Intraosseous delivery of lentiviral vectors for hemophilia A gene therapy

用于血友病 A 基因治疗的慢病毒载体骨内递送

• 批准号: 10450849
• 负责人: Carol H Miao
• 金额: $ 68.54万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10450849
• 关键词: Alpha Granule; Antibodies; Biomedical Engineering; Bispecific Antibodies; Blood Platelets; Blood coagulation; Bone Marrow; Bone Marrow Cells; Bypass; Canis familiaris; Cell Culture Techniques; Cells; Clinical; Clinical Trials; Complication; Data; Development; Disease susceptibility; Dose; Engraftment; Ensure; Erythropoiesis; F8 gene; Factor VIII; Factor Xa; Gene Transduction Agent; Gene Transfer; Genes; Hematopoietic stem cells; Hemophilia A; Hemorrhage; Hemostatic Agents; Human; Immune Tolerance; Immune response; Immune system; In Situ; Infusion procedures; Injury; Lentivirus Vector; Mediating; Megakaryocytes; Morbidity - disease rate; Mus; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Plasma; Platelet Activation; Production; Proteins; Protocols documentation; Quality of life; Reagent; Risk; Safety; Site; Therapeutic; Therapeutic Effect; Thrombocytopenia; Toxic effect; Transgenes; Transgenic Mice; Variant; Viral; antibody inhibitor; cost; delivery vehicle; effective therapy; efficacy evaluation; gene therapy; improved; in vivo; inhibitor; integration site; lentivirally transduced; mouse model; neutralizing antibody; new technology; novel; novel strategies; novel therapeutics; overtreatment; pediatric patients; preconditioning; promoter; side effect; stem cell gene therapy; targeted treatment; transduction efficiency

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.

项目摘要 A型血友病（HemA）是由因子VIII（FVIII）基因（在构建体中缩写为F8）缺陷引起的。 A型血友病患者的传统治疗是反复输注FVIII，这是昂贵的、不方便的、短期的， 不完全有效。此外，约25%的治疗患者发生抗FVIII免疫反应， 应答抑制性抗体（抑制剂）的发展显着增加发病率和降低寿命 这些患者的质量。血友病抑制物患者的治疗包括使用旁路剂或免疫治疗。 使用高剂量的凝血因子VIII诱导耐受（ITI）或使用生物工程因子XA（人源化因子）的新疗法 双特异性抗体（emicizumab; ACE 910）等。然而，频繁地注入昂贵的试剂， 需要，潜在的长期副作用仍需评估。与药物或蛋白质疗法相比， 基因治疗只需一生进行一次或多次治疗即可达到延长治疗效果。 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，但目前尚不容易获得。 大部分患者，包括儿科患者和具有抗AAV或抗FVIII抗体的患者。 最近，靶向巨核细胞（Megs）中FVIII表达的体外HSC基因治疗纠正了出血 甚至在抑制剂的存在下。这是因为FVIII储存在α颗粒中，仅在 血小板活化过程中的损伤部位，因此免受循环抑制剂的影响。但几 离体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（IO）输注携带FVIII转基因的慢病毒载体（LV）（IO-LV基因疗法）， Meg-specific promoter GpIbα（G-F8-LVs）.在这种体内基因治疗方案中，造血干细胞（HSC） 在原位被LV有效地转导，导致FVIII在Megs中表达并储存在血小板α颗粒中。 G-F8-LV的单次IO输注导致A型血友病小鼠中的表型校正。LV的体内递送可以避免 离体基因治疗遇到的许多困难和潜在毒性，包括低植入潜力 以及诱导血小板减少症的受试者的预处理，血小板减少症是 血友病患者体内基因治疗可以绕过这种显著的止血风险。 以前，我们已经证明了非常有希望的结果，以纠正血友病表型后，IO-LV基因 在有和没有预先存在的抑制剂的A型血友病小鼠中的治疗。为了开发一种临床上可行的 方案用于人类应用，我们建议提高该策略在HemA小鼠中的有效性和安全性。 此外，我们将使用新的人源化NSGW 41和人源化NSGW 42在人细胞中检查功效和安全性。 HemA/NSG/VWFRH/RH转基因小鼠。最后，我们已经开始了IO-LV基因疗法在HemA狗的研究。 我们将进一步改进该方案，以实现长期的表型校正，在有和没有 抑制剂的这些研究对于推动这项新的临床试验技术治疗癌症至关重要。 有和无抑制剂的A型血友病患者。