Genetic Modification of Human Hematopoietic Stem Cells with Pseudotyped rAAV

描述（由申请人提供）： 理想的干细胞基因治疗载体仍然没有确定。与用于将基因转移到造血干细胞（HSC）中的其他病毒载体不同，rAAV转导处于细胞周期的G0期的细胞，包括原始多能HSC，使得rAAV对于评估HSC基因转移特别有吸引力。最近，人骨髓被鉴定为天然AAV的共同位点，表明其作为天然靶标。在某些情况下，新的AAV衣壳的使用已经显示出极大地提高基因转移效率并克服表达开始的限制。我们以前已经表明，rAAV2优先转导原始HSC驻留在GO和能够多谱系和系列植入。在此，我们提出评估在AAV 1、5、7、8和9衣壳中假型化的rAAV 2基因组用于原始人HSC的转导，确定转导是否可以进一步改善。我们的初步数据表明，某些血清型有效地抑制了人HSC，能够长期植入，并在体内以高于用rAAV 2获得的水平稳健表达，因此需要评估用于HSC基因转移的假型rAAV。将用移植到免疫缺陷小鼠中的转导的人HSC在体内评价转导。具体目标是：1.通过假型rAAV对能够长期多谱系初次和连续植入的人HSC的转导的评价。将比较血清型的表达、初步和系列复溶、安全性和毒性。将利用具有相同基因组的衣壳之间的差异来剖析HSC转导的重要步骤。2.鉴定HSC中假型rAAV整合的染色体位点，这对评估遗传毒性潜力很重要。假型之间的连接位点的比较将提供深入了解整合位点选择的机制。3.转导的人HSC中AAV ITR的绝缘子功能的分析。我们将测试我们最近令人兴奋的发现，在HSC的背景下，AAV ITR的绝缘体样功能。当完成这项研究时，不仅要确定干细胞基因转移最强大和最安全的AAV血清型，而且还要阐明血清型之间差异的机制。在体内环境中对人HSC的直接评价将提供与任何治疗应用直接相关的临床前数据。描述HSC中AAV ITR的绝缘子功能将鉴定用于人HSC的安全、有效的基因转移载体，其具有大大降低的插入肿瘤发生的风险。与公共卫生的相关性：这项研究探索了一种新的基因转移载体系统，用于安全有效的造血干细胞治疗。这项工作的结果预计将确定用于治疗各种血液疾病（包括遗传性和感染性疾病）的最佳基因递送载体。

DESCRIPTION (provided by applicant): The ideal stem cell gene therapy vector remains as yet unidentified. Unlike other viral vectors for gene transfer into hematopoietic stem cells (HSC), rAAV transduces cells in the G0 phase of the cell cycle, including primitive multipotent HSC making rAAV particularly attractive for evaluation for HSC gene transfer. Recently the human marrow was identified as a common site for native AAV, indicating that it serves as a natural target. The use of novel AAV capsids has in some cases been shown to vastly improve gene transfer efficiencies and overcome limitations in onset of expression. We have previously shown that rAAV2 preferentially transduces primitive HSC residing in GO and capable of multilineage and serial engraftment. Here we propose to evaluate rAAV2 genomes pseudotyped in AAV 1,5,7,8&9 capsids for transduction of primitive human HSCs determine if transduction may be further improved. Our preliminary data indicates that certain serotypes efficiently transduce human HSC capable of long-term engraftment and robust expression in vivo at levels higher than that attained with rAAV2, necessitating the evaluation of pseudotyped rAAV for HSC gene transfer. Transductions will be evaluated in vivo with transduced human HSCs transplanted into immune-deficient mice. The specific aims are: 1. Evaluation of transduction of human HSC capable of longterm multi-lineage primary and serial engraftment by pseudotyped rAAV. Serotypes will be compared for expression, primary and serial reconstitution, safety and toxicity. Differences between capsids with identical genomes will be exploited to dissect steps important for HSC transduction. 2. Identification of chromosomal sites of pseudotyped rAAV integration in HSCs, important to assess genotoxic potential. Comparision of junction sites between pseudotypes will provide insight into mechanisms underlying intgeration site selection. 3. Analysis of insulator function of AAV ITRs in transduced human HSC. We will test our recent exciting discovery of insulator-like functions of AAV ITRs in the context of HSCs. When completed this research should not only identify the most robust and safest AAV serotype for stem cell gene transfer but would also elucidate mechanisms underying the differences between the serotypes. Direct evaluation of human HSC in an in vivo setting will should provide preclinical data directly relevant to any therapeutic application. Delineating insulator function of AAV ITRs in HSC would identify a safe, efficient gene transfer vector for human HSC with a greatly reduced risk of insertional oncogenesis. RELEVANCE TO PUBLIC HEALTH: This research explores a novel gene transfer vector system for safe and effective therapies for blood stem cells. The outcome of this work is expected to identify the optimal gene delivery vector for the treatment of a variety of blood diseases including inherited and infectious diseases.