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

基本信息

批准号：
10198928
负责人：
Shannon Elizabeth Boye
金额：
$ 53.63万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10198928
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 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

项目摘要

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 GUCY2D的突变，编码视网膜鸟苷酸环化酶1（RETGC1）的基因是主要原因常染色体显性锥体杆营养不良。 GuCy2D-Cord6患者出现视力丧失，异常在前十年内，色觉，恐惧症，视野损失和黄斑萎缩。杆变性和外围视野损失随后。基因置换疗法临床应用的重大进展对于由于GUCY2D中隐性突变（LCA1）而引起的LCA，但需要另一种方法来处理cord6，功能突变的增益会导致功能障碍和营养不良。我们的初步数据表明 1）具有AAV- CRISPR/CAS9导致视网膜结构/功能的后续丧失，从而表现出降低的RETGC1 分别在猕猴和鼠标中表达2）“ trans中的敲除 +完成”方法（其中用“硬化”的GuCy2E进行互补，gucy2e grna识别）保留残留在小鼠中的功能，3）基于AAV-Crispr/Cas9的GUCY2D编辑在R838S转基因（TG）中是治疗性的 CORD6的小鼠模型和4）通过定向进化标识的CAS9变体具有等位基因特异性 Gucy2d（R838）。我们将在以下目标中基于这些结果。 AIM 1将建立最佳基于AAV-CRISPR/CAS9的基因编辑的参数在两个R838S CORD6 TG小鼠系中。我们将建立最佳的AAV CAPSID/剂量，治疗的耐用性，治疗窗口和瞬态CAS9表达的可行性系统。 AIM 2将通过查找基于AAV-CRISPR/CAS9的基因编辑的安全/功效来查看。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。我们还将评估在编辑效率方面的区域差异并进行剂量范围研究。目标3意志比较“击倒 +完成”与“等位基因靶向”的方法进行比较。最佳来自AIM 1的AAV CAPSID和CAS9表达系统将用于测试GC2 - / - 小鼠中的KO +完成。等位基因靶向的编辑将在人源化的R838S CORD6小鼠中进行，这些cord6小鼠同时携带WT和R838S- 包含gucy2d的外显子13。方法将包括一个小说，工程的CAS9，专门编辑 R838S突变等位基因，包含与突变体不匹配但不匹配WT等位基因的GRNA，或使用替代品在突变体中发现的PAM位点，但没有WT等位基因。成功的方法将在猕猴中测试效率/安全性。我们的发现将确定最佳的衣壳/剂量，以及治疗年龄的治疗年龄基于CRISPR/CAS9的R838S GUCY2D在体内的破坏。我们将建立安全性和地区 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。在此外，我们还将确定将允许AAV-CRISPR/CAS9临床应用的材料和方法 CORD6以及其他主要遗传残留疾病的疗法。