Developing efficient AAV vectors for photoreceptor targeting via the vitreous

开发有效的 AAV 载体，用于通过玻璃体靶向光感受器

基本信息

批准号：
9275995
负责人：
Shannon Elizabeth Boye
金额：
$ 47.29万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-06-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9275995
关键词：
Acute Adhesions Affect Affinity Age related macular degeneration Anatomy Area Avastin Bar Codes Basement membrane Binding Capsid Cells Characteristics Clinic Clinical Trials Clinical Trials Design Complex Cone DNA Dependovirus Directed Molecular Evolution Discrimination Disease Dose Engineering Exhibits Fluorescence Gene Targeting Gene Therapy Agent Genes Geography Goals Heparin Binding Human Individual Inherited Injectable Inner Limiting Membrane Knock-in Mouse Knowledge Leber&apos s amaurosis Libraries Life Lucentis Mediating Membrane Protein Traffic Methodology Modeling Mus Mutation Operative Surgical Procedures Outpatients Patients Peripheral Pharmaceutical Preparations Phenotype Photoreceptors Polysaccharides Population Primates Procedures Publishing RPE65 protein Research Personnel Resource Sharing Retina Retinal Retinal Cone Retinal Detachment Retinal Diseases Route Serotyping Site Structural Models Structure Surface Testing Thick Threonine Tropism Tyrosine Variant Vertebrate Photoreceptors Vision Visual Acuity achromatopsia adeno-associated viral vector base clinically relevant cohort deep sequencing design disease-causing mutation extracellular gene replacement gene therapy gene therapy clinical trial heparin proteoglycan improved intravitreal injection mouse model mutant nonhuman primate novel patient population photoreceptor degeneration prevent public health relevance receptor retinal rods rho screening subretinal injection therapeutic gene therapeutic transgene trafficking transduction efficiency vector vector genome

项目摘要

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.

描述（由申请人提供）：最近发现，RPE65 LEBER先天性Amaurosis-2（LCA2）患者的视网膜下注射Adeno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光感受器（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。我们建议开发能够在更安全的玻璃体内注射下更安全的侵入性侵入性的新型AAV载体。这样一来，我们将克服视网膜基因治疗领域的一个主要障碍 - 如何在脆弱的，患病的视网膜中安全地向杆和圆锥体安全地输送基因，这些视网膜容易在手术引起的视网膜脱离时进一步损害。将安全靶向中央锥的能力尤其重要，因为这是负责急性，日光视觉的视网膜区域。通过玻璃体具有未修饰的AAV血清型的内部视网膜的转导取决于它们结合硫酸盐蛋白聚糖蛋白聚糖（HSPG）的能力，硫酸盐蛋白聚糖（HSPG）是内部限制膜（ILM）中存在的聚糖，这是一种基底膜，形成了玻璃体视网膜界面。我们的假设是，可以同时优化AAV CAPSID，以赋予内部限制膜的粘附，并在维持或获得感光受体的tropism中，并向外视网膜交通。为了实现这一目标，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 Capsid库）开发的新型变体，以便它们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配（GFP阳性）PR的小鼠模型来量化 - 小鼠中的Rho-GFP敲击。由于它们的眼部特征与人类最相似，因此在AIM 2中，我们将在非人类灵长类动物中筛选和测试向量，并确定将PR转导向小鼠视网膜中aav突变体赋予AAV突变体的衣壳图案是否在灵长类动物中也很重要。可排序的NHP PR将通过视网膜下注入载体，该向量被证明在NHP的杆和锥中具有AAV5-HGRK1-GFP的杆和锥体。在AIM 3中，将测试最有效的变体，以使其能够将视力恢复到CNGB3的鼠标模型 Achromomatia和Gucy2d Leber先天性症（LCA1）。结果可能会直接影响两种疾病的临床试验设计，并更广泛地应用于其他感光体介导的视网膜疾病。总而言之，这项研究的结果将改变基因治疗剂的施用方式，使其对遗传性视网膜疾病患者进行施用，将完全吹干的玻璃体视网膜手术转化为类似于与湿时代相关的黄斑变性药物，lucentis和avastin所需的门诊手术。这些载体可以在诊所而不是手术套件中给药，从而增加基因疗法对更大的患者人群的可及性。随着有资格管理研究代理的临床试验研究地点的数量将大大增加，因此获得基因疗法将有所改善。