CORE--DEVELOPMENT OF RIBOZYME GENE THERAPY

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AIDS, as a result of retroviral infection, represents an acquired genetic disease. Applications of gene therapy to HIV/AIDS hold great theoretic promise. To realize this promise, the expedited transition of this basic discovery from the laboratory bench to clinical application is urgently needed, and the basic strategies themselves must be refined. Hence, the goal of Project 9001 of this SPIRAT is two-fold: supporting a phase 1 trial of an HIV-1 leader sequence ribozyme and improving the ribozyme gene therapy strategy. We have shown that a hairpin ribozyme targeting the 5'- leader sequence of HIV-1 can inhibit the replication of diverse strains of HIV-1 in transient transfection. Stable T-cells conferred long-term resistance to infection of HIV-1 including clinical isolates. Recent demonstrations of the efficacy of ribozyme gene therapy in PBLs for the preclinical studies led to design of a phase I trial that is now RAC approved. To reach the goals of Project 9001, we will: (1) provide GMP vectors to Project 2 for this trial, and perform safety tests on the producer cells and viral supernatant; (2) evaluate the persistence and expression of the ribozyme gene in cells from patients and assist other follow-up studies for phase 1 trial in Project 2, including DNA and RNA PCR to quantitate the ribozyme and control vector in recovered PBLs; (3) evaluate the effects of cellular compartmental targeting strategies to improve ribozyme efficiency. This objective will be approached by cloning the HIV-1 Rev Responsive Elements (RRE) into the ribozyme transcription cassette to create a "fusion RNA" (serving not only as an RRE decoy but more importantly, targeting the ribozyme to the same cellular compartments as the HIV targets), and by cloning the ribozyme into HIV-2-based vectors so that the HIV packaging signal will also co-localize the ribozyme with HIV targets; (4) investigate the interactions between the leader sequence and other ribozymes or other anti-HIV gene products, including the RevM10 transdominant mutant protein (in collaboration with Dr. Gary Nabel) and the interferon inducible cellular gene RBP9-27 (in collaboration with Dr. George Pavlakis).