Whole body single AAV microgene therapy in canine DMD

 描述(由申请人提供) Duchenne肌营养不良症(DMD)是由肌营养不良蛋白缺乏引起的。基因替代疗法在治疗DMD方面大有可为。腺相关病毒(AAV)是肌肉基因治疗的主要病毒载体。然而，AAV的包装容量较小(~5kb)，不能携带全长的dystrophin基因(~12kb)。微基因是一种超小的人造肌营养不良蛋白基因。它包含三分之一的全长抗肌营养不良蛋白编码序列。许多实验室的研究表明，微量肌营养不良蛋白可以有效地改善肌营养不良蛋白缺陷小鼠的肌肉疾病。不幸的是，到目前为止，对大型哺乳动物的基因转换并不是非常成功。我们最近设计了一个新的∆R2-15/∆R18-19/∆R20-23/∆C微基因(简称∆R2µDys)，并用Y731F AAV-9单肌肉注射对成年抗肌营养不良犬进行了测试。两个月后，我们观察到炎症和纤维化显著减少。重要的是，偏心收缩引起的损伤--DMD的一个生理标志--显著减轻。我们的研究首次证明了微肌营养不良蛋白可以治疗大型哺乳动物肌肉中的营养不良症。我们的结果还表明，新开发的Y731F AAV-9∆R2µDys载体可能具有很大的翻译潜力。这项提案的首要目标是解决与未来临床翻译相关的关键问题。具体地说，我们将追求两个目标，包括(1)测试区域肌肉注射可以导致成年营养不良犬持续保护的假设。这组研究将使我们能够评估未来改善晚期轮椅患者生活质量治疗的可行性；以及(2)测试一次静脉注射可以导致年轻营养不良狗的全身改善的假设。DMD会影响身体的所有肌肉。这组研究将使我们能够确定系统干预是否可以从根本上改变受影响个人的疾病进程。综上所述，我们的研究将突破该领域的主要障碍，并提供急需的大型动物数据来指导未来的人类试验。

 DESCRIPTION (provided by applicant) Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency. Gene replacement therapy holds great promise to treat DMD. Adeno-associated virus (AAV) is the leading viral vector for muscle gene therapy. However AAV has a small packaging capacity (~ 5-kb) and it cannot carry the full-length dystrophin cDNA (~12-kb). A microgene is a super-small synthetic dystrophin gene. It contains one-third of the full-length dystrophin coding sequence. Studies from many laboratories suggest that micro-dystrophin can effectively ameliorate muscle disease in dystrophin-deficient mice. Unfortunately, translation to large mammals has so far not been very successful. We recently engineered a new ∆R2-15/∆R18-19/∆R20-23/∆C microgene (abbreviated as ∆R2 µDys) and tested it adult dystrophin-null dogs by single muscle injection using Y731F AAV-9. Two months later, we observed dramatic reduction of inflammation and fibrosis. Importantly, eccentric contraction-induced damage, a physiological hallmark of DMD, was significantly alleviated. Our study demonstrates for the first time that microdystrophin can treat dystrophinopathy in muscles of large mammals. Our results also suggest that the newly developed Y731F AAV-9 ∆R2 µDys vector may hold great translational potential. The overarching goal of this proposal is to address key questions related to future clinical translatio. Specifically, we will pursue two aims including (1) to test the hypothesis that regional intramuscular injection can lead to persistent protection in adult dystrophic dogs. This set of studies will allow us to evaluate the feasibility of life-quality improving therapy in late-stage wheelchair-bound patients in the future; and (2) to test the hypothesis that a single intravenous injection can lead to bodywide amelioration in young dystrophic dogs. DMD affects all muscles in the body. This set of studies will allow us to determine whether systemic intervention can radically change the disease course in affected individuals. Taking together, our studies will break through major barriers in the field and provide the much-needed large animal data to guide human trials in the future.