The aim of this research proposal is to understand how transcriptional activity and its modification can be used to direct HIV-derived lentiviral vector integration in hematopoietic stem cells. Recent awareness of known lentiviral insertion site preferences for transcriptionally active genomic sites and reports of insertional mutagenesis after Moloney murine leukemia virus vector transduction of stem cells have highlighted the need to more fully investigate the biology of proviral integration. I herein propose to develop in vitro and in vivo model systems to explore strategies that should ultimately minimize insertion related risks of HIV-lentiviral gene replacement therapy. We hypothesize that systematic modifications in the transcriptional profile of hematopoietic target cells may serve to """"""""attract"""""""" insertion events to specific genomic locations while minimizing mutagenic potential and transcriptional interference at others, such as oncogenes and tumor suppressor loci. Critically, the proposed in vivo studies will aim to reconcile the competing requirements for transcriptional profiling and efficient lentiviral gene transfer with the retention of stem cell properties, including target cell engraftment and long-term repopulation. These studies will provide mechanistic insight into how transcription, be it a surrogate, or a causally related event, affects proviral insertion, and will inform future studies for genomic targeting. The proposed mentored training program will help me acquire additional skills and gain understanding through the mentorship by Dr. David Kabat and Dr. Markus Grompe, and their scientific expertise in virology and stem cell biology, respectively. The proposal is further augmented by input and guidance from advisors/collaborators in the areas of hematopoiesis, DNA array technology, and biostatistics, as well as course work. A career advisory committee will help direct my progress to independence and career advancement. The comprehensive training environment at Oregon Health & Science University (OHSU) and the substantial institutional commitment to my career will allow me to accomplish the goals of this proposal.
