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，以纠正或消除患者体内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。我们建议通过测试治疗策略的关键要素来为这种治疗奠定基础，这些治疗策略旨在将特定的变化引入小鼠视杆光感受器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视杆光感受器（视紫红质表达的地方）的死亡，但最终会破坏视杆细胞和视锥细胞，导致暗光和色觉的丧失。即使是一小部分杆状细胞的成功修饰也可能延长有用视力的寿命，这可能会提供显着的临床效益。为了实现这些策略，我们产生了一组小鼠品系，每个品系都携带修饰的人类视紫红质- gfp融合基因，以取代正常小鼠视紫红质基因，为基因修饰提供可见标记。我们的主要假设是，针对视紫红质基因特定位点的双链断裂（DSBs）可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（ZFNs）来切割视紫红质- gfp靶基因的特定位点，并将其包装成重组腺相关病毒（rAAV），我们将在小鼠眼睛视网膜下注射。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），可以实现有效的视紫红质基因切割和稳健的修复。我们提出了三个特定的目标，旨在测试各种策略，以处理导致RP的显性视紫红质突变。在Aim 1中，我们将测试HR有效校正视紫红质基因显性突变的策略。在Aim 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Aim 3中，我们将测试一种敲除视紫红质基因表达的一般策略，通过修饰基因来刺激无义介导的衰变，从而消除mRNA。总的来说，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，这些方法将为治疗已知引起RP的显性视紫红质突变提供一般方法。

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.