Direct in vivo bone marrow transfer of lentiviral vector to correct hemophilia A

产品说明：本提案的目标是开发有效的基因治疗策略，通过直接体内骨髓转移表达人因子VIII（FVIII）的慢病毒载体（LV）来治疗血友病A（HemA）。目前使用FVIII重复输注治疗A型血友病患者成本高、不方便且不完全有效。此外，约25%的治疗患者发生抗FVIII免疫应答。高度期望可以实现长期表型校正而没有抗FVIII抗体形成的并发症的基因疗法治疗。骨髓中的干细胞（SC）是基于基因和细胞的治疗的理想靶点，因为它们能够自我更新，并且可以分化为血液和其他类型的细胞。骨内（IO）和新生儿递送慢病毒载体（LV）已显示有效地使小鼠骨髓细胞增殖。这些方法避免了离体SC基因转移遇到的困难，包括维持干细胞特性、细胞转移后移植潜力的丧失和离体细胞因子刺激的负面影响。此外，使用这些方法不需要使用潜在毒性的骨髓抑制剂对干细胞进行操作或对受试者进行预处理。以前，我们比较了使用两种不同启动子的LV的IO递送，这两种启动子是普遍存在的人延伸因子-1 α（EF 1 α）启动子（E-F8-LV）和人巨核细胞特异性糖蛋白1bα（GP 1b α）启动子（G-F8-LV）。我们证明，尽管SC可以被E-FVIII-LV有效地转导，但诱导了强大的抗FVIII免疫应答，其消除了循环中的功能性FVIII。相比之下，在G-FVIII-LV的单次IO递送后获得的FVIII的血小板特异性表达，导致血小板特异性表达的长期、部分校正。 存在和不存在既存抑制剂的动物中的HemA。我们的研究结果表明，血小板可能是递送FVIII的理想载体，因为储存在α颗粒中的FVIII受到中和抗体的保护，并且在出血期间，活化的血小板局部分泌FVIII以促进凝块形成。1）IO递送结合高表达FVIII变体的新G-DF 8-LV构建体，与抑制初始先天免疫应答的试剂组合，将导致HSC的更有效转导，从而允许更高数量的表达FVIII的血小板以及每个血小板更高水平的FVIII表达-从而导致有效治疗HemA; 2）HemA的表型校正也可以通过利用间充质SC（MSC）特异性巢蛋白启动子（有或没有免疫调节）的新N-F8-LV的IO递送来实现。3)通过比较E-F8-LV、G-F8-LV和N-F8-LV载体的新生儿分娩，我们将确定是否可以通过新生儿基因治疗实现HemA的长期表型校正。在研究结束时，我们将能够破译最有效的LV系统，用于指导BM SC基因疗法治疗HemA。

DESCRIPTION: The goal of this proposal is to develop effective gene therapy strategies to treat hemophilia A (HemA) by direct in vivo bone marrow transfer of lentiviral vectors (LV) expressing human factor VIII (FVIII). Current treatment of HemA patients with repeated infusions of FVIII is costly, inconvenient, and incompletely effective. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Gene therapy treatment that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired. Stem cells (SCs) in the bone marrow are ideal targets for gene- and cell-based therapy because they are capable of self-renewal and can differentiate into blood and other types of cells. Intraosseous (IO) and neonatal delivery of lentiviral vectors (LVs) have been shown to effectively transduce bone marrow cells in mice. These approaches avoid the difficulties encountered by ex vivo SC gene transfer including maintenance of stem cell properties, the loss of engraftment potential after cell transfer, and negative effects of ex vivo cytokine stimulation. Furthermore, no manipulation of stem cells or pre-conditioning of the subject using potentially toxic, myelosuppressive agents is required using these approaches. Previously we have compared IO delivery of LVs utilizing two different promoters, a ubiquitous human elongation factor-1α (EF1α) promoter (E-F8-LV) and a human megakaryocytic-specific glycoprotein 1bα (GP1bα) promoter (G-F8-LV). We demonstrated that although SCs can be efficiently transduced by E-FVIII-LV, robust anti-FVIII immune responses were induced that eliminated functional FVIII in the circulation. In contrast, platelet-specific expression of FVIII as obtained following a single IO delivery of G-FVIII-LV, leading to long- term, partial correction of HemA in animals both with and without pre-existing inhibitors. Our findings suggest that platelets may comprise an ideal vehicle for delivering FVIII as FVIII stored in α-granules is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. In the current proposal, we will test the hypothesis that: 1) IO delivery of a new G-DF8-LV construct incorporating a high-expressing FVIII variant, in combination with agents that suppress the initial innate immune response, will result in more efficient transduction of HSCs allowing both higher numbers of platelets expressing FVIII as well as higher levels of FVIII expression per platelet- thereby leading to effective treatment of HemA; 2) Phenotypic correction of HemA can also be achieved via IO delivery of a new N-F8-LV utilizing a mesenchymal SC (MSC)-specific nestin promoter (with or without immunomodulation). 3) By comparing neonatal delivery of E-F8-LV, G-F8-LV and N-F8-LV vectors, we will determine whether long-term phenotypic correction of HemA can be achieved by neonatal gene therapy. At the end of the study, we will be able to decipher the most effective LV system for directing BM SC gene therapy to treat HemA.