Therapeutic genome editing to treat Best disease

项目摘要 进行性视网膜营养不良，包括色素性视网膜炎和黄斑变性， 由显性负突变引起，这种疾病可以通过沉默突变等位基因来治愈。 直到最近，这项任务都是不可能完成的。然而，CRISPR/Cas9基因组编辑的出现提高了 通过选择性地使主要致病等位基因失活来治愈疾病的可能性令人兴奋， 保留正常的等位基因作为一个概念验证，我们将重点放在bestrophin上，这是一种由 在视网膜色素上皮细胞中表达的形成钙激活氯离子通道的BEST 1基因 （RPE）。最常见的BEST 1相关疾病被称为“最佳疾病”（BD），其由>200 不同的显性负性蛋白编码突变导致氯离子通道功能缺陷，亚 视网膜脂质积聚和黄斑萎缩。来自BD患者的诱导多能干细胞（iPSC） 发展成具有疾病表型的RPE，如异常氯通道传导和雌激素样蛋白 错误定位用双切割Cas9进行DNA切除是非常有效的，并且通过用引导物靶向Cas9， 我们提出，将RNA（gRNA）与疾病突变相同等位基因上的常见多态性（顺式）联系起来， 以消除疾病蛋白质。通过针对常见的多态性，我们希望治疗大多数BD 只有几对gRNA的患者。虽然BD的治疗编辑是有希望的，但许多令人生畏的 挑战依然存在。1.我们如何能确信，疾病等位基因的失活将治愈疾病？2. 我们如何在20-30 kb的基因组窗口内有效地识别cis中的多态性， 一个等位基因的双Cas9切除，而另一个等位基因保持完整？(Fig. 5）。3.什么是理想的方法， 将编辑DNA/RNA/蛋白质引入RPE以进行高效和特异性编辑？4.我们如何评估- 针对不同SNP的靶向编辑和将Cas9插入细胞的不同方法？5.我们能不能尽量减少- 使用Cas9的替代形式靶向DNA损伤？我们将系统地解决这些问题中的每一个 结合生物信息学、细胞生物学和生物工程学，其目标是： 目标1.确定等位基因特异性编辑的功效和BD相关RPE表型的拯救 使用荧光报告基因iPSC 目标2.测试来自10名BD患者的RPE中疾病等位基因的失活的等位基因特异性gRNA 目标3.确定最稳健的等位基因特异性编辑的保真度 BD是用于治疗性编辑的肥沃的试验场，因为RPE可以容易地衍生自iPSC用于体外培养。 研究，并且已经是细胞和基因治疗试验的目标。我们的研究也有更大的影响：我们的 方法适用于任何显性阴性遗传疾病，其中选择性去除单个 等位基因可能有治疗作用。例如，光感受器的显性负性疾病（例如，RHO），听觉 细胞、神经系统和肌肉是未来的潜在目标。

PROJECT ABSTRACT Progressive retinal dystrophies, including retinitis pigmentosa and macular degeneration, are frequently caused by dominant negative mutations in which the disease could be cured by the silencing the mutant allele. Until recently, this task was impossible. However, the advent of CRISPR/Cas9 genome editing raises the exciting possibility of curing the disease by selectively inactivating the dominant disease-causing allele, while preserving the normal allele. As a proof-of-concept, we will focus on bestrophin, a protein encoded by the BEST1 gene that forms a calcium-activated chloride channel expressed in the retinal pigment epithelium (RPE). The most common BEST1-related disease is called “Best disease” (BD), which is caused by >200 different dominant-negative protein coding mutations that result in defective chloride channel function, sub- retinal lipid accumulation, and macular atrophy. Induced pluripotent stem cells (iPSCs) from BD patients develop into RPE with disease phenotypes, such as abnormal chloride channel conductance and bestrophin mislocalization. DNA excision with dual cutting Cas9 is remarkably efficient, and by targeting Cas9 with guide RNAs (gRNA) to common polymorphisms on the same allele as (in cis with) the disease mutation, we propose to eliminate the disease protein. By targeting common polymorphisms, we hope to treat a majority of BD patients with just a few gRNA pairs. Although therapeutic editing for BD is promising, many daunting challenges remain. 1. How can we be confident that inactivation of the disease allele will cure the disease? 2. How do we efficiently identify polymorphisms in cis within a 20-30 kb genomic window that can be used for dual Cas9 excision of one allele, while leaving the other allele intact? (Fig. 5). 3. What are the ideal methods to introduce editing DNA/RNA/proteins into RPE for efficient and specific editing? 4. How do we assess the off- target editing for different SNPs and different methods of inserting Cas9 into cells? 5. Can we minimize off- target DNA damage using alternative forms of Cas9? We will systematically address each of these questions with a combination of bioinformatics, cell biology, and bioengineering with these aims: Aim 1. Determine the efficacy of allele-specific editing and the rescue of BD-associated RPE phenotypes using fluorescent reporter iPSCs Aim 2. Test allele-specific gRNAs for inactivation of disease alleles in RPE from 10 BD patients Aim 3. Determine the fidelity of the most robust allele-specific editing BD is a fertile testing ground for therapeutic editing, since RPE can readily be derived from iPSCs for in vitro studies, and is already the target of cell and gene therapy trials. Our studies also have larger implications: our methods are applicable to any dominant negative genetic disease where the selective removal of a single allele could be therapeutic. For instance, dominant negative disease of photoreceptors (e.g., RHO), auditory cells, nervous system, and muscle, are potential targets in the future.