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。骨骼肌是利用腺相关病毒载体（AAV）进行基因治疗的一个有吸引力的靶组织。早期临床研究使用重组腺相关病毒（rAAV）载体编码人类FIX基因，用于HB受试者肌内注射（IM），具有良好的安全性，但疗效较低。提高效率的一个策略是调动更大面积的肌肉。我们通过开发一种非侵入性的外周经静脉将AAV载体通过传入经静脉逆行外渗（ATVRX）输送到HB犬的骨骼肌中来实现这一目标。经测试的媒介剂量在安全交付给人类受试者的剂量范围内。在本应用中，我们建议通过使用天然存在的FIX变体FIX- padua或FIX- r338l进一步提高ATVRX用于HB基因治疗的治疗潜力。该FIX变体的特异性活性比野生型FIX高8倍。在HB犬中使用aav -6编码犬(c) FIX- padua通过ATVRX进入骨骼肌的早期数据显示，血浆中cFIX蛋白的表达量为正常FIX活性的10-30%，但抗原水平仅为1-3%。所使用的HB犬来自北卡罗来纳大学教堂山分校（UNC-CH）菌落，在FIX基因中含有错义突变。我们现在寻求在两种不同的犬HB模型中进行一项综合研究，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FIX的局部抗原产生；(2)克服骨骼肌对全功能蛋白进行所有翻译后FIX修饰的有限能力。这一提议有三个具体目标。目的1：确定AAV-6-cFIX-Padua经ATVRX给药至骨骼肌后的安全和治疗剂量范围。我们将进行剂量递增研究，以确定最小和最大载体剂量，从而实现可持续和安全的FIX治疗水平。目的2：确定两种犬HB模型中cFIX-Padua表达的免疫学特征：(a)使用来自阿拉巴马大学伯明翰分校（UAB）犬群体的抑制剂易感HB犬形成的高风险模型(b)来自UNC- CH犬群体的HB犬从先前注射的AAV-2-cFIX中表达亚治疗水平的野生型（WT） FIX。目标3。目的：测定重组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，并对使用这些FIX蛋白的AAV-FIX治疗的狗进行免疫挑战。因此，本提案的总体目标是建立一种新的治疗方案的有效性和安全性，该方案结合了优化的外周血管内递送rAAV到骨骼肌，使用有效的AAV血清型（AAV-6），并使用一种新型的高活性FIX作为转基因产品，应该在未来转化为患者护理。

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.