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: 7319736
• 负责人: Saswati Chatterjee
• 金额: $ 41.6万
• 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/7319736
• 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; Outcome; Pathogenicity; Pathology; Process; Property; Proto-Oncogenes; Public Health; Recommendation; Research; Research Personnel; Risk; SCID Mice; 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; gene therapy; gene transfer vector; human stem cells; improved; in vivo; insight; novel; pre-clinical; programs; reconstitution; 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尚未确定。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，包括原始多能 HSC，这使得 rAAV 对于 HSC 基因转移的评估特别有吸引力。最近，人类骨髓被确定为天然 AAV 的常见位点，表明它是天然靶标。在某些情况下，新型 AAV 衣壳的使用已被证明可以极大地提高基因转移效率并克服表达起始的限制。我们之前已经表明，rAAV2 优先转导位于 GO 中的原始 HSC，并且能够进行多谱系和连续移植。在这里，我们建议评估在 AAV 1,5,7,8&9 衣壳中假型化的 rAAV2 基因组，用于原始人类 HSC 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转导人 HSC，能够在体内长期植入并以高于 rAAV2 所达到的水平稳定表达，因此需要对假型 rAAV 进行 HSC 基因转移评估。将转导的人类造血干细胞移植到免疫缺陷小鼠体内，对转导进行体内评估。具体目标是： 1. 评估能够通过假型 rAAV 进行长期多谱系初级和连续植入的人类 HSC 转导。将比较血清型的表达、初级和系列重建、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对于 HSC 转导重要的步骤。 2. 鉴定 HSC 中假型 rAAV 整合的染色体位点，对于评估潜在的遗传毒性很重要。假型之间连接位点的比较将提供对整合位点选择的机制的深入了解。 3.转导的人HSC中AAV ITR的绝缘体功能分析。我们将在 HSC 的背景下测试我们最近令人兴奋的 AAV ITR 绝缘体样功能的发现。完成后，这项研究不仅应该确定用于干细胞基因转移的最强大和最安全的 AAV 血清型，而且还将阐明血清型之间差异的机制。在体内环境中对人类 HSC 的直接评估应提供与任何治疗应用直接相关的临床前数据。描绘 HSC 中 AAV ITR 的绝缘体功能将为人类 HSC 找到一种安全、有效的基因转移载体，并大大降低插入肿瘤发生的风险。与公众健康的相关性：这项研究探索了一种新型基因转移载体系统，用于安全有效的血液干细胞治疗。这项工作的成果预计将确定用于治疗包括遗传性疾病和传染病在内的多种血液疾病的最佳基因递送载体。