Developing efficient AAV vectors for photoreceptor targeting via the vitreous

描述（由申请人提供）：最近发现，在患有RPE 65 Leber先天性黑蒙-2（LCA 2）的患者中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丧失。由于绝大多数遗传性视网膜疾病是由感光细胞（PR）特异性基因突变引起的，因此显然需要开发可以更安全地靶向这些细胞的基因替代策略。我们建议开发新的AAV载体，能够转导PR后，一个更安全的，手术微创玻璃体内注射。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-如何安全地将基因传递到脆弱、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后容易进一步受损。将基因安全地靶向中央视锥细胞的能力尤其重要，因为这是视网膜上负责急性日光视觉的区域。用未修饰的AAV血清型通过玻璃体转导内视网膜取决于它们结合硫酸肝素蛋白聚糖（HSPG）的能力，HSPG是存在于内界膜（ILM）（形成玻璃体视网膜界面的基底膜）中的聚糖。我们的假设是，AAV衣壳可以同时优化，以赋予粘附到内界膜和交通到外视网膜，同时保持或获得感光细胞向性。为实现这一目标，我们提出以下三个目标。在目标1中，我们将测试通过合理设计和定向进化开发的新型变体（新型高度复杂的AAV衣壳文库）通过玻璃体包裹小鼠PR的能力。将使用具有可分选（GFP阳性）PR的小鼠模型-小鼠中的Rho-GFP敲除来定量相对转导效率。因为它们的眼部特征与人类最相似，在目标2中，我们将在非人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PR转导至AAV突变体的衣壳基序在灵长类动物中是否也很重要。可分选的NHP PR将通过视网膜下注射被证明在NHP的视杆和视锥中具有排他性活性的载体AAV 5-hGRK 1-GFP来产生。在目标3中，将测试最有效的变体恢复CNGB 3小鼠模型视力的能力。 色盲和GUCY 2D Leber先天性黑蒙（LCA 1）。结果可能会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。总之，这项研究的结果将改变基因治疗药物对遗传性视网膜疾病患者的治疗方式，将全面的玻璃体视网膜手术转化为门诊手术，类似于湿性年龄相关性黄斑变性药物Lucentis和Avastin所需的手术。这样的载体可以在诊所而不是手术室中施用，从而增加基因疗法对更大的患者群体的可及性。随着有资格管理研究药物的临床试验研究中心数量的大幅增加，基因治疗的可及性将得到改善。

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.