Intraosseous delivery of lentiviral vectors for hemophilia A gene therapy

项目总结 反复输注因子VIII的血友病A(HEMA)患者的当前治疗 构造中的F8)是昂贵的、不方便的，并且不完全有效。此外，约25%的接受治疗的患者 开发抗FVIII免疫反应(抑制剂)；治疗这些抑制剂患者特别具有挑战性。 基因治疗可以实现长期的表型纠正，而不会出现抗FVIII抗体的并发症 对于使用或不使用抑制剂的患者来说，形成是非常理想的。最近有研究表明，FVIII表达 异位注射巨核细胞(MEG)能有效防止HEMA小鼠尾部出血致死。体外HSC 即使在存在抑制剂的情况下，基因治疗也能纠正出血素质。这是因为FVIII 在MEGS中合成的物质储存在α颗粒中，并仅在血小板激活时在损伤部位释放， 因此不受循环抑制物的影响。然而，体外基因治疗存在一些局限性。第一, 保存在血小板中的FVIII(PFVIII)与血浆中的FVIII相比具有不同的时空可用性。PFVIII 在不同的止血小鼠模型中显示出不同的疗效及其对抑制剂的耐药性 在不同的设置中有所不同。PFVIII基因治疗的功能作用及其对抑制剂的耐药性 将需要仔细调查和界定。第二，体外HSC遇到了困难 基因治疗，特别是体外基因治疗所要求的预适应，对HEMA来说是非常不可取的。 病人。第三，研究表明高水平的pFVIII表达可诱导血小板凋亡。 体外基因治疗所使用的预适应方案会导致血小板减少。这个问题可能是 与pFVIII的表达相结合，导致显著的血小板减少，这构成了主要的止血作用 对HEMA抑制剂患者的风险。最近，慢病毒载体(LV)的骨内(IO)传递已被证明 有效转导小鼠骨髓细胞。我们建议使用这种新方法来交付F8- LVS进入BM，从而避免使用预适应方案，从而降低与 可安全有效地应用于临床。之前，我们演示了一首单曲 人脑磁特异性糖蛋白1bα(GP1bα)启动子(G-F8-LV)驱动的F8-LV的IO递送 血小板特异性FVIII的表达导致HEMA小鼠长期部分纠正 预先存在的抑制剂。在目前的提案中，我们将扩大针对血小板表达FVIII的研究 通过IO交付，旨在优化这一新技术并开发人性化的临床前协议 小鼠和大型动物模型(河马犬)。目的1.优化G-F8-LV在小鼠体内的IO传递，并检测 由此产生pFVIII的生物学效应。目的：优化pFVIII基因在人MEGs中的表达。目标3.评估 输注LV是否能有效地转导大动物模型中的HSC，以及输注犬F8是否能有效地转导HSCs (CF8)-LV可纠正HEMA犬的表型。

PROJECT SUMMARY Current treatment of hemophilia A (HemA) patients with repeated infusions of factor VIII (FVIII; abbreviated as F8 in constructs) is costly, inconvenient, and incompletely effective. In addition, ~25% of treated patients develop anti-FVIII immune responses (inhibitors); it is particularly challenging to treat these inhibitor patients. Gene therapy that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired for patients with or without inhibitors. Recently it was shown that FVIII expressed ectopically in megakaryocyte (Meg) is effective to prevent HemA mouse tail bleeding to death. Ex vivo HSC gene therapy corrected the bleeding diathesis even in the presence of inhibitors. This is because FVIII synthesized in Megs is stored in α-granules and only released at the injury sites during platelet activation, therefore protected from circulating inhibitors. However, several limitations exist for ex vivo gene therapy. First, FVIII stored in platelets (pFVIII) has different temporal-spatial availability compared with plasma FVIII. pFVIII was shown to have variable efficacy in different hemostasis mouse models and its resistance to inhibitors also varies in different settings. The functional roles of gene therapy delivered pFVIII and its resistance to inhibitors will need to be carefully investigated and defined. Second, there are difficulties encountered by ex vivo HSC gene therapy, in particular, preconditioning required by ex vivo gene therapy is highly undesirable for HemA patients. Third, it was shown that high levels of pFVIII expression can induce platelet apoptosis. Preconditioning regimens used by ex vivo gene therapy induce thrombocytopenia. This problem can be compounded with pFVIII expression, leading to significant thrombocytopenia, which poses a major hemostatic risk to HemA inhibitor patients. Recently, intraosseous (IO) delivery of lentiviral vector (LV) has been shown to effectively transduce bone marrow (BM) cells in mice. We propose to employ this new approach to deliver F8- LVs into BM, which avoids the use of preconditioning regimen, thereby decreasing the associated risk with thrombocytopenia and can be both safe and efficacious for clinical use. Previously, we demonstrated a single IO delivery of F8-LVs driven by a human Meg-specific glycoprotein 1bα (GP1bα) promoter (G-F8-LV) produced platelet-specific FVIII expression, leading to long-term, partial correction of HemA mice both with and without pre-existing inhibitors. In the current proposal, we will expand our studies targeting FVIII expression in platelets via IO delivery, aiming at optimizing this novel technology and developing preclinical protocols in humanized mice and large animal models (HemA dogs). AIM 1. Optimize IO delivery of G-F8-LVs in mice and examine the resulting biological efficacy of pFVIII. AIM 2. Optimize pFVIII gene expression in human Megs. AIM 3. Evaluate if IO delivery of LVs can effectively transduce HSCs in large animal models and if IO delivery of canine F8 (cF8)-LV can correct the phenotype in HemA dogs.