Intraosseous delivery of lentiviral vectors for hemophilia A gene therapy

项目总结 血友病A(HEMA)是由于凝血因子VIII(FVIII)基因(在结构中缩写为F8)缺失所致。 传统的治疗HEMA患者的方法是反复输注FVIII，这是一种昂贵、不便、短期、 而且不完全有效。此外，大约25%的接受治疗的患者产生了抗FVIII免疫。 回应。抑制性抗体(抑制剂)的发展显著增加了发病率并降低了寿命。 这些患者的质量。血友病抑制剂患者的治疗包括使用旁路药物或免疫 大剂量FVIII诱导耐受(ITI)或使用人源化生物工程因子Xa的新疗法 双特异性抗体(埃米珠单抗；ACE910)等。然而，频繁输注昂贵的试剂是 需要的，以及潜在的长期副作用仍需评估。与药物或蛋白质疗法相比， 基因治疗可以在一生中只需一次或几次治疗就能达到持久的治疗效果。 AAV介导的基因治疗在临床试验中显示出非常有希望的结果，但并不适用于 很大一部分患者包括儿科患者和抗AAV或抗FVIII抗体患者。 最近，针对巨核细胞FVIII表达的体外HSC基因治疗纠正了出血 即使在存在抑制剂的情况下也是如此。这是因为Fviii存储在α颗粒中，仅在 在血小板激活过程中的损伤部位，因此不受循环抑制物的影响。然而，有几个 体外基因治疗存在局限性。我们直接在血小板中长期表达FVIII的方法是 骨内输注慢病毒载体(IO-LV)(IO-LV基因治疗)携带FVIII转基因的人 脑磁图特异性启动子GPIBα(G-F8-LV)。在这个体内基因治疗方案中，造血干细胞(HSCs) LV能有效地原位转导FVIII，导致FVIII在MEGs中表达，并储存在血小板α颗粒中。 单次IO输注G-F8-LV可纠正HEMA小鼠的表型。体内注射LV可以避免 体外基因治疗遇到许多困难和潜在的毒性，包括低植入潜力 以及导致血小板减少的受试者的预适应，这是一种特别令人不快的并发症 血友病患者。体内基因治疗可以绕过这种显著的止血风险。 此前，我们已经证明了IO-LV基因后纠正血友病表型的非常有希望的结果 在HEMA小鼠中使用和不使用预先存在的抑制剂的治疗。为了开发一种临床上可行的 人类应用的协议，我们建议在HEMA小鼠中提高这一策略的有效性和安全性。 此外，我们还将检测新型人源化NSGW41和NSGW41在人体细胞中的有效性和安全性 HEMA/NSG/VWFRH/RH转基因小鼠。最后，我们启动了IO-LV基因治疗HEMA犬的研究。 我们将进一步改进方案，以实现长期的表型纠正在HEMA犬和没有 抑制剂。这些研究对于推动这项新技术的临床试验至关重要。 使用和不使用抑制剂的HEMA患者。

PROJECT SUMMARY Hemophilia A (HemA) results from a deficiency of factor VIII (FVIII) gene (abbreviated as F8 in constructs). Traditional treatment of HemA patients is repeated infusions of FVIII, which is costly, inconvenient, short-term, and incompletely effective. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Development of inhibitory antibodies (inhibitors) significantly increases morbidity and lowers life quality for these patients. Treatment for hemophilia inhibitor patients includes use of bypassing agents or immune tolerance induction (ITI) with high doses of FVIII or new therapies with bioengineered factor Xa, a humanized bispecific antibody (emicizumab; ACE910), and others. However, frequent infusions of costly reagent are required, and potential long-term side effects still need to be evaluated. Compared to drug or protein therapy, gene therapy can achieve a prolonged therapeutic effect with only one or several treatments over the lifetime. AAV-mediated gene therapy showed very promising results in clinical trials however is not accessible to a significant portion of patients including pediatric patients and patients with anti-AAV or anti-FVIII antibodies. Recently ex vivo HSC gene therapy targeting FVIII expression in megakaryocytes (Megs) corrected the bleeding diathesis even in the presence of inhibitors. This is because FVIII 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. Our approach to direct long-term expression of FVIII in platelets is intraosseous (IO) infusion of lentiviral vectors (LVs) (IO-LV gene therapy) carrying a FVIII transgene driven by a Meg-specific promoter GpIbα (G-F8-LVs). In this in vivo gene therapy protocol, hematopoietic stem cells (HSCs) were efficiently transduced by LVs in situ, resulting in FVIII expression in Megs and storage in platelet α-granules. A single IO infusion of G-F8-LV leads to phenotype correction in HemA mice. In vivo delivery of LVs can avoid many difficulties and potential toxicities encountered by ex vivo gene therapy including low engraftment potential and pre-conditioning of the subject which induces thrombocytopenia, a particularly undesirable complication for hemophilia patients. In vivo gene therapy can bypass this significant hemostatic risk. Previously we have demonstrated very promising results to correct hemophilia phenotype following IO-LV gene therapy in HemA mice both with and without pre-existing inhibitors. In order to develop a clinically feasible protocol for human applications, we propose to enhance the efficacy and safety of this strategy in HemA mice. Furthermore, we will examine the efficacy and safety in human cells using novel humanized NSGW41 and HemA/NSG/VWFRH/RH transgenic mice. Lastly, we have initiated the study of IO-LV gene therapy in HemA dogs. We will further improve the protocol to achieve long-term phenotypic correction in HemA dogs with and without inhibitors. These studies are essential for pushing forward this new novel technology for a clinical trial to treat HemA patients with and without inhibitors.