Vitreal delivery of novel AAV vectors for CNGA3 achromatopsia cone gene therapy

描述（由申请人提供）：我们建议探索利用aav基因传递来恢复和/或维持视网膜锥体结构、功能和视觉依赖行为的方法。我们的总体假设是，玻璃体内（IV）注射后适当设计的、基于基因的治疗方法将在临床上对各种视网膜疾病和视网膜功能障碍有用。开发静脉注射方案的需求是基于最近发表的来自当前NEI赞助的LCA2临床试验（NCT00481546）的15例患者的结果。单个或多个视网膜下（SR） AAV载体注射导致载体泡脱离中央凹，导致患者视力很少或没有改善，有些患者视力略有下降。相比之下，等量的视网膜下载体剂量未使中央凹脱离，则可使视力获得实质性和持续性的改善。因此，开发更安全的替代方法来传导中央凹锥是本建议的重点。为此，我们将测试载体注射的IV途径，并使用我们刚刚开发的新型AAV变体，以努力验证和优化在不脱离视网膜的情况下纠正2型色盲小鼠模型中锥体缺陷的能力。在这项研究中，我们将使用CNGA3突变的人类色盲小鼠模型，使用几种新型酪氨酸转苯丙氨酸和苏氨酸转缬氨酸突变的衣壳AAV载体作为玻璃体内注射的基因传递载体。虽然静脉注射载体可以在不引起视网膜脱离的情况下大面积转导内视网膜，但传统的玻璃体载体不能穿透内视网膜，转导外视网膜的杆状或锥状细胞。因此，开发和验证AAV载体的视网膜穿透特性是该领域的关键未满足需求，也是当前AAV介导的基因替代疗法的挑战。该项目的另一个关键因素是选择一种不仅具有遗传和表型特征良好的锥体变性动物模型，而且已经建立了对SR基因治疗有良好反应的动物模型，以便筛选这些新的AAV载体，以从玻璃体中恢复功能和结构锥体，可以参照既定的治疗标准进行自信的筛选。因此，我们将重点研究自然发生的人类色盲2（隐性CNGA3突变）小鼠模型，Cpfl5小鼠，其表现出与人类相似的锥体功能障碍/变性，并且对SR AAV载体基因治疗反应良好。该动物模型的成功治疗将为更安全的静脉治疗试验提供模板，不仅适用于人类色盲，也适用于其他影响视网膜中央凹功能的人类视网膜疾病，如晚期视网膜色素变性、视锥和视锥/视杆营养不良以及潜在的黄斑变性。

DESCRIPTION (provided by applicant): We propose to explore ways to use AAV-based gene delivery to restore and/or maintain retinal cone structure, function and vision-dependent behavior. Our overall hypothesis is that appropriately designed, gene-based therapies following intravitreal (IV) injection will be clinically useful for a wide variety of retinal diseases with cne dysfunctions. The need for developing the IV injection option is based on recently published results for 15 patients from the current NEI sponsored LCA2 clinical trial (NCT00481546). Single or multiple subretinal (SR) AAV vector injections resulting in vector blebs that detached the fovea resulted in little or no vision gain for the patient and in some a slight loss in visual acuiy. In contrast, equivalent subretinal vector doses that did not detach the fovea mediated substantial and persistent gains in vision. Therefore, developing alternative ways to more safely transduce foveal cones is the focus of this proposal. To that end, we will test the IV route of vector injection and employ novel AAV variants that we have just developed in an effort to validate and optimize the ability to correct cone defects in a mouse model of achromatopsia 2 without detaching the retina. For this investigation, we will use a mouse model of human achromatopsia with CNGA3 mutations using several novel tyrosine to phenylalanine plus threonine to valine mutant capsid AAV vectors as the gene delivery vehicles for intravitreal injection. Although IV injection of vector can transduce a large area of the inner retina without causing retinal detachment, conventional vectors in the vitreous cannot penetrate the inner retina and transduce rod or cone cells in outer retina. Developing and validating a retina-penetrating property for AAV vectors is therefore a key unmet need in the field and a challenge for current AAV-mediated gene replacement therapy. Another pivotal element of this project is the selection of an animal model that not only possesses a genetically and phenotypically well-characterized cone degeneration, but also has been established to respond well to SR gene-based therapy so that screening these novel AAV vectors for functional and structural cone rescue from the vitreous can be carried out with confidence with reference to an established therapeutic standard. We will therefore focus on a naturally occurring mouse model of human achromatopsia 2 (recessive CNGA3 mutations), the Cpfl5 mouse, that exhibits cone dysfunction/degeneration similar to that seen in humans and responds well to SR AAV vector gene therapy. Therapeutic success in this animal model will provide a template for safer IV treatment trials not only for human achromatopsia but also for other human retinal diseases affecting foveal function such as late stage retinitis pigmentosa, cone and cone/rod dystrophy and potentially macular degeneration.