Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-05-25
2004-01-06
Shukla, Ram R. (Department: 1635)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S091100, C435S366000, C536S023100, C536S024500
Reexamination Certificate
active
06673611
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to novel chemically modified nucleic acid molecules that are capable of modulating gene expression through a variety of mechanisms. Specifically, the invention concerns novel combinations of chemical modifications in an oligonucleotide which enhance nuclease resistance, binding affinity, and/or potency.
The following is a discussion of relevant art, none of which is admitted to be prior art to the present invention.
Since the discovery of the mechanisms underlying gene expression, specifically nucleic acid based transcription and translation, a great deal of effort has been placed on blocking or altering these processes for a variety of purposes, such as understanding biology, gene function, disease processes, and identifying novel therapeutic targets. Approaches involving nucleic acid molecules for modulating gene expression have gained popularity in recent years. For example, nucleic acid molecules have been designed which are capable of binding to specific mRNA sequences by Watson-Crick base pairing interaction and blocking translation (Crooke, 1996, Medicinal Res. Rev. 16, 319-344). Another approach involves complexation of DNA with triplex forming oligonucleotides to prevent transcription of bound DNA sequences thereby inhibiting gene expression (Kim et al., 1998, Biochemistry. 37, 2299-2304). The interaction of antisense oligonucleotides, 2-5A antisense chimera, or ribozymes with target RNA have been used to prevent gene expression. All of these nucleic acid molecules are highly specific to their matching target sequences and therefore can offer lower toxicity compared to traditional approaches such as chemotherapy.
The use of oligonucleotides for modulation of gene expression generally requires stabilization of oligonucleotides from degradation by nucleases that are present in biological systems. Cellular efficacy can be effected if the nucleic acid molecule is degraded before it reaches its desired target. Chemical modifications of nucleic acid molecules have been found to be advantageous in making them inaccessible to degradation by cellular nucleases. Uhlmann and Peyman, 1990, Chem. Reviews 90, 543, review the use of nucleoside modifications to stabilize antisense oligonucleotides. Besides improved stability, chemical modifications have also been shown to increase binding affinity, improve cellular penetration, and enhanced target specificity (Monia et al., 1993, J. Biol. Chem. 268, 14514-14522; Wu-Pong, 1994, BioPharm, 22-33).
One of the most studied and utilized chemical alteration in oligonucleotides has been backbone modifications such as phosphorothioates. Phosphorothioate oligonucleotides are nucleic acid molecules whose phosphodiester linkage has been modified by substituting a sulfur atom in place of an oxygen atom. In addition to increased nuclease resistance, phosphorothioate oligonucleotides are substrates for ribonuclease H (RNase H) (Monia, supra; Crooke et al., 1995, Biochem. J. 3112, 599-608). RNase H is an endonuclease which catalyzes the degradation of RNA in an RNA-DNA heteroduplex (Hostomsky et al., 1993 in Nucleases, Linn et al., eds., Cold Spring Harbor Laboratory Press, NY, 341-376). RNA/DNA heteroduplexes, called Okazaki fragments, are formed naturally during DNA replication. Therefore, the normal function of RNase H is to degrade the RNA portion of the heteroduplex to complete DNA replication. In experiments with
E. coli
RNase H, the phosphorothioate oligonucleotide activated the enzyme more efficiently (2-5 fold) compared to a standard phosphodiester containing oligonucleotide (Crooke, 1995, supra).
Binding of DNA to RNA is not as thermodynamically favorable as an RNA to RNA interaction (Altmann et al., 1996, Chimia 50, 168-176). Inoe & Ohtsuka, 1987, Nucleic Acids Research 115, 6131, first proposed an oligonucleotide with a central region consisting of oligodeoxynucleotides flanked by 2′-O-methyl modified nucleotide regions. The region of oligodeoxynucleotides in such a chimeric molecule is recognized by RNase H when bound to target RNA; and facilitates cleavage of target RNA by RNase H. (Inoe & Ohtsuka, 1987, FEBS Lett. 215, 327; Shibahara & Morisava, 1987, Nucleic Acids Res. 15, 4403). Such chimeric oligonucleotides were proposed to interact with target RNA more stably than an all DNA oligonucleotide.
Subsequent developments included the introduction of nuclease resistant modifications of the chimeric oligonucleotides, such as methylphosphonates (Tidd & Gibson, 1988, Anticancer Drug Design 3, 117), phosphorothioates (Agrawal & Pederson, 1990, Proc Nat. Acad. Sci. USA 87, 1407), and phosphoramidates (Potts & Runyun, 1991, Proc Nat. Acad. Sci. USA 88, 1516). Additionally, the flanking sequences have been modified with 2′-O-methyl and 2′-F-modifications (Cook, 1993, Antisense Research and Applications, CRC Press, 150-181).
Agrawal et al., U.S. Pat. No. 5,652,355, describe a phosphorothioate-containing nucleic acid molecule with at least two 2′-O-methyl modifications on the 5′ and 3′ ends.
Agrawal, U.S. Pat. No. 5,652,356, describes an oligonucleotide which consists of a region of 2′-O-substituted oligonucleotide located between two oligodeoxyribonucleotide regions. The DNA regions of this nucleic acid molecule consists of phosphorothioate modifications at every position.
Cook et al., U.S. Pat. No. 5,623,065, describe the use of a nucleic acid molecule which contains an RNase H cleavable region flanked by certain specifically modified nucleotides, for inhibition of gene expression of a ras gene.
Cook et al., U.S. Pat. No. 5,587,362, describe a nucleic acid molecule having “substantially chirally pure inter-sugar linkages”, for modulation of gene expression.
Ohtsuka et al., U.S. Pat. No. 5,013,830, describe mixed oligomers having a DNA region and a 2′-O-methyl modified region, useful for modulation of gene expression.
Walder et al., U.S. Pat. No. 5,491,133, describe a method for modulating gene expression using chimeric oligonucleotides with 3′-phosphodiester linkage modifications.
Cohen et al., U.S. Pat. No. 5,276,019, and Cohen et al., U.S. Pat. No. 5,264,423 describe the use of oligodeoxynucleotides of no more than 32 nucleotides in length, containing at least one phosphorothioate internucleoside linkage which are capable of preventing foreign nucleic acid replication.
Cohen et al., U.S. Pat. No. 5,286,717, describe an oligodeoxyribonucleotide with at least one phosphorothioate modification capable of inhibiting oncogenes.
Sproat et al., U.S. Pat. No. 5,334,711, describe 2′-O-R modified hammerhead and hairpin ribozymes, where R is alkyl, alkynyl or alkenyl.
Crooke et al., 1996, Exp. Opin. Ther. Patents 6, 855, list and discuss various patents and PCT publications in the field of antisense technology.
Sproat et al., U.S. Pat. No. 5,678,731, describe 2′-O-R modified oligonucleotides where R is alkyl, alkynyl or alkenyl.
Usman et al., U.S. Pat. No. 5,652,094, describe enzymatic nucleic acid molecules which include nucleic acid analogues or deoxyribonucleotides.
Joyce, International Publication No. WO 96/17086, describes a DNA enzyme capable of cleaving RNA.
Rossi et al., U.S. Pat. No. 5,144,019, describe chimeric hammerhead ribozymes with the binding arms and stem II region modified with deoxyribonucleotides.
Arrow et al., U.S. Pat. Nos. 5,989,912 and 5,849,902, describe three component chimeric antisense oligonucleotides.
Tullis, U.S. Pat. No. 5,919,619, describes methods for inhibiting target protein expression with specific antisense nucleic acid molecules.
Walder et al., U.S. Pat. No. 5,144,019, describe the use of specific oligodeoxynucleotides modified at the 3′-terminal internucleotide link as therapeutic agents by a method of hybridizing the modified oligonucleotide to a complementary sequence within a targeted mRNA and cleaving the mRNA within the RNA-DNA helix by the enzyme RNaseH to block the expression of the corresponding gene.
Crooke et al., U.S. Pat. No. 6,001,653, describe the use of specific antisense ol
Beigelman Leonid
Bellon Laurent
Haeberli Peter
Jarvis Thale
Karpeisky Alexander
McDonnell & Boehnen Hulbert & Berghoff
Shukla Ram R.
Sirna Therapeutics, Inc.
Zara Jane
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