Optimization of gene transfer safety and efficacy focusing on the NHP model

摘要：临床和基础实验室研究旨在开发高效、安全的造血细胞（包括干细胞和祖细胞）基因转导和离体操作策略，并利用遗传标记技术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试验，我们专注于优化基因转移到原始干细胞和祖细胞，以及了解和提高已建立的和新的载体系统的安全性。我们检索并分析了CD34+转导的祖细胞移植后对外周血群的克隆贡献。考虑到使用逆转录病毒转导的造血干细胞接受严重免疫缺陷基因治疗的患者发生白血病，我们对移植了MLV、HIV或SIV载体转导的细胞的恒河猴进行了大规模的逆转录病毒插入位点测序，并继续跟踪18年前移植了转导CD34+细胞的动物。预测逆转录病毒基因转移的长期安全性和实用性的独特资源。

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 on understanding and enhancing safety of established and new vector systems. We retrieve and analyze clonal contributions to peripheral blood populations following transplantation of CD34+ transduced progenitor cells. Given the occurence of leukemia in 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, HIV or SIV vectors, and we continue to follow animals transplanted up to 18 years ago with transduced CD34+ cells, a unique resource for predicting the long-term safety and utility of retroviral gene transfer. 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 utilizes 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, however, repeated dosing with escalating doses failed to ablate 100% of vector-containing cells, in contrast to our results with herpes TK/ganciclovir. We have discovered that expression level of the iCasp transgene impacts on ablation, and low-expressing cells persist. We have also documented upregulation of the anti-apoptotic gene product Bcl2 in cells persisting in vivo and in vitro following dimerizer exposure. We have begun applying our barcoding of individual hematopoietic stem and progenitor cell clones to investigate genotoxicity and develop a relevant preclinical model for assessing genotoxicity prior to clinical trials, since in vitro assays and murine models have not been ideally predictive. The quantitative assessment of oligoclonality in vivo, via our highly sensitive and quantitative barcoding approach, should allow relevant comparisons between highly genotoxic and non-genotoxic vectors as an initial model to validate this approach. The lack of appropriate rodent models for some human acquired and congenital diseases has limited investigations of pathophysiology and treatment. There are few natural disease models in monkeys. We have begun to apply several genome engineering approaches to develop rhesus macaque models for human diseases with no appropriate rodent models, specifically paroxysmal nocturnal hemoglobinuria, a serious hematologic disease with many questions remaining regarding the pathophysiology of clonal dominance of PNH stem cells and the pathways resulting in thrombosis, the most common cause of morbidity and mortality in PNH patients. Using the iCas/CRSPR approach, we have in vitro evidence for knockout of the rhesus PIG-A gene (the mutated gene in PNH), and we plan to move into in vivo studies in the next year. We will utilize mRNA transfection to deliver the knockout construct. We have also begun to utilize this technology to create a primate model of DNMT3 deficiency in rhesus HSPC. This gene is commonly mutated in human myeloid leukemias, as well as in older adults with clonal hematopoiesis but no leukemia.