Novel Therapy for Hemophilia B Using AAV-FIX Variants

项目1描述血友病B（HB）是由凝血因子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）进行基因治疗的有吸引力的靶组织。使用编码人FIX基因的重组腺相关病毒（rAAV）载体对HB受试者进行肌内（IM）注射的早期临床研究显示安全性良好，但疗效较低。提高效率的一个策略是锻炼更大面积的肌肉。我们通过开发经由传入经静脉逆行外渗（ATVRX）将AAV载体非侵入性外周经静脉递送至HB狗的骨骼肌来实现这一点。测试的载体剂量在安全递送给人类受试者的剂量范围内。在本申请中，我们提出通过使用天然存在的FIX变体FIX-帕多瓦或FIX-R338 L来进一步提高ATVRX用于HB基因治疗的治疗潜力。该FIX变体的比活性是野生型FIX的8倍。使用编码AAV-6的犬（c）FIX-帕多瓦通过ATVRX至骨骼肌的HB犬中的早期数据导致cFIX蛋白以血浆中正常FIX活性的10-30%表达，但抗原水平仅为1-3%。所用HB犬来自北卡罗来纳大学教堂山分校（UNC-CH）菌落，FIX基因存在错义突变。我们现在寻求在两种不同的HB犬模型中进行全面研究，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）降低止血功效所需的有效rAAV载体剂量，同时不增加FIX的局部抗原产生，和（2）克服骨骼肌在进行全功能蛋白的所有翻译后FIX修饰中的有限能力。该提案有三个具体目标。目的1：确定AAV-6-cFIX-Padua在通过ATVRX递送至骨骼肌后的安全和治疗剂量范围。我们将进行一项剂量递增研究，以确定将导致FIX可持续和安全治疗水平的最小和最大载体剂量。目的2：确定cFIX-Padua在两种犬HB模型中表达的免疫学特征：（a）使用来自伯明翰亚拉巴马大学（UAB）狗群的抑制剂易感HB犬的抑制剂形成的高风险模型（B）来自先前注射AAV-2-cFIX的表达亚治疗水平的野生型（WT）FIX的H-CH狗群的HB犬。目标3：目的：研究重组FIX-Padua蛋白的生物学功能和免疫原性。我们将通过一系列生物化学和功能动力学测定来表征FIX-帕多瓦，并在用这些FIX蛋白质进行AAV-FIX处理的狗中进行免疫挑战。因此，本提案的总体目标是建立一种新型治疗方案的有效性和安全性，该方案将rAAV向骨骼肌的优化外周血管内递送与有效AAV血清型（AAV-6）的使用以及新型高活性FIX作为转基因产品的使用相结合，该转基因产品应可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.