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

描述：本提案的目标是开发有效的基因治疗策略，通过直接在体内骨髓移植表达人因子VIII （FVIII）的慢病毒载体（LV）来治疗A型血友病（HemA）。目前对HemA患者反复输注FVIII的治疗是昂贵、不方便和不完全有效的。此外，约25%的治疗患者出现抗fviii免疫反应。基因治疗治疗，可以实现长期的表型纠正，而没有抗fviii抗体形成的并发症是非常需要的。骨髓中的干细胞（SCs）是基因和细胞治疗的理想目标，因为它们具有自我更新的能力，可以分化为血液和其他类型的细胞。慢病毒载体（lv）的骨内（IO）和新生儿分娩已被证明能有效地转导小鼠骨髓细胞。这些方法避免了体外SC基因转移所遇到的困难，包括维持干细胞特性、细胞转移后移植潜力的丧失以及体外细胞因子刺激的负面影响。此外，使用这些方法不需要对干细胞进行操作，也不需要使用潜在毒性的骨髓抑制剂对受试者进行预处理。之前，我们比较了使用两种不同启动子的lv的IO递送，一种是普遍存在的人延伸因子-1α (EF1α)启动子（E-F8-LV），另一种是人巨核细胞特异性糖蛋白1bα (GP1bα)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免疫反应，消除了循环中的功能性FVIII。相比之下，单次IO递送G-FVIII-LV后获得FVIII的血小板特异性表达，导致长期的部分纠正

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.