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

摘要 编码视网膜鸟苷酸环化酶-1（retGC 1）的基因GUCY 2D的突变是导致视网膜病变的主要原因。 常染色体显性视锥杆细胞营养不良。GUCY 2D-CORD 6患者存在视力丧失，异常 色觉、恐光症、视野丧失和黄斑萎缩。视杆变性和 周边视野丧失。基因替代治疗临床应用的重大进展 由于GUCY 2D（LCA 1）中的隐性突变，已经进行了LCA，但需要不同的方法来 治疗CORD 6，其中功能获得突变导致功能障碍和营养不良。初步数据显示， 1)用AAV选择性和有效的体细胞敲除GUCY 2D和Gucy 2 e（鼠同源物）， CRISPR/Cas9导致视网膜结构/功能的随后丧失，这表现为retGC 1减少 分别在猕猴和小鼠中表达，2）“敲除+反式互补”方法（其中 用不被Gucy 2 e gRNA识别的“硬化”Gucy 2 e进行互补， 3）GUCY 2D的基于AAV-CRISPR/Cas9的编辑在R838 S转基因（Tg）小鼠中是治疗性的。 C 0 RD 6的小鼠模型，和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将建立最佳 在两个R838 S C 0 RD 6 Tg小鼠系中进行基于AAV-CRISPR/Cas9的基因编辑的参数。我们将建立 最佳AAV衣壳/剂量、治疗的持久性、治疗窗和瞬时Cas9表达的可行性 系统.目的2将通过观察基于AAV-CRISPR/Cas9的基因编辑在猕猴中的安全性/有效性， 用于脱靶编辑和评估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 比较用于治疗CORD 6的“敲除+反式互补”与“等位基因靶向”方法。最优 来自Aim 1的AAV衣壳和Cas9表达系统将用于测试GC 2-/-小鼠中的KO +互补。 等位基因靶向编辑将在携带wt和R838 S-C 0 RD两者的人源化R838 S C 0 RD 6小鼠中进行。 包含GUCY 2D的外显子13。方法将包括一种新的工程化Cas9，它专门编辑 R838 S突变体等位基因、含有与突变体但不含野生型等位基因错配的gRNA，或利用替代的 在突变体中发现PAM位点，而不是野生型等位基因。成功的方法将在猕猴中进行测试， 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。 R838 S GUCY 2D的基于CRISPR/Cas9的体内破坏。我们将建立安全档案， 在具有基因组和临床相关性的物种中通过AAV-CRISPR/Cas9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， CORD 6以及其他显性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.