Developing Efficient and Safe Gene Transfer to Primate Hematopoietic Stem Cells

开发高效、安全的灵长类造血干细胞基因转移方法

• 批准号: 8557916
• 负责人: CYNTHIA E DUNBAR
• 金额: $ 193.43万
• 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/8557916
• 关键词: AP 1903 reagent; Ablation; Animal Model; Animals; Area; Autologous Transplantation; Avian Sarcoma; Bacteriophages; Bar Codes; Behavior; Biological Assay; Busulfan; CD34 gene; Cells; Chronic; Chronic Lymphocytic Leukemia; Clinical; Clonal Expansion; Cytotoxic Chemotherapy; EVI1 gene; Engineering; Engraftment; Event; Frequencies; Future; Ganciclovir; Gene Expression; Gene Transfer; Genes; Genetic; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Hypoxia; Immunologic Deficiency Syndromes; Individual; Inflammation; Laboratory Study; Large-Scale Sequencing; Length; Lentivirus Vector; Leukocytosis; Macaca mulatta; Methodology; Modeling; Modification; Mus; Mutagenesis; Non-Viral Vector; Output; Patients; Physiology; Population; Primates; Procedures; Proteins; Proto-Oncogenes; Recording of previous events; Retrieval; Retroviral Vector; Risk; SIV; Safety; Site; Stem cells; System; Techniques; Technology; Testing; Time; Transduction Gene; Transgenes; Transplantation; Virus; Work; base; caspase-9; cell behavior; cellular transduction; clinical application; cytokine; cytotoxic; design; gene therapy; gene therapy clinical trial; genotoxicity; improved; in vivo; insight; leukemia; leukemogenesis; murine retroviral vector; mutant; nonhuman primate; novel; overexpression; peripheral blood; preference; pressure; response; restriction enzyme; small molecule; stem; suicide gene; treatment duration; 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 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 discovered that transduction under hypoxic conditions can improve engraftment and long-term modification of hematopoietic stem cells. We have continued to utilized the LAM-PCR technology, most recently utilizing a high throughput modification, and a non-biased restriction-enzyme free procedure to improve the technology for insertion site retrieval and tracking. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. Given the occurence of leukemia in now seven patients receiving gene therapy for severe immunodeficiencies with retrovirally-transduced hematopoietic stem cells, 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. 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. BCL2A1 over-expressed in murine HSCs results in clonal primarily B cell leukemias, implicating this gene product for the first time as leukemogenic. We have also recently found a profound impact of ex vivo expansion of transduced CD34+ cells on clonal diversity in vivo, with a selection for MDS1/EVI1 insertions after prolonged culture prior to transplantation. In vivo, cytotoxic pressure with busulfan was shown to result in clonal dominance of cells containing vector insertions in specific genes. We have successfully developed two suicide gene strategies allowing ablation of vector-containing hematopoietic cells in vivo, following transplantation of transduced cells. The first utilizes an optimized and highly sensitive herpes tk mutant transgene, which is activated by ganciclovir. We have shown complete ablation of all detectable retrovirus vector containing cells with a non-toxic 21 day treatment course of ganciclovir in non-human primates transplanted 4-6 months previously, with stable vector marking levels pre ganciclovir. The second utilized an engineered inducible caspase 9 suicide gene which can be activated by the small molecule dimerizer AP1903. Stably engrafted animals had greater than 90% of their vector-containing cells ablated with short treatment courses of AP1903, and we continue to optimize this system. We have recently utilized "bar coded" lentiviral vectors as an alternative methodology for performing clonal tracking of HSCs and their progeny in vivo, avoiding the issues with bias and efficiency in attempts to quantify clonal contributions via insertion site tracking. This very powerful approach circumvents the non-quantitative retrieval of vector insertion sites with LAM-PCR or other insertion retrieval strategies, and is allowing detailed quantitative assessment of the output from and behavior of individual HSPC clones in vivo, in a relevant large animal model.

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载体以及基于SIV的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。我们已经发现，在低氧条件下的转导可以改善造血干细胞的植入和长期修饰。我们继续利用LAM-PCR技术，最近利用高吞吐量修改，以及无偏见的限制性酶 - 无酶程序来改善插入站点检索和跟踪的技术。在移植CD34+转导的祖细胞后，我们检索并分析了对外周血种群的克隆贡献。鉴于白血病的发生在现在的七名患者中，患有逆转录病毒转导的造血干细胞的严重免疫缺陷的患者，我们已经对恒河猕猴中的逆转录病毒插入部位进行了大规模测序，该猕猴用用MLV或SIV vectors to dromant型细胞进行了细胞。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。 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。在鼠HSC中过表达的Bcl2a1导致克隆的主要是B细胞白血病，首次将这种基因产物视为白血病。我们最近还发现，转导CD34+细胞的离体扩展对体内克隆多样性的深远影响，在移植前长时间培养后选择了MDS1/EVI1插入。 在体内，证明具有链球菌的细胞毒性压力会导致特定基因中含有载体插入的细胞的克隆优势。 我们已经成功地制定了两种自杀基因策略，允许在转导细胞移植后体内消融含有载体的造血细胞。 第一个利用了优化且高度敏感的疱疹TK突变体转基因，该转基因被Ganciclovir激活。我们已经显示了所有可检测的逆转录病毒载体完全消融，其中包含无毒的21天治疗过程的Ganciclovir在4-6个月前移植的非人类灵长类动物中的ganciclovir，稳定的矢量标记水平在Ganciclovir前。第二个利用了工程化的诱导caspase 9自杀基因，该基因可以被小分子二聚体AP1903激活。 稳定的植入动物的含量超过90％的含量的细胞，用AP1903的简短治疗过程消融，我们继续优化该系统。 我们最近利用“钢筋编码”的慢病毒载体作为对HSC进行克隆跟踪及其后代体内克隆跟踪的替代方法，避免了通过插入部位跟踪来量化克隆贡献的偏见和效率的问题。这种非常有力的方法规定了使用LAM-PCR或其他插入检索策略对向量插入位点的非定量检索，并允许对相关的大型动物模型对体内单个HSPC克隆的产出和行为进行详细的定量评估。