Overcoming limitations for AAV gene therapy

基因治疗是治疗肌营养不良症的一种很有前途的方法。我们的中心发挥了重要作用， 推进AAV在肌肉疾病中的应用。其中包括抗肌萎缩蛋白和DUX 4的广泛表征 表达和功能，发现AAV可用于全身性基因递送至肌肉，以及 产生了许多小的肌肉特异性表达盒（MSEC），微肌营养不良蛋白（μDys）和 RNAi盒。这些进展使DMD、LGMD、XL-MTM 1和 其他肌肉疾病我们还开发和推进了许多骨骼和心肌测试 用于肌肉基因疗法的平台和工具，包括3D DMD人iPSC衍生的工程化肌肉 组织和DMD大鼠模型。然而，目前的AAV- μDys疗法并不像所希望的那样有效，并且AAV- μDys疗法也不像所希望的那样有效。 FSHD和其他显性疾病的RNAi疗法尚未进入临床。在这里，我们提出两个 相关目的是解决AAV基因疗法的局限性：（A）测试用于心脏和心脏病的新AAV衣壳变体， 骨骼肌，新型微小肌营养不良蛋白、较大分裂内含肽肌营养不良蛋白和MSEC设计的迭代测试 以使免疫原性最小化并增加心肌和骨骼肌效力;（B）应用增强的 用于FSHD治疗中DUX 4转录物敲低的盒和载体。在目标1中，我们开发载体来改善 DMD临床干预受到治疗性肌营养不良蛋白的水平和功能的限制， 在某些患者中，由于以下原因， 在一些实施方案中，所述方法包括对载体和/或转基因的免疫反应。我们将比较多种亲肌性血清型， 显示肌肉转导显着增加，以递送已设计的新型转基因盒 用于降低免疫原性和增加效力，特别是在心肌中。一个主要的重点将包括 测试新的分裂内含肽AAV载体以产生小型和全长肌营养不良蛋白。我们还测试了一个对偶向量 通过过表达一种酶（RNR）来恢复心脏功能抑制的策略， dATP，一种小分子肌球蛋白激活剂。抗肌萎缩蛋白免疫反应的研究将得到加强， 筛查病人血细胞的免疫反应性在目标2中，我们将联合收割机与增强的 RNAi盒用于推进FSHD的基因治疗。这个实验计划结合了从 目的1利用项目2中的新FSHD动物模型和FSHD临床研究。遗传和表型变化 在FSHD的FLExDUX 4小鼠模型中，将通过AAV-DUX 4 RNAi的局部和全身递送来靶向， 而进一步的研究将在FSHD的哥廷根小型猪模型中评估这些AAV-DUX 4 RNAi载体， 在项目2中得到体现。DUX 4基因沉默和减轻小型猪中的炎症将是 根据项目2的临床结果，通过MRI、肌肉结构变化和生物标志物测量。在一起， 项目1和2中的实验策略将通过解决当前的基因治疗问题， 由于载体效力、免疫力、转基因表达和动物模型缺陷而导致的局限性。

Gene therapy is a promising treatment for the muscular dystrophies. Our Center has played major roles in advancing the use of AAV for muscle disease. These include extensive characterization of dystrophin and DUX4 expression and function, the discovery that AAV can be used for systemic gene delivery to muscle, and generation of numerous small, muscle-specific expression cassettes (MSECs), micro-dystrophins (μDys) and RNAi cassettes. These advances have enabled multiple gene therapy trials for DMD, LGMDs, XL-MTM1and other muscle disorders. We have also developed and advanced many skeletal and cardiac muscle testing platforms and tools useful for muscle gene therapies, including 3D DMD human iPSC-derived engineered muscle tissues and a DMD rat model. However, current AAV- μDys therapies are not as effective as hoped, and AAV- RNAi therapies for FSHD and other dominant disorders have not advanced to the clinic. Here we propose two related aims to address limitations of AAV gene therapies: (A) Testing new AAV capsid variants for cardiac and skeletal muscles, iterative testing of novel micro-dystrophins, larger split-intein dystrophins, and MSEC designs to minimize immunogenicity and increase cardiac and skeletal muscle potency; (B) Application of enhanced cassettes and vectors for DUX4 transcript knock-down in FSHD therapy. In Aim 1, we develop vectors to improve DMD clinical interventions that have been limited by the levels and functionality of therapeutic dystrophins, and by very high vector doses that have caused serious adverse events (SAEs) in some patients due to immunological reaction to the vector and/or transgene. We will compare multiple myotropic serotypes, which show significantly increased muscle transduction to deliver novel transgene cassettes that have been designed for reduced immunogenicity and increased potency, especially in cardiac muscle. A major focus will include the testing of novel, split-intein AAV vectors to produce mini- and full-length dystrophins. We also test a dual vector strategy to recover depressed function in the heart via overexpression of an enzyme (RNR) that elevates cardiac dATP, a small molecule myosin activator. Studies of immune response to dystrophin will be augmented by screening patient blood cells for immune reactivity. In Aim 2 we combine the myotropic vectors with enhanced RNAi cassettes to advance gene therapy for FSHD. This experimental plan combines vector developments from Aim 1 with new FSHD animal models and FSHD clinical studies from Project 2. Genetic and phenotypic changes in the FLExDUX4 mouse model of FSHD will be targeted via local and systemic delivery of AAV-DUX4 RNAi, while further studies will evaluate these AAV-DUX4 RNAi vectors in the Göttingen minipig model of FSHD that is being characterized in Project 2. DUX4 gene silencing and reduction of inflammation in the minipig will be measured by MRI, muscle structural changes, and biomarkers based on clinical results from Project 2. Together, experimental strategies in Projects 1 and 2 will facilitate the potential for gene therapy by addressing current limitations resulting from vector potency, immunity, transgene expression, and animal model deficiencies.