Rev is a regulatory protein essential to the replication of HIV-1. We have previously shown that a dominant negative mutant form of Rev, M10, protects against viral replication in human lymphocytes without affecting T cell function. Because this gene product has therapeutic potential for the treatment of acquired immunodeficiency syndrome (AIDS), we have developed vectors that express Rev M10 and inhibit the replication of HIV. Preliminary experiments using human lymphocytes indicate that expression of Rev M10 provides significant protection against HIV infection. Based on these and other studies, a clinical protocol has been developed that will determine whether Rev M10 can prolong the survival of T cells in HIV- infected individuals. This protocol was recently approved by the Recombinant DNA Advisory Committee of the NIH. The objective of this proposal is to provide a transition to human clinical studies with preclinical models and to optimize its safety and efficacy prior to the initiation of trials. The first goal is to ensure the safety and optimal expression of Rev M10 in T cells. This goal will be accomplished by appropriate quality control of retroviral vectors to be used in the trial. Certification and quality control testing will be conducted on the retroviral vector and the particle-mediated gene delivery system, to ensure that these agents are safe and standardized for use in patient testing. Testing of the producer cell line, preparation of cell banks, producer cell line characterization of both a master cell bank and a manufacturer's cell bank, testing of the retroviral vector and testing of final production cells will be performed. In addition, particle-mediated gene delivery will be adapted for human use.
The second aim of this project will be to evaluate the safety of Rev M10 expression in CD4+ human cells in a preclinical model. Serum biochemical parameters, analysis of replication-competent virus, organ pathology, and malignancy potential of transduced cells will be analyzed in SCID mice as part of the pre-clinical safety assessment. The safety of the particle-mediated gene delivery system will be evaluated in SCID and normal mice to optimize transduction efficiencies and determine whether there is associated toxicity.
The third aim i s to improve the utility and efficacy of the proposed treatment by the generation of improved retroviral vectors that show optimal expression for anti-viral effects, optimizing regulated expression in T cells using retroviral vectors, and by developing expression systems for hematopoietic stem cells. We will develop vectors that incorporate alternative enhancers and other regulatory elements to increase the expression of the protein in these cells. The vectors will be tested on T cells stimulated with anti- CD3/IL2 or anti-CD3/CD28 as proposed in the human trial. These essential studies will facilitate the initiation of human gene therapy trials for AIDS and allow them to proceed with greater safety and efficacy. At the same time that these studies serve as a paradigm for other gene therapy approaches to the treatment of AIDS, they will also provide fundamental information on the role of Rev in the pathogenesis of AIDS.
| Ranga, U; Woffendin, C; Verma, S et al. (1998) Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals. Proc Natl Acad Sci U S A 95:1201-6 |
| Flowers, C C; Woffendin, C; Petryniak, J et al. (1997) Inhibition of recombinant human immunodeficiency virus type 1 replication by a site-specific recombinase. J Virol 71:2685-92 |
| Fox, B A; Woffendin, C; Yang, Z Y et al. (1995) Genetic modification of human peripheral blood lymphocytes with a transdominant negative form of Rev: safety and toxicity. Hum Gene Ther 6:997-1004 |
