Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1996-06-17
2002-12-03
Wang, Andrew (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100
Reexamination Certificate
active
06489464
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to synthetic oligonucleotides that have an inhibitory effect upon pathogens. More particularly, the invention relates to anti-pathogen synthetic oligonucleotides having superior inhibitory properties relative to known oligonucleotides.
2. Summary of the Related Art
The use of synthetic oligonucleotides as anti-infecting agents has recently developed into a promising field. Agrawal, Trends in Biotechnology 10:152-158 (1992), reviews the development of antisense oligonucleotides as antiviral agents. Synthetic oligonucleotides now show considerable promise not only as antiviral agents, but as inhibitors of other pathogens as well. Rapaport et al., Proc. Natl. Acad. Sci. USA 89:8577-8580 (1992), discloses antimalarial activities of oligonucleotide phosphorothioates against Plasmodium falciparum.
Due to the great promise of synthetic oligonucleotides as anti-infective agents, considerable interest has developed in improving the pharmacological properties of such compounds. Many such efforts have involved introducing modified internucleoside linkages into oligonucleotides, thereby providing increased resistance to nucleolytic degradation and improving biostability. Agrawal et al., Proc. Natl. Acad. Sci. USA 85:7079:7083 (1988), teaches inhibition of human immunodeficiency virus (HIV) propagation by oligodeoxynucleotide phosphorothioates and phosphoramidites. Sarin et al., Proc. Natl. Acad. Sci. USA 85:7448-7451 (1988), discloses inhibition of HIV by oligodeoxynucleotide methylphosphonates. Padmapriya and Agrawal, Bioorganic & Medicinal Chemistry Letters 3:761-764 (1993), discloses oligonucleotides having novel methylphosphonothioate internucleoside linkages.
Goodchild and Zamecnik (U.S. Pat. No. 4,806,463) disclose antisense oligonucleotides that inhibit HTLV-III replication and protein expression. These oligonucleotides are targeted to highly conserved regions in the HTLV-III genome. Among the sites targeted are: a) the rRNA
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primer binding site, b) regions of the HTLV-III genome vicinal in the 5′ direction to the rRNA
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primer binding site, c) the tRNA
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primer binding site and regions of the HTLV-III genome vicinal in the 5′direction to the tRNA
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primer binding site, d) the mRNA donor splice sites, e) the mRNA acceptor splice sites, f) the initiator codon for the gag gene, g) the initiator condon for the env gene, h) the initiator codon for the tat gene, i) the initiator codon for the sor gene, j) the initiator codon for the 3′ orf gene, k) the cap nucleotide of the HTLV-III genome, l) the art gene or portions thereof, m) and the region of the HTLV-III genome encoding a grameshift.
Other modifications, not necessarily involving solely the use of modified phosphodiester internucleotide linkage, have included the introduction of chemical blocking structures at the 3′ end of oligonucleotides. Temsamani et al., Annals of the New York Academy of Sciences 660:318-320 (1992), teaches that 3′-capped oligonucleotide phosphorothioates have superior in vivo pharmacokinetics and bioability relative to uncapped oligonucleotide phosphorothioates. Tang et al., Nucleic Acids Res. 21:2729 (1993), teaches that oligonucleotides having 3′ terminal hairpin structures demonstrate superior biostability. Koga et al., J. Org. Chem. 56:3757-3759 (1991), reports improved nuclease resistance for alternating &agr;,&bgr;-oligothymidylates having alternating (3′ to 3′) and (5′ to 5′) internucleotide phosplhodiester linkages. Seliger et al., Nucleosides & Nucleotides 10:469-477 (1991), and Ortigao et al., Antisense Research & Development 2:129-146 (1992), disclose improved resistance to nucleolytic degradation for oligodeoxynucleotides having single terminal 3′ to 3′ and 5′ to 5′ linkage inversions.
Methods for synthesizing oligonucleotides having at least one 3′ to 3′ internucleoside linkage can involve the use of modified nucleoside monomers that allow 5′ to 3′ synthesis or the use of linkers from which dual 3′ to 5′ synthesis of oligonucleotides having 3′ to 3′ and 5′ to 5′ linkages using commercially available 5′ phosphoramidite nucleoside monomers. CLONETECHniques (April 1993) discloses synthesis of oligonucleotides having a single 3′ to 3′ internucleoside linkage using a commercially available branched linker. Horne and Dervan, J. Am. Chem. Soc. 112:2435-2437 (1990); Luebke and Dervan, Nucleic Acids Res. 20:3005-3009 (1992); and van de Sande et al., Science 241:551-557 (1988), disclose similar synthesis of oligonucleotides having a single 3′ to 3′ linkage for the purpose of studying alternate strand triple helix formation or parallel stranded DNA.
Certain potential problems for effective use of synthetic oligonucleotides as anti-infective therapeutics arise, however, from the nature of the target, rather than the oiligonucleotide, and are thus not addressed by improving the biostability of the oligonucleotide. One such problem is the potential for an infective agent to escape oligonucleotide-mediated therapy by mutation of the target sequence, thereby reducing the ability of the oligonucleotide to interact with the target sequence. For example, Lisziewicz et al., Proc. Natl. Acad. Sci. USA 89:11209-11213 (1992) teaches that a splice acceptor site antisense oligonucleotide initially suppressed HIV in infected MOLT-3 cells, but that after 25 days, viral breakthrough of the suppression was observed. This report suggests that combined or sequential treatment with oligonucleotides complementary to distinct targets may be useful in avoiding viral breakthrough.
There remains a need for additional means of providing biostability to synthetic oligonucleotides that inhibit pathogens. There also remains a need for new ways to avoid mutation-derived breakthrough of pathogens. Ideally, an oligonucleotide should be developed that overcomes both of these problems.
BRIEF SUMMARY OF THE INVENTION
The invention relates to synthetic oligonucleotides that have an inhibitory effect upon pathogens. The invention provides anti-pathogen synthetic oligonucleotides that have superior inhibitory properties, relative to known oligonucleotides. The superior inhibitory properties of oligonucleotides according to the invention are the result of the primary structural feature of such oligonucleotides, which is the linking together of two or more oligonucleotide sequences that are complementary to one or more essential gene of one or more pathogen.
In a first aspect, the invention provides oligonucleotides having two or more identical oligonucleotide sequences linked together, wherein each oligonucleotide sequence is complementary to the same target sequence of a pathogen. The target sequence is the sequence of a portion of a gene or regulatory sequence that is essential for the propagation of the pathogen. According to this aspect of the invention, the identical oligonucleotide sequences may be linked together in a 5′ to 3′ configuration or in a 3′ to 3′ configuration. In this latter configuration, the oligonucleotide is highly resistant to nucleolytic degradation.
In a second aspect, the invention provides oligonucleotides having two or more different oligonucleotide sequences linked together, wherein each oligonucleotide sequence is complementary to a different target sequence of the same pathogen. The different oligonucleotide sequences may be complementary to different portions of the same gene or regulatory sequence or they may be complementary to different genes and/or regulatory sequences. Oligonucleotides according to this aspect of the invention provide improved resistance to mutation-induced pathogen escape from the inhibitory effect of the oligonucleotide. The different oligonucleotide sequences may be linked together in 5′ to 3′ or 3′ to 3′ configurations, with the latter configuration providing greatly increased resi
Agrawal Sudhir
Meschwitz Susan
Hale and Dorr LLP
Hybridon, Inc.
Wang Andrew
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