Hemophilia A: Intrathymic Delivery for Tolerance and Longterm Expression of FVIII

描述（由申请方提供）：血友病A的当前标准治疗是静脉输注人凝血因子VIII（hFVIII）蛋白治疗，无论是预防性治疗还是出血发作期间。预防性和按需FVIII蛋白输注的主要缺点是在约25%的治疗个体中形成抗人FVIII抗体（抑制剂）。抑制剂形成是这种出血性疾病最困难的并发症之一，因为抑制剂中和hFVIII，从而阻碍hFVIII替代疗法。该项目的长期目标是开发一种疗法，可以避免抑制剂形成的严重问题并提供恒定量的VIII蛋白。该提案将利用胸腺的独特性质，通过胸腺内注射编码人FVIII的慢病毒载体来诱导对外源蛋白（hFVIII）的耐受性。胸腺内注射慢病毒载体有效地转导长寿命的胸腺基质细胞，其是持续hFVIII蛋白生产的靶细胞类型。由于胸腺的免疫学特性，这种方法应有助于hFVIII的长期表达而不被免疫系统消除。这一方法的普遍可行性得到了几项研究的支持。例如，MHC不相容的骨髓基质细胞被工程化以表达转基因并植入胸腺中不会经历免疫排斥，并且可以长期分泌转基因产物。40年的研究表明，胸腺内暴露于外源性抗原可诱导对随后外周移植物的耐受。关于递送系统，来源于猫免疫缺陷病毒（FIV）的非灵长类慢病毒载体提供了包括基质细胞在内的多种细胞和器官的优异转导效率。总之，这些结果表明，利用胸腺的免疫特权状态作为蛋白质产生的位点有可能彻底改变血友病A的治疗。该提案将探索胸腺内FIV-hFVIII载体注射以防止抑制剂形成。将使用FIV载体，因为它们有效地抑制非增殖细胞，包括构成胸腺基质的那些细胞，并稳定地整合到宿主基因组中，潜在地允许转基因的终身表达。另外的益处是病毒颗粒的低毒性、低免疫原性和在整合到宿主基因组中后缺乏任何病毒基因。将测试有效且相对无免疫原性的载体系统与胸腺内递送途径的组合将防止抑制剂形成并允许hFVIII蛋白从转导的胸腺基质分泌到外周血中的假设。为了证明这种方法用于长期hFVIII治疗的可行性，将在小鼠模型中在六个月内测量hFVIII蛋白和可能的抗hFVIII抗体，将评估FIV-hFVIII胸腺内注射对胸腺生成的可能影响，并将分析胸腺以外的器官的载体转导和hFVIII mRNA表达。来自这些实验的数据将表明胸腺内注射FIV-hFVIII载体是否将提供特别针对减少抑制剂形成的有价值的治疗替代方案。超出了本资助申请的范围，拟议的概念验证研究将成为未来研究的基础，包括胸腺内注射与静脉注射给药途径的比较以及胸腺内注射减少既存抑制剂的潜力。使用基因治疗来纠正血友病A引入了因子VIII蛋白的表达。因子VIII蛋白在血友病患者中含量低或缺失，因此对大多数患者来说是外源蛋白。暴露于任何外来蛋白质都有可能使受体患者对新蛋白质产生免疫反应。这种免疫反应中和了治疗性蛋白质，使其无效。大约25%的血友病A患者患有抑制物，这导致严重的并发症，包括不受控制的出血，慢性关节疾病和过早死亡。拟议的研究将评估和优化一种方法，以“教导”免疫系统新的治疗蛋白质不是外来的，这样就不会产生免疫反应，并且VIII因子蛋白质阻止不受控制的出血的功能将得到保留。

DESCRIPTION (provided by applicant): The current standard of care for hemophilia A is treatment with intravenous human factor VIII (hFVIII) protein infusions, either prophylactically or during bleeding episodes. A major disadvantage for both prophylactic and on- demand FVIII protein infusions is the formation of anti-human FVIII antibodies (inhibitors) in about 25% of treated individuals. Inhibitor formation is one of the most difficult complications of this bleeding disorder since inhibitors neutralize hFVIII and thus thwart hFVIII replacement therapy. The long-term goal of this project is to develop a therapy that can avoid the serious problem of inhibitor formation and provide constant amounts of FVIII protein. This proposal will exploit the unique properties of the thymus to induce tolerance to foreign proteins (hFVIII) by intrathymic injection of a lentiviral vector encoding human FVIII. Intrathymic injection of lentiviral vectors efficiently transduces the long-lived thymic stromal cells, which are the target cell types for sustained hFVIII protein production. Due to the immunological properties of the thymus, this approach should facilitate long-term expression of hFVIII without elimination by the immune system. The general feasibility of this approach is supported by several studies. For example, MHC-incompatible bone marrow stromal cells engineered to express a transgene and implanted into the thymus will not undergo immune rejection and can secrete the transgene product long-term. Work spanning forty years has shown that intrathymic exposure to foreign antigen can induce tolerance to a subsequent graft in the periphery. With respect to the delivery system, a non-primate lentiviral vector derived from Feline Immunodeficiency Virus (FIV) provides excellent transduction efficiency of a variety of cells and organs including stromal cells. Together, these results show that exploiting the immune privileged status of the thymus as a site for protein production has the potential to revolutionize treatment for hemophilia A. This proposal will explore intrathymic FIV-hFVIII vector injection to prevent inhibitor formation. FIV vectors will be used since they efficiently transduce non-proliferating cells including those that make up the thymic stroma, and stably integrate into the host genome potentially allowing lifelong expression of the transgene. Additional benefits are the viral particle's low toxicity, low immunogenicity and the lack of any viral genes after integration into the host genome. The hypothesis that the combination of an efficient and relatively non-immunogenic vector system with the intrathymic route of delivery will prevent inhibitor formation and allow secretion of hFVIII protein from transduced thymic stroma into peripheral blood will be tested. To demonstrate the feasibility of this approach for long-term hFVIII therapy, hFVIII protein and possible anti-hFVIII antibodies will be measured over six months in a mouse model, possible effects of FIV- hFVIII intrathymic injection on thymopoiesis will be assessed, and organs other than thymus will be analyzed for vector transduction and hFVIII mRNA expression. Data from these experiments will indicate whether intrathymic injection of FIV-hFVIII vectors will provide a valuable treatment alternative specifically aimed at reduced inhibitor formation. Exceeding the scope of this grant application, the proposed proof-of-concept studies will form the basis for future studies including a comparison of the intrathymic with the iv route of administration and the potential of intrathymic injection to reduce pre-existing inhibitors. The use of gene therapy to correct hemophilia A introduces the expression of the factor VIII protein. Factor VIII protein is low or missing in hemophilia patients and therefore a foreign protein for most patients. Exposure to any foreign protein runs the risk of the recipient patient mounting an immune response to the new protein. Such immune responses neutralize the therapeutic protein rendering it ineffective. Approximately 25% of all hemophilia A patients suffer from inhibitors, which leads to severe complications including uncontrolled bleeding, chronic joint disease and premature death. The proposed research will evaluate and optimize a means to "teach" the immune system that the new therapeutic protein is not foreign so that an immune response will not be generated and the function of the factor VIII protein to stop uncontrolled bleeding will be preserved.