Gene Transfer And Ex Vivo Manipulation Of Stem Cells

干细胞的基因转移和离体操作

• 批准号: 7969030
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 612.57万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7969030
• 关键词: Area; Autologous Transplantation; Avian Sarcoma; Bacteriophages; Biological Assay; CD34 gene; Cells; Characteristics; Child; Clinical; Clonal Expansion; Engraftment; Event; Fibroblasts; France; Frequencies; Future; Gene Transfer; Genes; Genetic; Genomics; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Immunologic Deficiency Syndromes; Laboratory Study; Large-Scale Sequencing; Length; Lentivirus Vector; Leukocytosis; Macaca mulatta; Modeling; Modification; Mutagenesis; Non-Viral Vector; Pharmaceutical Preparations; Physiology; Population; Primates; Production; Proto-Oncogenes; Recording of previous events; Regenerative Medicine; Retroviral Vector; Risk; SIV; Site; Source; Stem cells; Techniques; Technology; Transduction Gene; Transplantation; Virus; Work; base; cell behavior; cellular transduction; clinical application; gene therapy; genotoxicity; improved; in vivo; induced pluripotent stem cell; insight; leukemia; leukemogenesis; murine retroviral vector; novel; overexpression; peripheral blood; preference; safety study; stem; vector

Summary: Clinical and basic laboratory studies are directed at developing efficient and safe gene transduction and ex vivo manipulation strategies for hematopoietic cells, including stem and progenitor cells, and using genetic marking techniques to answer important questions about in vivo hematopoiesis. In the rhesus model, shown to be the only predictive assay for human clinical results, we have focused on optimizing gene transfer to primitive stem and progenitor cells, and using genetic marking techniques to understand stem cell behavior in vivo. We have continued to further enhance gene transfer efficiency into rhesus engrafting cells, resulting in early levels of marked cells as high as 50-80%, with stable levels of 5-35% in all lineages, a range with clinical utility. These levels can be achieved with traditional amphotropic MLV vectors, as well as with novel SIV-based lentiviral vectors. We have developed avian sarcoma leukocytosis virus (ASLV) vectors and site-specific non-viral vectors based on phage for hematopoietic target cell applications, due to more favorable insertion site profiles. ASLV can transduce rhesus long-term repopulating cells, as first demonstrated in our in vivo autologous transplantation model. We have continued to utilized the LAM-PCR technology to identify and track clonal contributions to peripheral blood populations following transplantation of CD34+ tranduced progenitor cells. Given the occurence of leukemia in two children receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells in France, we have performed large scale sequencing of retroviral insertion sites in rhesus macaques transplanted with cells transduced either with MLV or SIV vectors. The insertion site analysis shows non-random preference for insertions within genes for both MLV and SIV, with SIV insertions distributed evenly over the length of genes and particularly being found in highly gene rich chromosomal regions. MLV instead targets the region around transcriptional start sites. Over 49 common integration sites, or genes or genomic areas with more than one integration event have been found. These highly non-random events indicate either a strong non-random preference for integration at these sites, or an in vivo engraftment or survival/proliferative advantage for these clones. 14 independent insertions were localized to the MDS1/EVI1 locus, an area previously implicated in spontaneous leukemias and in retroviral mutagenesis with replication competent viruses. We have found no MDS1/EVI1 insertions using SIV or ASLV vectors. SIV and ASLV vectors have a significantly lower rate of insertion clusters in proto-oncogenes as compared to MLV. These findings have important implications for future gene therapy clinical applications. We continue to explore the mechanism of clonal expansion and leukemogenesis in primitive transduced hematopoietic cells, now using overexpression vectors to study the impact of BCL2A1 and MDS1/EVI1 on immortalization or transformation. We have begun to study the impact of specific insertion events on the ability to derive human iPS cells via vector transduction of differentiated fibroblasts.

摘要：临床和基础实验室研究致力于开发有效且安全的基因转导，以及用于包括茎和祖细胞在内的造血细胞的体内操纵策略，并使用遗传标记技术来回答有关体内造血的重要问题。在恒河类模型中，被证明是人类临床结果的唯一预测分析，我们专注于优化基因转移到原始的茎和祖细胞，并使用遗传标记技术来了解体内的干细胞行为。我们继续进一步提高基因转移效率到恒河猴开采细胞中，导致标记细胞的早期水平高达50-80％，在所有谱系中，稳定水平为5-35％，这是临床实用性的范围。这些水平可以通过传统的两性MLV载体以及新型的SIV基慢病毒载体来达到这些水平。由于更有利的插入部位剖面，我们基于造血靶细胞施加的噬菌体，基于噬菌体的噬菌体开发了禽肉瘤白细胞增多病毒（ASLV）载体和特异性非病毒载体。正如我们的体内自体移植模型中最初证明的那样，ASLV可以转导恒湿性的长期再现细胞。我们继续利用LAM-PCR技术来识别和跟踪CD34+ tand骨祖细胞移植后对外周血种群的克隆贡献。鉴于两名儿童在法国接受逆转录病毒转导的造血干细胞的严重免疫缺陷的儿童中发生了严重的免疫缺陷，我们在恒河猴猕猴中对逆转录病毒插入部位进行了大规模测序，该猕猴用MLV或SIV vectors或Siv vectors转导的细胞移植了细胞。插入位点分析显示，MLV和SIV中基因内插入的非随机偏好，SIV插入均匀分布在基因的长度上，尤其是在高度基因丰富的染色体区域中发现的。 MLV取而代之的是针对转录起始站点周围的区域。已经发现了超过49个常见的集成位点，或具有多个集成事件的基因或基因组区域。这些高度非随机的事件表明，在这些位点积分的非随机偏好，或者是体内植入的偏爱，或者是这些克隆的体内植入或生存/增殖优势。 14独立插入位于MDS1/EVI1基因座，该区域以前涉及自发性白血病和逆转录病毒诱变，并具有复制能胜任的病毒。 我们没有使用SIV或ASLV向量发现MDS1/EVI1插入。 与MLV相比，SIV和ASLV载体在原始基因中的插入簇速率明显较低。这些发现对未来的基因治疗临床应用具有重要意义。我们继续探索原始转导的造血细胞中克隆扩张和白血病的机制，现在使用过表达载体研究BCL2A1和MDS1/EVI1对永生或转化的影响。我们已经开始研究特定插入事件对通过分化成纤维细胞的载体转导推导人IPS细胞能力的影响。