Optimizing Gene Therapies in Large Animal Models of Retinal Degeneration

7.项目摘要/摘要 光感受器(PR)和视网膜色素上皮(RPE)是突变的主要位置 许多形式的遗传性视网膜变性，目前尚无治疗方法。因此，a 视觉研究领域的主要目标是开发向这些细胞运送基因的改进技术。 该建议旨在通过创建腺相关(AAV)载体来解决这一问题 靶向PR和RPE进入犬的玻璃体。我们选择了这只狗，因为它是一只相关的 与人类有着相似的眼球体积和视网膜结构的大型动物模型，以及天然的- 发生突变，导致与患者中发现的视网膜疾病表型相似的疾病。 自然产生的AAVs的血清型不能穿透视网膜的结构障碍，因此 到目前为止，向PR和RPE提供治疗性DNA需要将病毒载体注射到 视网膜下间隙。视网膜下注射需要一根针穿过视网膜，以及短暂的 视网膜脱离，将RPE与PR分开。这种显微外科技术需要普通的 麻醉，将病毒载体运送到视网膜/RPE的有限区域，并已在临床前相关 黄斑中心凹下视网膜变薄和黄斑裂孔形成的安全性研究和人体临床试验。 最近，AAV载体已经被优化，以增加玻璃体内注射后PR和RPE的靶向性 交付，这将提供比载体更容易、更安全和更广泛的PR和RPE靶向 通过视网膜下途径输送。然而，当在小鼠身上进行测试时，已经取得了成功的结果 大型动物由于视网膜结构的差异，这些载体的效率很低。基于强首字母 结果在老鼠身上，我们将使用类似的定向进化方法，从高度多样化的混合物中进行选择 突变的AAVs文库，具有穿越结构障碍并感染 狗的玻璃体外视网膜。生成的载体将通过包装GFP和分析来表征 表达配置文件。然后我们将测试增加狗转导的遗传特征是否也 足以改善非人灵长类外部视网膜的感染。此外，我们将使用深度 排序，以分析几轮选择，以阐明哪些步骤减少了多样性，并 确定定向进化的最终结果是否可以从最初几轮中预测出来。被认同的小说 变异将用于在RCD1狗身上测试纠正性基因疗法，RCD1狗是一种由 杆状特异性PDE6基因的突变，以及RPE65突变犬，Leber先天性心脏病的模型 由于RPE的遗传缺陷而导致的黑色素症。然后我们将测量玻璃体内注射是否能提供 通过评估视网膜结构和功能，与视网膜下注射相比，具有同等或更好的抢救作用。 这些实验将为Noval提供多样化的培训经验和宝贵的临床前数据 视网膜中的基因治疗。

7. Project Summary/Abstract Photoreceptors (PRs) and retinal pigment epithelium (RPE) are the primary location of mutations underlying many forms of inherited retinal degeneration for which there is currently no treatment. Therefore, a major goal in the field of vision research is to develop improved technology for gene delivery to these cells. This proposal aims to address this problem by creating novel adeno-associated (AAV) vectors capable of targeting PRs and RPE when delivered into the canine vitreous. We have selected the dog as it is a relevant large animal model that shares similar globe volume and retinal structure with humans, as well as naturally- occurring mutations that result in phenotypically similar retinal diseases as those identified in patients. Naturally occurring serotypes of AAVs are unable to penetrate structural barriers of the retina, and therefore therapeutic delivery of DNA to PRs and RPE has required until now that the viral vector be injected into the subretinal space. Subretinal injections entail the passage of a needle through the retina as well as a transient retinal detachment that separates the RPE from the PRs. This microsurgical technique requires general anesthesia, delivers the viral vector to a limited area of the retina/RPE, and has been associated in preclinical safety studies and human clinical trials with retinal thinning under the fovea and the formation of macular holes. Recently, AAV vectors have been optimized for increased targeting of PRs and RPE following intravitreal delivery, which would provide easier, safer and more extensive targeting of the PRs and RPE than vectors delivered via the subretinal route. Successful results have been achieved in mice, however, when tested in large animals these vectors were inefficient due to differences in retinal structure. Based on strong initial results in mice, we will use a similar directed evolution approach to select, from a mixture of highly diverse libraries of mutated AAVs, individual variants with the novel ability to traverse structural barriers and infect the dog outer retina from the vitreous. Resulting vectors will be characterized by packaging GFP and analyzing expression profiles. We will then test whether the genetic features that increase transduction in dogs are also sufficient to improve infection of the non-human primate outer retina. Furthermore, we will use deep sequencing to analyze rounds of selection, in order to elucidate at which steps diversity is reduced and to determine if the final outcome of directed evolution can be predicted from the initial rounds. The identified novel variants will be used to test corrective gene therapies in the rcd1 dog, a model of PR disease caused by a mutation in the rod-specific PDE6¿ gene, as well as in the RPE65 mutant dog, a model of Leber's congenital amaurosis due to a genetic defect in the RPE. We will then measure whether intravitreal injections provide equivalent or better rescue compared to subretinal injections by evaluating retinal structure and function. Together, these experiments will provide a diverse training experience and valuable preclinical data for novel gene therapy treatments in the retina.