Preclinical evaluation of a homing endonuclease gene therapy for adRP in models of P23H retinopathy.

常染色体显性遗传性视网膜色素变性(ADRP)是一种致盲的眼病，可导致视杆细胞和 最终形成锥形感光器。ADRP的很大一部分是由视紫红质(RHO)基因突变引起的， 北美最常见的突变导致视紫红质P23H突变。患者通常 出现中晚期疾病，此时许多杆状感光细胞已经退化。 我们将使用巨核酸酶基因组编辑工具(Rho1-2)来评估基因组编辑，该工具专用于 几种临床前P23H ADRP动物模型中导致P23H突变的序列。我们将交付AAV5- 视网膜下注射GRK1-Rho1-2感染光感受器。我们将测量疗效，Rho1-2的能力 改变视杆变性和功能衰退。我们将定量测定Rho1-2效率，比例 在具有插入/缺失(INDELs)的P23H等位基因中，具有INDELs的P23H等位基因与WT等位基因的比例具有特异性， 以及它的安全性，在整个人类基因组中的其他基因中没有Indels(非靶标编辑)。 在Pig P23H adrp模型中，我们将评估Rho1-2的编辑效率，因为猪和 人类的眼睛大小和视网膜结构与猪的视锥有着丰富的视觉条纹有关。我们将使用相同的 临床上用于测量视网膜功能(全场视网膜电信号)和结构的非侵入性工具， (光谱域OCT)。我们将使用两个现有的转基因猪株系，它们含有完整的P23H hRHO基因， 但它们的时间进程不同，导致杆和杆功能的快速或缓慢损失。我们会配合治疗 时间与杆结构和功能衰退的自然历史，比较Rho1-2在早期的编辑效果-， 中晚期P23H ADRP，确定其暂时性治疗窗。在人性化的adrp小鼠模型中 我们将评估Rho1-2编辑的效率和特异性，因为它们包含WT和P23H人类等位基因 适当的基因组环境。在人类视网膜有机体中，我们将评估Rho1-2编辑的安全性(非靶标 切割整个基因组)，因为它们包含人类的光感受器以及人类的其余部分 相关背景下的基因组.. 综上所述，我们的实验结果将决定Rho1-2是否应该进一步在 人体临床试验，并将确定这些试验的终点。

Autosomal dominant Retinitis pigmentosa (adRP) is a blinding eye disease that results in loss of rods and eventually cone photoreceptors. A large fraction of adRP is caused by mutations in the rhodopsin (Rho) gene, and the most common mutation in North America causes a P23H mutation in rhodopsin. Patients typically present with mid- to late-stage disease when many rod photoreceptors already have degenerated. We will evaluate genome editing using a meganuclease genome editing tool (Rho1-2) that is specific for the sequence causing the P23H mutation in several preclinical animal models of P23H adRP. We will deliver AAV5- GRK1-Rho1-2 via subretinal injection to infect photoreceptors. We will measure efficacy, the ability of Rho1-2 to alter rod degeneration and functional decline. We will quantitatively determine Rho1-2 efficiency, the proportion of P23H alleles with insertions/delections (indels), specificity the proportion of P23H to WT alleles with indels, and its safety, the absence of indels in other genes throughout the human genome (off target editing). In Pig P23H adRP models we will evaluate Rho1-2 editing efficacy because of the similarities between pigs and humans in eye size and retinal structure and because the pig has a cone rich visual streak. We will use the same non-invasive tools used in the clinic to measure retinal function (full field electroretinograms) and structure, (spectral domain OCT). We will use two existing transgenic pig lines that harbor the entire P23H hRHO gene, but differ in their time course, resulting in a fast or slow loss of rods and rod function. We will match treatment time with the natural history of rod structural and functional decline, compare Rho1-2 editing efficacy in early-, mid- and late-stage P23H adRP and define its temporal treatment window. In Humanized adRP mouse models we will evaluate Rho1-2 editing efficiency and specificity because they contain WT and P23H human alleles in the appropriate genomic context. In human retinal organoids we will evaluate Rho1-2 editing safety (off-target cutting throughout the genome) because they contain human photoreceptors as well as the rest of the human genome in a relevant context.. Taken together the results of our experiments will determine if Rho1-2 should move forward for eventual use in human clinical trials and will define endpoints for those trials.