Gene Transfer And Ex Vivo Manipulation Of Hematopoietic

基因转移和造血的离体操作

• 批准号: 6966938
• 负责人: CYNTHIA E DUNBAR
• 金额: --
• 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/6966938
• 关键词: CD34 molecule; Macaca mulatta; Retroviridae; blood disorder chemotherapy; cell population study; gene therapy; genetic markers; hematopoiesis; hematopoietic stem cells; hematopoietic tissue transplantation; human tissue; immunotherapy; myocardium; nonhuman therapy evaluation; platelet derived growth factor; radiation therapy; regeneration; stem cell transplantation; tissue /cell culture

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. The high levels have allowed us to continue to track clonal contributions to hematopoiesis for the first time in a large animal model. We have utilized a new technology that allows simultaneous assessment of multiple clonal contributions to peripheral blood populations. We have found a different population of engrafting cells that contribute for the first 1-2 months post-transplantation, that are then replaced by a very stable set of 30-100 clones that contribute to all lineages for over 6-7 years in some animals. We have investigated the impact of cytokine therapy, radiation, and chemotherapy on the in vivo behavior of stem cell clones, using this powerful methodology. Prolonged cytokine treatment with either G-CSF or SCF does not significantly alter the number of stem cell clones contributing to hematopoiesis, nor result in detectable clonal exhaustion or recruitment. In contrast, treatment with low dose total body irradiation or with busulfan results in a significant decrease in stem cell clones contributing to peripheral blood lineages. We have investigated the lineage contributions of individual stem and progenitor cell clones, asking whether clones contribute equally to each lineage such as granulocytes, T cells, B cells, dendritic cells and mast 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. Thus far, no animal has developed leukemia or any hematologic disorder, in a cohort of 46 animals followed greater than 1 year, and for a median now of 4.5 years. 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. These findings have important implications for future gene therapy clinical applications. We have continued to investigate the transforming properties of the novel rabaptin/PDGFRbeta fusion protein a murine model, and broadened our investigation of activated tyrosine kinases in myeloproliferative syndromes to patients with hypereosinophilic syndromes. We have also investigated the activity of the tyrosine kinase inhibitor imatinib on T lymphocytes. Using the non-human prrimate model, we have investigated immune reconstitution in detail following transplantation with purified CD34+ cells versus whole mobilized peripheral blood, as well as cells ex vivo expanded for 4 days in the presence of cytokines. We find significant improvements in immune reconstitution using ex vivo expanded cells.

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的茎和祖细胞，并使用遗传标记技术了解体内的干细胞行为。我们继续进一步增强。基因转移效率转移到恒河猴上，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。高水平使我们能够在大型动物模型中首次追踪对造血的克隆贡献。我们使用了一项新技术，该技术允许同时评估对外围血液种群的多个克隆贡献。我们发现，在移植后最初的1-2个月贡献了不同的雕刻细胞，然后用非常稳定的30-100个克隆替换，这些克隆在某些动物中有助于所有谱系超过6 - 7年。我们使用这种强大的方法研究了细胞因子疗法，放射疗法和化学疗法对干细胞克隆体内行为的影响。用G-CSF或SCF长时间的细胞因子治疗不会显着改变导致造血的干细胞克隆的数量，也不会导致可检测的克隆疲劳或募集。相比之下，低剂量的总体辐照或用链球素治疗导致干细胞克隆显着降低，导致外周血谱系。我们已经研究了单个茎和祖细胞克隆的谱系贡献，询问克隆是否同等贡献每个谱系，例如粒细胞，T细胞，B细胞，B细胞，树突状细胞和肥大细胞。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。到目前为止，没有动物患有白血病或任何血液学疾病，在46只动物的队列中，遵循的是1年以上，并且中位数为4。5年。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。这些发现对未来的基因治疗临床应用具有重要意义。我们继续研究新型Rabaptin/PDGFRBETA融合蛋白A的转化特性A鼠模型，并扩大了我们对髓增生性综合征中活性酪氨酸激酶对嗜嗜酸疾病综合症患者的研究。我们还研究了酪氨酸激酶抑制剂伊马替尼对T淋巴细胞的活性。使用非人类培养子模型，我们在用纯化的CD34+细胞移植后详细研究了免疫重建，与整个动员的外周血相比，在存在细胞因子的情况下，体内膨胀了4天。我们发现使用离体扩展的细胞对免疫重建的显着改善。