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

摘要 编码视网膜鸟苷环化酶-1(RetGC1)的基因GUCY2D的突变是导致 常染色体显性遗传性视锥视杆细胞营养不良。GUCY2D-CORD6患者出现视力丧失，异常 在第一个十年内，色觉障碍、畏光、视野丧失和黄斑萎缩。杆状变性和 随之而来的是周围视野丧失。基因替代疗法在临床应用中的重大进展 对于由于GUCY2D(LCA1)隐性突变导致的LCA，已经做出了研究，但需要不同的方法来 治疗CORD6，功能突变的获得会导致功能障碍和营养不良。我们的初步数据显示， 1)GUCY2D和Gucy2e(小鼠同源物)与AAV-1选择性高效的体细胞敲除 CRISPR/Cas9导致视网膜结构/功能的随后丧失，表现为retGC1减少 分别在猕猴和小鼠中表达，2)反式敲除互补方法(其中 补充是用Gucy2e不识别的硬化的Gucy2e进行的)保存视网膜 3)基于AAV-CRISPR/Cas9的GUCY2D编辑对R838S转基因(TG)具有治疗作用 通过定向进化鉴定的CORD6小鼠模型和4)Cas9变异体显示出等位基因特异性 GUCY2D(R838S)。我们将在这些成果的基础上，实现以下目标。目标1将建立最优的 基于AAV-CRISPR/Cas9的两个R838S CORD6转基因小鼠系的基因编辑参数。我们将建立 最佳AAV衣壳/剂量、治疗持久性、治疗窗口和瞬时表达Cas9的可行性 系统。AIM 2将通过观察来评估基于AAV-CRISPR/Cas9的猕猴基因编辑的安全性/有效性 用于非目标编辑和评估AAV载体插入和CAS9长期表达的潜在影响。 我们还将评估编辑效率的地区差异，并进行剂量范围研究。目标3将 比较“反式”和“等位基因靶向”治疗CORD6的“基因敲除互补”方法。最优的 来自AIM 1的AAV衣壳蛋白和Cas9表达系统将用于检测GC2-/-小鼠的KO互补。 针对等位基因的编辑将在同时携带wt和R838S的人源化R838S CORD6小鼠身上进行。 含有GUCY2D的外显子13。方法将包括一部小说，工程Cas9，专门编辑 R838S突变等位基因，包含与突变等位基因不匹配但不包含wt等位基因的gRNA，或替代基因的利用 在突变体中发现了PAM位点，但没有wt等位基因。成功的方法将在猕猴身上进行测试 效率/安全。我们的发现将确定治疗AAV的最佳衣壳/剂量和治疗年龄- 基于CRISPR/Cas9的R838S GUCY2D体内干扰我们将建立安全概况，并建立区域 AAV-CRISPR/Cas9在具有基因组和临床相关性的物种中的基因编辑效率。在……里面 此外，我们将确定允许AAV-CRISPR/CAS9临床应用的材料和方法 CORD6以及其他主要遗传性视网膜疾病的治疗。

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.