Thesis

Determining the efficacy of human trim cyp in inhibiting a broad spectrum of HIV viruses when it is stably expressed in CD34- derived macrophages

Hematopoietic stem cells genetically modified to express anti-HIV genes can repopulate the immune system, replacing HIV target cells such as CD4+ T lymphocytes and macrophages. If such cells express therapeutic genes that halt early steps in the viral life cycle, HIV replication can be suppressed prior to reverse- transcription and integration. The HIV host restriction factor, huTrimCyp, was recently developed and does not occur naturally in humans. It is believed that huTrimCyp inhibits HIV shortly after viral entry by binding to capsid proteins and sequestering them for degradation. My data indicates th a t T-cell lines transduced with a lentivirus vector expressing huTrimCyp showed no short-term or long- term cytotoxicity, as well as exhibited a stable gene expression for a period greater than six weeks in vitro. Furthermore, transduced T-cell lines showed an inhibition of R5- tropic HIV strain greater than 5 f o l d when compared to cells expressing ZsGreen alone as determined by flow analysis of HIV-reporter infected MOLT4 T-cell line. I have performed in-vitro experiments in CD34+ hematopoietic stem cells in order to determine the feasibility of pursuing huTrimCyp as an anti-HIV gene therapy candidate in vivo. I have shown that huTrimCyp is capable of a fivefold reduction in supernatant p24 levels in CD34+ derived macrophages when infected with dual tropic HIV-1 (89.6) and a threefold reduction when infected with R5-tropic HIV-1 (NFNS-X), compared to macrophages expressing my control vector.

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