CRISPR-Cas Editing as a Genetic Cure for Autosomal Dominant Polycystic Kidney Disease

项目总结 常染色体显性遗传性多囊肾病是世界上最常见的单基因疾病 影响了65万美国人。ADPKD是由PKD1或PKD2的突变引起的，这些突变引发了细胞增殖和 肾小管上皮细胞分泌液体进入囊内，双侧增大肾脏并导致 进行性肾功能减退。ADPKD无法治愈，大多数患者需要肾脏置换 在55岁之前以透析或移植的形式进行治疗以维持生命。基因治疗可以恢复 功能水平的多囊蛋白-1(PKD1)或多囊蛋白-2(PKD2)基因产物提供了一种很有前途的、创新的 ADPKD的治疗方法可能是彻底治愈的。然而，ADPKD的发展 由于缺乏有效的向肾小管上皮细胞输送DNA的载体，基因治疗受到了阻碍 以及一刀切的基因编辑解决方案。与我们的学术合作伙伴斯坦福大学合作， 我们开发了几种腺相关病毒(AAV)衣壳变异体，可以有效地转导肾脏 活着。该项目目标是对这些新型AAV衣壳进行引线优化，并使用它们来 建立CRISPR-Cas编辑的治疗ADPKD基因的可行性。我们的研究将集中在 PKD1是一种概念验证，因为PKD1占所有临床ADPKD病例和PKD1突变的85% 与PKD2相比，肾功能衰竭(更严重的疾病)的发病更早。在具体目标1中，我们 将使用碱基或基序编辑来灭活人和人PKD13‘UTR内的miR-17基序 体外培养小鼠ADPKD肾小管上皮细胞。临床前研究表明，移除或 抑制miR-17基序是一种逆转和减弱ADPKD的突变不可知论方法 进步。我们将评估几个CRISPR编辑程序，指导RNA和交付工具以实现最佳 编辑效率。我们将使用我们已发表的膀胱发生的三维细胞培养模型来评估表型。 以及CRISPR编辑引起的基因表达变化。在具体目标2中，我们将在体内进一步优化 通过测试肾动脉内注射的各种手术改进来交付我们的AAV衣壳变体 方法和测试新的启动子。这种交付方法是一种有吸引力的翻译方法，因为 血管可以通过传统的导尿术进入，以允许在人体内进行微创分娩 不需要手术。我们将使用从这些研究中确定的优化交付方法来交付 最有效地编辑从AIM#1到ADPKD小鼠模型的疗法。在这些研究结束时， 我们希望证明我们的ADPKD基因治疗方法的可行性，并确定一个领先的候选者来 临床前工业使能研究进展(研究性新药应用)。我们的第二期沙田公路 提案将专注于完成这些支持IND的研究，使我们能够提交IND申请和 启动临床试验。这种潜在的治疗ADPKD的方法解决了一个巨大的未得到满足的需求 鉴于缺乏根治性治疗选择，并有可能从根本上改变护理标准。

PROJECT SUMMARY Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic disorder in the world and affects 650,000 Americans. ADPKD is caused by mutations in Pkd1 or Pkd2 that trigger proliferation and fluid secretion by renal tubular epithelial cells into cysts, which bilaterally enlarge the kidney and lead to progressive loss of kidney function. There is no cure for ADPKD with most patients requiring renal replacement therapy in the form of dialysis or transplantation by age 55 years to sustain life. Gene therapy to restore functional levels of the polycystin-1 (Pkd1) or polycystin-2 (Pkd2) gene products offers a promising, innovative therapeutic approach for ADPKD that could serve as an outright cure. However, the development of ADPKD gene therapy has been hindered by the lack of an efficient DNA delivery vehicle to renal tubular epithelial cells and by a one-size-fits-all gene editing solution. In collaboration with our academic partner, Stanford University, we developed several adeno-associated virus (AAV) capsid variants that efficiently transduce the kidney in vivo. The goal of this project is to perform lead optimization for these novel AAV capsids and use them to establish feasibility for a curative ADPKD gene therapy with CRISPR-Cas editing. Our studies will focus on Pkd1 as a proof-of-concept because Pkd1 accounts for 85% of all clinical ADPKD cases and Pkd1 mutations are associated with an earlier onset of kidney failure (more severe disease) than Pkd2. In Specific Aim #1, we will use base or prime editing to inactivate the miR-17 motif within the 3’ UTR of Pkd1 in both human and mouse ADPKD renal tubular epithelial cells in vitro. Preclinical studies have shown that removing or suppressing this miR-17 motif is a mutation-agnostic approach that reverses and attenuates ADPKD progression. We will evaluate several CRISPR editors, guide RNAs, and delivery vehicles to achieve optimal editing efficiency. We will use our published 3-D cell culture model of cystogenesis to evaluate the phenotypic and gene expression changes induced by CRISPR editing. In Specific Aim #2, we will further optimize in vivo delivery of our AAV capsid variants by testing various surgical modifications to the intra-renal artery injection method and testing new promoters. This delivery method is an attractive translational approach because the vessels can be accessed via conventional catheterization to allow for minimally invasive delivery in humans without requiring surgery. We will use the optimized delivery method identified from these studies to deliver the most efficient editing therapeutics from Aim #1 to ADPKD mouse models. At the conclusion of these studies, we expect to demonstrate feasibility for our ADPKD gene therapy approach and identify a lead candidate to advance for preclinical IND-enabling studies (Investigational New Drug application). Our Phase II STTR proposal will focus on completing these IND-enabling studies to enable us to file our IND application and initiate clinical trials. This potentially, curative therapeutic approach for ADPKD addresses a large unmet need given the lack of curative treatment options and has the potential to fundamentally alter the standard of care.