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旨在为造血细胞（包括干细胞和祖细胞）开发高效、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。在被证明是人类临床结果的唯一预测分析的恒河猴模型中，我们专注于优化原始干细胞和祖细胞的基因转移，并使用遗传标记技术来了解干细胞的体内行为。我们继续进一步提高恒河猴移植细胞的基因转移效率，标记细胞的早期水平高达50-80%，所有谱系的稳定水平为5-35%，具有临床实用性的范围。这些水平可以通过传统的双嗜性 MLV 载体以及基于 SIV 的慢病毒载体来实现。由于具有更有利的插入位点特征，我们开发了禽肉瘤白细胞增多病毒（ASLV）载体和基于噬菌体的用于造血靶细胞应用的位点特异性非病毒载体。正如我们的体内自体移植模型首次证明的那样，ASLV 可以转导恒河猴长期再生细胞。我们发现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，最近利用高通量修饰和无偏见的无限制性酶程序来改进插入位点检索和跟踪技术。我们检索并分析了 CD34+ 转导祖细胞移植后对外周血群体的克隆贡献。鉴于目前有 7 名接受逆转录病毒转导造血干细胞治疗严重免疫缺陷的患者出现白血病，我们对移植了 MLV 或 SIV 载体转导细胞的恒河猴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 对基因内的插入具有非随机偏好，SIV 插入均匀分布在基因长度上，特别是在基因高度丰富的染色体区域中发现。 MLV 的目标是转录起始位点周围的区域。 这些高度非随机事件表明在这些位点整合的强烈非随机偏好，或者这些克隆的体内植入或存活/增殖优势。 14 个独立插入位于 MDS1/EVI1 基因座，该区域先前与自发性白血病和具有复制能力的病毒的逆转录病毒诱变有关。 我们没有发现使用 SIV 或 ASLV 载体的 MDS1/EVI1 插入。 与 MLV 相比，SIV 和 ASLV 载体在原癌基因中的插入簇率明显较低。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BCL2A1和MDS1/EVI1对永生化或转化的影响。 BCL2A1 在小鼠 HSC 中过度表达会导致克隆性原发性 B 细胞白血病，首次表明该基因产物具有致白血病作用。我们最近还发现转导的 CD34+ 细胞的离体扩增对体内克隆多样性具有深远的影响，在移植前长时间培养后选择 MDS1/EVI1 插入。 在体内，白消安的细胞毒性压力被证明会导致含有特定基因中载体插入的细胞的克隆优势。 我们已经成功开发了两种自杀基因策略，允许在移植转导细胞后体内消除含有载体的造血细胞。 第一个利用优化且高度敏感的疱疹 tk 突变体转基因，由更昔洛韦激活。我们已经证明，在 4-6 个月前移植的非人灵长类动物中，使用更昔洛韦无毒的 21 天治疗疗程，可以完全消除所有可检测到的含有逆转录病毒载体的细胞，并且在更昔洛韦之前具有稳定的载体标记水平。第二种利用工程诱导型 caspase 9 自杀基因，该基因可以被小分子二聚体 AP1903 激活。 稳定移植的动物在 AP1903 的短期治疗过程中，超过 90% 的含有载体的细胞被消融，我们将继续优化该系统。 我们最近利用“条形码”慢病毒载体作为对 HSC 及其后代进行体内克隆追踪的替代方法，避免了通过插入位点追踪来量化克隆贡献的尝试中的偏差和效率问题。这种非常强大的方法避免了使用 LAM-PCR 或其他插入检索策略对载体插入位点进行非定量检索，并且允许在相关大型动物模型中对体内单个 HSPC 克隆的输出和行为进行详细的定量评估。