Gene Transfer And Ex Vivo Manipulation Of Stem Cells

总结：临床和基础实验室研究的目的是为造血细胞（包括干细胞和祖细胞）开发有效和安全的基因转导和离体操作策略，并使用遗传标记技术来回答有关体内造血的重要问题。在恒河猴模型中，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干细胞和祖细胞，并使用遗传标记技术来了解干细胞在体内的行为。我们继续进一步提高基因转移到恒河猴移植细胞中的效率，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%，具有临床实用性。这些水平可以用传统的嗜中性MLV载体以及新型的基于SIV的慢病毒载体来实现。我们已经开发了禽肉瘤白细胞增多症病毒（ASLV）载体和位点特异性非病毒载体的基础上的噬菌体造血靶细胞的应用，由于更有利的插入位点配置文件。在我们的体内自体移植模型中首次证明，ASLV可以使恒河猴长期再生细胞。我们继续利用LAM-PCR技术来鉴定和追踪CD 34+转导祖细胞移植后对外周血群体的克隆贡献。鉴于在法国两名接受逆转录病毒转导的造血干细胞基因治疗的严重免疫缺陷儿童发生白血病，我们对移植了MLV或SIV载体转导细胞的恒河猴中的逆转录病毒插入位点进行了大规模测序。插入位点分析显示MLV和SIV基因内插入的非随机偏好，SIV插入在基因长度上均匀分布，特别是在基因丰富的染色体区域中发现。相反，MLV靶向转录起始位点周围的区域。这些高度非随机的事件表明在这些位点的整合具有强烈的非随机偏好，或者这些克隆具有体内植入或存活/增殖优势。14个独立的插入定位于MDS 1/EVI 1基因座，该基因座是先前涉及自发性白血病和具有复制能力的病毒的逆转录病毒诱变的区域。 使用SIV或ASLV载体，我们没有发现MDS 1/EVI 1插入。 与MLV相比，SIV和ASLV载体在原癌基因中具有显著较低的插入簇率。这些发现对未来基因治疗的临床应用具有重要意义。我们继续探索原始转导造血细胞克隆扩增和白血病发生的机制，现在使用过表达载体来研究BCL 2A 1和MDS 1/EVI 1对永生化或转化的影响。我们已经开始研究特定插入事件对通过分化的成纤维细胞的载体转导获得人iPS细胞的能力的影响。

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.