HIGH TRANSDOMINANT REVM10 PROTEIN-LEVELS ARE REQUIRED TO INHIBIT HIV-1 REPLICATION IN CELL-LINES AND PRIMARY T-CELLS - IMPLICATION FOR GENE-THERAPY OF AIDS

Expression of antiviral genes in CD4(+) T cells has been proposed as a strategy for gene therapy of AIDS. Over the past years, we and others have developed retroviral vectors encoding the RevM10 protein, a domi nant-negative mutant of the HIV-1 Rev trans-activator protein. We coul d demonstrate gene transfer and inhibition of HIV-1 replication in cul tured T cell lines and primary T cells. However, little is known about the levels of the antiviral protein required to achieve a therapeutic effect, particularly in primary cells. In this report, we compare dif ferent vector designs with regard to expression of the antiviral gene to develop an optimal vector for clinical applications. Our results de monstrate that intracellular steady-state RevM10 protein levels expres sed from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV) or mouse embryonic stem cell virus (MESV) promot ers located in the long terminal repeat (LTR) were uniformly higher th an from internal promoters (eg CMV, PGK). Analysis of selected vectors in acutely and chronically HIV-infected cell lines suggested that thr eshold levels of RevM10 expression are required to achieve inhibition of HIV replication. LTR-driven RevM10 expression also yielded high ste ady-state protein levels in activated primary T cells resulting in inh ibition of HIV replication, and there was no apparent difference betwe en the MoMLV, MPSV and MESV-LTR vectors. However, RevM10 expression wa s down-regulated in resting primary cells and consequently anti-HIV ef ficacy was significantly reduced. Taken together, the data suggest tha t relatively high steady-state levels of RevM10 protein are required t o achieve inhibition of HIV replication and that the MPSV- and MESV-de rived retroviral vectors show no advantage over the MoMLV-based vector s for expression of anti-HIV genes in human T cells.