SCGE Disease Models Studies Supplement: Correction of RHO P23H adRP

项目摘要 这项工作的广泛和长期目标是建立一个标准来评估安全和 体细胞基因组编辑技术的功效。这些目标将在#年得到解决 Rho引起的视网膜色素变性的实验性治疗背景 P23H变异体。这项提案的具体目标是完善治疗方案 这一疗法的窗口，必须编辑以保存视力的最小细胞数， 以及治疗策略的总体安全性。这些目标将通过 使用AAV5递送特异性靶向的工程巨核酸酶 显性P23H等位基因，保留仅有一个碱基差异的野生型等位基因。这 试剂将在经过验证的、进展迅速的猪模型中进行评估，该模型含有 人Rho P23H转基因。之前优化过的剂量，可产生持久的 应答将通过视网膜下注射进行，并将对动物进行32次跟踪 数周内通过临床眼科检查、全视野检查重复测量随时间推移的有效性 视网膜电流图(FfERG)和光学相干断层扫描(OCT)。在…的末尾 实验中，眼睛将通过免疫组织化学(IHC)进行检查，以确定 区域，必须编辑的最小杆数，以保存锥体功能和 因此，为了挽救视力。这些实验将在#年的两个不同阶段进行。 疾病进展被假设为代表最后的可治疗状态和 第一个不可治疗的状态，以便能够为登记的标准提供指导 患者在未来的临床试验中。同时，动物将受到类似的对待，然后 在注射后两周处死，以确定邻近组织的范围 AAV5载体将迁移的。这些动物也将被用来确定 在非预期组织中发生的编辑级别，包括生殖系。所有的动物 将被用来获得关于治疗的一般生理反应的数据。

Project Summary The broad, long-term objective of this work is to establish a rubric to evaluate safety and efficacy of somatic cell genome editing technologies. These goals will be addressed in the context of an experimental therapy for retinitis pigmentosa caused by the RHO P23H variant. The specific aims of this proposal address refinement of the therapeutic window for this therapy, the minimum number of cells that must be edited to save vision, and the general safety of the therapeutic strategy. These aims will be met through the use of AAV5 to deliver an engineered meganuclease that specifically targets the dominant P23H allele while saving the wildtype allele that differs by only one base. This reagent will be evaluated in a validated, rapidly progressing swine model that harbors a human RHO P23H transgene. A previously optimized dose that produces a durable response will be delivered via subretinal injection and the animals will be followed for 32 weeks to repeatedly measure effectiveness over time via clinical eye exam, Full-Field Electroretinogram (ffERG), and Optical Coherence Tomography (OCT). At the end of the experiment, eyes will be examined via immunohistochemistry (IHC) to determine the regional, minimum number of rods that must be edited to save cone function and therefore to save vision. These experiments will be performed at two different stages in disease progression that are hypothesized to represent the last treatable state and the first non-treatable state so that guidance can be provided for criteria of enrollment of patients in a future clinical trial. In parallel, animals will be similarly treated and then sacrificed at two weeks post injection to determine the range of adjacent tissues to which the AAV5 vector will migrate. These animals will also be used to determine the level of editing that occurs in non-intended tissues, including the germline. All animals will be used to obtain data on general physiological response to the therapy.