Genetic targeting of proviral integration in stem cells

描述(申请人提供)：这项研究提案的目的是了解如何利用转录活性及其修饰来指导HIV衍生的慢病毒载体在造血干细胞中的整合。最近人们意识到慢病毒插入位点对转录活性基因组位点的偏好，以及Moloney鼠白血病病毒载体转导干细胞后插入突变的报道，这些都突显了更全面地研究前病毒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，以探索最终应将HIV-慢病毒基因替代治疗的插入相关风险降至最低的策略。我们假设，对造血靶细胞转录图谱的系统性修改可能有助于将插入事件“吸引”到特定的基因组位置，同时最大限度地减少突变潜力和其他地方的转录干扰，如癌基因和肿瘤抑制基因。关键的是，拟议的体内研究将旨在协调转录图谱和有效慢病毒基因转移的相互竞争的要求与保留干细胞特性，包括靶细胞植入和长期再繁殖。这些研究将提供对转录如何影响前病毒插入的机械性见解，无论是代理转录还是因果相关事件，并将为未来的基因组靶向研究提供信息。拟议的导师培训计划将帮助我获得额外的技能，并通过David Kabat博士和Markus Grompe博士的指导以及他们在病毒学和干细胞生物学方面的科学专业知识获得理解。该提案还得到了顾问/合作者在以下领域的投入和指导 造血学，DNA阵列技术，生物统计学，以及课程作业。职业咨询委员会将帮助我在独立和职业发展方面取得进步。俄勒冈健康与科学大学(OHSU)全面的培训环境以及机构对我职业生涯的坚定承诺将使我能够实现这项提议的目标。

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.