Precision genome editing in vivo to treat retinal diseases

体内精准基因组编辑治疗视网膜疾病

基本信息

批准号：
10565189
负责人：
Krzysztof Palczewski
金额：
$ 60.23万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10565189
关键词：
220kDa rod outer segment rim protein Address Adenine Alleles Animal Model Benchmarking Biological Assay Blindness COVID-19 CRISPR/Cas technology Cell Line Cell physiology Cells Charge Color Complex Coomassie blue DNA DNA Double Strand Break DNA Integration Data Deletion Mutation Derivation procedure Development Disease Engineering Enzymes Eye FDA approved Genes Genetic Genome Genomic DNA Genomics Guide RNA Hereditary Disease Histology Human Imaging Techniques Inherited Insertion Mutation Label Laboratories Leber&apos s amaurosis Link Lipids Mass Spectrum Analysis Membrane Messenger RNA Methods Modeling Multienzyme Complexes Mus Mutate Mutation Nucleosides Penetration Peptides Photoreceptors Phototransduction Point Mutation Preclinical Testing Protein Analysis Protein Binding Domain Proteins Publications Publishing Quality of life RNA RNA Stability RNA delivery RNA vaccine RPE65 protein Reagent Reporter Retina Retinal Degeneration Retinal Diseases Retinitis Pigmentosa Rhodopsin Ribonucleoproteins Route Safety Stargardt&apos s disease Structure of retinal pigment epithelium System Technology Testing Therapeutic Therapeutic Intervention Therapy Evaluation Toxic effect Viral Visual Cortex Visual impairment Western Blotting amino group autosome base base editing base editor cell type clinical translation delivery vehicle disease-causing mutation effective therapy experimental study functional restoration gene augmentation therapy gene function gene therapy genome editing human disease human model immunoregulation improved in vivo innovation insertion/deletion mutation interest lipid nanoparticle mouse model mutant mutation correction next generation sequencing novel particle photoreceptor degeneration preclinical study prime editing prime editor protein expression repaired research clinical testing response retinol isomerase risk minimization screening success therapeutic genome editing tool transcriptome transversion mutation two photon microscopy two-photon visual cycle

项目摘要

SUMMARY Inherited retinal disorders are a genetically heterogeneous group of blinding diseases that have significant impact on quality of life. Therapeutic approaches have lagged significantly behind initial identification of the genetic bases for these diseases. However, there are some striking successes; e.g., RPE65 gene augmentation therapy was the first FDA-approved gene therapy for any genetically inherited disease. Clinical translation of current CRISPR-Cas9 technology has been impeded by its low editing efficiency, error-prone homology-directed repair (HDR), and substantial indel formation. Precision genome editing is an advanced, innovative CRISPR-Cas9- associated genome-editing tool that addresses the limitations of typical CRISPR-Cas9 implementation. Adenine base editors (ABEs) enable conversion of a point mutation independently of Cas9-induced double-stranded DNA breaks and HDR. When base editing is not applicable (e.g., due to transversion mutations, large deletions, or insertions), prime editing technology offers feasible alternatives. Genome editing is highly specific; however, prolonged expression of base editors could lead to undesired off-target alterations throughout the genome and transcriptome. We hypothesize that transient delivery of genome editors via RNPs and synthetic RNAs can achieve the same high editing rates as those for genome editors delivered via viral transduction with reduced off-target and bystander editing. Accordingly, we propose two thematically linked aims. Aim 1. Correct inherited retinal disease-causing mutations in the rhodopsin gene (RhoE150K/E150K) associated with autosomal recessive retinitis pigmentosa (RP) via adenine base editing. Delivery of ABEs will be optimized in the thoroughly characterized RhoE150K/E150K mouse model of RP. Proposed approaches will provide a platform for ABEs to be quickly adapted to any suitable RPE or retinal mutation. Aim 2. Repair the ABCA4 protein in Abca4PV/PV mice by prime editing. Using the PE3b prime editor and two concurrent stabilized engineered prime-editing guide RNAs (epegRNA), we will restore functional ABCA4 protein in Abca4PV/PV mice that carry double allelic mutations in photoreceptors and the RPE. Using immunoblotting and next-generation sequencing for detecting rescued Abca4, and two-photon imaging techniques to detect A2E, we will optimize genome editing efficiency in this animal model to improve prime-editing technology and its application to treat inherited retinal diseases. For both aims, we will test various means to deliver the editors transiently: (i) cell-penetrating peptides fused to editors in purified ribonucleoprotein (RNP)-editing complexes; (ii) Coomassie-lipid tags on purified RNP-editing complexes; (iii) viral-like particles containing RNP-editing complexes; or (iv) lipid nanoparticles containing stabilized mRNAs of genome-editing materials for intracellular expression. These delivery systems will be optimized first in engineered chromogenic cell lines. The efficacy of base and prime editing in mice will be benchmarked against the level of expression of RPE65 in the rd12 animal model of Leber congenital amaurosis.

概括遗传性视网膜疾病是一组具有显着影响的遗传异质性致盲疾病。关于生活质量。治疗方法明显落后于基因的初步鉴定这些疾病的基础。然而，也有一些引人注目的成功；例如，RPE65 基因增强疗法是 FDA 批准的第一个针对任何遗传性疾病的基因疗法。当前的临床翻译 CRISPR-Cas9技术因其编辑效率低、同源定向修复容易出错而受到阻碍（HDR），以及大量的插入缺失形成。精准基因组编辑是一种先进、创新的 CRISPR-Cas9- 相关的基因组编辑工具，解决了典型 CRISPR-Cas9 实施的局限性。腺嘌呤碱基编辑器 (ABE) 能够独立于 Cas9 诱导的双链 DNA 进行点突变的转换中断和 HDR。当碱基编辑不适用时（例如，由于颠换突变、大量缺失或插入），主要编辑技术提供了可行的替代方案。基因组编辑具有高度特异性；然而，碱基编辑器的延长表达可能会导致整个基因组出现不希望的脱靶改变，转录组。我们假设通过 RNP 和合成 RNA 瞬时传递基因组编辑器可以实现与通过病毒转导传递的基因组编辑器相同的高编辑率，并且减少脱靶和旁观者编辑。因此，我们提出两个主题相关的目标。目标 1. 纠正视紫红质基因 (RhoE150K/E150K) 中引起遗传性视网膜疾病的突变通过腺嘌呤碱基编辑与常染色体隐性色素性视网膜炎（RP）相关。 ABE 的交付将在彻底表征的 RP RhoE150K/E150K 小鼠模型中进行优化。拟议的方法将为 ABE 提供一个快速适应任何合适的 RPE 或视网膜突变的平台。目标 2. 通过 Prime 编辑修复 Abca4PV/PV 小鼠中的 ABCA4 蛋白。使用 PE3b prime 编辑器和两个同时稳定的工程引物编辑引导RNA (epegRNA)，我们将恢复功能性ABCA4蛋白在光感受器和 RPE 中携带双等位基因突变的 Abca4PV/PV 小鼠中。使用免疫印迹和新一代测序技术用于检测获救的 Abca4，双光子成像技术用于检测 A2E，我们将优化该动物模型的基因组编辑效率，以改进prime编辑技术及其应用于治疗遗传性视网膜疾病。为了实现这两个目标，我们将测试各种瞬时传递编辑器的方法：(i) 融合细胞穿透肽纯化核糖核蛋白 (RNP) 编辑复合物中的编辑器； (ii) 纯化 RNP 编辑上的考马斯脂质标签复合体； (iii) 含有 RNP 编辑复合物的病毒样颗粒；或(iv)含有脂质纳米颗粒稳定基因组编辑材料的 mRNA 以进行细胞内表达。这些输送系统将首先在工程显色细胞系中进行优化。碱基和引物编辑在小鼠中的功效将是以 Leber 先天性黑蒙 rd12 动物模型中 RPE65 的表达水平为基准。