Optimizing Gene Therapies in Large Animal Models of Retinal Degeneration

7.项目总结/摘要 光感受器（PRs）和视网膜色素上皮（RPE）是突变的主要位置 它是许多遗传性视网膜变性的基础，目前还没有治疗方法。因此 视觉研究领域的一个主要目标是开发用于将基因递送到这些细胞的改进技术。 该提议旨在通过创建能够在细胞内表达的新型腺相关（AAV）载体来解决该问题。 当递送到犬玻璃体时靶向PR和RPE。我们选择了狗，因为它是一个相关的 大型动物模型，其具有与人类相似的地球仪体积和视网膜结构，以及天然的- 发生突变，导致与患者中鉴定的视网膜疾病表型相似的视网膜疾病。 天然存在的AAV血清型不能穿透视网膜的结构屏障，因此， 到目前为止，将DNA治疗性递送至PR和RPE需要将病毒载体注射到视网膜中。 视网膜下间隙视网膜下注射需要针穿过视网膜以及短暂的视网膜内注射。 视网膜脱离，将RPE与PR分离。这种显微外科技术需要一般的 麻醉，将病毒载体递送到视网膜/RPE的有限区域，并且已经在临床前 安全性研究和人类临床试验与视网膜变薄下凹和黄斑裂孔的形成。 最近，AAV载体已经被优化用于在玻璃体内注射后增加PR和RPE的靶向。 递送，这将提供比载体更容易、更安全和更广泛的PR和RPE靶向 通过视网膜下途径递送。然而，当在小鼠中进行测试时， 大型动物由于视网膜结构的差异，这些载体效率低下。基于强初始化 结果在小鼠中，我们将使用类似的定向进化方法来选择，从高度多样化的混合物中， 突变的AAV文库，具有穿越结构屏障并感染宿主细胞的新能力的个体变体， 狗的外层视网膜从玻璃体。所得载体将通过包装GFP和分析GFP来表征。 表达谱。然后，我们将测试增加狗的转导的遗传特征是否也 足以改善非人灵长类动物外层视网膜的感染。此外，我们将使用深度 测序以分析多轮选择，以阐明在哪些步骤多样性降低， 确定定向进化的最终结果是否可以从最初的几轮预测。鉴定的新型 变异体将用于在rcd 1狗中测试矫正基因疗法，rcd 1狗是由一种 在杆特异性PDE 6？基因突变，以及RPE 65突变狗，Leber先天性 由于视网膜色素上皮的遗传缺陷而导致的黑蒙。然后，我们将测量玻璃体内注射是否提供 通过评估视网膜结构和功能，与视网膜下注射相比，等同或更好的补救措施。 这些实验将为新型药物提供多样化的培训经验和宝贵的临床前数据 视网膜的基因治疗

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.