Engineering Novel AAV Vectors for Retinal Gene Therapy

描述(申请人提供)：基因疗法在治疗和潜在治愈各种疾病方面具有巨大的潜力，特别是在包括青光眼、老年性黄斑变性和光感受器疾病在内的许多视网膜疾病中。然而，基因传递技术需要在细胞靶向、效率和安全性方面有重大改进，才能将动物研究中有希望的发现转化为临床。特别是，对于视网膜基因治疗来说，转导一种跨越整个视网膜的单细胞类型来传递和分泌一种通用的神经保护因子，以保护受到从视网膜神经节细胞到光感受器等不同视网膜疾病影响的大量神经元，将是非常有利的。此外，理想情况下，这种单细胞类型应该可以通过玻璃体内注射获得，因为视网膜下注射对视网膜更具侵入性和破坏性。不幸的是，没有一种载体能够有效地感染满足这些需求的细胞类型--穆勒胶质细胞。基于腺相关病毒(AAV)的载体在许多视网膜疾病模型中被证明是非常有前途的，但不幸的是它们也不能感染Muller细胞。我们开发了新的定向进化技术来产生具有新特性的AAV新突变体，包括改变的受体结合，并建议进化能够有效地进行Muller细胞转导的突变体。同时，还将探索MULER细胞转导AAV的基本机制。最后，将结果转化为光感受器疾病模型。这项工作中开发的新方法将对一些视网膜疾病的增强型病毒基因输送载体的分子工程产生普遍影响。

DESCRIPTION (provided by applicant): Gene therapy has vast potential for treating and potentially curing a wide variety of disorders, in particular in a number of retinal diseases including glaucoma, age-related macular degeneration, and photoreceptor diseases. However, gene delivery technologies require significant improvements in cellular targeting, efficiency, and safety before promising findings in animal studies can be translated to the clinic. In particular, for retinal gene therapy it would be highly advantageous to transduce a single cell type that spans the entire retina for the delivery and secretion of a general neuroprotective factor throughout the retina to protect numerous populations of neurons that are affected by different retinal diseases, from retinal ganglion cells to photoreceptors. In addition, ideally this single cell type should be accessible from an intravitreal injection, as subretinal injections are more invasive and disruptive to the retina. Unfortunately, there is no vector capable of efficiently infecting the cell type that meets these needs, Muller glia. Vectors based on adeno-associated virus (AAV) have proven themselves to be highly promising in numerous retinal disease models, but they are also unfortunately incapable of Muller cell infection. We have developed novel directed evolution technology to generate new mutants of AAV with new properties, including altered receptor binding, and we propose to evolve variants capable of efficient Muller cell transduction. In parallel, the basic mechanisms of AAV transduction of Muller cells will be explored. Finally, results will be translated to photoreceptor disease model. The novel approaches developed in this work will have general impact for the molecular engineering of enhanced viral gene delivery vehicles for a number of retinal diseases.