Genetic Modification of Human Peripheral Blood Lymphocytes with a Transdominant Negative Form of Rev: Safety and Toxicity

Abstract

A transdominant mutant form of the rev gene, M10, confers resistance to infection by the human immunodeficiency virus (HIV) in vitro and is currently under investigation as a potential intervention in acquired immunodeficiency syndrome (AIDS). In this report, we examine three issues relevant to the safety of autologous transfer of human T cells genetically modified with Rev M10. First, the potential for malignant transformation was assessed in vitro using interleukin-2 (IL-2) dependence and fibroblast transformation assays, and tumorigenicity was evaluated in severe combined immunodeficient (SCID) mice. Possible toxicity was evaluated by pathologic analysis following adoptive transfer of genetically modified human T cells into SCID mice. Second, methods were developed that permit T cell activation required for gene transfer but do not allow replication of endogenous HIV. Third, T cell function was evaluated in peripheral blood lymphocytes (PBL) of HIV-seropositive donors transduced with Rev M10 and compared to a negative control mutant, ΔRev M10. By all criteria, no oncogenicity or toxicity was observed. Human T cells transduced with these vectors did not grow in the absence of IL-2 in vitro, and no tumors were observed following transplantation of genetically modified human cells into recipient SCID mice. Histopathological analysis of heart, lung, liver, spleen, and kidney of animals 1–21 weeks following adoptive transfer of gene-modified human T cells revealed no significant abnormalities. Additionally, no differences were observed in the pattern of cytokine secretion in enriched human PBL expressing Rev M10 compared to ΔRev M10. These results suggest that human T cells genetically modified with Rev M10 or ΔRev M10 may be administered to patients with minimal toxicity. Expression of Rev M10, a transdominant mutant form of the Rev gene, in T cell lines confers resistance to HIV in vitro. Isertion of this Rev M10 gene into PBL appears to be nontoxic and well-tolerated by SCID mice. These results demonstrate that genetic modification of T cells by an antiviral gene can be performed safely and without overt toxicity. This finding encourages the development of therapeutic strategies to genetically protect T cells to prolong their survival in HIV-infected individuals.