Rhodopsin Gene Correction and Gene Knockout in Rod Cells

描述（由申请人提供）：开发精确修饰人类基因组的方法，以纠正或消除患者中受其影响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。我们建议通过测试旨在将特定变化引入小鼠视杆细胞视紫红质基因的治疗策略的关键要素，为此类治疗奠定基础。视紫红质基因的显性突变导致最常见的遗传性致盲疾病视网膜色素变性（RP）的最常见形式。这种进行性神经退行性疾病开始于视杆细胞的死亡，视紫红质在视杆细胞中表达，但最终破坏视杆细胞和视锥细胞，导致昏暗光和色觉的丧失。即使是一小部分视杆细胞的成功修饰也可能延长有用视力的寿命，这可以提供显著的临床益处。为了追求这些策略，我们已经产生了一组小鼠品系，每个小鼠品系携带修饰的人视紫红质-GFP融合基因代替正常小鼠视紫红质基因，以提供用于基因修饰的可见标记。我们的总体假设是，双链断裂（DSB）针对特定位点的视紫红质基因可以用来纠正或敲除显性视紫红质突变的功能。为此，我们已经构建了六个锌指核酸酶（ZFN）切割视紫红质-GFP靶基因中的特定位点，并将ZFN包装成重组腺相关病毒（rAAV），我们将其注射到视网膜下小鼠眼睛中。我们的初步实验表明，有效的视紫红质基因切割和强大的修复发生同源重组（HR）和非同源末端连接（NHEJ）。我们提出了三个具体的目标，旨在测试各种策略，用于处理显性视紫红质突变，导致RP。在目标1中，我们将测试通过HR有效校正视紫红质基因中的显性突变的策略。在目标2中，我们将测试通过NHEJ有效敲除视紫红质基因中的显性突变的策略。在目标3中，我们将测试通过修饰基因以刺激无义介导的衰变，从而消除mRNA来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视杆细胞功能并最终导致其死亡的显性突变的一般治疗策略的关键要素。如果成功的话，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。 公共卫生相关性：视紫红质基因的显性突变导致最常见的遗传性致盲疾病视网膜色素变性，这是一种进行性神经退行性疾病，最终破坏视杆细胞和视锥细胞，导致昏暗光和色觉丧失。我们建议在小鼠视杆细胞中的视紫红质基因的特定位点上进行双链断裂，以纠正或敲除显性视紫红质突变的功能。如果成功的话，这些方法将为治疗已知引起视网膜色素变性的显性视紫红质突变提供通用方法。

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. PUBLIC HEALTH RELEVANCE: Dominant mutations in the rhodopsin gene cause the most common form of the most common hereditary blinding disease, retinitis pigmentosa, which is a progressive neurodegenerative disorder that ultimately destroys rod and cone cells, leading to loss of both dim-light and color vision. We propose to deliver double-strand breaks to specific sites in the rhodopsin gene in mouse rods cells in order to correct or knockout the function of dominant rhodopsin mutations. If successful, these approaches would provide general methods for treating the dominant rhodopsin mutations that are known to cause retinitis pigmentosa.