Developing gene editing platforms for retinal degeneration.

摘要 遗传性视网膜营养不良(IRD)是一组在常染色体上遗传的异质性孤儿疾病 除了线粒体传递外，还有显性、隐性或X连锁的模式，所有这些都会导致 功能性视力，常进展为失明。作为一个群体，IRDS是由于280多个基因的突变造成的。 目前，只有一种FDA批准的基因疗法可以治疗这一视网膜变性大家族。素数 Editing是一种新的多功能基因组编辑工具，允许所有12个碱基到碱基的改变，插入多达44个碱基 长的，最多80个碱基的缺失。主编有能力纠正89%的已知基因变异 与人类疾病有关，但仍处于体内使用的婴儿期。我们的长期目标是优化 IRD的主要编辑平台。基于脂质的纳米颗粒(LNPs)是一个模块化的平台，可以 封装和交付基因组编辑。以信使核酸酶形式递送核酸酶是一种最佳的选择 与DNA切割机制相关的瞬时蛋白表达而不是持续表达的策略 通过病毒载体。LNPs能够快速有效地将mRNA输送到视网膜色素上皮， 然而，它们转染光受体的能力有限，而光感受器是靶向许多基因所必需的。 与红斑狼疮相关。我们假设，通过利用噬菌体展示技术，我们将分离出有希望的 目标多肽将装饰我们的LNP并有效地将主要的编辑货物运送到 光感受器。我们的主要目标是产生多肽靶向LNPs，从而导致PRIME的细胞特异性传递 编辑用于治疗IRD的组件。为实现这一目标，我们提出以下具体目标：1) 优化体内噬菌体展示生物扫描以鉴定允许 基于光感受器特异性脂质纳米粒的基因传递，2)分析多肽靶向的机制 LNP进入光感受器，以及3)在两个方面评估主要编辑的有效性和任何相关的毒性 IRD的啮齿动物模型。到目前为止，我们已经确定了可以将LNPs导向光感受器的新型多肽 并确定LNPs可以将所有主要的编辑成分打包在一起，并导致 在体外和体内对报告基因进行高效的初级编辑。这个项目的成功完成将导致 开发针对细胞的基因编辑平台，这将促进IRD的治疗。

ABSTRACT Inherited retinal dystrophies (IRDs) are a heterogenous group of orphan diseases, inherited in an autosomal dominant, recessive or X-linked pattern in addition to mitochondrial transmission, all leading to the loss of functional vision and often progressing to blindness. As a group, IRDs are due to mutations in over 280 genes. Currently, there is only one FDA approved gene therapy for this large family of retinal degenerations. Prime editing, a new versatile genome editing tool, allows for all 12 base-to-base changes, insertions up to 44 bases long and deletions of up to 80 bases. Prime editors are capable of correcting 89% of known genetic variants associated with human disease, but are still in their infancy for in-vivo use. Our long-term goal is to optimize prime editing platforms for IRDs. Lipid based nanoparticles (LNPs) are a modular platform that can encapsulate and deliver genome editors. Delivering nucleases as mRNA has been an optimal alternative strategy for transient protein expression rather than persistent expression of DNA cutting machinery associated with viral vectors. LNPs are capable of rapid and efficient delivery of mRNA to the retinal pigment epithelium, however, they have limited capacity to transfect photoreceptors, which is necessary to target the many genes associated with IRDs. We hypothesize that by employing phage display techniques, we will isolate promising targeting peptides which will decorate our LNPs and effectively deliver prime editing cargo to the photoreceptors. Our main goal is to generate peptide-targeted LNPs that lead to cell-specific delivery of prime editing components for the treatment of IRDs. To achieve this goal, we propose the following specific aims: 1) Optimize in-vivo phage display biopanning for the identification of targeting peptide moieties that allow for photoreceptor-specific lipid nanoparticle-based gene delivery, 2) Dissect the mechanism of peptide-targeted LNP entry into photoreceptors, and 3) Evaluate the efficacy, and any associated toxicity, of prime editing in two rodent models of IRD. Thus far, we have identified novel peptides that can steer LNPs toward photoreceptor gene delivery and determined that LNPs can package all prime editing components together and lead to efficient prime editing of reporter genes in-vitro and in-vivo. Successful completion of this project will lead to the development of cell-specific gene editing platforms that will advance treatment for IRDs.