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

• 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
• 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' 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' 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

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种具有重大影响的遗传性异构疾病群 关于生活质量。治疗方法已显着落后于遗传的初始鉴定 这些疾病的基础。但是，取得了一些惊人的成功。例如，RPE65基因增强疗法 是第一个针对任何遗传遗传疾病的FDA批准的基因疗法。电流的临床翻译 CRISPR-CAS9技术受到其低编辑效率，易用错误的同源性维修的阻碍 （HDR）和实质性的indel形成。精确基因组编辑是一种先进的创新CRISPR-CAS9- 相关的基因组编辑工具，可以解决典型的CRISPR-CAS9实施的局限性。腺嘌呤 基本编辑器（ABE）可以独立于Cas9诱导的双链DNA转换点突变 休息和HDR。当不适用基础编辑时（例如，由于横向突变，大删除或 插入），主要编辑技术提供了可行的替代方案。基因组编辑是高度特异性的。然而， 基本编辑器的长时间表达可能会导致整个基因组中不希望的脱靶变化，并且 转录组。我们假设通过RNP和合成RNA的基因组编辑器的短暂传递可以 达到与通过病毒转导传递的基因组编辑者相同的高编辑率，并降低 脱离目标和旁观者编辑。因此，我们提出了两个主题联系的目标。 AIM 1。在视紫红质基因（Rhoe150k/e150k）中正确遗传性视网膜疾病突变 通过腺嘌呤碱基编辑，与常染色体隐性视网膜炎色素炎（RP）相关。交付AB 将在RP的RHOE150K/E150K小鼠模型中进行优化。建议的方法将 提供一个平台，使ABS快速适应任何合适的RPE或视网膜突变。 AIM 2。通过Prime编辑在ABCA4PV/PV小鼠中修复ABCA4蛋白。使用PE3B Prime编辑器和两个 并发稳定工程的原始编辑指南RNA（Epegrna），我们将恢复功能性ABCA4蛋白 在ABCA4PV/PV小鼠中，在感光体和RPE中携带双重等位基因突变。使用免疫印迹和 用于检测被救出的ABCA4的下一代测序，以及用于检测A2E的两光子成像技术，我们 将优化该动物模型中的基因组编辑效率，以改善主要编辑技术及其 用于治疗遗传性视网膜疾病的应用。 对于这两个目的，我们将测试各种手段以瞬时交付编辑器：（i）融合的细胞穿透肽 纯化的核糖核蛋白（RNP）编辑复合物中的编辑； （ii）纯化的RNP编辑上的coomassie-lipid标签 复合物； （iii）含有RNP编辑复合物的病毒样颗粒；或（iv）含有含有的脂质纳米颗粒 稳定基因组编辑材料的mRNA用于细胞内表达。这些输送系统将是 首先在工程的发色细胞系中进行了优化。小鼠基础和主要编辑的功效将是 在Leber先天性症状的RD12动物模型中反对RPE65表达水平的基准测试。