Preclinical evaluation of a homing endonuclease gene therapy for adRP in models of P23H retinopathy.

P23H 视网膜病变模型中 adRP 归巢核酸内切酶基因疗法的临床前评估。

基本信息

批准号：
10587797
负责人：
RONALD G GREGG
金额：
$ 52.37万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10587797
关键词：
Accounting Affect Alleles Animal Euthanasia Animal Model Animals Behavior Biological Models Blindness Canis familiaris Cells Clinic Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Cone Development Disease Double Strand Break Repair Electroretinography Engineering Eye Eye diseases Family suidae Frequencies GRK1 gene Gene Expression Gene Mutation Genes Genome Genomics Goals Homing Human Human Genome Intervention Measures Methodology Modality Modeling Morphology Mus Mutate Mutation Natural History Night Blindness Nonhomologous DNA End Joining North America Organoids Outcome Patient Selection Patients Photoreceptors Pre-Clinical Model Proteins Rare Diseases Research Personnel Rest Retina Retinal Cone Retinal Degeneration Retinal Diseases Retinitis Pigmentosa Rhodopsin Rod Safety Selection Criteria Site Specificity Structure Technology Therapeutic Time Transgenic Organisms Vertebrate Photoreceptors Viral Packaging Visual alternative treatment autosome deep sequencing efficacy evaluation efficacy testing endonuclease experimental study functional decline gene therapy genome editing human model humanized mouse in silico individualized medicine insertion/deletion mutation interest mouse model mutant neglect nonhuman primate nuclease off-target site optimal treatments porcine model pre-clinical preclinical evaluation retinal rods safety assessment subretinal injection timeline tool zinc finger nuclease

项目摘要

Autosomal dominant Retinitis pigmentosa (adRP) is a blinding eye disease that results in loss of rods and eventually cone photoreceptors. A large fraction of adRP is caused by mutations in the rhodopsin (Rho) gene, and the most common mutation in North America causes a P23H mutation in rhodopsin. Patients typically present with mid- to late-stage disease when many rod photoreceptors already have degenerated. We will evaluate genome editing using a meganuclease genome editing tool (Rho1-2) that is specific for the sequence causing the P23H mutation in several preclinical animal models of P23H adRP. We will deliver AAV5- GRK1-Rho1-2 via subretinal injection to infect photoreceptors. We will measure efficacy, the ability of Rho1-2 to alter rod degeneration and functional decline. We will quantitatively determine Rho1-2 efficiency, the proportion of P23H alleles with insertions/delections (indels), specificity the proportion of P23H to WT alleles with indels, and its safety, the absence of indels in other genes throughout the human genome (off target editing). In Pig P23H adRP models we will evaluate Rho1-2 editing efficacy because of the similarities between pigs and humans in eye size and retinal structure and because the pig has a cone rich visual streak. We will use the same non-invasive tools used in the clinic to measure retinal function (full field electroretinograms) and structure, (spectral domain OCT). We will use two existing transgenic pig lines that harbor the entire P23H hRHO gene, but differ in their time course, resulting in a fast or slow loss of rods and rod function. We will match treatment time with the natural history of rod structural and functional decline, compare Rho1-2 editing efficacy in early-, mid- and late-stage P23H adRP and define its temporal treatment window. In Humanized adRP mouse models we will evaluate Rho1-2 editing efficiency and specificity because they contain WT and P23H human alleles in the appropriate genomic context. In human retinal organoids we will evaluate Rho1-2 editing safety (off-target cutting throughout the genome) because they contain human photoreceptors as well as the rest of the human genome in a relevant context.. Taken together the results of our experiments will determine if Rho1-2 should move forward for eventual use in human clinical trials and will define endpoints for those trials.

常染色体显性遗传性色素性视网膜炎 (adRP) 是一种致盲性眼病，会导致视杆细胞和视杆细胞丧失。最终形成视锥细胞光感受器。 adRP 的很大一部分是由视紫红质 (Rho) 基因突变引起的，北美最常见的突变会导致视紫质发生 P23H 突变。患者通常当许多视杆光感受器已经退化时，就会出现中晚期疾病。我们将使用大范围核酸酶基因组编辑工具 (Rho1-2) 来评估基因组编辑，该工具专门针对在 P23H adRP 的几种临床前动物模型中引起 P23H 突变的序列。我们将提供 AAV5- GRK1-Rho1-2通过视网膜下注射来感染光感受器。我们将测量功效，即 Rho1-2 的能力改变视杆细胞退化和功能衰退。我们将定量测定Rho1-2的效率、比例具有插入/缺失 (indels) 的 P23H 等位基因的数量，特异性 P23H 与具有 indels 的 WT 等位基因的比例，及其安全性，整个人类基因组中其他基因中不存在插入缺失（脱靶编辑）。在猪 P23H adRP 模型中，我们将评估 Rho1-2 编辑功效，因为猪和猪之间的相似性猪的眼睛大小和视网膜结构与人类相似，而且猪具有丰富的视锥细胞视觉条纹。我们将使用相同的临床上用于测量视网膜功能（全视野视网膜电图）和结构的非侵入性工具，（谱域 OCT）。我们将使用两个现有的含有整个 P23H hRHO 基因的转基因猪品系，但它们的时间进程不同，导致视杆细胞和视杆细胞功能的快速或缓慢丧失。我们会配合治疗时间与视杆细胞结构和功能衰退的自然史，比较早期的 Rho1-2 编辑功效，中期和晚期 P23H adRP 并定义其时间治疗窗口。在人源化 adRP 小鼠模型中我们将评估 Rho1-2 编辑效率和特异性，因为它们包含 WT 和 P23H 人类等位基因适当的基因组背景。在人类视网膜类器官中，我们将评估 Rho1-2 编辑安全性（脱靶切割整个基因组），因为它们含有人类光感受器以及人类的其余部分相关背景下的基因组.. 总而言之，我们的实验结果将决定 Rho1-2 是否应进一步用于最终用途人体临床试验并将确定这些试验的终点。