Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-01-14
2001-10-16
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S325000, C435S320100, C435S455000, C514S04400A, C424S093100, C424S093210, C536S023100
Reexamination Certificate
active
06303334
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a medical method of treatment. In particular, the present invention concerns the use of a transdominant negative integrase gene to make at least one mammalian cell resistant to a retroviral infection, to methods for their production, to pharmaceutical delivery methods which include these genes, and to pharmaceutical methods of treatment. More particularly, the novel transdominant negative integrase gene alone or combined with another gene that confers protection from human immunedeficiency virus (HIV) such as, for example, a transdominant negative rev gene and/or a ribozyme that cleaves HIV ribonucleic acid (RNA) is useful in treating acquired immunedeficiency syndrome (AIDS).
AIDS results from HIV infection which depletes CD4
+
T cells. Currently, there is no effective treatment for AIDS. One of the most attractive and least exploited targets for the therapy of AIDS is the viral integrase (Brown P. O., “Integration of Retroviral DNA”. In:
Current Topics in Microbiology of Immunology,
157:19-48 (1990)). The life cycle of retroviruses is dependent on integration into the host chromosome. For HIV, integration is necessary for viral replication (LaFemina R. L., et al.,
Journal of Virology,
66:7414-7419 (1992) and Sakai H., et al.,
Journal of Virology,
67:1169-1174 (1993)). This process is mediated by integrase, a viral protein. One approach to inhibition of the essential integration process is to express some form of integrase that will block the integrase function of the incoming HIV. This is based on the concept of “pathogen-derived resistance” disclosed by Sanford J. C. and Johnston S. A.,
J. Theor. Biol.,
113:395-405 (1985). Pathogen-derived resistance is based on the strategy that expression of certain genes from pathogens inhibit replication of such pathogens. Various examples of this concept have been disclosed including: retroviral envelope genes (Robinson H. L., et al.,
Journal of Virology,
40:745-751 (1981)); coat protein genes of plant viruses (Wilson T. M. A.,
Proc. Natl. Acad. Sci. USA,
90:3134-3141 (1993)); envelope glycoprotein genes of herpes viruses (Petrovskis E. A., et al.,
Journal of Virology,
62:2196-2199 (1988)); transdominant negative HIV rev (Malim M. H., et al.,
Cell,
58:205-214 (1989)); over-expression of HIV tar sequences as “decoys” (Sullenger B. A., et al.,
Cell,
63:601-608 (1990)); and transdominant negative mutants of HIV gag. In the case of HIV, this concept of pathogen-derived resistance was later termed “intracellular immunization” (Baltimore D.,
Nature,
335:395-396 (1988)).
However, prior to the present invention, there was no suggestion that retroviral integrase could be made to exert a transdominant negative phenotype. In fact, since integrase enters a cell with the virion and is presumed to remain part of the preintegration complex, it was regarded by many as an unlikely candidate for being amenable to interference by a transdominant negative mutant. In a recent review on gene therapy of AIDS (Yu M., et al.,
Gene Therapy,
1:13-26 (1994)), many possibilities were discussed with no mention of integrase as a target. Additionally, prior to the present invention, there was no good technology for exploring the possibility of a transdominant negative integrase. Retroviral integrases are made as part of polyprotein precursors (the “gag-pol precursor”) in infected cells. There is no natural expression of a retroviral integrase in a mammalian cell without many other viral proteins that are part of the precursor. Although there is a report of expression of Rous sarcoma virus integrase alone (Mumm S. R., et al.,
Virology,
189:500-510 (1992)), expression of HIV integrase has been problematic. This problem has been attributed to a rev-responsive element within the integrase gene (Cochrane, et al.,
J. Virol.,
65:5305-5313 (1991)). Holler T. P., et al.,
Gene,
136:323-328 (1993) reported the synthesis of genes coding for wild-type (“NdeI”) and an inactive mutant (“D116N:) integrase for expression in
E. coli.
Thus, an object of the present invention is the expression of HIV integrase in mammalian cells. Efficient expression of HIV integrase was achieved in the present invention by employing a synthetic gene for expression in mammalian cells (Seq ID No.: 1). That this was successful was an unpredictable and surprising result, since the synthetic gene used was synthesized to optimize codon usage for the bacterium
E. coli.
Bacterial genes could have sequences recognized by mammalian cells as splice sites or methylation sites for inactivation of the gene, making the successful expression of a bacterial gene in mammalian cells highly unpredictable.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to a method of using a transdominant negative integrase gene to make at least one cell resistant to a retroviral infection.
In a preferred embodiment of the first aspect of the invention, the retroviral infection is selected from the group consisting of: HIV; HTLV I; HTLV II; equine infections anemia virus; bovine leukemia virus; murine retrovirus; and avian leukosis virus, and the cell is selected from the group consisting of a mammalian cell and an avian cell.
In a most preferred embodiment of the first aspect of the invention, the retroviral infection is HIV and the cell is a human cell.
In a second aspect, the present invention is directed to a method for introducing a transdominant negative integrase gene into at least one mammalian cell to make said cell resistant to a retroviral infection, comprising introducing the DNA into at least one mammalian cell by a process of delivery selected from the group consisting of:
a) use of calcium phosphate co-precipitation;
b) in a complex of cationic liposomes:
c) electroporation;
d) receptor mediated endocytosis;
e) naked DNA;
f) transduction by a viral vector; and
g) particle-mediated gene transfer (Cheng L., et al.,
Proc. Natl. Acad. Sci. USA,
90:4455-4459 (1993) and Yang N-S.,
Critical Reviews in Biotechnology,
12:335-356 (1992)).
In a preferred embodiment of the second aspect of the invention, the transdominant negative integrase gene is HIV integrase and the mammalian cell is a human cell.
In a most preferred embodiment of the second aspect of the invention, the human cell is selected from the group consisting of a CD4
+
T lymphocyte, a monocyte, and a hematopoietic progenitor cell, and the viral vector is selected from the group consisting of: retrovirus; adenovirus; adeno-associated virus; and herpes virus.
In a third aspect, the present invention is directed to a method for introducing a transdominant negative integrase gene into at least one mammalian cell to make said cell resistant to a retroviral infection comprising inserting said transdominant negative integrase gene into a vector and expressing transdominant negative integrase in said at least one mammalian cell.
In a preferred embodiment of the third aspect of the invention, the transdominant negative integrase gene is HIV integrase; the vector is selected from the group consisting of: pRSV/IN-NdeI and pRSV/IN-D116N; and the mammalian cell is 293-CD4.
In a more preferred embodiment of the third aspect of the invention, the transdominant negative integrase gene is HIV integrase; the vector is pRSV/IN-NdeI; and the mammalian cell is 293-CD4.
In a most preferred embodiment of the third aspect of the invention, the transdominant negative integrase gene is HIV integrase; the vector is pRSV/IN-NdeI; and the mammalian cell is a human cell selected from the group consisting of a CD4
+
T lymphocyte, a monocyte, and a hematopoietic progenitor cell.
In a fourth aspect, the present invention is directed to a method of constructing the pRSV/IN-NdeI vector comprising inserting the HIV integrase gene into the pRcRSV vector to afford the pRSV/IN-NdeI vector.
In a fifth aspect, the present invention is directed to a vector for production of HIV transdominant negative integrase comprising a DNA sequence encoding HIV integrase.
In a preferred embo
Holler Tod Paul
Meyer Annette
Nabel Gary Jan
Post Leonard
Clark Deborah J. R.
Merchant & Gould P.C.
Warner & Lambert Company
Wilson Michael C.
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