Rhodopsin Gene Correction and Gene Knockout in Rod Cells

描述(申请人提供)：开发精确修改人类基因组的方法，纠正或消除患者体内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元素来为此类治疗奠定基础，这些治疗策略旨在将特定的变化引入小鼠视杆感光细胞中的视紫红质基因。视紫红质基因的显性突变导致最常见的遗传性失明疾病--视网膜色素变性(RP)。这种进行性的神经退行性疾病始于视紫红质表达的视杆感光细胞的死亡，但最终破坏了视杆细胞和视锥细胞，导致暗光和色觉丧失。成功地改造即使是一小部分视杆细胞也可能延长有用视力的寿命，这可能会带来显著的临床益处。为了实施这些策略，我们建立了一组小鼠品系，每个品系携带一个修饰的人视紫红质-GFP融合基因来代替正常的小鼠视紫红质基因，为基因修饰提供可见的标记。我们的主要假设是，针对视紫红质基因特定位置的双链断裂(DSB)可以用于纠正或敲除显性视紫红质突变的功能。为此，我们构建了六个锌指核酸酶(ZFN)来切割视紫红质-GFP靶基因中的特定位点，并将ZFN包装成重组腺相关病毒(RAAV)，我们将其注射到小鼠的视网膜下。我们的初步实验表明，通过同源重组(HR)和非同源末端连接(NHEJ)，有效的视紫红质基因切割和强大的修复发生。我们提出了三个特定的目标，旨在测试不同的策略，以处理导致RP的显性视紫红质突变。在目标1中，我们将测试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，我们将测试一种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意义介导的衰退，从而消除信使核糖核酸。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，这些方法将提供治疗已知引起RP的显性视紫红质突变的一般方法。 与公共卫生相关：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，导致暗光和色觉丧失。我们建议将双链断裂传递到小鼠视紫红质基因的特定位置，以纠正或敲除显性视紫红质突变的功能。如果成功，这些方法将提供治疗已知导致视网膜色素变性的显性视紫红质突变的一般方法。

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.