Developing efficient AAV vectors for photoreceptor targeting via the vitreous

描述（由申请人提供）：最近发现，在RPE65 Leber先天性黑朦-2 （LCA2）患者的视网膜下注射腺相关病毒（AAV）导致中央视网膜厚度和中央视力下降。由于绝大多数遗传性视网膜疾病是由光感受器（PR）特异性基因突变引起的，因此显然需要开发能够更安全地靶向这些细胞的基因替代策略。我们建议开发一种新的AAV载体，能够在更安全、手术微创的玻璃体内注射后转导pr。这样，我们将克服视网膜基因治疗领域的一个主要障碍——如何安全地将基因传递到脆弱的、患病的视网膜中的视杆细胞和视锥细胞，这些视网膜在手术诱导的视网膜脱离中容易进一步受损。将基因安全地定位到中央视锥细胞的能力尤其重要，因为这是视网膜负责急性日光视力的区域。未经修饰的AAV血清型通过玻璃体的内视网膜转导取决于它们结合硫酸肝素蛋白多糖（HSPG）的能力，硫酸肝素蛋白多糖存在于内限制膜（ILM）中，这是形成玻璃体视网膜界面的基膜。我们的假设是，AAV衣壳可以同时被优化，在保持或获得光感受器的趋向性的同时，赋予内层限制膜的粘附性和通往外层视网膜的交通。为实现这一目标，我们提出以下三个目标。在Aim 1中，我们将测试通过合理设计和定向进化（新颖的，高度复杂的AAV衣壳库）开发的新变体通过玻璃体转导小鼠pr的能力。相对转导效率将使用具有可分类（gfp阳性）pr的小鼠模型-小鼠Rho-GFP敲除。由于它们的眼部特征与人类最相似，在Aim 2中，我们将在非人类灵长类动物中筛选和测试载体，并确定在小鼠视网膜中赋予AAV突变体PR转导的衣壳基序在灵长类动物中是否也很重要。可分类的NHP pr将通过视网膜下注射一种被证明在NHP的杆状体和锥状体中具有独家活性的载体AAV5-hGRK1-GFP来创建。在Aim 3中，将测试最有效的变体恢复CNGB3小鼠模型视力的能力

DESCRIPTION (provided by applicant): It was recently found that subretinal injection of Adeno associated virus (AAV) in patients with RPE65 Leber congenital amaurosis-2 (LCA2) led to a loss of central retinal thickness and central visual acuity. Because the vast majority of inherited retinal diseases are caused by mutations in photoreceptor (PR)-specific genes, there is an obvious need to develop gene replacement strategies that can more safely target these cells. We propose to develop novel AAV vectors capable of transducing PRs following a safer, surgically less invasive intravitreal injection. In so doing, we will overcome a major hurdle in th field of retinal gene therapy - how to safely deliver genes to rods and cones in fragile, diseased retinas that are prone to further damage upon surgically-induced retinal detachment. The ability to safely target genes to central cones is especially significant because this is the area of the retina responsible for acute, daylight vision. Transduction of inner retina via the vitreous with unmodified AAV serotypes depends on their ability to bind heparin sulfate proteoglycan (HSPG), a glycan present in the inner limiting membrane (ILM), a basement membrane that forms the vitreoretinal interface. Our hypothesis is that the AAV capsid can be simultaneously optimized to confer adhesion to the inner limiting membrane and traffic to the outer retina while maintaining or gaining photoreceptor tropism. To achieve this goal we propose the following three aims. In Aim 1, we will test novel variants developed via rational design and directed evolution (novel, highly complex AAV capsid libraries) for their ability to transduce mouse PRs via the vitreous. Relative transduction efficiency will be quantified using a mouse model with sortable (GFP-positive) PRs- the Rho-GFP knock in mouse. Because their ocular characteristics are most similar to human, in Aim 2, we will screen and test vectors in non-human primate and determine whether capsid motifs that confer PR transduction to AAV mutants in mouse retina are also important in primate. Sortable NHP PRs will be created via subretinal injection of a vector proven to have exclusive activity in rods and cones of NHP, AAV5-hGRK1-GFP. In Aim 3, the most efficient variants will be tested for their ability to restore vision to mouse models of CNGB3 achromatopsia and GUCY2D Leber congenital amaurosis (LCA1). Results may directly impact clinical trial designs for both diseases and be applied more broadly to other photoreceptor-mediated retinal disease. In summary, results of this study would transform the way gene therapy agents are administered to inherited retinal disease patients, converting a full blown vitreoretinal surgery into an outpatient procedure akin to that required for wet age related macular degeneration drugs, Lucentis and Avastin. Such vectors could be administered in clinic rather than a surgical suite, thereby increasing accessibility of gene therapies to much larger patient populations. Access to gene therapies would improve as the number of clinical trial study sites qualified to administer study agent would greatly increase.