This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction of genes able to inhibit HIV replication into hematopoietic stem cells offers the potential for long-lived immune reconstitution. However, multiple ethical and practical considerations significantly constrain the ability to address basic questions regarding stem cell gene therapy for AIDS in human clinical trials. Experiments in nonhuman primates therefore offer the opportunity to rigorously address these issues in an in vivo experimental model. A number of recent reports have highlighted the potential utility of small interfering RNA (siRNA) molecules to inhibit viral replication. In collaboration with J. Rossi, we initiated inhibition studies of various SHIV strains with siRNA targeting Tat and Rev. We obtained two siRNA (one targeting both Tat and Rev and the other targeting only Rev) and a control sequence, each transcriptionally regulated by the polymerase III promoter U6. Using transient transfection both si(I) and si(II) strongly inhibited HIV-1 (as previously shown), SHIV 89.6p and SHIV Hxbc2 3.2P (viruses with homologous envelopes). Using a second generation construct expressing the site I and site II interfering RNAs as a hairpin sequence (shRNA) has demonstrated even more potent inhibition of SHIV replication by over 1000-fold. These encouraging results support further studies of the utility of siRNA to inhibit SHIV replication in vivo in the macaque model.