Developing gene editing platforms for retinal degeneration.

开发视网膜变性的基因编辑平台。

基本信息

批准号：
10522389
负责人：
Gaurav Sahay
金额：
$ 64.84万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10522389
关键词：
Affect Age Amyloidosis Bacteriophages Biological Biological Assay Blindness COVID-19 Cell Line Cells DNA DNA delivery Data Deoxyribonucleases Dependovirus Development Docking Electroretinography Encapsulated FDA approved Family Fluorescence-Activated Cell Sorting Frequencies Gene Delivery Genes Genetic Genome Goals Green Fluorescent Proteins In Vitro Inherited Lead Leucine Zippers Libraries Ligands Link Measurement Measures Mediating Messenger RNA Mitochondria Modeling Mouse Protein Mus Mutation Neural Retina Patients Pattern Peptide Receptor Peptides Persons Phage Display Photoreceptors Population Prealbumin Proteins Publishing RNA Interference Rare Diseases Reporter Genes Retina Retinal Degeneration Retinal Dystrophy Rodent Model Structure of retinal pigment epithelium Surface System Techniques Thick Toxic effect Transfection Viral Vector Vision Western Blotting base deep sequencing efficacy evaluation experimental study functional loss gene therapy genetic variant genome editing human disease in vivo infancy inherited retinal degeneration knock-down lipid nanoparticle mRNA delivery mouse model nanoparticle delivery novel nuclease photoreceptor degeneration prime editing prime editor protein expression receptor receptor mediated endocytosis screening sex subretinal injection tool transmission process uptake

项目摘要

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.

抽象的遗传性视网膜营养不良症 (IRD) 是一组异质性孤儿疾病，以常染色体遗传方式遗传。除了线粒体传递之外，显性、隐性或 X 连锁模式都会导致功能性视力并经常进展至失明。作为一个整体，IRD 是由 280 多个基因的突变引起的。目前，针对这一视网膜变性大家族，只有一种 FDA 批准的基因疗法。主要的编辑，一种新的多功能基因组编辑工具，允许所有 12 个碱基到碱基的改变，插入多达 44 个碱基长且删除多达 80 个碱基。 Prime 编辑器能够纠正 89% 的已知遗传变异与人类疾病有关，但在体内使用仍处于起步阶段。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) 是一个模块化平台，可以封装并提供基因组编辑器。以 mRNA 形式提供核酸酶是最佳替代方案瞬时蛋白表达策略，而不是与 DNA 切割机制相关的持续表达与病毒载体。 LNP 能够快速有效地将 mRNA 递送至视网膜色素上皮，然而，它们转染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 相关。我们假设通过采用噬菌体展示技术，我们将分离出有希望的靶向肽，它将装饰我们的 LNP 并有效地将主要编辑货物传递到光感受器。我们的主要目标是生成肽靶向 LNP，从而实现细胞特异性递送prime 用于治疗 IRD 的编辑组件。为了实现这一目标，我们提出以下具体目标：1）优化体内噬菌体展示生物淘选，以鉴定靶向肽部分，从而允许基于光感受器特异性脂质纳米颗粒的基因传递，2）剖析肽靶向机制 LNP 进入光感受器，以及 3) 评估两种启动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，我们已经鉴定出可以引导 LNP 朝向光感受器的新型肽基因传递并确定 LNP 可以将所有主要编辑组件包装在一起并导致体外和体内报告基因的高效主编辑。该项目的成功完成将导致开发细胞特异性基因编辑平台，以推进 IRD 的治疗。