Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1999-09-07
2004-02-03
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S002600, C530S300000, C530S327000, C435S235100, C435S320100, C435S252300, C435S069100, C536S023100, C536S023700, C424S093600
Reexamination Certificate
active
06686333
ABSTRACT:
TECHNICAL FIELD
The present invention relates to nucleic acid and peptide compositions which inhibit HIV replication in a mammalian cell. The present invention further relates to methods of inhibiting HIV replication in a mammalian cell by administering the compositions of the invention.
BACKGROUND OF THE INVENTION
Human immunodeficiency virus type (HIV-1) encodes a potent transactivator, Tat. Subsequent to the integration of viral DNA, a major function of Tat is to transactivate the viral long terminal repeat (LTR) to regulate the production of viral mRNA (Arya SK, et al. (1985),
Science,
229:69-73; Sadaie MR, et al. (1988),
Science
239:910-913). Tat's mechanism of action has been implicated to be at both transcription initiation and elongation (Kashanchi F, et al. (1994),
Nature
367:295-299; Xhou Q, et al. (1995),
EMBO J,
14:321-328; Chiang C-M, et al. (1995),
Science,
267:531-536).
The use of transdominant mutants of peptides derived from the 86-amino acid Tat protein has been suggested as a means to inhibit HIV replication in vivo. Since the pharmaceutical utility of transdominant mutants of minimal length is generally desired, attempts have been made to define the elements of Tat which are necessary and sufficient to inhibit Tat function. Such attempts have been discredited or implicated nearly full length regions of the Tat protein.
Tat structure comprises an amino-terminal domain, a cysteine-rich domain, a core region, and a basic domain. The Tat core domain is a stretch of eleven amino acids between the cysteine-rich and basic domain. The core domain is conserved in all HIV isolates. Kashanchi et al. (
Nature,
367:295-299 (1994)) reported that the lysine at position 41 of the core was critical for transactivation in vivo.
Green et al. reported that Tat peptides spanning amino acids 37-62 (and including the core domain) could act as transactivators (
Cell,
55:1179-1188 (1988)). Green et al. also reported that peptides 37-62 and 37-72 having substitutions at positions 41, 46, or 47 and 46/47 inhibit Tat transactivation of the HIV LTR in vivo (
Cell,
58:215-223 (1989)). Further, Green et al. state that they believed that a double substitution of amino acids 41 and 47 in any peptide backbone would be a good antagonist (WO 89/12461; PCT/US89/02404). However, as set forth below, subsequent studies by independent researchers have raised substantial uncertainties regarding the findings and suggestions of Green et al.
Frankel et al. (
Proc. Natl. Acad. Sci. USA,
86:7397-7401 (1989)) investigated transactivation of an HIV-1 LTR-CAT gene construct using synthetic peptides from the Tat protein. In sharp contrast to the studies of Green et al., Frankel et al. reported that the transactivation activity of Tat residues 37-62 as reported by Green et al. was inconsistent with their findings. To resolve the apparent discrepancy, Frankel et al. synthesized and tested Tat 37-62 and found that the peptide failed to have any detectable activity under four different assay conditions. Moreover, Frankel et al. note that core domains lacking a complete amino-terminal domain failed to exhibit any inhibitory effect at 20 &mgr;g/ml and state that it seemed unlikely that these peptides could be used to specifically block Tat function in vivo. Id. at page 7400.
Pearson et al. (
Proc Nat/Acad Sci USA,
87:5079-5083 (1990)) studied peptides and mutant peptides derived from the Tat protein to determine the essential features of peptides having the inhibitory transdominant phenotype. In agreement with the findings of Frankel et al., Pearson et al. further discount the findings of Green et al., and instead teach that their mutagenesis studies suggest that both an intact amino terminus and cysteine-rich domain are required for the inhibitory transdominant phenotype.
Further contradicting the findings of Green et al., Mehtali and Sorg (Australian Patent No. 52803/93) report that a Tat variant with a lysine to alanine substitution at position 41 (as in the mutants of Greene et al.) gives completely contradictory results to the study of Greene et al Indeed, instead of inhibiting transactivation, the variant of Mehtali and Sorg appeared to act in cooperation with the native Tat protein and increased transactivation 72% over the control level.
Accordingly, the thrust of the prior art is that the inhibitory transdominant phenotype of a core domain peptide requires an intact amino terminus, as well as a cysteine-rich domain. However, what is needed in the art is an inhibitory transdominant soluble Tat peptide of minimal length for in vitro and in vivo applications. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
In one aspect, the present invention relates to an isolated transdominant soluble Tat peptide. The transdominant soluble Tat peptide comprises a transdominant peptide sequence Cys-Phe-Xaa
39
-Xaa
40
-Xaa
41
-Gly-Leu-Gly-Ile-Ser-Xaa
47
-Gly-Xaa
49
-Lys (SEQ ID NO:1), wherein Xaa
39
is an amino acid residue selected from the group consisting of: Leu, Met, Ile, Thr, Gln, and Val; Xaa
40
is an amino acid residue selected from the group consisting of: Thr, Arg, Lys, and Asn; Xaa
41
is an amino acid residue exclusive of Lys; Xaa
47
is an amino acid residue selected from the group consisting of: Tyr and His; Xaa
49
is an amino acid residue selected from the group consisting of: Arg and Lys. The transdominant peptide sequence comprises an amino acid residue substitution at a position selected from the group consisting of: 44, 46, 47, and combinations thereof. Additionally, the transdominant soluble Tat peptide lacks an intact amino-terminal domain or an intact cysteine-rich domain.
In some embodiments, the transdominant peptide sequence comprises a single amino acid residue substitution at position 44. In other embodiments, the transdominant peptide sequence comprises a single amino acid residue substitution at position 46 or 47. Generally, the transdominant soluble Tat peptide is no longer than 25 amino acid residues in length. In preferred embodiments, the amino acid at position 44 is an alanine residue. Typically, the transdominant peptide sequence is substituted only at position 44.
In another aspect, the present invention relates to an isolated nucleic acid sequence encoding a transdominant soluble Tat peptide. The transdominant soluble Tat peptide comprises a transdominant peptide sequence having the sequence Cys-Phe-Xaa
39
-Xaa
40
-Xaa
41
-Gly-Leu-Gly-Ile-Ser-Xaa
47
-Gly-Xaa
49
-Lys (SEQ ID NO:1), wherein Xaa
39
is an amino acid residue selected from the group consisting of: Leu, Met, Ile, Thr, Gln, and Val; Xaa
40
is an amino acid residue selected from the group consisting of: Thr, Arg, Lys, and Asn; Xaa
41
is an amino acid residue exclusive of Lys; Xaa
47
is an amino acid residue selected from the group consisting of: Tyr and His; Xaa
49
is an amino acid residue selected from the group consisting of: Arg and Lys. The transdominant peptide sequence comprises an amino acid residue substitution at a position selected from the group consisting of: 44, 46, 47, and combinations thereof. Additionally, the transdominant soluble Tat peptide lacks an intact amino-terminal domain or an intact cysteine-rich domain.
In a further aspect, the present invention is directed to an expression vector. The expression vector comprises a nucleic acid encoding a transdominant soluble Tat peptide comprising a transdominant peptide sequence having the sequence Cys-Phe-Xaa
39
-Xaa
40
-Xaa
41
-Gly-Leu-Gly-Ile-Ser-Xaa
47
-Gly-Xaa
49
-Lys (SEQ ID NO:1), wherein Xaa
39
is an amino acid residue selected from the group consisting of: Leu, Met, Iie, Thr, Gln, and Val; Xaa
40
is an amino acid residue selected from the group consisting of: Thr, Arg, Lys, and Asn; Xaa
41
is an amino acid residue exclusive of Lys; Xaa
47
is an amino acid residue selected from the group consisting of: Tyr and His; Xaa
49
is an amino acid residue selected from the group consisting of: Arg and Lys. The transdominant peptide sequence comprises an amino acid residue substitution at a position
Brady John
Kashanchi Fatah
Sadaie Mohammad Reza
Kam Chih-Min
Low Christopher S. F.
The United States of America as represented by the Department of
Townsend & Townsend & Crew LLP
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