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.sup.+ 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.
USPTO Backgrounds
USPTO Backgrounds