Joseph P. Dougherty
Robert Wood Johnson Medical School
Dept. of Pharmacology
Piscataway, NJ 08854
HIV-1 replication. gene therapy and retroviral vectors
Our recent HIV-1 studies have focused upon investigating HIV-1 reverse transcription and recombination. DNA primer strand transfers are essential for HIV-1 replication, and recombination influences the rapid evolution of HIV- 1. The accepted mechanism of retroviral reverse transcription proposes that two primer strand transfers occur during replication. Given the diploid nature of the retroviral virion, the question arises concerning the nature of these primer strand transfers, namely, whether they are intra- or intermolecular. A system was developed to examine the nature of primer transfer events during HIV-1 replication. Both intra- and intermolecular primer transfer were observed. and occurred with similar frequencies during minus strand synthesis. Plus-strand primer transfer was primarily intramolecular. A high rate of homologous recombination occurred during minus-strand DNA synthesis. Recombination occurred at a rate of 3 cross-overs per genome per replication cycle. These results suggest that both viral genomic RNAs serve as templates during HIV-1 reverse transcription and that primer strand transfers as well as recombination contribute to the rapid genetic variation of HIV-1.
Primary lymphocytes play a key role in immune surveillance and regulation. Moreover, they are long-lived and easy to obtain. Because of these considerations, they are potentially important targets for somatic cell gene therapy. Using retroviral vectors, we have developed efficient gene transfer procedures for introducing genes into primary lymphocytes. The procedures do not perturb the primary lymphocytes, since after transplantation into recipient hosts, they home to the appropriate lymphoid organs, persist for long periods of time, and express the transferred genes at significant levels. Thus, we are in a position to genetically manipulate lymphocytes and are using this technology to develop procedures for treating autoimmune diseases, such as multiple sclerosis.