length.
10. The oligonucleotide composition of claim 1, wherein the
oligonucleotides are greater than 25 nucleomonomers in length.
11. The oligonucleotide composition of claim 2, wherein at least
one of the antisense oligonucleotides is complementary in sequence
to its target nucleotide sequence.
12. The oligonucleotide composition of claim 2, wherein the
antisense oligonucleotides activate RNase H.
13. The oligonucleotide composition of claim 1, wherein at least
one of the oligonucleotides comprise at least one modified
internucleoside linkage.
14. The oligonucleotide composition of claim 1, wherein at least
one of the oligonucleotides comprise at least one modified sugar
moiety.
15. The oligonucleotide composition of claim 1, further comprising
a pharmaceutically acceptable carrier.
16. The oligonucleotide composition of claim 1, wherein the
oligonucleotide composition achieves a level of inhibition of
protein synthesis the same as or higher than the level of
inhibition achieved by the most effective individual
oligonucleotide of the composition.
17. The oligonucleotide composition of claim 1, wherein the
individual oligonucleotides are not separately tested for their
ability to inhibit protein synthesis prior to their incorporation
into the composition.
18. The oligonucleotide composition of claim 1, wherein the
oligonucleotide composition results in greater than about 80%
inhibition of protein synthesis.
19. A method of inhibiting protein synthesis in a cell comprising
contacting the cell with at least 3 different oligonucleotides
targeted to at least three different nucleotide sequences within a
target gene, wherein (i) the oligonucleotides bind to their target
nucleotide sequence with high affinity and (ii) the
oligonucleotides are GC enriched, to thereby inhibit protein
synthesis.
20. The method of claim 19, wherein the oligonucleotides are
antisense oligonucleotides.
21. The method of claim 19, wherein the oligonucleotides are
double-stranded RNA oligonucleotides.
22. The method of claim 19, wherein the method is performed in a
high-throughput format.
23. A method of identifying function of a gene encoding a protein
comprising: contacting the cell with at least 3 different
oligonucleotides targeted to at least three different nucleotide
sequences within a target gene, wherein (i) the oligonucleotides
bind to their target nucleotide sequence with high affinity and
(ii) the oligonucleotides are GC enriched, and assaying for a
change in a detectable phenotype in the cell resulting from the
inhibition of protein expression, to thereby determine the function
of a gene.
24. The method of claim 23, wherein the oligonucleotides are
antisense oligonucleotides.
25. The method of claim 23, wherein the oligonucleotides are
double-stranded RNA oligonucleotides.
26. The method of claim 23, wherein the method is performed in a
high-throughput format.
27. A method of making the oligonucleotide composition of claim 1,
comprising: combining at least 3 different oligonucleotides
targeted to at least three different nucleotide sequences within a
target gene, wherein (i) the oligonucleotides bind to their target
nucleotide sequence with high affinity and (ii) the
oligonucleotides are GC enriched, and wherein, the individual
oligonucleotides are not separately tested for their ability to
inhibit protein synthesis prior to their incorporation into the
composition.
28. The method of claim 27, wherein the oligonucleotides are
antisense oligonucleotides.
29. The method of claim 27, wherein the oligonucleotides are
double-stranded RNA oligonucleotides.
30. An oligonucleotide composition comprising at least 3 different
double-stranded RNA oligonucleotides targeted to at least three
different nucleotide sequences within a target gene.
31. A method of inhibiting protein synthesis in a cell comprising
contacting the cell with at least 3 different double-stranded RNA
oligonucleotides targeted to at least three different nucleotide
sequences within a target gene.
32. A method of identifying function of a gene encoding a protein
comprising: contacting the cell with at least 3 different
double-stranded RNA oligonucleotides targeted to at least three
different nucleotide sequences within a target gene and assaying
for a change in a detectable phenotype in the cell resulting from
the inhibition of protein expression, to thereby determine the
function of a gene.
33. A method of making an oligonucleotide composition comprising:
combining at least 3 different double-stranded RNA oligonucleotides
targeted to at least three different nucleotide sequences within a
target gene wherein, the individual oligonucleotides are not
separately tested for their ability to inhibit protein synthesis
prior to their incorporation into the composition.
Description
RELATED APPLICATIONS
[0001] This application claims the priority of U.S. provisional
patent application No. 60/353,381, filed on Feb. 1, 2002. This
application also 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. The entire contents of the aforementioned
applications are hereby expressly incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Antisense and double-stranded RNA oligonucleotides are
promising therapeutic agents and useful research tools for
elucidating gene function. However, it is often difficult to
achieve efficient inhibition of protein synthesis using such
compositions.
[0003] In order to maximize their therapeutic activity, it would be
of great benefit to improve upon the prior art antisense and
double-stranded RNA oligonucleotides by enhancing the efficiency
with which they inhibit protein synthesis.
SUMMARY OF THE INVENTION
[0004] The instant invention is based, at least in part, on the
discovery of antisense and double-stranded oligonucleotide
compositions that provide improved inhibition of gene expression.
In particular, the oligonucleotide compositions of the present
invention make use of combinations of antisense or double-stranded
oligonucleotides.
[0005] In one aspect, the invention pertains to an oligonucleotide
composition comprising at least 3 different oligonucleotides
targeted to at least three different nucleotide sequences within a
target gene, wherein (i) the oligonucleotides bind to their target
nucleotide sequence with high affinity and (ii) the
oligonucleotides are GC enriched.
[0006] In one embodiment, the oligonucleotides are antisense
oligonucleotides.
[0007] In another embodiment, the oligonucleotides are
double-stranded RNA oligonucleotides.
[0008] In one embodiment, the oligonucleotide compositions bind to
their target nucleotide sequence with a Tm of at least about
60.degree. C.
[0009] In one embodiment, the oligonucleotides have a GC content of
at least about 20%.
[0010] In one embodiment, the composition comprises at least about
4 antisense oligonucleotides targeting at least four different
nucleic acid sequences. In another embodiment, the composition
comprises at least about 5 oligonucleotides targeting at least five
different nucleic acid sequences. In still another embodiment, the
composition comprises at least about 6 oligonucleotides targeting
at least six different nucleic acid sequences.
[0011] In one embodiment, the oligonucleotides are at least about
25 nucleomonomers in length. In another embodiment, the
oligonucleotides are greater than about 25 nucleomonomers in
length.
[0012] In one embodiment, at least one of the antisense
oligonucleotides is complementary in sequence to its target
nucleotide sequence. In another embodiment, the antisense
oligonucleotides activate RNase H.
[0013] In one embodiment, at least one of the oligonucleotides
comprise at least one modified internucleoside linkage.
[0014] In another embodiment, at least one of the oligonucleotides
comprise at least one modified sugar moiety.
[0015] In one embodiment, the composition further comprises a
pharmaceutically acceptable carrier.
[0016] In one embodiment, the oligonucleotide composition achieves
a level of inhibition of protein synthesis the same as or higher
than the level of inhibition achieved by the most effective
individual oligonucleotide of the composition.
[0017] In one embodiment, the individual oligonucleotides are not
separately tested for their ability to inhibit protein synthesis
prior to their incorporation into the composition. In this respect,
the present invention represents a substantial and unrecognized
improvement over the state of the art.
[0018] In one embodiment, the oligonucleotide composition results
in greater than about 80% inhibition of protein synthesis.
[0019] In another aspect, the invention pertains to a method of
inhibiting protein synthesis in a cell comprising contacting the
cell with at least 3 different oligonucleotides targeted to at
least three different nucleotide sequences within a target gene,
wherein (i) the oligonucleotides bind to their target nucleotide
sequence with high affinity and (ii) the oligonucleotides are GC
enriched, to thereby inhibit protein synthesis.
[0020] In one embodiment, the oligonucleotides are antisense
oligonucleotides. In another embodiment, the oligonucleotides are
double-stranded RNA oligonucleotides.
[0021] In one embodiment, the method is performed in a
high-throughput format.
[0022] In still another aspect, the invention pertains to a method
of identifying function of a gene encoding a protein comprising:
contacting the cell with at least 3 different oligonucleotides
targeted to at least three different nucleotide sequences within a
target gene, wherein (i) the oligonucleotides bind to their target
nucleotide sequence with high affinity and (ii) the
oligonucleotides are GC enriched, and assaying for a change in a
detectable phenotype in the cell resulting from the inhibition of
protein expression, to thereby determine the function of a
gene.
[0023] The relative amounts of these different oligonucleotides may
optionally be different. That is, the three or more different
oligonucleotides may be present in equimolar concentrations, or
non-equimolar concentrations.
[0024] In one embodiment, the oligonucleotides are antisense
oligonucleotides. In another embodiment, the oligonucleotides are
double-stranded RNA oligonucleotides.
[0025] In one embodiment, the method is performed in a
high-throughput format.
[0026] In another aspect, the invention pertains to a method of
making the oligonucleotide composition, comprising: combining at
least 3 different oligonucleotides targeted to at least three
different nucleotide sequences within a target gene, wherein (i)
the oligonucleotides bind to their target nucleotide sequence with
high affinity and (ii) the oligonucleotides are GC enriched, and
wherein the individual oligonucleotides are not separately tested
for their ability to inhibit protein synthesis prior to their
incorporation into the composition.
[0027] In one embodiment, the oligonucleotides are antisense
oligonucleotides. In another embodiment, the oligonucleotides are
double-stranded RNA oligonucleotides.
[0028] In another aspect, the invention pertains to an
oligonucleotide composition comprising at least 3 different
double-stranded RNA oligonucleotides targeted to at least three
different nucleotide sequences within a target gene.
[0029] In still another aspect, the invention pertains to a method
of inhibiting protein synthesis in a cell comprising contacting the
cell (or cell lysate) with at least 3 different double-stranded RNA
oligonucleotides targeted to at least three different nucleotide
sequences within a target gene.
[0030] In yet another aspect, the invention pertains to a method of
identifying function of a gene encoding a protein comprising:
contacting the cell with at least 3 different double-stranded RNA
oligonucleotides targeted to at least three different nucleotide
sequences within a target gene and assaying for a change in a
detectable phenotype in the cell resulting from the inhibition of
protein expression, to thereby determine the function of a
gene.
[0031] In another aspect, the invention pertains to a method of
making an oligonucleotide composition comprising combining at least
3 different double-stranded RNA oligonucleotides targeted to at
least three different nucleotide sequences within a target gene
wherein, the individual oligonucleotides are not separately tested
for their ability to inhibit protein synthesis prior to their
incorporation into the composition.
DRAWINGS
[0032] FIG. 1 shows a summary of the results of about 30 antisense
inhibition experiments against about thirty different genes in cell
culture. Oligonucleotide compositions comprising mixtures of
oligonucleotides (with the worst 10% of target genes removed) are
compared with the best individual oligonucleotides and data for all
individual oligonucleotides in the percent inhibition observed.
[0033] FIG. 2 shows ultramer data for a mixture of siRNA complexes
targeting p53.
[0034] FIG. 3 shows ultramer data for a mixture of siRNA complexes
targeting GTP20.
[0035] FIG. 4 shows ultramer data for a mixture of siRNA complexes
targeting Cbfa-1.
[0036] FIG. 5 shows ultramer data for a mixture of siRNA complexes
targeting PTP mu.
[0037] FIG. 6 shows ultramer data for a mixture of siRNA complexes
targeting PTP-PEST.
[0038] FIG. 7 shows ultramer data for a mixture of siRNA complexes
targeting PTP eta.
DETAILED DESCRIPTION OF THE INVENTION
[0039] Although inhibition of protein synthesis could be achieved
with certain antisense and double-stranded RNA oligonucleotides of
the prior art, multiple transfections were required to identify
effective oligonucleotides. The instant invention advances the
prior art, inter alia, by providing oligonucleotide compositions
that enhance the efficiency with which protein synthesis is
inhibited and methods of making and using these improved
oligonucleotide compositions.
[0040] Methods of stabilizing oligonucleotides, particularly
antisense oligonucleotides, by formation of a duplex with a
complementary oligonucleotide, are disclosed in co-pending
application No. U.S. ______, filed on the same day as the present
application, bearing attorney docket number "SRI-020," and entitled
"Double-Stranded Oligonucleotides." This application and all of its
teachings is hereby expressly incorporated herein by reference in
its entirety.
[0041] Antisense and Double-Stranded RNA Oligonucleotide
Compositions
[0042] Antisense or double-stranded RNA oligonucleotides for
incorporation into compositions of the invention inhibit 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. 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).
[0043] 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.
[0044] 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.
[0045] The term "oligonucleotide" includes two or more
nucleomonomers covalently coupled to each other by linkages or
substitute linkages. An oligonucleotide may comprise, for example,
between a few (e.g.,7, 10, 12, 15) or a few hundred ( e.g., 100,
200, 300, or 400) nucleomonomers. For example, an oligonucleotide
of the invention preferably comprises between about 10 and about 50
nucleomonomers, between about 15 and about 40, or between about 20
and about 30 nucleomonomers. In one embodiment, an oligonucleotide
comprises about 25 nucleomonomers. In another embodiment, an
oligonucleotide comprises greater than about 25 nucleomonomers.
[0046] 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 or
analogs 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.
[0047] 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.
[0048] 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").
[0049] 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.
[0050] The term "nucleomonomer" includes bases 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. The term "second moiety" as used herein
includes 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.
[0051] Sugar moieties include natural, unmodified sugars, e.g.,
monosaccharides (such as pentoses, e.g., ribose), modified sugars
and sugar analogs. Possible modifications of nucleomonomers
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 group as an ether, an amine, a thiol, or
the like. 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=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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] The term "heteroatom" includes atoms of any element other
than carbon or hydrogen. Preferred heteroatoms are nitrogen,
oxygen, sulfur and phosphorus.
[0065] The term "hydroxy" or "hydroxyl" includes groups with an
--OH or --O.sup.- (with an appropriate counterion).
[0066] 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.
[0067] 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.0-3C(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.
[0068] 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.
[0069] 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.
[0070] The term "ester" includes compounds and moieties which
contain a carbon or a heteroatom bound to an oxygen atom which is
bonded to the carbon of a carbonyl group. The term "ester" includes
alkoxycarboxy groups such as methoxycarbonyl, ethoxycarbonyl,
propoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, etc.
[0071] The term "base" includes the known purine and pyrimidine
heterocyclic bases, deazapurines, and analogs (including heterocycl
substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives
(e.g., 1-alkenyl-, 1-alkynyl-, 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.
[0072] 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).
[0073] The term "nucleotide" includes nucleosides which further
comprise a phosphate group or a phosphate analog.
[0074] 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.
[0075] As used herein, the term "linkage" includes a naturally
occurring, unmodified phosphodiester moiety
(--O--(PO.sub.2--)--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., such as
phosphorothioate, phosphorodithioate, and P-ethyoxyphosphodiester,
P-ethoxyphosphodiester, P-alkyloxyphosphotriester,
methylphosphonate, and nonphosphorus containing linkages, e.g.,
such as 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).
[0076] Oligonucleotides of the invention comprise 3' and 5' termini
(except for circular oligonucleotides). 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., hydrogen phosphonate, phosphoramidite, or
PO.sub.3.sup.2-). "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.
[0077] 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 can
comprise 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.
[0078] In one embodiment, an oligonucleotide may comprise a 5'
phosphate group or a group larger than a phosphate group.
[0079] 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).
[0080] One skilled in the art will recognize that the length of an
RNAi oligonucleotide corresponds to a region of complementarity to
the target in the antisense stranded, and the RNAi may be longer,
if, for example the RNAi is of a hairpin design.
[0081] 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.
[0082] 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). 1
[0083] 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.
[0084] 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.
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). 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.
[0085] In one embodiment, when included in an RNase H activating
antisense oligonucleotide, 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 oligonucleotide such that the
specificity of the chimeric antisense oligonucleotide is not
altered when compared with the specificity of a chimeric antisense
oligonucleotide 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
directed against the target than by an oligonucleotide that does
not comprise an affinity enhancing agent that is directed against
the target.
[0086] In one embodiment, the oligonucleotides of the invention are
GC enriched. As used herein the term "GC enriched" includes
oligonucleotides that have a relatively high percent GC content.
For example, in one embodiment an oligonucleotide of the invention
has at least about 20%, at least about 30%, at least about 40% GC
content. In another embodiment, an oligonucleotide of the invention
has at least about 50%, at least about 60%, or at least about 70%
GC content.
[0087] In one embodiment, the oligonucleotides of the invention are
at least about 25 nucleomonomers in length. In one embodiment, the
antisense oligonucleotides of the invention are greater than about
25 nucleomonomers in length. In one embodiment, an antisense
oligonucleotide of the invention is at least about 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.
[0088] Double-Stranded RNA Oligonucleotides
[0089] Double-stranded RNA (double-stranded RNA or RNAi
(double-stranded RNA interference)) is a double-stranded RNA
oligonucleotide that can be used to inhibit protein synthesis in a
cell (see, e.g., WO 01/3 6646A1; Elbashir et al. 2001. Genes &
Deveolpment 15:188; Elbashir et al. 2001. Nature 411:494; Elbashir
et al. 2001 EMBO. 20:6877). Double-stranded RNA may be formed by a
single, self-complementary strand or two separate complementary
strands. Duplex formation can occur either inside or outside the
cell containing the target gene.
[0090] 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.
[0091] 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.
[0092] 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.
[0093] 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 Al; Shuey, et al., "RNAi: gene-silencing in
therapeutic intervention." Drug Discov. Today Oct. 15,
2002;7(20):1040-6; Aoki, et al., "Clin. Exp. Pharmacol. Physiol.
Jan. 30, 2003 (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. Feb. 10, 2003(2):125-33.
[0094] 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. Jan. 15,
2003;31(2):700-7; Cottrell, et al., "Silence of the strands: RNA
interference in eukaryotic pathogens." Trends Microbiol. Jan. 11,
2003;(1):37-43; Links, "Mammalian RNAi for the masses." Trends
Genet. Jan. 19, 2003;(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. Oct. 12,
2002;(5):301-9; Links, "RNAi and related mechanisms and their
potential use for therapy." Curr. Opin. Chem. Biol. Dec. 6,
2002;(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. Jan. 15, 2003;31(2):700-7.).
[0095] Double-stranded RNA molecule comprises a nucleotide sequence
which is substantially identical to at least part of the target
gene. In one embodiment, a double-stranded RNA molecule comprises a
nucleotide sequence which is at least about 100% identical to a
portion of the target gene. In another embodiment, a
double-stranded RNA molecule comprises a nucleotide sequence which
is at least about 95% identical to a portion of the target gene. In
another embodiment, a double-stranded RNA molecule comprises a
nucleotide sequence which is at least about 90% identical to a
portion of the target gene. In another embodiment, a
double-stranded RNA molecule comprises a nucleotide sequence which
is at least about 80% identical to a portion of the target gene. In
another embodiment, a double-stranded RNA molecule comprises a
nucleotide sequence which is at least about 60% identical to a
portion of the target gene. In another embodiment, a
double-stranded RNA molecule comprises a nucleotide sequence which
is at least about 100% identical to a portion of the target
gene.
[0096] 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). In a preferred embodiment, the length of the
target gene sequence aligned for comparison purposes is at least
about 25 nucleotide residues, at least about 50, at least about
100, at least about 150, at least about 200, or at least about 300
or more nucleotide residues are aligned. The nucleotides at
corresponding nucleotide positions are then compared. 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.
[0097] 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.
[0098] 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 internet
website.
[0099] In one embodiment, the oligonucleotides of the invention are
identical to a target nucleic acid sequence over at least about 80%
of the length of the oligonucleotide. In another embodiment,
oligonucleotides of the invention are identical to a target nucleic
acid sequence over at least about 90-95% of the length of the
oligonucleotide. In another embodiment, oligonucleotides of the
invention are identical to a target nucleic acid sequence over the
entire length of the oligonucleotide.
[0100] In yet another embodiment, a sequence of a double-stranded
RNA molecule of the invention hybridizes to at least a portion of
the target gene 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. 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.
[0101] Antisense Oligonucleotides
[0102] As used herein, the term "antisense oligonucleotide"
includes oligonucleotides which comprise a nucleotide sequence
which is specifically interferes with the synthesis of the target
polypeptide. In general, antisense oligonucleotides of the
invention 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 antisense oligonucleotides of the
invention bind to nucleic acid molecules, they can bind to any
region of the nucleic acid molecule, including e.g., introns,
exons, 5', or 3' untranslated regions. For example, antisense
oligonucleotides that work as steric blockers preferentially bind
within a splice junction, 5' untranslated region, or the start
region of a nucleic acid target molecule. Antisense
oligonucleotides that work by 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.
[0103] Antisense oligonucleotides of the invention may or may not
be complementary to their target sequence. Without being limited to
any particular mechanism of action, an antisense oligonucleotide
used in an oligonucleotide composition of the invention that can
specifically hybridize with a nucleotide sequence within the target
gene (i.e., is 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).
[0104] Without being limited to any particular mechanism of action,
the antisense oligonucleotides used in an oligonucleotide
composition of the invention that can not specifically hybridize
with a nucleotide sequence within the target gene (are not
complementary to a nucleotide sequence within the target gene) may
achieve their affects based on, e.g., (1) the secondary structure
of the oligonucleotide; (2) hybridization to a different nucleotide
sequence; (3) binding to proteins or other molecules that may
affect the target gene; or (4) modulating oligonucleotide
degradation products which themselves can affect cellular
functions.
[0105] In one embodiment, at least two of the antisense
oligonucleotides in an oligonucleotide composition of the invention
inhibit protein synthesis via the same mechanism. In another
embodiment, at least two of the antisense oligonucleotides in an
oligonucleotide composition inhibit protein synthesis via a
different mechanism. In yet another embodiment, all of the
antisense oligonucleotides present in an oligonucleotide
composition inhibit protein synthesis via the same mechanism. The
oligonucleotide compositions of the present invention may comprise
antisense oligonucleotides which rely simultaneously on several of
these modes of action.
[0106] The antisense oligonucleotides used in an oligonucleotide
composition of the invention may be of any type, e.g., including
morpholino oligonucleotides, RNase H activating oligonucleotides,
or ribozymes.
[0107] In one embodiment, antisense oligonucleotides of the
invention are substantially complementary to a target nucleic acid
sequence. Percent complementarity is determined analogously to
percent identity. For example, when a position in a test nucleotide
sequence is occupied by a nucleotide that is complementary to the
corresponding position in the reference sequence, then the
molecules are complementary at that position. 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.
[0108] In one embodiment, the antisense oligonucleotides of the
invention are complementary to a target nucleic acid sequence over
at least about 80% of the length of the oligonucleotide. In another
embodiment, antisense oligonucleotides of the invention are
complementary to a target nucleic acid sequence over at least about
90-95% of the length of the oligonucleotide. In another embodiment,
antisense oligonucleotides of the invention are complementary to a
target nucleic acid sequence over the entire length of the
oligonucleotide.
[0109] Antisense oligonucleotides 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). More
preferably, the RNase H activating region comprises about nine
contiguous deoxyribose containing nucleomonomers. Preferably, the
contiguous nucleomonomers are linked by a substitute linkage, e.g.,
a phosphorothioate linkage.
[0110] The language "non-activating region" includes a region of an
antisense oligonucleotide, 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.
[0111] Antisense oligonucleotides 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 non-ionic
phosphorodiamidate intersubunit linkages. An example of a 2 subunit
morphilio oligonucleotide is shown below. 2
[0112] 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.
[0113] A variety of nucleotides of different lengths may be used.
In one embodiment, an oligonucleotide of the invention is greater
than about 25 nucleomonomers in length. In one embodiment, an
oligonucleotide of the invention is at least about 10, 12, 14, 16,
18, 20, 22, 24, 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 oligonucleotide of the invention is less than about
25 nucleomonomers in length, particularly about 21 to 23. In yet
another embodiment, an oligonucleotide of the invention is about
10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30
nucleomonomers in length. In another embodiment, an oligonucleotide
of the invention is at most about 26, 27, 28, 29, 30, at most about
40, at most about 50, or at most about 60, at most about 70, at
most about 80, or at most about 90 nucleomonomers in length.
[0114] Preferred nucleomonomers in some aspects are
ribonucleotides, including 2'-O-methyl ribonucleotides and other
2'-modified RNA molecules.
[0115] Oligomers of the invention may also comprise a DNA gap or a
phosphorothioate DNA gap.
[0116] In some aspects, the present invention relates to
compositions and methods comprising at least about 4, 5, 6, 7, 8,
9, or 10 antisense oligonucleotides targeting at least four, five,
six, seven, eight, nine, or ten different nucleic acid
sequences.
[0117] Selection of Oligonucleotide Sequences
[0118] Once the target protein is selected and the nucleotide
sequence which encodes it is determined, the sequence of an
oligonucleotide for inclusion in the compositions of the invention
is determined. The sequence of the target gene is analyzed and
oligonucleotides are chosen by a process including both elimination
and selection steps. In one embodiment, oligonucleotides which have
more than 3 of any nucleotide (A, U, C, or G) occurring
consecutively within the oligonucleotide are eliminated. In another
embodiment, oligonucleotides having dinucleotide repeats (e.g.,
AUAU, ACAC, AGAG, UCUC, UGUG, or CGCG) are eliminated. In another
embodiment, oligonucleotides are chosen that target nucleotide
sequences of the target gene that are preferably at least about 25
nucleotides apart. In another embodiment, oligonucleotides are
chosen that comprise between 4 and 10 (inclusive) of each base,
such that the base composition of the oligonucleotides is similar.
In another embodiment, the percentage of bases in the
oligonucleotide which are G or C is greater than 50%. In one
embodiment, when oligonucleotides are designed to be complementary
to a chosen target sequence, preferably, they are 100%
complementary to the target sequence. In another embodiment, an
oligonucleotide preferably has greater than 2 mismatches to other,
non-target genes. This can be tested by one of ordinary skill in
the art, e.g., using available alignment programs and public
databases, e.g., the National Institutes of Health internet
website.
[0119] Oligonucleotide Compositions of the Invention
[0120] This invention relates to oligonucleotide compositions
including more than one individual oligonucleotide molecule. The
individual oligonucleotide molecules of the composition target at
least one target nucleotide sequence of a single target gene. For
example, in one embodiment, at least two of the oligonucleotides
present in the composition target the same nucleotide sequence in
the same target gene e.g., the oligonucleotides comprise different
chemistries but target (e.g., specifically hybridize to) the same
sequence of bases in a target nucleic acid molecule. In another
embodiment, at least two of the oligonucleotides present in the
composition target different nucleotide sequences in the same
target gene (e.g., the oligonucleotide composition comprises one
oligonucleotide targeting a nucleotide sequence in the promoter of
a gene and another oligonucleotide targeting a nucleotide sequence
in the portion of the coding sequence of the target nucleic acid
molecule or the oligonucleotide composition comprises at least two
different oligonucleotides that target two different nucleotide
sequences in the coding region of the target nucleic acid
molecule).
[0121] The number of oligonucleotides used in an oligonucleotide
composition of the invention can vary from as few as about 2
oligonucleotides to greater than about 20 oligonucleotides. In one
embodiment, at least about 3-4 different oligonucleotides are used
in the oligonucleotide composition. In another embodiment, at least
about 5-6 different oligonucleotides are used in the
oligonucleotide composition. In a further embodiment, at least
about 7-8 different oligonucleotides are used in the
oligonucleotide composition. In one embodiment, greater than about
8 different oligonucleotides are used in an oligonucleotide
composition of the invention. In a preferred embodiment, the number
of different oligonucleotides in the oligonucleotide composition is
chosen so as to use the minimum number of different
oligonucleotides that effectively inhibit synthesis of the target
protein.
[0122] The different oligonucleotides used in an oligonucleotide
composition of the invention can each be present at the same
concentration or can be present in different concentrations. For
example, more desirable oligonucleotides (e.g., those that are more
inexpensive or easier to synthesize) may be present at higher
concentrations than less desirable oligonucleotides.
[0123] Preferably, the oligonucleotides in a composition are either
all double-stranded RNA oligonucleotides or all antisense
oligonucleotides.
[0124] It will be understood that the individual oligonucleotides
of the invention can be synthesized to comprise different
chemistries. For example, in one embodiment, a composition of the
invention can comprise at least one oligonucleotide that is
optionally GC enriched. In another embodiment, a composition of the
invention comprises at least one oligonucleotide that binds to its
target with high affinity. In another exemplary embodiment, a
composition of the invention comprises at least one that is at
least about 25 nucleomonomers in length. In one embodiment, an
oligonucleotide of the invention comprises an oligonucleotide that
is GC enriched and binds to its target with high affinity. Thus, as
shown by this example, one of skill in the art will recognize that
given the teachings of the specification, multiple variations of
the individual oligonucleotides present in improved oligonucleotide
compositions of the invention can be made.
[0125] Making Oligonucleotide Compositions
[0126] In one embodiment, an individual oligonucleotide is not
individually tested for its ability to inhibit protein synthesis
prior to its inclusion into a composition of the invention.
[0127] In another embodiment, an individual oligonucleotide for
inclusion in an oligonucleotide composition inhibits protein
synthesis by about 20% when tested individually. In another
embodiment, an individual oligonucleotide for inclusion in an
oligonucleotide composition inhibits gene expression by about 30%
when tested individually. In another embodiment, an individual
oligonucleotide for inclusion in an oligonucleotide composition
inhibits gene expression by about 40% when tested individually. In
another embodiment, an individual oligonucleotide for inclusion in
an oligonucleotide composition inhibits gene expression by about
50% when tested individually. In another embodiment, an individual
oligonucleotide for inclusion in an oligonucleotide composition
inhibits gene expression by about 60% when tested individually.
Preferably, an individual oligonucleotide for inclusion in an
oligonucleotide composition inhibits gene expression by less than
about 40% when tested individually.
[0128] In one embodiment, an oligonucleotide composition of the
invention inhibits gene expression to an extent that is greater
than the level of inhibition of gene expression achieved by any of
the individual oligonucleotides of the oligonucleotide composition
acting alone. In another embodiment, the oligonucleotide
composition achieves a level of inhibition of protein synthesis the
same as or higher than the level of inhibition achieved by the most
effective individual oligonucleotide of the composition. In one
embodiment, an oligonucleotide composition of the present invention
is at least about 80% effective at inhibiting gene expression. In
another embodiment, an oligonucleotide composition of the present
invention is at least about 90%-95% effective at inhibiting gene
expression. In another embodiment, an oligonucleotide composition
of the present invention is at least about 99% effective at
inhibiting gene expression.
[0129] The subject compositions greatly increase the efficiency of
the inhibition of protein synthesis because the ability of an
individual oligonucleotide to inhibit protein synthesis does not
have to be tested prior to its inclusion in an oligonucleotide
composition of the invention. Accordingly, only one transfection
need be done to effectively inhibit protein synthesis. Thus, in one
embodiment, an oligonucleotide composition of the invention is
contacted with a cell or population of cells prior to testing the
ability of the individual oligonucleotides of the composition to
inhibit target gene expression. In another embodiment, an
oligonucleotide composition of the invention is contacted with a
cell or population of cells subsequent to testing the ability of
the individual oligonucleotides of the composition to inhibit
target gene expression.
[0130] To achieve inhibition of gene expression, an oligonucleotide
composition of the invention is contacted with a cell (or cell
lysate). In one embodiment, the oligonucleotides of an
oligonucleotide composition are contacted with a cell
simultaneously. In an alternative embodiment, the oligonucleotides
of an oligonucleotide composition can be brought into contact with
a cell at different times. For example, at least one of the
oligonucleotides can be contacted with a cell at a different time
from the other oligonucleotides. In yet another example, each of
the oligonucleotides of an oligonucleotide composition is contacted
with a cell sequentially so that each of the oligonucleotides of an
oligonucleotide composition comes into contact with the cell at a
different time. As such, the compositions of the instant invention
can be formulated for separate administration of the
oligonucleotides. Preferably, a cell is contacted with
oligonucleotides of the invention such that the level of inhibition
of protein synthesis (e.g., as measured either directly (by
measuring the decrease in the amount of the target protein
produced) or, for example, by measuring the disappearance of a
phenotype associated with the presence of the target protein, by
measuring a reduction in the amount of mRNA produced from the
target gene, or by measuring in increase in the level of
degradation of the mRNA) is greater than that observed when
individual nucleotides of the invention are tested
individually.
[0131] The number of oligonucleotides used to contact a cell can
vary from as few as 2 oligonucleotides to greater than about 20
oligonucleotides. In one embodiment, at least about 2-3 different
oligonucleotides are contacted with a cell. In another embodiment,
at least about 4-5 different oligonucleotides are used to contact
the cell. In a further embodiment, at least about 6-7 different
oligonucleotides are contacted with a cell.
[0132] 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., eds. 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.
[0133] 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. By incrementally adjusting the concentrations and
identities of the oligonucleotides in the oligonucleotide
composition and monitoring the resulting change in reporter gene
expression, it is possible to optimize the oligonucleotide
composition.
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] Uptake of Oligonucleotides by Cells
[0140] 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. 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.
[0141] Oligonucleotide compositions of the invention can be
contacted with cells in vitro or in vivo. 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.
[0142] 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
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).
[0143] Conjugating Agents
[0144] 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).
[0145] 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.
[0146] Encapsulating Agents
[0147] Encapsulating agents entrap oligonucleotides within
vesicles. In another embodiment, 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] Complexing Agents
[0152] Complexing agents bind to the oligonucleotide by a strong
but non-covalent attraction (e.g., an electrostatic, van der Waals,
pi-stacking interaction, etc.). 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.
[0153] 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.31 ,
Br.sup.-, I.sup.-, F.sup.-, acetate, trifluoroacetate, sulfate,
nitrite, and nitrate.
[0154] 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
(JBL, San Luis Obispo, Calif.). Cationic liposomes may comprise the
following: N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
chloride (DOTMA),
N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP), 3p-[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).
[0155] 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.
[0156] 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.
[0157] 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).
[0158] 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).
[0159] 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.
[0160] In one 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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).
[0165] 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.
[0166] 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
GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: ##) or a portion or variant
thereof that facilitates transport of an oligonucleotide into a
cell.
[0167] 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.
[0168] 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.
[0169] 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).
[0170] 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.1-C.sub.20 alkenyl chains, C.sub.1-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).
[0171] 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).
[0172] Targeting Agents
[0173] 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.
[0174] 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).
[0175] 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.
[0176] 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.
[0177] Liposomes are naturally targeted to the liver, spleen, and
reticuloendothelial system. By coupling liposomes to various
ligands such as antibodies are protein A, they can be 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).
[0178] Assays of Oligonucleotide Stability
[0179] Preferably, the 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, unmodified 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.
[0180] One way in which substantial stability can be demonstrated
is 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).
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.
[0186] 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.
[0187] 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.
[0188] Oligonucleotide Synthesis
[0189] Oligonucleotides of the invention can be synthesized by any
methods 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.
[0190] 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.
[0191] 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.
[0192] 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 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
can be purchased commercially.
[0193] 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.
[0194] 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.
[0195] 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 (DN
Glover Ed. 1985); Oligonucleotide Synthesis (M J Gait Ed, 1984;
Nucleic Acid Hybridisation (BD Hames and S J Higgins eds. 1984); A
Practical Guide to Molecular Cloning (1984); or the series, Methods
in Enzymology (Academic Press, Inc.)).
[0196] Uses of Oligonucleotides
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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).
[0204] The invention also relates to a selecting oligonucleotides
for the methods described herein in which in which many oligomers
are screened (e.g., from about 10-20 to significantly greater
numbers as may be found in a combinatorial library), after which
the more efficacious oligomers are chosen and combined to produce a
composition of the invention. Thus, inhibition of greater than 95%,
90%, 85%, 80%, 70%, or 60% may be achieved.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] Administration of Oligonucleotide Compositions
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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).
[0222] 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.
[0223] 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.
[0224] 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.
[0225] 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.
[0226] 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.
[0227] 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
(S0750), 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.
[0228] 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 (C8-C12) mono, di, and
tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty
alcohols, polyglycolized glycerides, saturated polyglycolized
C8-C10 glycerides, vegetable oils and silicone oil.
[0229] 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.
[0230] 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.
[0231] 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.
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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, cows, pigs, horses, dogs,
cats, mice, rats, and transgenic non-human animals.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] Treatment of Diseases or Disorders
[0240] 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
(e.g., ICAM-1 related disorders, Psoriasis, Ulcerative Colitus,
Crohn's disease), viral diseases (e.g., HIV, Hepatitis C), and
cardiovascular diseases.
[0241] 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 95/22553) influenza virus (WO 94/23028), and
malignancies (WO 94/08003). Other examples of clinical uses of
antisense oligonucleotides 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.
[0242] 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)).
[0243] The invention is further illustrated by the following
examples, which should not be construed as further limiting.
EXAMPLES
Example 1
[0244] Ability of Oligonucleotide Compositions to Inhibit CDK2 in
A549 Cells.
[0245] In this example, the ability of 5 different antisense
oligonucleotides individually was compared with the ability of all
5 of the antisense oligonucleotides transfected at one time for
their ability to inhibit the expression of CDK2 in A549 cells. The
sequences of the 5 antisense oligonucleotides used were:
Oligonucleotide 1 GCAGUAUACCUCUCGCUCUUGUCAA (SEQ ID NO: ##);
oligonucleotide 2 UUUGGAAGUUCUCCAUGAAGCGCCA (SEQ ID NO: ##);
oligonucleotide 3 GUCCAAAGUCUGCUAGCUUGAUGGC (SEQ ID NO: ##);
oligonucleotide 4 CCCAGGAGGAUUUCAGGAGCUCGGU (SEQ ID NO: ##);
oligonucleotide 5 UAGAAGUAACUCCUGGCCACACCAC (SEQ ID NO: ##);reverse
control AACUGUUCUCGCUCUCCAUAUGACG (SEQ ID NO: ##).
[0246] For transfection with antisense oligonucleotides A549 cells
were maintained in DMEM with high glucose (Gibco-BRL) supplemented
with 10% Fetal Bovine Serum, 2 mM L-Glutamine, and 1.times.
penicillin/streptomycin.
[0247] On the day before transfection 24-well plates were seeded
with 30,000 A549 cells per well. The cells were approximately 60%
confluent at the start of transfection, and were evenly distributed
across the plate. On the day of transfection, a 10.times. stock of
Lipofectamine 2000 (Invitrogen) was prepared in Opti-MEM (serum
free media, Gibco-BRL). The diluted lipid was allowed to stand at
room temperature for 15 minutes. The optimal conditions for
transfection of A549 cells were determined to be 25 nM
oligonucleotide complexed with 1 ug/mL Lipofectamine 2000. A
10.times. stock of each oligonucleotide to be used in the
transfection was also prepared in Opti-MEM (10.times. concentration
of oligonucleotide is 0.25 uM). Equal volumes of the 10.times.
Lipofectamine 2000 stock and the 10.times. oligonucleotide
solutions were mixed well and incubated for 15 minutes at room
temperature to allow complexation of the oligonucleotide and lipid.
The resulting mixture was 5.times.. After the 15 minutes of
complexation, four volumes of full growth media was added to the
oligonucleotide/lipid complexes to make a 1.times. solution. The
media was aspirated from the cells, and 0.5 mL of the 1.times.
oligonucleotide/lipid complexes was added to each well. The cells
were not permitted to dry out during the changing of media. The
cells were incubated for 16-24 hours at 37.degree. C. in a
humidified CO.sub.2 incubator. Cell pellets were harvested for
protein determination or RNA isolation. The Tables below show the
results of the experiment.
1 Ratio of CDK2 expression Oligonucleotide to GAPDH expression
Standard Deviation No transfection 1.481 0.242 FITC 1.004 0.203 1
0.233 0.041 2 0.231 0.058 3 0.198 0.015 4 0.193 0.065 5 0.673 0.232
Reverse Control 0.749 0.079 Oligonucleotide 0.137 0.012 Composition
Percent Inhibition Compared to Reverse Oligonucleotide Control No
transfection 0 (-98%) FITC 0 (-34%) 1 69% 2 69% 3 74% 4 74% 5 10%
Reverse Control 0% Oligonucleotide Composition 82%
[0248] The levels of expression of CDK2 were normalized to levels
of GAPDH. No transfection or transfection with a fluorescent
control oligonucleotide (which targets luciferase) showed levels of
1 or higher. A reverse sequence control oligonucleotide gave a
level of about 0.8. Each of the individual oligonucleotides (1-5)
showed inhibition in CDK2 expression (with levels ranging from
about 0.2 (about 70% inhibition compared to the reverse control) to
0.65 (10% inhibition compared to the reverse control) for
oligonucleotide number 5). All five of the oligonucleotides
transfected at once gave a level of less than about 0.2, about 82%
inhibition compared to the reverse control. Thus, using only one
transfection, an oligonucleotide composition comprising five
different antisense oligonucleotides can be used to efficiently
inhibit protein synthesis.
Example 2
[0249] Summary of Results of Experiments in Which Oligonucleotide
Compositions were Tested on Thirty Different Genes.
[0250] FIG. 1 shows a summary of the results of about 30 antisense
inhibition experiments against about thirty different genes in cell
culture. Antisense was transfected as described in Example 1 and
inhibition analyzed by Taqman real time PCR using standard methods.
In each case the antisense inhibition was determined by comparison
to a control oligonucleotide of the same chemistry that was not
antisense to the target gene. Antisense compositions comprised 5-8
antisense oligonucleotides that had been designed against each
gene, and individual oligonucleotides where compared to the
mixtures of 5 or more antisense oligonucleotides. For three target
genes the mixtures did not work well, and these data were
eliminated from the analysis of the mixtures. Remarkably, the
mixtures inhibited approximately as well (81-vs 84%) as the best
individual oligonucleotide. The average inhibition of all
individual oligonucleotides was much lower (56%), with a much
higher variation. Thus, using the mixtures allows one to obtain
high inhibition in the vast majority of cases (.about.90% of the
target genes) without first screening through individual
oligonucleotides to select those which work best. Also, as
evidenced by the increased variation in the results obtained when
individual oligonucleotides were used, in many cases the mixture
was better than the best individual oligonucleotide.
Example 3
[0251] Ultramer Data for a Mixture of siRNA Complexes Targeting
p53.
[0252] HeLa cells were transfected with 50 nM siRNA complexed with
1 ug/mL of Lipofectamine 2000 for 24 hours. After 24 hours, cells
were lysed and RNA isolated for analysis by RT-PCR. Seven siRNA
complexes were transfected that target a unique site of the p53
gene and a mixture of all seven siRNAs (equal concentrations of
each) called the "siRNA ultramer." The best siRNA complex inhibited
the target by 87% and the ultramer inhibited 69% compared to
average of the controls.
2 P53 sequences (Antisense, Sense): siRNA1: CUGACUGCGGCUCCUCCAUTT
(SEQ ID NO:##) AUGGAGGAGCCGCAGUCAGTT (SEQ ID NO:##) siRNA2:
CUCACAACCUCCGUCAUGUTT (SEQ ID NO:##) ACAUGACGGAGGUUGUGAGTT (SEQ ID
NO:##) siRNA3: GACCAUCGCUAUCUGAGCATT (SEQ ID NO:##)
UGCUCAGAUAGCGAUGGUCTT (SEQ ID NO:##) siRNA4: GUACAGUCAGAGCCAACCUTT
(SEQ ID NO:##) AGGUUGGCUCUGACUGUACTT (SEQ ID NO:##) siRNA5:
ACCUCAAAGCUGUUCCGUCTT (SEQ ID NO:##) GACGGAACAGCUUUGAGGUTT (SEQ ID
NO:##) siRNA6: CCUCAUUCAGCUCUCGGAATT (SEQ ID NO:##)
UUCCGAGAGCUGAAUGAGGTT (SEQ ID NO:##) siRNA7: CCCUUCUGUCUUGAACAUGTT
(SEQ ID NO:##) CAUGUUCAAGACAGAAGGGTT (SEQ ID NO:##)
Example 4
[0253] Ultramer Data for a Mixture of siRNA Complexes Targeting
GTP20.
[0254] Human Mesenchymal Stems cells (hMSC) were transfected with 2
ug/mL Lipofectamine 2000 complexed to 400 nM siRNA (total
concentration, for clarity in the mixture each individual oligomer
was at 80 nM). Five siRNA duplexes targeted to GTP20 (TD), one
composition matched control duplex (CD) and an equimolar mixture of
each of the 5 oligos ("Mixture") were transfected continuously for
24 hours and RNA was harvested using the RNA Catcher (Sequitur,
Inc. Natick, Mass.). Expression of GTP20 mRNA was quantified by
Taqman and normalized to GAPDH. Inhibition of 70% or greater
relative to the control duplex was achieved using TD5 (70%) and the
Ultramer (76%).
[0255] Human mesenchymal stem cells were plated at 15,000 per well
in 48 well dishes and transfected 24 hours later. Lipofectamine
2000 was diluted in Opti-MEM to a 10.times. concentration of 20
ug/mL and incubated for 15 minutes. Following incubation, lipid was
complexed to siRNA duplexes by addition of 10.times. lipid to an
equal volume of 10.times. (4 uM) siRNA, and incubated for 15
minutes. 5.times. lipid/siRNA complexes were diluted to 1.times. by
the addition of MSC Differentiation Media. 250 ul of each 1.times.
siRNA treatment was added per well of 48 well dish. Each treatment
was applied to triplicate wells. Osteoblastic differentiation of
MSC was induced approximately 4 hours after transfection. Cells
were differentiated for 4 days prior to RNA isolation.
Example 5
[0256] Ultramer Data for a Mixture of siRNA Complexes Targeting
Cbfa-1.
[0257] Human Mesenchymal Stems cells (hMSC) were transfected with 2
ug/mL Lipofectamine 2000 complexed to 400 nM siRNA (total
concentration, in mixture each individual duplex was at 80 nM).
Five targeted duplexes (TD), five control duplexes (CD), one
equimolar mixture of all 5 duplexes ("Mixture") and one control
Mixture(UC) were transfected continuously for 72 hours. RNA was
harvested 96 hours after transfection using the RNA Catcher.
Expression of Cbfa-1 mRNA was quantified by Taqman and normalized
to GAPDH. Inhibition of 70% or greater relative to the average of
the control duplexes was achieved using TD4 (74%). The Mixture
inhibited 70% relative to the Mixture Control.
[0258] Human mesenchymal stem cells were plated at 15,000 per well
in 48 well dishes and transfected 24 hours later. Lipofectamine
2000 was diluted in Opti-MEM to a 10.times. concentration of 20
ug/mL and incubated for 15 minutes. Following incubation, lipid was
complexed to siRNA duplexes by addition of 10.times. lipid to an
equal volume of 10.times. (4 uM) siRNA, and incubated for 15
minutes. 5.times. lipid/siRNA complexes were diluted to 1.times. by
the addition of MSC Differentiation Media. 250 ul of each 1.times.
siRNA treatment was added per well of 48 well dish. Each treatment
was applied to triplicate wells. Osteoblastic differentiation of
MSC was induced approximately 4 hours after transfection. Cells
were differentiated for 4 days prior to RNA isolation. The
following antisense sequences of Cbfa-1 siRNA duplexes were used
(corresponding sense sequences where the complementary sequence
with a 2 nt TT 3' overhang, T's are DNA, all other nucleotides are
RNA):
3 TD1 (s18883): AUUUAAUAGCGUGCUGCCATT (SEQ ID NO:##) TD2 (s18885):
CUGUAAUCUGACUCUGUCCTT (SEQ ID NO:##) TD3 (s18887):
AAUAUGGUCGCCAAACAGATT (SEQ ID NO:##) TD4 (s18889):
GUCAACACCAUCAUUCUGGTT (SEQ ID NO:##) TD5 (s18891):
AGGUUUAGAGUCAUCAAGCTT (SEQ ID NO:##) CD1 (s18884):
ACCGUCGUGCGAUAAUUUATT (SEQ ID NO:##) CD2 (s18886):
CCUGUCUCAGUCUAAUGUCTT (SEQ ID NO:##) CD3 (s18888):
AGACAAACCGCUGGUAUAATT (SEQ ID NO:##) CD4 (s18890):
GGUCUUACUACCACAACUGTT (SEQ ID NO:##) CD5 (s18892):
CGAACUACUGAGAUUUGGATT (SEQ ID NO:##)
Example 6
[0259] Ultramer Data for a Mixture of siRNA Complexes Targeting PTP
mu.
[0260] Efficacy of all phosphorothioate DNA 25 nt antisense
oligonucleotides targeted against PTP mu mRNA in human lung
carcinoma (A549) cells. Potent inhibition of mRNA was obtained
following a 16 hour transfection of A549 cells with 25 nM oligo.
AS: antisense oligonucleotide; RC: reverse control; MIX: mixture of
individual AS oligomers (total oligomer concentration of 25 nM).
Target mRNA quantity was normalized to GAPDH.
[0261] A549 cells at passage 3 were plated at 25,000 cells/well in
48 well plates and incubated overnight in a humidified 5% CO.sub.2
chamber (37.degree. C.). A 250 nM solution of AS oligomer in
Optimem-1 (Gibco BRL) was mixed with an equal volume of 10 ug/mL
lipofectamine 2000 (InVitrogen) in Optimem-I (lipid solution was
pre-incubated at 25 C. for 15 minutes). Oligomer-lipid complexes
were formed by incubation at room temperature for 15 minutes. 4
volumes of DMEM plus 10% fetal serum medium was added to the
complexes and 250 ul of the diluted suspension was added to cells.
The final concentration of oligomer was 25 nM. Following a 16 h
transfection, cells ware washed with PBS and poly A+ mRNA was
isolated using Sequitur's mRNA Catcher. mRNA was quantified by real
time RT-PCR (Taqman); automated data collection was with an ABI
prism.RTM. sequence detection system. Data are normalized to GAPDH
mRNA. Oligonucleotide sequences: AS1, CAUUCACCAGCAUGAGAGAACCUGA
(SEQ ID NO: ##); AS2, TCCCAGAGGCATTCACCAGCATGAG (SEQ ID NO: ##);
AS3, UCCAGAUAGGAUUCCCCAGUGGCCC (SEQ ID NO: ##); AS4,
CUGGUCAGGAGCACACUAAUCUCAU (SEQ ID NO: ##); AS5,
AGUCAAGGUGUUCACUUGCUCCCAA (SEQ ID NO: ##); AS6,
AAGUACUAAUGGCCAGUUCUGCCC (SEQ ID NO: ##); AS7,
CCCUGUAACCAGAGCCUGUCUCCUG (SEQ ID NO: ##); AS8,
GAGCUGGUCACCUUGAUUUCCUUCA (SEQ ID NO: ##); AS9,
CCAGGCAAGUCCCAAGUGUCCUCAU (SEQ ID NO: ##); AS10,
GAUGUCCUAACACCUUCACCUCAUC (SEQ ID NO: ##); MIX, equimolar solution
of AS1 through AS10.
Example 7
[0262] Ultramer Data for a Mixture of siRNA Complexes Targeting
PTP-PEST
[0263] Efficacy of 25 nt phosphorothioate DNA antisense
oligonucleotides targeted against PTP-PEST mRNA in Human Umbilical
Vein Endothelial Cells (HuVEC). Inhibition of mRNA was obtained
following a 4 hour serum-free transfection of cells with 200 nM
oligo followed by a 14 h incubation in serum-containig medium. AS:
antisense oligonucleotide; RC: reverse control; Mixture: mixture of
individual AS oligomers (total oligo concentration of 200 nM).
Target mRNA quantity is normalized to GAPDH.
[0264] HuVEC cells at passage 3 were plated at 25,000 cells/well in
48 well plates and incubated overnight in a humidified 5% CO.sub.2
chamber (37.degree. C.). A 2000 nM solution of AS oligomer in
Optimem-I (Gibco BRL) was mixed with an equal volume of 100 ug/mL
Lipofectin (Gibco BRL) in Optimem-I (lipid solution was
pre-incubated at 25.degree. C. for 30 minutes). Oligomer-lipid
complexes were formed by incubation at room temperature for 30
minutes. 4 volumes of Optimem-I (serum-free) was added to the
complexes and 250 ul of the diluted suspension was added to cells.
Four hours later, the transfection complexes were aspirated and
replaced with 250 ul of EGM-2 complete serum medium
(Clonetics/Biowhittaker). Following a 16 h transfection, cells ware
washed with PBS and poly A+ mRNA was isolated using an mRNA Catcher
(Sequitur, Inc.). mRNA was quantified by real time RT-PCR (Taqman);
automated data collection was with an ABI prism.RTM. sequence
detection system.
[0265] Data are normalized to GAPDH mRNA. AS1,
CCCAUUGUGGUCAGGACUCUUCAUGU (SEQ ID NO: ##); AS2,
UUCCCAUCUCAAAUUCU-CGGCAGGCU (SEQ ID NO: ##); AS3,
UGGCACAAAUGGCACCUGUUCUUCCU (SEQ ID NO: ##); RC,
GACUCCUUUAAGUAGGUCUCCCAGG- U (SEQ ID NO: ##). MIX, equimolar
solution of AS1, AS2, and AS3.
Example 8
[0266] Ultramer Data for a Mixture of siRNA Complexes Targeting
PTP-eta.
[0267] Efficacy of all phosphorothioate DNA 25 nt antisense
oligonucleotides targeted against PTP-eta mRNA in Normal Rat Kidney
(NRK) cells. Inhibition of mRNA was obtained following an overnight
transfection of cells with 25 nM oligo. AS: antisense
oligonucleotide; RC: reverse control; Mix: mixture of individual AS
oligomers (total oligomer concentration of 25 nM). Target mRNA
quantity is normalized to GAPDH.
[0268] NRK cells at passage 5 were plated at 25,000 cells/well in
48 well plates and incubated overnight in a humidified 5% CO2
chamber (37.degree. C.). A 250 nM solution of AS oligomer in
Optimem-I (Gibco BRL) was mixed with an equal volume of 10 ug/mL
Lipofectamine 2000 (InVitrogen) in Optimem-I (lipid solution was
pre-incubated at 25 C. for 30 minutes). Oligomer-lipid complexes
were formed by incubation at room temperature for 15 minutes. 4
volumes of complete DMEM plus 5% bovine calf serum were added to
the complexes and 250 ul of the diluted suspension was layered onto
cells. The final oligomer concentration was 25 nM. Following a 16 h
incubation, cells ware washed with PBS and poly A+ mRNA was
isolated using Sequitur's mRNA Catcher*. mRNA was quantified by
real time RT-PCR (Taqman*); automated data collection was with an
ABI prism.RTM. sequence detection system.
[0269] Data are normalized to GAPDH mRNA. AS 1,
ACCUGUGCACACAACCUGGCCCUGGU (SEQ ID NO: ##); AS2,
ACAGUAUACCGCAGCGUGUUUCCCUU (SEQ ID NO: ##); AS3,
GUCUCAUUGACUGUUCCCAAGGUGAU (SEQ ID NO: ##); AS4,
GCUCUACAAUCUGCAUCCGGUAAG- AU (SEQ ID NO: ##); AS5,
UCUGUGCCAUCUGCUGCUUGAGAAUU (SEQ ID NO: ##); AS6,
UGUUCACAGCUCGGAUGUCAGAAACU (SEQ ID NO: ##);RC,
UAAGAGUUCGUCGUCUACCGUGUCUU (SEQ ID NO: ##); MIX, equimolar solution
of AS1 through AS6
[0270] Equivalents
[0271] 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.
Sequence CWU 1
1
58 1 10 PRT Artificial Sequence Description of Artificial Sequence
Illustrative peptide 1 His Ile Trp Leu Ile Tyr Leu Trp Ile Val 1 5
10 2 16 PRT Drosophila sp. 2 Arg Gln Ile Lys Ile Trp Phe Gln Asn
Arg Arg Met Lys Trp Lys Lys 1 5 10 15 3 27 PRT Artificial Sequence
Description of Artificial Sequence Synthetic peptide 3 Gly Trp Thr
Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys
Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 4 25 PRT Human
immunodeficiency virus 4 Cys Phe Ile Thr Lys Ala Leu Gly Ile Ser
Tyr Gly Arg Lys Lys Arg 1 5 10 15 Arg Gln Arg Arg Arg Pro Pro Gln
Cys 20 25 5 15 PRT Human immunodeficiency virus 5 Cys Gly Arg Lys
Lys Arg Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10 15 6 19 PRT
Human immunodeficiency virus 6 Cys Leu Gly Ile Ser Tyr Gly Arg Lys
Lys Arg Arg Gln Arg Arg Pro 1 5 10 15 Pro Gln Cys 7 37 PRT Human
immunodeficiency virus 7 Gly Cys Phe Ile Thr Lys Ala Leu Gly Ile
Ser Tyr Gly Arg Lys Lys 1 5 10 15 Arg Arg Gln Arg Arg Arg Pro Pro
Gln Gly Ser Gln Thr His Gln Val 20 25 30 Ser Leu Ser Lys Gln 35 8
25 RNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 8 gcaguauacc ucucgcucuu gucaa 25 9 25 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 9 uuuggaaguu cuccaugaag cgcca 25 10 25 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 10 guccaaaguc ugcuagcuug auggc 25 11 25 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 11 cccaggagga uuucaggagc ucggu 25 12 25 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 12 uagaaguaac uccuggccac accac 25 13 25 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 13 aacuguucuc gcucuccaua ugacg 25 14 21 DNA
Artificial Sequence Description of DNA/RNA hybrid Synthetic
oligonucleotide 14 cugacugcgg cuccuccaut t 21 15 21 DNA Artificial
Sequence Description of DNA/RNA hybrid Synthetic oligonucleotide 15
auggaggagc cgcagucagt t 21 16 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 16
cucacaaccu ccgucaugut t 21 17 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 17
acaugacgga gguugugagt t 21 18 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 18
gaccaucgcu aucugagcat t 21 19 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 19
ugcucagaua gcgaugguct t 21 20 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 20
guacagucag agccaaccut t 21 21 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 21
agguuggcuc ugacuguact t 21 22 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 22
accucaaagc uguuccguct t 21 23 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 23
gacggaacag cuuugaggut t 21 24 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 24
ccucauucag cucucggaat t 21 25 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 25
uuccgagagc ugaaugaggt t 21 26 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 26
cccuucuguc uugaacaugt t 21 27 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 27
cauguucaag acagaagggt t 21 28 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 28
auuuaauagc gugcugccat t 21 29 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 29
cuguaaucug acucugucct t 21 30 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 30
aauauggucg ccaaacagat t 21 31 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 31
gucaacacca ucauucuggt t 21 32 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 32
agguuuagag ucaucaagct t 21 33 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 33
accgucgugc gauaauuuat t 21 34 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 34
ccugucucag ucuaauguct t 21 35 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 35
agacaaaccg cugguauaat t 21 36 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 36
ggucuuacua ccacaacugt t 21 37 21 DNA Artificial Sequence
Description of DNA/RNA hybrid Synthetic oligonucleotide 37
cgaacuacug agauuuggat t 21 38 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 38
cauucaccag caugagagaa ccuga 25 39 25 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 39
tcccagaggc attcaccagc atgag 25 40 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 40
uccagauagg auuccccagu ggccc 25 41 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 41
cuggucagga gcacacuaau cucau 25 42 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 42
agucaaggug uucacuugcu cccaa 25 43 24 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 43
aaguacuaau ggccaguucu gccc 24 44 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 44
cccuguaacc agagccuguc uccug 25 45 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 45
gagcugguca ccuugauuuc cuuca 25 46 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 46
ccaggcaagu cccaaguguc cucau 25 47 25 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 47
gauguccuaa caccuucacc ucauc 25 48 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 48
cccauugugg ucaggacucu ucaugu 26 49 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 49
uucccaucuc aaauucucgg caggcu 26 50 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 50
uggcacaaau ggcaccuguu cuuccu 26 51 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 51
gacuccuuua aguaggucuc ccaggu 26 52 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 52
accugugcac acaaccuggc ccuggu 26 53 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 53
acaguauacc gcagcguguu ucccuu 26 54 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 54
gucucauuga cuguucccaa ggugau 26 55 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 55
gcucuacaau cugcauccgg uaagau 26 56 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 56
ucugugccau cugcugcuug agaauu 26 57 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 57
uguucacagc ucggauguca gaaacu 26 58 26 RNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 58
uaagaguucg ucgucuaccg ugucuu 26
* * * * *