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: 9195405
• 负责人: Carol H Miao
• 金额: $ 66.3万
• 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 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9195405
• 关键词: Address; Alpha Granule; Animal Model; Animals; Antibodies; Antibody Formation; Apoptosis; Automobile Driving; Biological; Blood Circulation; Blood Platelets; Bone Marrow; Bone Marrow Cells; Bypass; CD34 gene; Canis familiaris; Cells; Cessation of life; Clinical; Complication; Development; Disease susceptibility; Dose; Elongation Factor; Engraftment; Ensure; F8 gene; Factor VIII; Factor VIIa; Gene Expression; Gene Transfer; Glycoproteins; Hematopoietic stem cells; Hemophilia A; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; Immune Tolerance; Immune response; In Vitro; Infusion procedures; Injury; Lentivirus Vector; Megakaryocytes; Modeling; Morbidity - disease rate; Mus; Patients; Pattern; Phenotype; Plasma; Platelet Activation; Platelet Count measurement; Property; Protocols documentation; Quality of life; Regimen; Resistance; Risk; Role; Safety; Site; Staging; Stem cells; Tail; Testing; Therapeutic; Therapeutic Effect; Thrombocytopenia; Variant; Viral; cytokine; effective therapy; gene therapy; humanized mouse; improved; in vitro Assay; in vivo; integration site; mouse model; new technology; novel; novel strategies; pre-clinical; preconditioning; prevent; promoter; response; vector

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.

项目摘要 目前对血友病A（HemA）患者的治疗是反复输注因子VIII（FVIII;缩写为 F8）是昂贵的、不方便的并且不完全有效的。此外，约25%的治疗患者 产生抗FVIII免疫应答（抑制剂）;治疗这些抑制剂患者尤其具有挑战性。 可实现长期表型校正而不伴有抗FVIII抗体的基因治疗 对于具有或不具有抑制剂的患者，都高度期望形成。最近的研究表明，FVIII表达 异位巨核细胞（Meg）能有效防止HemA小鼠尾部出血死亡。离体HSC 基因治疗纠正了出血素质，即使在存在抑制剂的情况下。这是因为FVIII 在Megs中合成的β-淀粉酶储存在α-颗粒中，并且仅在血小板活化期间在损伤部位释放， 因此免受循环抑制剂的影响。然而，离体基因治疗存在一些限制。第一、 与血浆FVIII相比，储存在血小板中的FVIII（pFVIII）具有不同的时空利用度。pFVIII 在不同的止血小鼠模型中显示出不同的功效，并且其对抑制剂的抗性也 在不同的环境中会有所不同。基因治疗载体pFVIII的功能及其对抑制剂的耐受性 将需要仔细研究和定义。二是离体HSC遇到的困难 基因治疗，特别是离体基因治疗所需的预处理对于HemA是非常不希望的 患者第三，已显示高水平的pFVIII表达可诱导血小板凋亡。 预处理方案用于体外基因治疗诱导血小板减少症。这个问题可以 与pFVIII表达复合，导致显著的血小板减少，这构成了主要的止血措施， A型血友病抑制剂患者的风险。最近，慢病毒载体（LV）的骨内（IO）递送已经显示出， 有效地抑制小鼠骨髓（BM）细胞。我们建议采用这种新方法来提供F8- LV进入BM，避免使用预处理方案，从而降低与 本发明的组合物可用于治疗血小板减少症，并且对于临床使用可安全且有效。之前，我们演示了一个 由产生的人Mega特异性糖蛋白1bα（GP 1b α）启动子（G-F8-LV）驱动的F8-LV的IO递送 血小板特异性FVIII表达，导致长期，部分纠正HemA小鼠， 预先存在的抑制剂。在目前的提案中，我们将扩大我们的研究，针对血小板中的FVIII表达 通过IO递送，旨在优化这种新技术并开发人源化的临床前方案， 小鼠和大型动物模型（HemA狗）。AIM 1.优化G-F8-LV在小鼠中的IO递送，并检查G-F8-LV在小鼠中的表达。 产生pFVIII的生物学功效。AIM 2.优化人Megs中的pFVIII基因表达。AIM 3.评价 如果IO递送LV可以有效地在大型动物模型中使HSC增殖，并且如果IO递送犬F8 （cF 8）-LV可以纠正HemA犬的表型。