Genetic Modification of Human Hematopoietic Stem Cells with Pseudotyped rAAV

用假型 rAAV 对人类造血干细胞进行基因改造

• 批准号: 7617236
• 负责人: Saswati Chatterjee
• 金额: $ 41.5万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617236
• 关键词: Animal Model; Biological Assay; Blood; CD34 gene; Capsid; Cell Cycle; Cells; Chromatin; Chromosomes; Communicable Diseases; Data; Disease model; Engraftment; Enhancers; Evaluation; G0 Phase; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genetic; Genome; Goals; Hematological Disease; Hematopoietic stem cells; Human; Immune; Immune response; Immunity; In Vitro; Inherited; Insertional Activations; Investigation; Lead; Liver; Marrow; Modeling; Modification; Monitor; Morphologic artifacts; Mus; NOD/SCID mouse; Outcome; Pathogenicity; Pathology; Process; Property; Proto-Oncogenes; Recommendation; Research; Research Personnel; Risk; Safety; Serotyping; Shuttle Vectors; Site; Staging; Stem cell transplant; Stem cells; System; Testing; Therapeutic; Toxic effect; Transgenes; Transplant Recipients; Transplantation; Tropism; Viral Vector; Work; Xenograft Model; base; cell type; cellular transduction; conditioning; effective therapy; gene therapy; gene transfer vector; human stem cells; improved; in vivo; insight; novel; pre-clinical; premature; programs; public health research; reconstitution; self-renewal; therapeutic gene; transgene expression; tumorigenesis; vector

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.

描述(由申请人提供)： 理想的干细胞基因治疗载体仍未确定。与其他将基因转移到造血干细胞(HSC)的病毒载体不同，rAAV转导处于细胞周期G0期的细胞，包括原始的多潜能HSC，这使得rAAV特别适合于评估HSC的基因转移。最近，人的骨髓被确认为天然AAV的常见部位，表明它是一个天然的靶点。在某些情况下，使用新型AAV衣壳已被证明极大地提高了基因转移效率，并克服了表达开始时的限制。我们先前已经证明，rAAV2优先转导存在于GO中的原始HSC，并具有多系和连续植入的能力。在这里，我们建议评估在AAV1，5，7，8和9衣壳中假型的rAAV2基因组用于原始人类HSCs的转导，以确定是否可以进一步改进转导。我们的初步数据表明，某些血清型能够有效地转导人HSC，能够长期植入并在体内以高于rAAV2的水平表达，因此有必要对伪型rAAV进行HSC基因转移的评估。转导将在体内进行评估，将转导的人HSCs移植到免疫缺陷小鼠体内。本研究的具体目的是：1.利用伪型rAAV技术对具有长期多系原代和系列化植入能力的人HSC进行转导研究。血清型将在表达、原发和序列重组、安全性和毒性方面进行比较。具有相同基因组的衣壳之间的差异将被用来剖析对HSC转导重要的步骤。2.鉴定HSCs中假型rAAV整合的染色体位置，这对评估遗传毒性潜力具有重要意义。比较不同伪型之间的连接位点将有助于深入了解聚集位点选择的机制。3.AAV ITRs在转导的人HSC中的绝缘体功能分析我们将在HSC的背景下测试我们最近发现的AAV ITR的绝缘子样功能。完成后，这项研究不仅应该确定最强大和最安全的AAV血清型，用于干细胞基因转移，而且还将阐明不同血清型之间的差异机制。在活体环境中对人类HSC的直接评估将提供与任何治疗应用直接相关的临床前数据。阐明AAV ITRs在HSC中的绝缘体功能将为人类HSC寻找一种安全、有效的基因转移载体，并极大地降低插入癌的风险。与公众健康相关：这项研究探索了一种新的基因转移载体系统，用于安全有效地治疗血液干细胞。这项工作的结果有望确定用于治疗包括遗传性和传染性疾病在内的各种血液疾病的最佳基因输送载体。