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。我们中心在以下方面发挥了重要作用 推进AAV在肌肉疾病中的应用。这些研究包括对dystrophin和DUX4的广泛表征 表达和功能，发现AAV可用于全身基因传递到肌肉，以及 产生大量小的肌肉特异性表达盒(MSEC)、微肌营养不良蛋白(μDys)和 RNAi盒式磁带。这些进展使DMD、LGMD、XL-MTM1和XL-MTM1能够进行多种基因治疗试验 其他肌肉疾病。我们还开发和改进了许多骨骼肌和心肌测试 用于肌肉基因治疗的平台和工具，包括3D DMD人类IPSC来源的工程化肌肉 组织和DMD大鼠模型。然而，目前的aav-μDys疗法并不像希望的那样有效，aav- 针对FSHD和其他显性疾病的RNAi治疗尚未进入临床。在这里，我们提出两个建议 相关目的是解决AAV基因疗法的局限性：(A)测试新的AAV衣壳变异体对心脏和心脏的影响 骨骼肌，新型微肌营养不良蛋白的迭代测试，更大的分裂内联蛋白营养不良蛋白，以及MSEC设计 将免疫原性降至最低，并提高心肌和骨骼肌的效力；(B)应用增强的 FSHD治疗中DUX4转录本下调的磁带和载体。在目标1中，我们开发向量来改进 DMD临床干预受到治疗性肌营养不良蛋白水平和功能的限制，以及 通过极高的媒介剂量在一些患者中引起严重不良事件(SAE)，这是由于 对载体和/或转基因的免疫反应。我们将比较多种嗜肌血清型，它们 显示显著增加的肌肉转导，以交付已设计的新型转基因盒 用于降低免疫原性和提高效力，特别是在心肌中。一个主要的焦点将包括 测试新的分裂内含子AAV载体以产生微小和全长的抗肌营养不良蛋白。我们还测试了一个对偶向量 通过过表达提高心脏功能的一种酶(RNR)来恢复心脏受抑制的功能的策略 DATP，小分子肌球蛋白激活剂。对抗肌营养不良蛋白免疫反应的研究将得到加强 筛查患者血细胞的免疫反应性。在目标2中，我们将肌力向量与增强的 RNAi盒用于推进FSHD的基因治疗。这一实验计划结合了来自 目标1新的FSHD动物模型和项目2中的FSHD临床研究：遗传和表型变化 在FSHD的FlexDUX4小鼠模型中，将通过局部和系统递送AAV-DUX4 RNAi来靶向FSHD， 虽然进一步的研究将在FSHD的Göttingen小型猪模型中评估这些AAV-DUX4 RNAi载体，但 DUX4基因沉默和减轻小型猪的炎症将是项目2的特征。 根据项目2的临床结果，通过MRI测量肌肉结构变化和生物标记物。 项目1和项目2中的实验策略将通过解决当前的 由于载体效力、免疫力、转基因表达和动物模型缺陷造成的限制。

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.