DEVELOPMENT OF AAV-CRISPR/CAS9-BASED THERAPIES FOR CONE ROD DYSTROPHY

基于 AAV-CRISPR/CAS9 的锥杆营养不良疗法的开发

• 批准号: 10412033
• 负责人: Shannon Elizabeth Boye
• 金额: $ 52.11万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10412033
• 关键词: Ablation; Age; Alleles; Atrophic; Attention; Benchmarking; Blindness; CRISPR therapeutics; CRISPR/Cas technology; Capsid; Code; Color Visions; Complement; Cone; Data; Dependovirus; Deterioration; Directed Molecular Evolution; Disease; Dose; Engineering; Exhibits; Exons; Functional disorder; Gene Mutation; Genes; Genomics; Goals; Guide RNA; Homologous Gene; Human; Inherited; Knock-in Mouse; Knock-out; Lateral; Macaca; Methods; Mus; Mutation; Nucleotides; OLFM4 gene; Onset of illness; Pathologic; Patients; Peripheral; Phenotype; Photophobia; Photoreceptors; Pilot Projects; Primates; Reagent; Reporting; Retina; Retinal Diseases; Rod; Safety; Site; Specificity; Structure; System; T cell response; Testing; Therapeutic; Therapeutic Intervention; Transgenic Mice; Transgenic Model; Transgenic Organisms; Variant; Virus; Visual Acuity; Visual Fields; adeno-associated viral vector; autosomal dominant mutation; base; base editing; clinical application; clinically relevant; cone-rod dystrophy; experience; gain of function mutation; gene replacement; gene replacement therapy; guanylate cyclase 1; improved; in vivo; internal control; macula; mature animal; mouse model; mutant; nonhuman primate; novel; preservation; regional difference; sight restoration; somatic cell gene editing; vector

ABSTRACT Mutations in GUCY2D, the gene encoding retinal guanylate cyclase-1 (retGC1), are the leading cause of autosomal dominant cone-rod dystrophy. GUCY2D-CORD6 patients present with loss of visual acuity, abnormal color vision, photophobia, visual field loss and macular atrophy within the first decade. Rod degeneration and peripheral visual field loss follow. Significant progress towards clinical application of gene replacement therapy for LCA due to recessive mutations in GUCY2D (LCA1) has been made, but a different approach is needed to treat CORD6 where gain of function mutations cause dysfunction and dystrophy. Our preliminary data show that 1) selective and efficient somatic knock-out of GUCY2D and Gucy2e (murine homologue) with AAV- CRISPR/Cas9 results in a subsequent loss of retinal structure/function that manifests from reduced retGC1 expression in macaque and mouse, respectively, 2) a ‘knock-out + complementation in trans’ approach (wherein complementation is performed with ‘hardened’ Gucy2e not recognized by Gucy2e gRNA) preserves retinal function in mice, 3) AAV-CRISPR/Cas9- based editing of GUCY2D is therapeutic in a R838S transgenic (Tg) mouse model of CORD6, and 4) Cas9 variants identified by directed evolution exhibit allele specificity for GUCY2D(R838S). We will build upon these results in the following Aims. Aim 1 will establish the optimal parameters for AAV-CRISPR/Cas9-based gene editing in two R838S CORD6 Tg mouse lines. We will establish the optimal AAV capsid/dose, durability of therapy, treatment window, and feasibility of transient Cas9 expression systems. Aim 2 will evaluate safety/efficacy of AAV-CRISPR/Cas9-based gene editing in macaque by looking for off-target editing and assessing the potential impact of AAV vector insertions and long-term Cas9 expression. We will also evaluate regional differences in editing efficiency and conduct dose-ranging studies. Aim 3 will compare ‘knock out + complementation in trans’ vs. ‘allele-targeted’ approaches for treating CORD6. The optimal AAV capsids and Cas9 expression system from Aim 1 will be used to test KO + complementation in GC2-/- mice. Allele-targeted editing will be performed in humanized R838S CORD6 mice that carry both the wt and R838S- containing exon 13 of GUCY2D. Approaches will include a novel, engineered Cas9 that specifically edits the R838S mutant allele, gRNAs containing mismatches to mutant but not wt allele, or utilization of an alternative PAM site found in the mutant, but not wt allele. Successful approaches will be tested in macaque for efficiency/safety. Our findings will identify the optimal capsid/dose, and treatment age for therapeutic AAV- CRISPR/Cas9-based disruption of R838S GUCY2D in vivo. We will establish the safety profile, and regional efficiencies of gene editing by AAV-CRISPR/Cas9 in a species with both genomic and clinical relevance. In addition, we will identify materials and approaches that will allow clinical application of AAV-CRISPR/Cas9 therapies for CORD6 as well as other dominantly inherited retinal diseases.

抽象的 编码视网膜鸟苷酸环化酶-1 (retGC1) 的基因 GUCY2D 的突变是导致 常染色体显性锥杆营养不良。 GUCY2D-CORD6 患者出现视力丧失、视力异常 最初十年内出现色觉、畏光、视野丧失和黄斑萎缩。杆变性和 随后出现周边视野丧失。基因替代疗法临床应用取得重大进展 已经针对 GUCY2D (LCA1) 隐性突变引起的 LCA 进行了治疗，但需要采用不同的方法 治疗 CORD6，其中功能获得突变导致功能障碍和营养不良。我们的初步数据表明 1) 用AAV-选择性且高效地体细胞敲除GUCY2D和Gucy2e（鼠同源物） CRISPR/Cas9 导致视网膜结构/功能随后丧失，表现为 retGC1 减少 分别在猕猴和小鼠中表达，2）“敲除+反式互补”方法（其中 与 Gucy2e gRNA 不识别的“硬化”Gucy2e 进行互补）可保留视网膜 小鼠中的功能，3) 基于 AAV-CRISPR/Cas9 的 GUCY2D 编辑在 R838S 转基因 (Tg) 中具有治疗作用 CORD6 的小鼠模型，以及 4) 通过定向进化鉴定的 Cas9 变体表现出等位基因特异性 GUCY2D(R838S)。我们将在这些成果的基础上实现以下目标。目标 1 将建立最优 两个 R838S CORD6 Tg 小鼠品系中基于 AAV-CRISPR/Cas9 的基因编辑的参数。我们将建立 最佳 AAV 衣壳/剂量、治疗持久性、治疗窗以及瞬时 Cas9 表达的可行性 系统。目标 2 将通过观察来评估基于 AAV-CRISPR/Cas9 的基因编辑在猕猴中的安全性/有效性 用于脱靶编辑并评估 AAV 载体插入和长期 Cas9 表达的潜在影响。 我们还将评估编辑效率的区域差异并进行剂量范围研究。目标3将 比较治疗 CORD6 的“敲除 + 反式互补”与“等位基因靶向”方法。最优的 来自 Aim 1 的 AAV 衣壳和 Cas9 表达系统将用于测试 GC2-/- 小鼠中的 KO + 互补。 等位基因靶向编辑将在携带 wt 和 R838S- 的人源化 R838S CORD6 小鼠中进行 含有 GUCY2D 的外显子 13。方法将包括一种新颖的、工程化的 Cas9，专门编辑 R838S 突变等位基因、包含与突变等位基因错配但不包含 wt 等位基因错配的 gRNA，或利用替代方案 在突变体中发现了 PAM 位点，但在 wt 等位基因中没有发现。成功的方法将在猕猴身上进行测试 效率/安全。我们的研究结果将确定治疗性 AAV 的最佳衣壳/剂量和治疗年龄 基于 CRISPR/Cas9 的体内 R838S GUCY2D 破坏。我们将建立安全概况和区域 AAV-CRISPR/Cas9 在具有基因组和临床相关性的物种中进行基因编辑的效率。在 此外，我们将确定允许 AAV-CRISPR/Cas9 临床应用的材料和方法 CORD6 以及其他显性遗传性视网膜疾病的疗法。