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.

总结 遗传性视网膜疾病是一组遗传异质性致盲疾病， 生活质量的影响。治疗方法明显落后于遗传学的初步鉴定。 这些疾病的基础。然而，也有一些惊人的成功;例如，RPE 65基因增强治疗 这是FDA批准的第一种基因疗法，用于治疗任何遗传性疾病。当前临床翻译 CRISPR-Cas9技术因其编辑效率低，易于出错的同源定向修复而受到阻碍 (HDR)和大量的indel形成。精准基因组编辑是一种先进的、创新的CRISPR-Cas9- 相关的基因组编辑工具，解决了典型的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转化点突变 中断和HDR。当基础编辑不适用时（例如，由于颠换突变、大缺失或 插入），prime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， 碱基编辑器的延长表达可能导致整个基因组中不希望的脱靶改变， 转录组我们假设通过RNP和合成RNA瞬时递送基因组编辑器可以 实现与通过病毒转导递送的基因组编辑器相同的高编辑率， 脱靶编辑和旁观者编辑。因此，我们提出两个主题相关的目标。 目标1.纠正遗传性视网膜疾病引起的视紫红质基因突变（RhoE 150 K/E150 K） 通过腺嘌呤碱基编辑与常染色体隐性视网膜色素变性（RP）相关。ABE的交付 将在充分表征的RhoE 150 K/E150 K RP小鼠模型中进行优化。拟议的办法将 为ABE快速适应任何合适的RPE或视网膜突变提供平台。 目标2.通过引物编辑修复Abca 4PV/PV小鼠中的ABCA 4蛋白。使用PE 3b主编辑器和两个 同时稳定的工程引物编辑指导RNA（epegRNA），我们将恢复功能性ABCA 4蛋白 在光感受器和RPE中携带双等位基因突变的Abca 4PV/PV小鼠中。使用免疫印迹， 下一代测序用于检测拯救的Abca 4，双光子成像技术检测A2 E，我们 将在这种动物模型中优化基因组编辑效率，以改善引物编辑技术及其 用于治疗遗传性视网膜疾病。 对于这两个目标，我们将测试各种手段来瞬时递送编辑器：（i）融合到 （ii）纯化的核糖核蛋白（RNP）编辑复合物上的考马斯脂质标签 （iii）含有RNP-编辑复合物的病毒样颗粒;或（iv）含有RNP-编辑复合物的脂质纳米颗粒。 用于细胞内表达的基因组编辑材料的稳定mRNA。这些输送系统将是 首先在工程显色细胞系中优化。碱基编辑和引物编辑在小鼠中的功效将被证实。 以Leber先天性黑蒙的rd 12动物模型中RPE 65的表达水平为基准。