Developing efficient AAV vectors for photoreceptor targeting via the vitreous

描述(申请人提供)：最近发现RPE65 Leber先天性黑色素2(LCA2)患者视网膜下注射腺相关病毒(AAV)导致中心视网膜厚度和中心视力丧失。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针对这些细胞的基因替换策略。我们建议开发新型的AAV载体，能够在更安全、手术创伤更小的玻璃体内注射后转导PR。通过这样做，我们将克服视网膜基因治疗领域的一个主要障碍-如何安全地将基因输送到脆弱的、患病的视网膜中的视杆和视锥，这些视杆和视锥在手术诱导的视网膜脱离时容易受到进一步的损害。将基因安全地定位到中央视锥细胞的能力尤其重要，因为这是视网膜中负责急性日光视觉的区域。通过未修饰AAV血清型的玻璃体转导视网膜内取决于它们与硫酸肝素蛋白多糖(HSPG)的结合能力，HSPG是一种存在于内界膜(ILM)中的多糖，内界膜是形成玻璃体-视网膜界面的基底膜。我们的假设是，AAV衣壳可以同时优化，在保持或获得光感受器趋向性的同时，赋予内界膜黏附和向外视网膜运输。为了实现这一目标，我们提出了以下三个目标。在目标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衣壳文库)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选(GFP阳性)PR的小鼠模型进行量化-Rho-GFP敲击小鼠。由于它们的眼睛特征与人类最相似，在目标2中，我们将在非人类灵长类动物中筛选和测试媒介，并确定在小鼠视网膜中向AAV突变体传递PR的衣壳基序是否在灵长类动物中也很重要。可分类的NHP PR将通过视网膜下注射被证明在NHP、AAV5-hGRK1-GFP的视杆和视锥中具有独家活性的载体来创建。在Aim 3中，将测试最有效的变体是否具有恢复CNGB3小鼠模型视力的能力 色觉减退与先天性黑色素沉着(LCA1)结果可能直接影响这两种疾病的临床试验设计，并更广泛地应用于其他光感受器介导的视网膜疾病。总而言之，这项研究的结果将改变遗传性视网膜疾病患者接受基因治疗药物的方式，将全面的玻璃体视网膜手术转变为门诊手术，类似于湿性老年性黄斑变性药物Lucentis和阿瓦斯丁所需的程序。这样的载体可以在临床上使用，而不是在手术室中使用，从而增加了更多患者群体对基因治疗的可及性。随着有资格管理研究试剂的临床试验研究地点的数量将大大增加，获得基因疗法的机会将会增加。

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.