440. A Non-Human Primate Model for Lentivirus-Mediated Anti-HIV RNAi Strategies

AIDS remains a significant health problem worldwide despite the advent of highly active antiretroviral therapy (HAART). Although substantial efforts have been made to develop a vaccine there is still no cure and alternative strategies are needed to treat HIV infection and to control its spread. Our goal is to evaluate lentiviral vectors that inhibit HIV replication by RNA interference (RNAi) in a non-human primate SHIV model to develop a hematopoietic stem cell (HSC) gene therapy for AIDS. SHIV89.6 P is a chimeric virus comprised of an SIV genome that contains the tat, rev and env genes of HIV and infects both T lymphocytes and macrophages. Infection of non-human primates with SHIV89.6P results in significant decreases in CD4+ T cells as early as 4 weeks post infection, and is currently the best large animal model available to test gene therapy strategies for AIDS. We present here data showing RNAi-mediated inhibition of SHIV89.6 P replication using a lentiviral vector that expresses a short hairpin RNA targeted to both tat and rev transcripts in a hybrid cell line and in macrophages differentiated from Macaque CD34+ cells. To determine the effectiveness of an anti tat/rev shRNA to inhibit SHIV89.6P in vitro, a human T cell/B cell hybrid cell line (CEMx174) was transduced with a lentiviral vector expressing a short-hairpin RNA (shRNA) targeted to both HIV tat and rev sequences that also contained either a GFP reporter gene or a MGMT(G156A) resistance gene at MOIs of 1.3 and 3 respectively. Polyclonal populations of CEMx174 cells transduced with the GFP and MGMT(G156A) vectors were challenged with a 2.15|[times]|103 TCID50 dose of SHIV 89.6P. Expression of both tat and rev transcripts was reduced 88% and 97% respectively in these cultures as measured by real-time PCR and replication of SHIV was inhibited as evidenced by inhibition of p27 production. Although others reported a block to transduction of M. mulatta CD34+ cells with an HIV-based lentiviral vector, we observed efficient transduction rates (|[ldquo]| 50%) of M. nemestrina CD34+ cells, comparable to transduction rates observed in human CD34+ cells (|[ldquo]| 60%). Macaque CD34+ cells transduced with the shRNA expressing lentiviral vector were not impaired in their ability to differentiate into macrophages in vitro and SHIV replication was inhibited in these transduced macrophages as evidenced by reduced p27 levels following challenge with SHIV. In summary, we have shown efficient HIV-based lentiviral transduction of M. nemestrina cells and efficient inhibition of SHIV infection by expressng a shRNA targeted against HIV tat and rev thereby providing a useful model to test lentiviral-mediated anti-HIV RNAi stem cell gene therapy in vivo.