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

 产品说明： 本提案的目的是开发有效的基因疗法，通过直接体内骨髓（BM）转移或通过新生儿静脉内递送使用表达人因子VIII（FVIII）的慢病毒（LV）或泡沫病毒载体（FV）来治疗血友病A（HemA）。目前A型血友病患者的治疗方法是重复输注FVIII，成本高且不方便，约25%的患者产生抗FVIII抗体。非常需要能够实现长期表型校正而不形成抗体的基因治疗。骨髓造血干细胞（HSC）是基因治疗的理想靶点，因为它们能够自我更新，并能分化为血液和其他类型的细胞。已经显示LV的骨内（IO）递送和LV和FV的新生儿递送有效地使小鼠中的BM细胞增殖。这些方法避免了离体基因治疗遇到的困难，包括细胞转移后干细胞特性和植入潜力的丧失。此外，不需要干细胞的体外操作或受试者的预处理。 之前，我们比较了使用驱动人FVIII（F8）表达的两种不同启动子（普遍存在的人延伸因子-1 α（EF1 α）启动子（E-F8-LV）和人巨核细胞特异性糖蛋白1b α（GP1b α）启动子（G-F8-LV））的LV IO递送。尽管HSC可以被E-F8-LV有效地转导，但诱导了强烈的抗FVIII免疫应答，其消除了循环中的功能性FVIII。与此相反，血小板特异性FVIII的表达， 在单次IO输送G-FVIII-LV后实现，导致长期部分纠正 有和没有预先存在的前体的动物中的HemA 1。我们的研究结果表明，血小板可能是递送FVIII的理想载体：1）储存在α颗粒中的FVIII可免受高滴度中和抗体的影响; 2）即使是相对少量的局部分泌FVIII的活化血小板也足以促进出血期间有效的血凝块形成。 1）进一步优化的G-DF8-LV和G-DF8-FV构建体的IO递送，所述构建体结合了更高表达的FVIII变体，与抑制初始先天免疫应答的试剂组合，将导致HSC的更有效转导和更高水平的FVIII表达-从而导致HemA的有效治疗; 2）通过比较由更有效的普遍存在的PGK启动子指导的P-F8-LV与G-F8-LV（或相应的FV）的新生儿递送，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; 3）我们将通过在人源化小鼠中使用最佳载体来建立用于人类应用的主要研究的证据。在研究结束时，我们将能够破译最有效的LV或FV系统，指导BM HSC基因治疗治疗HemA。重要的是，这些研究是专门设计的，为在A型血友病犬中使用该递送平台的平行临床前研究铺平道路，并最终为最佳设计的临床试验铺平道路。

 DESCRIPTION: The goal of this proposal is to develop effective gene therapy to treat hemophilia A (HemA) by either direct in vivo bone marrow (BM) transfer or by neonatal intravenous delivery using lentiviral (LV) or Foamy viral vectors (FV) expressing human factor VIII (FVIII). Current treatment of HemA patients with repeated FVIII infusions is costly and inconvenient, and ~25% of patients develop anti-FVIII antibodies. Gene therapy that can achieve long-term phenotypic correction without antibody formation is highly desired. BM hematopoietic stem cells (HSC) are ideal targets for gene therapy because they are capable of self-renewal and can differentiate into blood and other types of cells. Intraosseous (IO) delivery of LVs and neonatal delivery of LVs and FVs have been shown to effectively transduce BM cells in mice. These approaches avoid the difficulties encountered by ex vivo gene therapy including loss of stem cell properties and engraftment potential after cell transfer. Furthermore, no in vitr manipulation of stem cells or pre-conditioning of subjects is required. Previously, we have compared IO delivery of LVs utilizing two different promoters driving expression of human FVIII (F8), a ubiquitous human elongation factor-1α (EF1α) promoter (E-F8-LV) and a human megakaryocytic-specific glycoprotein 1bα (GP1bα) promoter (G-F8-LV). Although HSCs can be efficiently transduced by E-F8-LV, robust anti-FVIII immune responses were induced that eliminated functional FVIII in the circulation. In contrast, platelet-specific FVIII expression was achieved 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 inhibitors1. Our findings suggest that platelets may comprise an ideal vehicle for delivering FVIII: 1) FVIII stored in α-granules is protected from high-titer neutralizing antibodies; and 2) even relatively small numbers of activated platelets that locally excrete FVIII are sufficient to promote efficient clot formation during bleeding. In the current proposal, we will test the hypothesis that: 1) IO delivery of a further optimized, G-DF8-LV and G-DF8-FV constructs that incorporate a higher-expressing FVIII variant, in combination with agents that suppress the initial innate immune response, will result in more efficient transduction of HSCs and higher levels of FVIII expression - thereby leading to effective treatment of HemA; 2) By comparing neonatal delivery of P- F8-LV directed by a more potent, ubiquitous PGK promoter vs. G-F8-LV (or corresponding FVs), we will determine whether long-term phenotypic correction of HemA can be achieved by neonatal gene therapy; 3) We will establish proof of principal studies for human applications by using the best vectors in humanized mice. At the end of the study, we will be able to decipher the most effective LV or FV system for directing BM HSC gene therapy to treat HemA. Importantly, these studies are specifically designed to pave the way for parallel pre- clinical studies using this delivery platform in HemA dogs and, ultimately, to an optimally designed clinical trial.