Rhodopsin Gene Correction and Gene Knockout in Rod Cells

视杆细胞中的视紫红质基因校正和基因敲除

基本信息

批准号：
8655854
负责人：
JOHN H WILSON
金额：
$ 38.34万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-03-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8655854
关键词：
Affect Alleles Biological Assay Cells Cessation of life Cleaved cell Clinical Color Visions DNA Detection Disease Double Strand Break Repair Elements Engineering Exons Eye Fluorescence Foundations Gene Expression Gene Targeting Gene-Modified Genes Genomics Human Human Genome Individual Inherited Introns Knock-out Light Messenger RNA Methods Modification Mus Mutation Neurodegenerative Disorders Nonsense Codon Nonsense-Mediated Decay Patients Photoreceptors Population Reading Frames Reagent Recombinant adeno-associated virus (rAAV)Resistance Retina Retinal Cone Retinitis Pigmentosa Rhodopsin Signal Transduction Site Techniques Testing Vision Zinc Fingers adeno-associated viral vector design fusion gene gene correction homologous recombination human disease knockout gene method development mouse genome mutant nuclease prevent public health relevance repaired research study retinal rods subretinal injection treatment strategy

项目摘要

DESCRIPTION (provided by applicant): Development of methods for precise modification of the human genome, to correct or eliminate disease alleles in the very cells they affect in patients, would have enormous impact on our approach to the treatment of many human diseases. We propose to lay the foundation for such therapies by testing key elements of treatment strategies designed to introduce specific changes into the rhodopsin gene in rod photoreceptor cells in mice. Dominant mutations in the rhodopsin gene cause the most common form of the most common hereditary blinding disease, retinitis pigmentosa (RP). This progressive neurodegenerative disorder begins with the death of rod photoreceptors, where rhodopsin is expressed, but ultimately destroys rod and cone cells, leading to loss of both dim-light and color vision. Successful modification of even a fraction of rod cells would likely extend the lifetime of useful vision, which could provide significant clinical benefit. To pursue these strategies, we have generated a set mouse lines that each carry a modified human rhodopsin-GFP fusion gene in place of the normal mouse rhodopsin gene to provide visible markers for gene modification. Our overarching hypothesis is that double-strand breaks (DSBs) targeted to specific sites in the rhodopsin gene can be used to correct or knockout the function of dominant rhodopsin mutations. To that end, we have constructed six zinc-finger nucleases (ZFNs) to cleave specific sites in the rhodopsin-GFP target genes, and packaged the ZFNs into recombinant adeno-associated virus (rAAV), which we will inject subretinally in mouse eyes. Our initial experiments demonstrate that efficient rhodopsin-gene cleavage and robust repair occur by both homologous recombination (HR) and nonhomologous end joining (NHEJ). We propose three Specific Aims designed to test various strategies for dealing with the dominant rhodopsin mutations that cause RP. In Aim 1, we will test strategies for efficient correction of dominant mutations in the rhodopsin gene by HR. In Aim 2, we will test strategies to efficiently knock out dominant mutations in the rhodopsin gene by NHEJ. In Aim 3, we will test a general strategy for knocking out rhodopsin gene expression by modifying the gene to stimulate nonsense-mediated decay, thereby eliminating the mRNA. Overall, the proposed studies will test key elements of general treatment strategies designed to correct or eliminate dominant mutations that compromise the function of rod cells and ultimately cause their death. If successful, these approaches would provide general methods for treating the dominant rhodopsin mutations known to cause RP.

描述（由申请人提供）：开发精确修饰人类基因组的方法，以纠正或消除它们影响患者的细胞中的疾病等位基因，将对我们治疗许多人类疾病的方法产生巨大影响。我们建议通过测试治疗策略的关键要素，为此类疗法奠定基础，这些策略旨在将特定变化引入小鼠视杆感光细胞的视紫红质基因。视紫红质基因的显性突变会导致最常见的遗传性致盲疾病——色素性视网膜炎 (RP)。这种进行性神经退行性疾病始于表达视紫质的视杆细胞死亡，但最终破坏了视杆细胞和视锥细胞，导致弱光和色觉丧失。即使是一小部分视杆细胞的成功修饰也可能会延长有用视力的寿命，这可能会带来显着的临床益处。为了实现这些策略，我们生成了一组小鼠品系，每个品系都携带修饰的人视紫红质-GFP融合基因来代替正常的小鼠视紫红质基因，从而为基因修饰提供可见的标记。我们的总体假设是，针对视紫红质基因中特定位点的双链断裂（DSB）可用于纠正或敲除视紫红质显性突变的功能。为此，我们构建了六种锌指核酸酶（ZFN）来切割视紫红质-GFP靶基因中的特定位点，并将ZFN包装到重组腺相关病毒（rAAV）中，我们将其注射到小鼠眼睛视网膜下。我们的初步实验表明，同源重组（HR）和非同源末端连接（NHEJ）可以实现有效的视紫红质基因切割和稳健修复。我们提出了三个具体目标，旨在测试处理导致 RP 的显性视紫红质突变的各种策略。在目标 1 中，我们将测试通过 HR 有效纠正视紫红质基因显性突变的策略。在目标 2 中，我们将测试通过 NHEJ 有效敲除视紫红质基因显性突变的策略。在目标 3 中，我们将测试敲除视紫红质基因表达的一般策略，方法是修改基因以刺激无义介导的衰变，从而消除 mRNA。总体而言，拟议的研究将测试一般治疗策略的关键要素，这些策略旨在纠正或消除损害视杆细胞功能并最终导致其死亡的显性突变。如果成功，这些方法将为治疗已知导致 RP 的显性视紫红质突变提供通用方法。