Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-06-03
2002-03-12
McGarry, Sean (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S023100, C536S024100, C435S325000, C435S366000, C435S372300, C435S375000
Reexamination Certificate
active
06355790
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a modified tRNA molecule which inhibits the full length reverse transcription of HIV-1 and HIV-1 replication. The invention further relates to a method for inhibiting the replication of HIV-1 in human cells.
BACKGROUND OF THE INVENTION
A retrovirus designated human immuno-deficiency virus (“HIV”) is the causative agent of the complex disease termed Acquired Immune Deficiency Syndrome (“AIDS”) and is a member of the lentivirus family of retroviruses. M. A. Gonda, et al.,
Science
227, 173, (1985); P. Sonigo, et al.,
Cell
42, 369, (1985). The complex disease AIDS includes progressive destruction of the immune system and degeneration of the central and peripheral nervous systems. The HIV virus was previously known or referred to as LAV, HTLV-III, or ARV. The Centers for Disease Control (“CDC”) reported the number of AIDS deaths for the first six months of 1996 was 22,000 nationwide.
tRNA functions in living cells mainly as a vehicle to translate genetic information stored in mRNA into amino acid sequence in proteins. Cellular tRNAs are recognized by many cellular proteins including 5′ and 3′ tRNA processing enzymes (Altman, S.,
Cell
23, 1-5 (1981)) and tRNA aminoacyl transferases (aa-tRNA synthetase) (Sampson, J. R., et al.,
Biochemistry
29, 2523-2532 (1990); Behlen, L. S., et al.,
Biochemistry
29, 2515-2523 (1990)). Most of these enzymes recognize both the anticodon region and specific features of the tridimensional structure of tRNA (Sampson, J. R., et al.,
Biochemistry
29, 2523-2532 (1990)). There are two different human tRNA
Lys
genes. One of these, tRNAlys-UUU (Roy, K. L., et al.,
Nuc. Acids Res.
10, 7313-7322 (1982); Richter-Cook, N. J., et al.,
J. Biol. Chem.
267, 15952-15957 (1992)), has a complementary sequence to the HIV-1 RNA genome in the region of the prime binding sequence (PBS), and is used as the primer for HIV-1 reverse transcription.
Human immunodeficiency virus type-1 (“HIV-1”) relies on multiple human cellular factors for its own replication (Steffy, K., et al.,
Microbiol Rev
55(2), 193-205 (1991)). In particular, tRNA
Lys3
is utilized by HIV-1 at the earliest step of its life cycle (Weiss, S., et al.,
Gene
111(2), 183-97 (1992), Das, A. T., et al.,
FEBS Lett.
341, 49-53 (1994); Arts, E., et al.,
J. Biol. Chem.
269, 14672-14680 (1994); Barat, C., et al.,
Embo J
8(11), 3279-85 (1989); Barat, C., et al.,
Nucleic Acids Res
19(4), 751-7 (1991); Khan, R., et al.,
J Biol Chem
267(10), 6689-95 (1992)). During the first step of reverse transcription, the single stranded plus-sense RNA genome is copied into minus-sense cDNA beginning at the 3′ end of a partially annealed tRNA
Lys3
primer (Peliska, J. A., et al.,
Science
258, 1112-1118 (1992)). Several reports indicated that p66 of the HIV-1 RT p51/p66 heterodimer recognizes and binds to the tRNA
Lys3
anticodon region (DeVico, A. L., et al.,
J. Biol. Chem.
266, 6774-6779 (1991); Sarih-Cottin, L., et al.,
J Mol Biol
226(1), 1-6 (1992); Rhim, H., et al.,
J Virol
65(9), 4555-64 (1991); Kohlstaedt, L. A., et al.,
Proc. Natl. Acad. Sci. USA
89, 9652-9656 (1992)) and may help unwind the acceptor stem (Kohlstaedt, L. A., et al.,
Proc. Natl. Acad. Sci. USA
89, 9652-9656 (1992)) in the presence of NCp7 protein (Barat, C., et al.,
Nucleic Acids Res
19(4), 751-7 (1991)). Another report demonstrated that excess wild type tRNA
Lys3
primer inhibited the DNA polymerase activity of a recombinant HIV-1 RT, p66/p51 heterodimeric form (Bordier, B., et al.,
Nucleic Acids Res
18(3), 429-36 (1990)). This effect was ascribed to the anticodon region of tRNA
Lys3
primer (Bordier, B., et al.,
Nucleic Acids Res
18(3), 429-36 (1990)). The necessary role for tRNA
Lys3
in HIV-1 reverse transcription, its specific affinity for HIV-1 RT, and its association with HIV-1 virions suggests that mutated derivatives of the tRNA
Lys3
primer might interfere with the viral replication cycle.
HIV-1 specific ribozymes, antisense RNA, and RNA decoys have been proposed as potential therapeutic reagents for HIV-1 (Chatterjee, S., et al.,
Science
258, 1485-1488 (1992); Sullenger, B. A., et al.,
Cell
63, 601-608 (1990); Ojwang, J. O., et al.,
PNAS
89, 10802-10806 (1992)).
Presently, a triple-drug therapy regimen is the most effective approach to controlling the AIDS virus. The cocktail therapy consists of a combination of a protease inhibitor called indinavir with two reverse-transcriptase inhibitors known as AZT and 3TC. The triple drug therapy results in a decrease in measured levels of virus in both blood and lymphatic tissues. However, a proportion of those treated, whose viruses had developed resistance to one or more of the cocktail's reverse-transcriptase inhibitors as a result of previous treatment, fail to respond. Some studies have projected that combination therapy can virtually eliminate the AIDS virus from those patients who respond within two or three years. However, other studies indicate that minimal residual virus is sufficient to cause relapse. In addition, existing pharmacologic therapies leave residual HIV genes lurking in cells in the form of latent “provirus.” As long as the cells remain alive, these genes can be transcribed, rekindling virus production. Therefore, drug therapy may need to be maintained throughout the patient's life. The therapy may have long term adverse side effects and treatments can cost over $15,000 annually for each patient. Thus, there is a continuing need to develop additional approaches to controlling HIV infection which can be used alone or in combination with existing therapies.
SUMMARY OF THE INVENTION
One aspect of the present invention relates to a modified tRNA
Lys3
molecule for inhibiting HIV template replication. The tRNA molecule has an acceptor stem having 3′ and 5′ segments which form a secondary structure. Furthermore, the 3′ segment hag a nucleic acid sequence with reduced complementarity to the HIV-1 primer binding site compared to the 3′ segment of a wild type tRNA
Lys3
. The 5′ segment has sufficient homology with the 3′ segment to maintain the secondary structure of the acceptor stem.
Another aspect of the present invention relates to a tRNA
Lys3
molecule which is modified to inhibit the interaction of the tRNA
Lys3
molecule with HIV-1 reverse transcriptase or HIV-1 RNA template. In this aspect of the invention, the tRNA
Lys3
molecule is modified in a region other than its acceptor stem.
The present invention also provides a method of inhibiting HIV infectivity in human cells by introducing the modified tRNA
Lys3
molecules of the invention into human cells.
REFERENCES:
Huang, Y. et al. J. of Virology, Dec. 1994. pp. 7676-7683. vol. 68. No. 12.*
Anderson, W.F. Human Gene Therapy. Nature. vol. 392. Supp. Apr. 30, 1998. pp. 25-30.*
Wohrl, B.M. et al. J. of Biological Chemistry. vol. 268. No. 18. Jun. 25, 1993. pp. 13617-13624.*
Gobbers, E. et al. J. of Virological Methods. vol. 66. (1997). pp. 293-301.*
Roy et al., “Nucleotide Sequence of a Segment of Human DNA Containing the Three tRNA Genes,”Nuc. Acids Res., 10(22):7313-7322 (1982).
Chatterjee et al., “Dual-Target Inhibition of HIV-1 in Vitro by Means of an Adeno-Associated Virus Antisense Vector,”Science, 258:1485-1488 (1992).
Tuerk et al., “RNA Pseudoknots that Inhibit Human Immunodeficiency Virus Type 1 Reverse Transcriptase,”Proc. Natl. Acad. Sci. U.S.A., 89:6988-6992 (1992).
Essink et al., “Structural Requirements for the Binding of tRNA3Lysto Reverse Transcriptase of the Human Immunodeficiency Virus Type 1,”J. Biol. Chem., 270(40):23867-23874 (1995).
Wakefield et al., “Construction of a Type 1 Human Immunodeficiency Virus that Maintains a Primer Binding Site Complementary to tRNAHis,”J. Virol., 70(2):966-975 (1996).
Biasolo et al., “A New Antisense tRNA Construct for the Genetic Treatment of Human Immunodeficiency Virus Type 1 Infection,”J. Virol., 70(4):2154-2161 (1996).
Harrich et al., “A Critical Role for the TAR Element in Promoting Efficient Human Immunodefic
Li Xinqiang
Rosenblatt Joseph D.
McGarry Sean
Nixon & Peabody LLP
Shibuya Mark L.
University of Rochester
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