Gene Silencing and Gene Editing in Phototransduction

项目摘要 基因增强疗法不能解决常染色体显性(AD)疾病，在这种疾病中， 表达有缺陷的蛋白质。为了治疗这些突变，例如主要形式的视紫红质 (Rho)相关的视网膜色素变性RP(ADRP)，唯一的治疗方法是沉默(RNAi)或编辑突变体 等位基因。精确的CRISPR治疗性编辑被预测是治疗几个 显性疾病，但利用CRISPR的分子方法发展缓慢 因为同源定向修复只发生在细胞的S和G2期 周而复始。为了克服这些限制，我们建立了一种通用的编辑技术，它使用 2引导RNAs(GRNAs)在细胞周期的任何阶段针对所有150个显性Rho突变。 为了对这种特定物种的技术进行临床前评估，我们已经取代了幼稚的 小鼠染色体Rho与人Rho突变序列(人源化Rho)。如果成功， 我们的治疗性编辑策略中使用的病毒载体可以直接在人体试验中重复使用 无需进一步修改的ADRP，从而避免了FDA的障碍并加快了 这项研究要在临床环境下进行。我们的临床前ADRP模型也可以用于测试 未来基于基因和药物的治疗方法。这项拟议的研究具有创新性，因为它引入了 新方法和模型系统作为精准医学方法的第一步 发展ADRP治疗。这些方法也有可能适用于基于 靶向与其他显性疾病相关的基因的DNA序列。

Project Summary Gene-augmentation therapy cannot address autosomal-dominant (ad) disorders, in which a defective protein is expressed. To treat these mutations, such as the dominant form of rhodopsin (RHO)-related retinitis pigmentosa RP (adRP), the only cure is to silence (RNAi) or edit the mutant allele. Precise CRISPR therapeutic editing is predicted to be the best means of treating several dominant disorders, but molecular methods harnessing CRISPR have been slow to develop because homology-directed repair (HDR) occurs only during the S and G2 phases of the cell cycle. To overcome these limitations, we have established a versatile editing technology that uses 2 guide RNAs (gRNAs) to target all 150 dominant RHO mutations at any phase of the cell cycle. To conduct preclinical evaluation of this species-specific technology, we have replaced the naïve mouse chromosomal Rho with human RHO mutant sequences (humanized RHO). If successful, the viral vectors used in our therapeutic editing strategy could be directly reused in human trials for adRP without further modification, thus avoiding FDA hurdles and accelerating translation of this research to a clinical setting. Our preclinical adRP model could also be useful for testing of future gene- and drug-based therapies. The proposed research is innovative, as it introduces new methods and model systems as the initial steps of a precision medicine approach toward developing adRP treatment. These methods are also potentially adaptable to therapies based on targeting DNA sequences of genes linked to other dominant disorders.