Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-25
2004-01-13
LeGuyader, John L. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024310, C536S024330
Reexamination Certificate
active
06677445
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to antisense oligonucleotides, and more particularly to chimeric antisense oligonucleotides which exhibit high resistance to endo- and exonucleases, high sequence specificity, and the ability to activate RNAse H, as evidenced by efficient and long-lasting knockout of target mRNA. Also provided are formulations of the oligonucleotides with carrier molecules which provide efficient transfection into cells.
BACKGROUND OF THE INVENTION
The use of antisense oligonucleotides to specifically inhibit the function of targeted genes has been the subject of extensive research, due to its promise in selective antiviral and anticancer therapy. Many studies have been directed to the design of oligonucleotide analogs having an optimal combination of properties, including stability (i.e. resistance to cellular nucleases), cellular uptake, DNA/RNA binding affinity and specificity, and efficiency of inhibition. Because the phosphodiester linkages of native nucleic acids are degraded by endo- and exonucleases, many early studies were directed to designing nuclease-resistant analogs. Phosphorothioates are one such class of compounds, which are relatively stable in vivo and retain the ability to activate RNAse H, the primary mechanism by which antisense oligonucleotides deactivate target RNA. However, the use of phosphorothioates presents several disadvantages, including a high level of non-specific binding to other cellular components, often leading to unwanted side effects, and reduced binding affinity for RNA.
Oligomeric ribonucleotides substituted at the 2′-oxygen show high RNA binding affinities and, in comparison to the unsubstituted ribonucleotides, reduced sensitivity to endogenous nucleases. Although 2′-O-methyl substituted ribonucleotides provide greater binding affinity than those having larger substituents (e.g. ethyl, propyl, pentyl, allyl), the larger substituents are reported to confer greater exonuclease resistance (see, for example, Monia et al.,
J. Biol. Chem
. 271(24): 14533, 1996). Arrow et al. (U.S. Patent No. 5,849,902) stated that “2′-O-methyl bases with phosphodiester linkages are degraded by exonucleases and so are not suitable for use in cell or therapeutic applications of antisense.” Phosphorothioate and phosphotriester linkages were recommended by the latter group as having greater stability, even though they presented the disadvantages of reduced binding affinity, more difficult synthesis (phosphotriester) and/or greater toxicity (phosphorothioate).
Therefore, there is still a need for antisense oligonucleotide compositions with optimal combinations of antisense activity, target binding affinity, biocompatibility, and stability.
SUMMARY OF THE INVENTION
The present invention includes, in one aspect, a chimeric oligonucleotide having the formula 5′-W-X
1
-Y-X
2
-Z-3′, where W represents a 5′-O-alkyl nucleotide, such as a 5′-O-alkyl thymidine; each of X
1
and X
2
represents a block of seven to twelve phosphodiester-linked 2′-O-alkyl ribonucleotides; Y represents a block of five to twelve phosphorothioate-linked deoxyribonucleotides; and Z represents a blocking group effective to block nuclease activity at the 3′ end of the oligonucleotide. In one embodiment, Z is a 3-to-3′ linked nucleotide. In further embodiments, the alkyl groups of the 5′-O-alkyl nucleotide and/or the 2′-O-alkyl ribonucleotides are methyl groups. In still further embodiments, groups W and/or Z are linked to X
1
and X
2
, respectively, via phosphodiester linkages, phosphotriester, phosphorothioate, or phosphoramidate linkages. Preferably, W is linked via a phosphodiester or phosphorothioate linkage, and Z is linked via a relatively nuclease-resistant linkage; i.e. a phosphotriester, phosphorothioate, or phosphoramidate linkage.
In specific embodiments, the segment X
1
-Y-X
2
of the chimeric oligonucleotide has a sequence represented by any of SEQ ID NOs: 1-24 disclosed herein.
In another aspect, the invention provides a therapeutic composition which comprises an oligonucleotide as described above in a pharmaceutically acceptable vehicle. In preferred embodiments, the vehicle includes a lipid-cationic peptoid conjugate or “lipitoid”. One class of lipid-cationic peptoid conjugates includes compounds of the formula:
L-linker-[N(CH
2
CH
2
NH
2
)CH
2
(C═O)—N(CH
2
CH
2
R)CH
2
(C═O)—N(CH
2
CH
2
R)CH
2
(C═O)]
3
—NH
2
,
where the lipid group L is a fatty acid-derived group, such as a phospholipid group (i.e. ROOCCH
2
CH(COOR)CH
2
OP(O)
2
O—), having fatty alkyl or alkenyl chains between about 8 and 24 carbon atoms in length, or a steroid-derived group, such as a cholesteryl group, and the portion of the molecule to the right of the linker is the peptoid segment. In the peptoid segment, R is selected from alkyl (branched or unbranched), aminoalkyl, and aralkyl. As used herein, “aralkyl” refers to an alkyl, preferably lower alkyl, substituent which is further substituted with an aryl group; one example is a benzyl group. “Aryl” refers to a substituted or unsubstituted monovalent aromatic radical having a single ring (e.g., benzene) or two condensed rings (e.g., naphthyl). This term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole. By “substituted” is meant that one or more ring hydrogens in the aryl group is replaced with a substituent, preferably selected from a halide, a lower alkyl or lower alkoxy group, halomethyl, or haloethyl.
In specific embodiments, R is isopropyl or 4-methoxyphenyl. A single lipitoid may include different groups R, or they may be the same within the molecule.
The linker may be a direct bond, or it may be a substantially linear linking group, such as an oligopeptide or an alkyl chain, of any effective length. The linker may also be an alkyl chain having one or more heteroatom-containing linkages, selected from the group consisting of ester, amide, carbonate, carbamate, disulfide, peptide, and ether, at either terminus of the chain or intervening between alkyl bonds. In selected embodiments, the linker is from 2 to about 30 atoms, or from 3 to about 15 atoms, in length.
In another aspect, the invention provides a method of inhibiting expression of a target gene in a subject, which comprises administering to the subject, in a pharmaceutically acceptable vehicle, an amount of a chimeric oligonucleotide effective to specifically hybridize to all or part of a selected target nucleic acid sequence derived from the gene, where the chimeric oligonucleotide has a structure as described above. In one embodiment, the target nucleic acid sequence is a mRNA derived from the target gene. In specific embodiments, the segment X
1
-Y-X
2
of the chimeric oligonucleotide has a sequence represented by any of SEQ ID NOs: 1-24 disclosed herein. In further embodiments, the vehicle includes a lipid-cationic peptoid conjugate such as described above.
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Innis Michael A.
Reinhard Christoph J.
Zuckermann Ronald N.
Blackburn Robert P.
Chiron Corporation
Collier Steven W.
Gorthey LeeAnn
LeGuyader John L.
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