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）：开发用于精确修饰人类基因组的方法，以纠正或消除患者影响的细胞中的疾病等位基因，将对我们对许多人类疾病的治疗方法产生巨大影响。我们建议通过测试旨在将特定变化引入小鼠Rod感光细胞中的视紫红质基因引入特定变化的治疗策略的关键要素，从而为这种疗法奠定基础。视紫红质基因中的显性突变引起最常见的遗传性盲疾病，视网膜炎色素炎（RP）。这种进行性神经退行性疾病始于杆光感受器的死亡，在该杆状蛋白被表达，但最终会破坏杆和锥细胞，从而导致昏暗的光和色觉丧失。即使是一小部分杆细胞的成功修饰可能会延长有用视力的寿命，这可能会提供重大的临床益处。为了采用这些策略，我们生成了一条固定的小鼠系，每种小鼠系列携带经过修改的人类Rhopopsin-GFP融合基因代替正常小鼠视opsin基因，以提供可见的标记以进行基因修饰。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）可用于纠正或敲除优势紫红蛋白突变的功能。为此，我们已经构建了六个锌指核酸酶（ZFN），以切开视opsin-GFP靶基因的特定位点，并将ZFN包装到重组腺相关病毒（RAAV）中，我们将在小鼠眼中缩放地注射。我们的最初实验表明，通过同源重组（HR）和非同源末端连接（NHEJ）进行有效的视紫红质裂解和健壮的修复。我们提出了三个特定的目标，旨在测试各种策略，以应对引起RP的主要视紫红质突变。在AIM 1中，我们将通过HR测试策略，以有效校正Rhopopsin基因中的显性突变。在AIM 2中，我们将测试NHEJ在Rhodopsin基因中有效淘汰显性突变的策略。在AIM 3中，我们将通过修饰基因以刺激废话介导的衰减，从而消除mRNA来测试敲除视紫红质基因表达的一般策略。总体而言，拟议的研究将测试旨在纠正或消除损害杆细胞功能并最终导致死亡的普通治疗策略的关键要素。如果成功，这些方法将为治疗已知引起RP的主要视紫红质突变提供一般方法。