Molecular Chimerism Therapy for Hemophilia A

描述（由申请方提供）：血友病A是一种X连锁隐性遗传性出血性疾病，由凝血因子VIII（FVIII）缺乏或功能缺陷引起。目前尚无治愈血友病A的方法，患者在出血时接受FVIII浓缩物或重组蛋白输注。虽然这种治疗方案显著增加了血友病患者的预期寿命，但它不方便，并且具有潜在的严重并发症，例如在大约25%的患者中发生的FVIII抑制性抗体的产生，使他们难以进一步治疗。本研究的目的是评价编码靶向造血干细胞（HSC）的修饰人FVIII转基因的逆转录病毒载体在小鼠血友病A模型中的疗效。HSC是血友病A基因治疗的有吸引力的靶细胞群，因为它们易于进行离体遗传修饰，并且允许在接受者的一生中在循环外周血细胞中持续表达FVIII转基因的可能性。此外，靶向HSC的潜在益处是诱导对FVIII转基因产物的免疫耐受的可能性。近二十年来，我们的实验室一直在设计和优化逆转录病毒载体，用于HSC生物学和基因治疗建模的基因转移研究。特别是，我们的MSCV（鼠干细胞病毒）逆转录病毒载体正在美国进行的几项HSC基因治疗试验中使用。然而，在法国X连锁严重联合免疫缺陷病的临床试验中出现的不良事件要求重新评估逆转录病毒诱导突变的风险。因此，基于我们最近通过基于MSCV的HSC定向基因递送在血友病A小鼠中实现临床相关的FVIII血浆水平的成功，我们的具体目的是：（1）进一步优化FVIII转基因序列，以在造血细胞中更有效地分泌并降低蛋白质的免疫原性;（2）开发非清髓性HSC移植预处理方案，允许足够水平的转基因分子嵌合体，长期治疗性FVIII产生和耐受性诱导;和（3）产生生物学上更安全的FVIII逆转录病毒载体-在其长末端重复序列内缺乏转录调控元件，并且侧翼有增强子/启动子阻断元件-显示降低的HSC遗传毒性。

DESCRIPTION (provided by applicant): Hemophilia A is an X-linked recessive genetic bleeding disorder caused by a deficiency or functional defect in coagulation factor VIII (FVIII). There is currently no cure for hemophilia A and patients receive infusion of FVIII concentrates or recombinant proteins at the time of bleeding. Although this treatment regimen has increased the life expectancy of hemophiliacs significantly, it is inconvenient and has potentially serious complications such as the development of inhibitory antibodies to FVIII, which occurs in approximately 25% of patients, rendering them refractory to further treatment. The objective of this research is to evaluate the curative efficacy of retroviral vectors encoding modified human FVIII transgenes targeted to hematopoietic stem cells (HSCs) in a murine hemophilia A model. HSCs are an attractive target cell population for hemophilia A gene therapy because they are readily accessible for ex vivo genetic modification and allow for the possibility of sustained expression of a FVIII transgene in circulating peripheral blood cells for the recipient's lifetime. Moreover, a potential benefit of targeting HSCs is the possibility of inducing immunological tolerance to the FVIII transgene product. For almost two decades, our laboratory has been designing and optimizing retroviral vectors for gene transfer studies of HSC biology and gene therapy modeling. In particular, our MSCV (murine stem cell virus) retroviral vector is in use in several HSC gene therapy trials currently underway in the United States. However, the emergence of adverse events in a French clinical trial for X- linked severe combined immunodeficiency disease demands a reevaluation of the risks of retroviral-induced mutagenesis. Therefore, building upon our recent success at achieving clinically-relevant FVIII plasma levels in hemophilia A mice by MSCV-based HSC-directed gene delivery, our Specific Aims are: (1) To further optimize FVIII transgene sequences for more efficient secretion in hematopoietic cells and decreased immunogenicity of the protein; (2) To develop nonmyeloablative HSC transplant conditioning regimens that allow sufficient levels of transgene molecular chimerism for long-term therapeutic FVIII production and tolerance induction; and (3) To create biologically safer FVIII retroviral vectors - devoid of transcriptional regulatory elements within their long terminal repeats and flanked by enhancer/promoter-blocking elements - displaying reduced HSC genotoxicity.