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

• 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
• 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' 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

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（申请人提供）：最近发现，RPE65 Leber 先天性黑蒙-2（LCA2）患者视网膜下注射腺相关病毒（AAV）会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，因此显然需要开发能够更安全地针对这些细胞的基因替代策略。我们建议开发新型 AAV 载体，能够在更安全、手术侵入性较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——如何将基因安全地传递到脆弱、患病视网膜中的视杆细胞和视锥细胞，这些视网膜在手术引起的视网膜脱离后容易受到进一步损伤。将基因安全地定位到中央视锥细胞的能力尤其重要，因为这是负责敏锐的日光视觉的视网膜区域。未经修饰的 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) 中的聚糖，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 衣壳可以同时进行优化，以赋予内界膜粘附性和外视网膜交通性，同时保持或获得光感受器向性。为了实现这一目标，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变体（新颖、高度复杂的 AAV 衣壳库），以了解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选（GFP 阳性）PR 的小鼠模型（小鼠中的 Rho-GFP 敲入）进行量化。由于它们的眼部特征与人类最相似，因此在目标 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将 PR 转导给 AAV 突变体的衣壳基序在灵长类动物中是否也很重要。可排序的 NHP PR 将通过视网膜下注射载体来创建，该载体已被证明对 NHP 的视杆细胞和视锥细胞具有独特的活性，即 AAV5-hGRK1-GFP。在目标 3 中，将测试最有效的变体恢复 CNGB3 小鼠模型视力的能力 全色盲和 GUCY2D Leber 先天性黑蒙 (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给药方式，将全面的玻璃体视网膜手术转变为类似于湿性年龄相关性黄斑变性药物 Lucentis 和 Avastin 所需的门诊手术。这种载体可以在临床而不是手术室中施用，从而增加了基因治疗对更多患者群体的可及性。随着有资格使用研究药物的临床试验研究中心数量的大幅增加，获得基因疗法的机会将会得到改善。