Therapeutic genome editing to treat Best disease

项目摘要 进行性视网膜营养不良，包括视网膜色素变性和黄斑变性是常见的 由显性负突变引起的，在这种突变中，疾病可以通过沉默突变等位基因来治愈。 直到最近，这项任务还是不可能完成的。然而，CRISPR/Cas9基因组编辑的出现提高了 通过选择性地灭活显性致病等位基因来治愈疾病的令人兴奋的可能性，而 保留正常的等位基因。作为概念验证，我们将重点介绍Bestrophin，这是一种由 形成钙激活氯离子通道的Best1基因在视网膜色素上皮中的表达 (RPE)。最常见的与BEST1相关的疾病被称为“最佳疾病”(BD)，由&gt；200引起 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 视网膜脂肪堆积，黄斑萎缩。BD患者的诱导多能干细胞(IPSCs) 发展为有疾病表型的RPE，如氯离子通道电导异常和贝斯特芬 本地化错误。用双切割Cas9进行DNA切除是非常有效的，并且通过使用GUIDE靶向Cas9 RNAs(GRNA)在与疾病突变相同的等位基因上的常见多态，我们建议 以消除疾病蛋白质。通过针对常见的多态，我们希望治疗大多数BD 只有几对gRNA的患者。尽管BD的治疗性编辑前景看好，但许多令人望而生畏的 挑战依然存在。1.我们怎样才能确信灭活疾病等位基因就能治愈疾病？ 我们如何有效地在20-30 kb的基因组窗口内识别可用于 两个Cas9切除了一个等位基因，而另一个等位基因保持不变？(图5)。3.理想的方法是什么？ 将编辑DNA/RNA/蛋白质引入RPE以进行高效和特定的编辑？4.我们如何评估 针对不同SNP和将Cas9插入到细胞中的不同方法进行目标编辑？5.我们能否将 使用其他形式的Cas9靶向DNA损伤？我们将系统地解决这些问题 结合生物信息学、细胞生物学和生物工程的这些目标： 目的1.确定等位基因特异性编辑的有效性和拯救BD相关的RPE表型 使用荧光报告程序iPSCs 目的2.检测10例BD患者RPE中疾病等位基因失活的等位基因特异性gRNAs 目标3.确定最健壮的等位基因特定编辑的保真度 BD是治疗编辑的肥沃试验田，因为RPE可以很容易地从IPSCs中衍生出来，用于体外实验 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负性遗传病，其中选择性地移除单个 等位基因可能具有治疗作用。例如，显性光感受器阴性疾病(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.