Safety and Efficacy of Systemic Gene Therapy in Informative Models for DMD

 描述（由申请人提供）：杜氏肌营养不良症（DMD）是由蛋白质肌营养不良蛋白的遗传缺陷引起的。该项目的总体目标是， 这是DMD基因治疗的合理临床试验，最近的研究表明，在营养不良的小鼠和狗中，单剂量的新型载体对肌纤维具有前所未有的保护作用。为了使针对转基因产物的免疫反应的风险最小化，我们用肌营养不良蛋白代替肌营养不良蛋白。系统优化的合成utrophin minigene与肌肉靶向载体AAV 9一起使用，以实现新生动物中的系统性基因转移。在随后生长至成熟的mdx小鼠中，经处理肌肉的组织学外观恢复正常;在目标1中，我们现在测试这种水平的肌纤维保护是否会在广泛的临床相关测定中纠正肌肉功能。我们使用适当的方法来研究小鼠的自发活动，运动类型，自愿跑步时间和距离，以及抓握测试中的前肢和后肢肌肉力量。我们测定运动后CPK的正常化。最后，我们建立了功能的重组utrophin在离体肌肉，包括膈肌和心脏，死后。为了解决临床试验中潜在受试者面临的主要安全性问题，在目标2中，我们将注意力转向免疫应答，使用由犬中天然存在的缺失提供的独特疾病模型。索引突变被确定在德国短毛指针（GSHPMD），并显示由我们的实验室导致的同源重组事件之间的铁蛋白样假基因侧翼的整个犬肌营养不良蛋白基因。我们系统地研究了在该模型中对重组肌营养不良蛋白和肌营养不良蛋白的免疫应答，以严格检验以下假设：在缺乏中枢（胸腺）免疫耐受的情况下，宿主T细胞将侵入AAV转导的肌纤维，从而消除重组肌营养不良蛋白表达并引起临床上严重的肌炎，其加剧GSHPMD的临床过程。我们进一步假设中枢免疫耐受将保护用重组utrophin转导的肌纤维，从而促进治疗功效。最后，在目标3中，我们在营养不良犬中进行全身载体递送的剂量探索研究，以估计随后用于临床前疗效试验的适当剂量。根据我们迄今为止在小鼠中的研究，这将需要在个体狗中使用前所未有的AAV滴度，并将包括载体毒性的筛选试验。

 DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is caused by inherited deficiency of the protein dystrophin. The overall aim of this project is to build towards rational clinical trials of gene therapy in DMD, following recent studies showing unprecedented protection of muscle fibers by a single dose of a novel vector in dystrophic mice and dogs. To minimize the risk of an immunological reaction against the transgene product, we have substituted utrophin for dystrophin. A systematically optimized, synthetic utrophin minigene is used with a muscle-targeting vector, AAV9, to achieve systemic gene transfer in newborn animals. In mdx mice following subsequent growth to maturity, the histological appearance of the treated muscle is restored to normal; in Aim 1 we now test whether this level of muscle fiber protection will correct muscle function in a wide range of clinically relevant assays. We use appropriate methods to study locomotor activity of the mice, types of movement, voluntary running times and distances, as well as forelimb and hindlimb muscle strength in grip tests. We assay for normalization of the CPK following exercise. Finally, we establish the function of the recombinant utrophin in isolated muscles, including the diaphragm and the heart, post mortem. To address the major safety issue facing potential subjects in clinical trials, in Aim 2 we turn ou attention to the immune response, using a unique disease model afforded by a naturally occurring deletion in the dog. The index mutation was identified in a German Short Haired Pointer (GSHPMD), and was shown by our lab to result from a homologous recombination event between ferritin-like pseudogenes flanking the entire canine dystrophin gene. We systematically investigate the immunological response to recombinant dystrophin and utrophin in this model to rigorously test the hypothesis that in the absence of central (thymic) immunological tolerance, host T cells will invade AAV-transduced muscle fibers, thereby eliminating recombinant dystrophin expression and causing a clinically severe myositis that exacerbates the clinical course of GSHPMD. We further hypothesize that central immunological tolerance will protect muscle fibers transduced with recombinant utrophin, thereby facilitating therapeutic efficacy. Finally, in Aim 3 we conduct a dose-finding study of systemic vector delivery in the dystrophic dog to estimate the appropriate dose for later use in a preclinical tria of efficacy. This will require the use of unprecedented titers of AAV in individual dogs, based on our studies thus far in mice, and will include screening tests for vector toxicity.