Novel Therapy for Hemophilia B Using AAV-FIX Variants

项目1的说明 血友病B(HB)是一种由凝血因子IX(FIX)缺乏引起的X连锁疾病。骨骼 肌肉是用腺相关病毒载体(AAV)进行基因治疗的有吸引力的靶组织。早-相 利用编码人FIX基因的重组腺相关病毒载体进行临床研究 肌肉注射(IM)的HB受试者具有极好的安全性，但疗效较低。 提高效率的一种策略是转导更大面积的肌肉。我们通过以下方式实现了这一点 经外周静脉途径无创地将AAV载体导入HB犬骨骼肌 传入经静脉逆行输精管外纤维(ATVRX)。测试的媒介剂量在这些范围内 安全地交付给人类受试者。在这项应用中，我们建议进一步提高治疗潜力 通过使用自然产生的FIX变异体FIX-PADUA或FIX-R338L，将ATVRX用于乙肝基因治疗。此修复程序 变异体的比活性是野生型修复的8倍。使用HB犬的早期数据 AAV-6编码犬(C)通过ATVRX将PaduA固定到骨骼肌导致cFIX蛋白的表达 血浆中正常FIX活性的10-30%，但只有1-3%的抗原水平。使用的HB犬来自 北卡罗来纳大学教堂山分校(UNC-CH)的殖民地，并在FIX基因中存在错义突变。 我们现在试图在两种不同的HB犬模型上进行全面的研究，以评估HB的疗效和 CFIX-PADUA的安全性。我们假设，使用具有高比活性的FIX蛋白将使我们能够(1) 降低止血效果所需的有效rAAV载体剂量，同时不增加局部 FIX抗原的产生和(2)克服了骨骼肌在完成所有后处理时的有限能力 全功能蛋白质的翻译修复修饰。这项提议有三个具体目标。目标1： AAV-6-cFIX-PADUA肌肉注射后的安全剂量和治疗剂量范围 通过ATVRX。我们将进行剂量递增研究，以确定最小和最大媒介剂量， 将导致可持续和安全的治疗水平的FIX。目的2：确定猪瘟病毒的免疫学特征 CFIX-PaduA在两种犬乙肝模型中的表达：(A)使用抑制剂形成抑制剂的高危模型。 来自阿拉巴马大学伯明翰分校(UAB)狗群的俯卧HB狗(B)来自北卡罗来纳大学的HB狗- 表达先前注射AAV-2-cFIX的野生型(WT)FIX亚治疗水平的CH犬群体。 目的3.检测重组FIX-Padua蛋白的生物学功能和免疫原性。我们会 用一系列生化和功能动力学方法鉴定FIX-PADUA并进行免疫学研究 挑战-用这些修复蛋白修复治疗犬的AAV。因此，这项提案的总体目标是 确定一种结合了优化的外围设备的新型治疗方案的有效性和安全性 使用一种有效的AAV血清型(AAV-6)将rAAV血管内传递到骨骼肌，以及 使用一种新的高度活性的FIX作为转基因产品，应该可以翻译到患者护理中 未来。

Description of Project 1 Hemophilia B (HB) is an X-linked disease resulting from the deficiency of clotting factor IX (FIX). Skeletal muscle is an attractive target tissue for gene therapy using adeno-associated viral vectors (AAV). Eariy-phase clinical studies, using recombinant adeno-associated virus (rAAV) vectors encoding the human FIX gene for intramuscular (IM) injection of HB subjects, were associated with an excellent safety profile, but low efficacy. One strategy for increasing efficiency is to transduce larger areas of muscle. We accomplished this by developing a non-invasive, peripheral transvenular delivery of AAV vectors to skeletal muscle of HB dogs via afferent transvenular retrograde extravasafion (ATVRX). Vector doses tested were within the range of those safely delivered to human subjects. In this application we propose to further improve the therapeutic potential of ATVRX for HB gene therapy by using a naturally occurring FIX variant, FIX-Padua or FIX-R338L. This FIX variant is associated with an 8-fold higher specific activity than the wild-type FIX. Eariy data in HB dogs using AAV-6-encoding canine (c) FIX-Padua to skeletal muscle via ATVRX resulted in expression of a cFIX protein at 10-30% of normal FIX activity in plasma, but with only 1-3% antigen levels. The HB dogs used were from University of North Carolina-Chapel Hill (UNC-CH) colony and harbor a missense mutation in the FIX gene. We now seek to carry out a comprehensive study in two distinct dog models of HB to assess the efficacy and safety of cFIX-Padua. We hypothesize that the use of a FIX protein with high specific activity will allow us to (1) lower the effective rAAV vector doses required for hemostatic efficacy and at the same time not increase local antigen production of FIX and (2) overcome the limited capacity of skeletal muscle in performing all the post- translational FIX modifications for a fully functional protein. There are 3 specific aims to this proposal. Aim 1: To determine the safe and therapeutic dose range of AAV-6-cFIX-Padua following delivery to skeletal muscle via ATVRX. We will perform a dose-escalation study to define the minimal and maximal vector doses that would result in sustainable and safe therapeutic levels of FIX. Aim 2:To determine the immunological profile of expression of cFIX-Padua in two canine HB models: (a) High risk model for inhibitor formafion using inhibitor- prone HB dogs from the University of Alabama at Birmingham (UAB) dog colony (b) HB dogs from the UNC- CH dog colony expressing subtherapeutic levels of wild-type (WT) FIX from previous injections of AAV-2-cFIX. Aim 3. To determine the biological function and immunogenicity of recombinant FIX-Padua protein. We will characterize FIX-Padua by a series of biochemical and functional kinetic assays and carry out immunologic challenges in AAV-FIX treated dogs with these FIX proteins. Thus, the overall goal of this proposal is to establish the efficacy and safety of a novel therapeutic protocol that combines an optimized peripheral intravascular delivery of rAAV to skeletal muscle with the use of an effective AAV serotype (AAV-6), and the use of a novel highly active FIX as the transgene product that should be translatable to patient care in the future.