Genetic targeting of proviral integration in stem cells

描述（由申请人提供）：本研究计划的目的是了解转录活性及其修饰如何用于指导hiv衍生慢病毒载体在造血干细胞中的整合。最近对已知慢病毒插入位点对转录活性基因组位点的偏好的认识，以及Moloney小鼠白血病病毒载体转导干细胞后插入突变的报道，都突出了更充分研究原整合生物学的必要性。在此，我建议开发体外和体内模型系统，以探索最终将hiv慢病毒基因替代疗法插入相关风险降至最低的策略。我们假设，造血靶细胞转录谱的系统修饰可能有助于“吸引”插入事件到特定的基因组位置，同时最大限度地减少其他基因（如癌基因和肿瘤抑制基因座）的致突变性和转录干扰。至关重要的是，拟议的体内研究将旨在协调转录谱和高效慢病毒基因转移与保留干细胞特性（包括靶细胞植入和长期再生）的竞争要求。这些研究将提供关于转录是如何影响前病毒插入的机制，无论是替代的，还是因果相关的事件，并将为未来的基因组靶向研究提供信息。在David Kabat博士和Markus Grompe博士的指导下，以及他们在病毒学和干细胞生物学方面的专业知识，将帮助我获得额外的技能和理解。顾问/合作者在下列领域的投入和指导进一步充实了该提案

DESCRIPTION (provided by applicant): 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.