Chimeric molecules targeted to viral RNAs

Details Chimeric molecules targeted to viral RNAs Chimeric molecules targeted to viral RNAs

1997-10-14

2001-08-07

McGarry, Sean (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S199000, C514S04400A, C514S046000, C514S047000, C536S023100, C536S024100, C536S025200

Reexamination Certificate

active

06271369

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of cleaving specific strands of RNA.
BACKGROUND OF THE INVENTION
Antisense oligonucleotides hold considerable promise both as research tools for inhibiting gene expression and as agents for the treatment of a myriad of human diseases. However, the targeted destruction of RNA using antisense oligonucleotides has been difficult to achieve in a versatile, efficient, and reliable manner.
The potential of oligonucleotides as chemotherapeutic agents has been long appreciated. Levene and Stollar (
Peogr. Allergy,
12:161 (1968)) found that tetra-and pentanucleotides partially inhibited the binding of nucleic acids to systemic lupus erythematosus sera, while Shen (
Cherm. Inemat. Ed,
9:678 (1970)) suggested the design of high affinity oligonucleotide inhibitors of similar antigen-antibody complexes. Miller, Ts'o, and associates were the first to attempt to capitalize on nucleic acid hybridization through the preparation of a series of trinucleotides modified through phosphotriester, 2′-O-methyl or methylphosphonate substitution (Miller, et al.,
Biochemisty,
13:4887). These short, modified DNA sequences, complementary to t-RNA anticodon regions, were found to be able to inhibit protein translation. Zamecnik and Stephenson (
Proc. Natl. Acad. Sci. U.S.A.,
74:280 (1978)) used a similar strategy to synthesize a 21 deoxyribonucleotide sequence which inhibited the replication of Rous sarcoma virus.
These early studies were precursors to what is now a burgeoning area of scientific research, i.e. the use of antimessenger/antisense polynucleotides to specifically regulate gene expression. In 1986, additional enthusiasm for this approach was generated by the demonstration that human immunodeficiency virus (HIV) could be inhibited through the use of antisense oligonucleotides (Zamecnik, et al.,
Proc. Natl. Acad Sci U.S.A.,
83:7706 (1986)). Although the mechanism of action of such antisense reagents is complex and not well understood, it has been demonstrated that complexes of target messenger RNA and complementary oligo-&bgr;-deoxynucleotides are degraded in vivo by the enzyme RNase H, which is present in both eukaryotes and prokaryotes. However, it has been found that many modified antisense oligonucleotides synthesized to improve delivery, cell penetration, or stability will form hybrids with sense strands of RNA but will not act as substrates for RNase H.
Another antisense mechanism which has been found to be operative to some extent is the inhibition of protein synthesis by the passive mechanism of hybridization arrest. By hybridizing an antisense oligonucleotide to a target RNA sequence, the translation of the RNA molecule containing the target sequence can be prevented, thereby inhibiting synthesis of the protein encoded by the RNA molecule. However, because the hybridization of an antisense oligonucleotide to its target ribonucleotide sequence is reversible, this technique cannot totally prevent the translation of the target RNA sequence.
Considerable effort has therefore been directed to the development of oligonucleotides which are able to induce chemical alteration or strand scission of a target RNA molecule. Thus, oligonucleotides have been modified with photoreactive agents such as psoralen or porphyrin (Lee, et al.,
Nucleic Acids Res.,
16:10681 (1988)); oxidative nuclease metal ion complexes such as porphyrin-iron (Doan, et al.,
Biochemistry,
25:6736 (1986)); phenanthroline-copper (Chen, et al.,
Proc. Natl. Acad. Sci. USA,
83:7147 (1986)) and ethylene diamine tetraacetic acid-iron (Dreyer, et al,
Proc. Natl. Acad. Sci USA,
82:963 (1985)); nucleases such as staphylococcal nuclease (Corey, et al.,
J. Am. Chem. Soc,
111:8523 (1989)) and RNase P (Li, et al.,
Pro. Natl. Acad. Sci. USA,
89:3185 (1992)); and catalytic rRNA sequences (Rossi, et al.,
Pharmac. Ther.,
50:245 (1991)). Some of these references discuss methods of digesting RNA molecules having a specific nucleotide sequence.
Ying Li et al. (
Proc. of the Nat. Acad. of Sci., U.S.A.,
89:3185-3189 (1992)), for example, showed that RNase P can be used to cleave specific strands of RNA to which antisense oligonucleotides having an ACCA sequence were annealed. Oligonucleotides with an ACCA sequence at one end, referred to as “external guide sequences” (EGS's), were hybridized to a specific sequence on an RNA molecule. The RNA molecule with the bound EGS thereby became a substrate for RNase P and was specifically cleaved by RNase P.
Another method of digesting RNA at a specific location with an antisense oligonucleotide and an RNase was demonstrated by Minshull et al. (
Nucleic Acids Research,
14:6433-6451 (1986)). Minshull cleaved a specific RNA molecule by first hybridizing an antisense DNA oligonucleotide to the RNA molecule and then treating the hybridized molecule with RNase H. Since RNase H specifically digests DNA/RNA-hybrids, the RNA strand of the hybridized molecule was digested by RNase H.
Corey et al. (
J Am. Clien. Soc.,
111:8523-8525 (1989)) also discussed a method of targeting a polynucleotide for destruction by a nuclease. Corey fused an antisense oligonucleotide to a nonspecific nuclease. When the oligonucleotide was then hybridized to a polynucleotide with which it could anneal, the nuclease specifically cleaved the targeted polynucleotide strand. This approach has not been applied in vivo, however, due to the great difficulties involved in passing a molecule as large as a nuclease into an intact living cell.
A number of nonenzymatic strategies for targeting a specific polynucleotide sequence for cleavage are also known to the prior art. Many of these involve covalently binding a chemical moiety that has polynucleotide cleavage activity to an antisense oligonucleotide. A method disclosed by Chi-Hong Chen (
Proc. Nat. Acad. Sci. U.S.A.,
83:7147-7151 (1986)) demonstrated such a strategy. In this method, a 1,10-phenanthroline-copper ion was attached to the 5′ end of an oligonucleotide that was complementary to a target polynucleotide sequence. The modified oligonucleotide was hybridized to the complementary target sequence, and cupric ion and 3-mercaptopropionic acid were then added to the reaction mixture. In this environment, the 1,10-phenanthroline-copper ion cleaved the target polynucleotide.
In addition, various non-specific means of cleaving polynucleotides have been identified. For example, the latent endonuclease 2-5A-dependent RNase has been found to cleave RNA in the presence of the unusual 2′,5′-phosphodiester-linked trimeric oligoadenylate 2-5A (ppp5′A2′p5′A2′p5′A) (Kerr, et al.,
Proc. Natl. Acad. Sci. USA,
15:9846 (1978)). Cells and tissues examined from reptilian, avian, and mammalian species have been found to contain basal levels of 2-5A-dependent endonuclease, which cleaves RNA after sequences containing UN (where N stands for A, U, G, or C). This enzyme is part of what has been termed the 2-5A system (Williams, et al.,
The
2-5
A System: Molecular and Clinical Aspects of the Interferon-Related Pathway,
(Alan R. Liss, Inc., New York (1985)), which is believed to mediate certain actions of interferon such as the inhibition of encephalomyocarditis virus replication. The 2-5A system also has been hypothesized to play a role in the regulation of cell growth (Etienne-Smekins, et al.,
Proc. Natl. Acad. Sci. USA,
80:4609 (1983)) and cell differentiation (Krause et al.,
Eur. J. Biochem.,
146:611 (1985)).
SUMMARY OF THE INVENTION
In one aspect, the present invention comprises a method of specifically cleaving a strand of RNA, comprising the steps of:
a. hybridizing the strand of RNA with a chimeric molecule to form a complex of the strand and the chimeric molecule, the chimeric molecule comprising an antisense oligonucleotide moiety that is complementary to the strand of RNA and an activator of 2-5A-dependent RNase attached to the antisense oligonucleotide; and
b. reacting the complex in the presence of 2-5A-dependent RNase, thereby specifically cleaving the stra

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