Developing gene editing platforms for retinal degeneration.

摘要 遗传性视网膜营养不良（IRD）是一组异质性孤儿疾病，在常染色体上遗传， 显性、隐性或X-连锁模式，除了线粒体传递，所有这些都导致了 功能性视力，并经常发展为失明。作为一个群体，IRD是由于超过280个基因的突变。 目前，只有一种FDA批准的基因疗法用于这个视网膜变性的大家族。Prime 编辑，一种新的多功能基因组编辑工具，允许所有12个碱基到碱基的变化，插入多达44个碱基 长的和多达80个碱基的缺失。Prime编辑器能够纠正89%的已知遗传变异 与人类疾病相关，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（LNP）是一种模块化平台， 封装和交付基因组编辑器。以mRNA的形式递送核酸酶是一种最佳的替代方案， 瞬时蛋白质表达而不是持续表达DNA切割机制相关的策略 with viral病毒vector载体. LNP能够快速有效地将mRNA递送至视网膜色素上皮， 然而，它们抑制光感受器的能力有限，而光感受器是靶向许多基因所必需的。 与IRD有关。我们假设，通过采用噬菌体展示技术，我们将分离出有前途的， 靶向肽，其将装饰我们的LNP并有效地将主要编辑货物递送到 光感受器我们的主要目标是产生肽靶向的LNP，导致细胞特异性递送引发剂， 编辑用于治疗IRD的组件。为达致这个目标，我们提出以下具体目标： 优化体内噬菌体展示生物淘选，以鉴定靶向肽部分， 光受体特异性脂质纳米粒的基因传递，2）剖析肽靶向的机制， LNP进入光感受器，以及3）在两种情况下评估初编辑的功效和任何相关毒性 IRD的啮齿动物模型。到目前为止，我们已经确定了新的肽，可以引导LNP向感光细胞， 基因递送，并确定LNP可以将所有主要编辑组分包装在一起， 体外和体内报告基因的有效引物编辑。该项目的成功完成将导致 开发细胞特异性基因编辑平台，将推进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.