Gene Silencing and Gene Editing in Phototransduction

光转导中的基因沉默和基因编辑

基本信息

批准号：
10246786
负责人：
Stephen H Tsang
金额：
$ 39.04万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10246786
关键词：
Ablation Address Adverse effects Affect Alleles American Base Sequence Biological Models CRISPR interference CRISPR therapeutics CRISPR/Cas technology Cell Culture Techniques Cell Cycle Clinical Clustered Regularly Interspaced Short Palindromic Repeats Codon Nucleotides DNA Sequence Defect Development Disease FDA approved Feasibility Studies Funding Future G2 Phase Gene Expression Regulation Gene Silencing Gene therapy trial Genes Genomics Goals Guide RNA Human Inherited Investigational New Drug Application Knock-in Link Longevity Mediating Methods Modeling Modification Molecular Mus Mutation Pathology Pharmaceutical Preparations Phase Phenocopy Phototransduction Pre-Clinical Model Preclinical Testing Proteins RNA Interference RNA Interference Therapy Research Resources Retina Retinitis Pigmentosa Rhodopsin S Phase Safety Supplementation Technology Testing Therapeutic Toxic effect Transgenic Mice Translational Research Viral Vector adeno-associated viral vector base blind clinically relevant experimental study gain of function mutation gene augmentation therapy gene replacement gene therapy humanized mouse in vivo Model individualized medicine innovation loss of function mouse model mutant novel pre-clinical precision medicine preclinical evaluation repaired restoration therapeutic gene therapeutic genome editing treatment response vector

项目摘要

Project Summary Gene-augmentation therapy cannot address autosomal-dominant (ad) disorders, in which a defective protein is expressed. To treat these mutations, such as the dominant form of rhodopsin (RHO)-related retinitis pigmentosa RP (adRP), the only cure is to silence (RNAi) or edit the mutant allele. Precise CRISPR therapeutic editing is predicted to be the best means of treating several dominant disorders, but molecular methods harnessing CRISPR have been slow to develop because homology-directed repair (HDR) occurs only during the S and G2 phases of the cell cycle. To overcome these limitations, we have established a versatile editing technology that uses 2 guide RNAs (gRNAs) to target all 150 dominant RHO mutations at any phase of the cell cycle. To conduct preclinical evaluation of this species-specific technology, we have replaced the naïve mouse chromosomal Rho with human RHO mutant sequences (humanized RHO). If successful, the viral vectors used in our therapeutic editing strategy could be directly reused in human trials for adRP without further modification, thus avoiding FDA hurdles and accelerating translation of this research to a clinical setting. Our preclinical adRP model could also be useful for testing of future gene- and drug-based therapies. The proposed research is innovative, as it introduces new methods and model systems as the initial steps of a precision medicine approach toward developing adRP treatment. These methods are also potentially adaptable to therapies based on targeting DNA sequences of genes linked to other dominant disorders.

项目摘要基因增强疗法无法解决常染色体主导（AD）疾病，其中表达有缺陷的蛋白质。处理这些突变，例如视紫红质的主要形式（RHO）相关的色素性视网膜炎RP（ADRP），唯一的治愈方法是沉默（RNAi）或编辑突变体等位基因。预计精确的CRISPR热编辑将是治疗几种的最佳方法主导疾病，但是利用CRISPR的分子方法发展缓慢因为同源性修复（HDR）仅在细胞的S和G2阶段发生循环。为了克服这些限制，我们建立了一种使用的多功能编辑技术 2指导RNA（GRNA）在细胞周期的任何阶段靶向所有150个主要的RHO突变。为了对该物种特定技术进行临床前评估，我们取代了幼稚的小鼠染色体Rho具有人类Rho突变序列（人源化RHO）。如果成功，我们的治疗编辑策略中使用的病毒矢量可以直接在人类试验中重复使用对于ADRP而没有进一步修改，因此避免了FDA障碍和加速翻译这项研究到临床环境。我们的临床前ADRP模型也可用于测试未来的基因和基于药物的疗法。拟议的研究是创新的，因为它介绍了新方法和模型系统作为精确医学方法的初始步骤开发ADRP治疗。这些方法也可能适应基于靶向与其他显性疾病相关的基因的DNA序列。