Novel Therapy for Hemophilia B Using AAV-FIX Variants

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。骨骼肌是用腺相关病毒载体(AAV)进行基因治疗的极具吸引力的靶向组织。使用编码人类FIX基因的重组腺相关病毒(RAAV)载体肌肉注射乙肝受试者(IM)的早期临床研究具有极好的安全性，但疗效较低。提高效率的一种策略是转导更大面积的肌肉。我们开发了一种通过传入逆行外渗(ATVRX)将AAV载体非侵入性地经外周静脉输送到HB犬骨骼肌的方法来实现这一点。测试的媒介剂量在安全传递给人类受试者的范围内。在这项应用中，我们建议通过使用自然产生的FIX变体FIX-PADUA或FIX-R338L来进一步提高ATVRX用于乙肝基因治疗的治疗潜力。这种FIX变异体比野生型FIX的比活性高8倍。在HB犬的早期数据中，通过ATVRX将AAV-6编码的犬(C)Fix-Padua通过ATVRX注射到骨骼肌，导致cFIX蛋白在血浆中的表达水平是正常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)殖民地，FIX基因存在错义突变。我们现在试图在两种不同的HB犬模型上进行一项全面的研究，以评估cFIX-Padua的有效性和安全性。我们假设，使用具有高比活性的FIX蛋白将允许我们：(1)降低止血效果所需的有效rAAV载体剂量，同时不增加FIX的局部抗原产生；(2)克服骨骼肌在执行完整功能蛋白的所有翻译后FIX修饰时的有限能力。这项提议有三个具体目标。目的1：确定AAV-6-cFIX-PADUA经ATVRX肌肉注射后的安全和治疗剂量范围。我们将进行剂量递增研究，以确定将导致可持续和安全的FIX治疗水平的最小和最大载体剂量。目的：确定cFIX-PADUA在两种犬乙肝模型中的表达情况：(A)使用阿拉巴马大学伯明翰分校(UAB)犬群中倾向于抑制剂的HB犬建立的高风险模型；(B)来自UNC-CH犬群的表达AAV-2-cFIX野生型(WT)FIX亚治疗水平的HB犬。目的3.检测重组FIX-Padua蛋白的生物学功能和免疫原性。我们将通过一系列生化和功能动力学分析来表征FIX-PADUA，并用这些FIX蛋白在AAV-FIX处理的狗身上进行免疫学挑战。因此，这项建议的总体目标是建立一种新的治疗方案的有效性和安全性，该方案将优化的外周血管内rAAV递送到骨骼肌与使用有效的AAV血清型(AAV-6)相结合，并使用一种新型的高活性FIX作为转基因产品，在未来应该可以转化到患者护理中。

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). Early-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 extravasations (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. Early 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 formation 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.