Gene Editing and Silencing in Phototransduction

光转导中的基因编辑和沉默

基本信息

批准号：
10668139
负责人：
Stephen H Tsang
金额：
$ 40.61万
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 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10668139
关键词：
Ablation Acceleration Address Alleles American Biological Markers Biological Models Biological Process Blindness CRISPR therapeutics Cell Cycle Cell Line Cells Clinical Clustered Regularly Interspaced Short Palindromic Repeats Codon Nucleotides Complementary DNA DNA Sequence Disease Effectiveness Engineering Episome Future G2 Phase Gene Silencing Genes Guide RNA Health Heterozygote Human In Vitro Inherited Injections Left Lentivirus Vector Light Link Mediating Methods Modeling Modification Molecular Mus Mutation Nucleotides Organoids Pathology Patients Pharmaceutical Preparations Phototransduction Pre-Clinical Model Proteins RNA Interference RNA Sequences Research Resistance Retina Retinitis Pigmentosa Rhodopsin S phase Safety Single Nucleotide Polymorphism Specificity Supplementation System Techniques Technology Testing Therapeutic Time Toxic effect Transcription Initiation Site Transgenes Translational Research Translations Treatment Efficacy United States Validation Variant Viral Vector autosome bench-to-bedside translation cDNA delivery caucasian American clinically relevant comparative efficacy cost cost effective design dominant genetic mutation effective therapy efficacy evaluation efficacy validation gene augmentation therapy humanized mouse improved in vivo innovation mouse model mutant novel phase I/IIa trial pre-clinical precision medicine preclinical evaluation prime editing promoter repaired rho screening therapeutic genome editing tool treatment strategy

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 devised a novel method that capitalizes on single nucleotide polymorphism (SNP) heterozygosity to create a system applicable to multiple mutations, referred to hereafter as SNP editing. To allow for immediate translation of editing strategies targeting human- specific sequences, we rely on our recently engineered, humanized RHO adRP mouse model (RHOP347L). We aim to assess and compare our proposed SNP editing with our previously established ablate-and-replace strategy capable of overcoming challenges associated with the mutation-specific targeting of traditional CRISPR- Cas9 ablation in dominantly-inherited conditions. SNP editing, on the other hand, relies on the ablation of the mutant gene in an allele-specific manner via CRISPR-Cas9 targeting of heterozygous SNPs, leaving intact the WT allele to support normal function. By testing these techniques on our novel mouse line, we will validate our cost-effective SNP editing and ablate-and-replace strategies and evaluate the in vivo toxicity of SNP editing. Our critical and innovative modification promises to significantly reduce costs by overcoming the need to engineer, validate and obtain FDA-approval for all CRISPR components for each mutation (a major limitation of mutation- specific CRISPR/Cas9. 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.

项目摘要基因增强疗法无法解决常染色体主导（AD）疾病，其中有缺陷的蛋白质是表达。治疗这些突变，例如视网膜蛋白（Rho）相关的视网膜炎的主要形式 RP（ADRP），唯一的治疗方法是沉默（RNAi）或编辑突变等位基因。精确的CRISPR热编辑是预测是治疗几种主要疾病的最佳手段，但是分子方法正在利用 CRISPR的发展缓慢，因为同源指导的维修（HDR）仅在S和G2期间发生细胞周期的阶段。为了克服这些局限性，我们设计了一种新颖的方法，可以利用单核苷酸多态性（SNP）杂合性，以创建适用于多个突变的系统，称此之后是SNP编辑。为了立即翻译针对人类的编辑策略特定序列，我们依靠我们最近设计的人源化RHO ADRP小鼠模型（RHOP347L）。我们旨在评估和比较我们提出的SNP编辑与先前建立的烧蚀和替代能够克服与传统CRISPR的突变针对特定于突变有关的挑战的策略 CAS9在主要生存条件下消融。另一方面，SNP编辑依赖于消融通过杂合SNP的CRISPR-CAS9靶向，以等位基因特异性的方式突变基因，使得完整 WT等位基因支持正常功能。通过在我们的新颖鼠标线上测试这些技术，我们将验证我们的具有成本效益的SNP编辑和消融策略，并评估SNP编辑的体内毒性。我们的批判性和创新性修改有望通过克服设计需要大幅度降低成本为每个突变验证并获得所有CRISPR成分的FDA批准（突变的主要局限性特定CRISPR/CAS9。为了对该规格特定技术进行临床前评估，我们替换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，在我们的治疗编辑策略中使用的病毒矢量可以直接在没有ADRP试验中进一步的修改，从而避免了FDA障碍并加速了这项研究为临床环境的翻译。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A novel mutation and phenotypes in phosphodiesterase 6 deficiency.

DOI：
10.1016/j.ajo.2008.06.017
发表时间：
2008-11
期刊：
AMERICAN JOURNAL OF OPHTHALMOLOGY
影响因子：
4.2
作者：
Tsang, Stephen H.;Tsui, Irena;Chou, Chai Lin;Zernant, Jana;Haamer, Eneli;Iranmanesh, Reza;Tosi, Joaquin;Allikmets, Rando
通讯作者：
Allikmets, Rando

Retinal and choroidal angiogenesis: a review of new targets.

视网膜和脉络膜血管生成：新靶点综述。

DOI：
10.1186/s40942-017-0084-9
发表时间：
2017
期刊：
International journal of retina and vitreous
影响因子：
2.3
作者：
Cabral T;Mello LGM;Lima LH;Polido J;Regatieri CV;Belfort R Jr;Mahajan VB
通讯作者：
Mahajan VB

Calpain-5 gene expression in the mouse eye and brain.

DOI：
10.1186/s13104-017-2927-8
发表时间：
2017-11-21
期刊：
BMC research notes
影响因子：
1.8
作者：
Schaefer K;Mahajan M;Gore A;Tsang SH;Bassuk AG;Mahajan VB
通讯作者：
Mahajan VB

Bevacizumab Injection in Patients with Neovascular Age-Related Macular Degeneration Increases Angiogenic Biomarkers.

DOI：
10.1016/j.oret.2017.04.004
发表时间：
2018-01
期刊：
Ophthalmology. Retina
影响因子：
0
作者：
Cabral T;Lima LH;Mello LGM;Polido J;Correa ÉP;Oshima A;Duong J;Serracarbassa P;Regatieri CV;Mahajan VB;Belfort R Jr
通讯作者：
Belfort R Jr

Gain-of-function mutations in a member of the Src family kinases cause autoinflammatory bone disease in mice and humans

DOI：
10.1073/pnas.1819825116
发表时间：
2019-06-11
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
影响因子：
11.1
作者：
Abe, Koichiro;Cox, Allison;de Angelis, Martin Hrabe
通讯作者：
de Angelis, Martin Hrabe