Ultrasound-programmable gene editing in kidneys

项目摘要 超过60种遗传疾病与肾功能不全和单基因肾病有关 约占终末期肾病的15%。虽然CRISPR机器具有显著的治疗潜力， 为了纠正致病性肾突变，目前缺乏临床相关技术， 高效地将CRISPR构建体递送到肾脏，并在复杂的三维空间中精确控制基因编辑， 肾组织这个催化工具开发项目将建立肾允许和超声（US）敏感 氟纳米材料能够成像引导基因编辑核糖核蛋白（RNP）进入肾脏 小管这项技术将为整个领域建立一个强大的工具。这一战略的基础是我们的 发现了一个含氟化合物佐剂家族，或称“FTags”，它与蛋白质可逆地相互作用， 加载到声响应氟纳米乳液中并从声响应氟纳米乳液中进行US可编程递送， 破坏蛋白质的结构或生物活性。我们小组发表的研究表明， 可以使用临床诊断US在组织中进行外部引导和激活， 在体外和体内三维组织中时空控制递送功能蛋白。 利用这一进展，并受到最近发现的启发，可变形纳米颗粒可以被动地 积累在肾小管中，我们将设计这些氟纳米载体，将碱基编辑RNP递送到 肾小管影响单核苷酸多态性的基因修复;侧重于常染色体显性遗传 多囊性肾病（ADPKD）作为示例性应用。为了实现这一目标，在目标1中，我们执行严格的 Cas9：sgRNA RNP的方法学优化过程中的蛋白质-FTag相互作用的生物化学分析 基地编辑目标2将在离体猪中配对全组织荧光和B模式/多普勒US成像 肾脏，以机械地研究载体的肾脏定位，并优化同步 引导和声激活。从这些研究中获得的生物物理学见解将用于改进 纳米乳液制剂，以实现隔室特异性肾定位，并优化声激活 使用临床相关的US压力测量肾脏。在目标3中，初步体内研究将定量评估 使用RFP报告鼠模型在肾组织中进行US程序化基因编辑的效率。并行递送 使用Cas9碱基编辑器的测定将测试PKD 1中单核苷酸多态性修复的特异性和功效。 基因，以及由此产生的ADPKD表型模型中的囊肿形成的调节。将对结果进行基准测试 与目前的脂质体RNP递送系统进行比较以评价性能。这些高风险/高- 奖励研究将为临床相关的、成像引导的和定量的基因编辑提供基础。 利用便携式和非侵入性诊断US的人类肾脏疾病工具。我们还希望获得 可能允许未来开发针对其他遗传性肾脏疾病的疗法的机制见解， 以及探测肾功能的新型诊断模式和分子生物传感技术。

PROJECT SUMMARY Greater than 60 genetic diseases are linked to impared kidney function, and single-gene kidney disorders account for ~15% of end-stage renal disease. While CRISPR machinery holds significant therapeutic potential to correct pathogenic renal mutations, there is currently a lack of clinically-relevant technologies available to efficiently deliver CRISPR constructs to kidneys and precisely control gene editing in complex three-dimensional renal tissue. This catalytic tool development project will establish renal-permissive and ultrasound (US) sensitive fluorine nanomaterials capable of imaging-guided delivery of gene-editing ribonucleoproteins (RNPs) into renal tubules. This technology will establish a powerful tool for the entire field. Fundamental to this strategy is our discovery of a family of fluorochemical adjuvants, or ‘FTags’, that reversibly interface with proteins to enable their loading into, and US programmable delivery from, acousto-responsive fluorous nanoemulsions, without compromising the protein’s structure or bioactivity. Published studies from our group show that FTagged proteins can be externally guided and activated in tissues using clinical diagnostic US to provide on-demand and spatiotemporally controlled delivery of functional proteins in three-dimensional tissues in vitro and in vivo. Leveraging this advance, and inspired by recent findings that deformable nanoparticles can passively accumulate in kidney tubules, we will engineer these fluorous nanovectors to deliver base editing RNPs into kidney tubules to affect gene repair of single-nucleotide polymorphisms; focusing on autosomal dominant polycystic kidney disease (ADPKD) as an exemplary application. To achieve this, in aim 1 we perform rigorous biochemical analysis of protein-FTag interactions en route to its methodologic optimization for Cas9:sgRNA RNP base editors. Aim 2 will pair whole tissue fluorescence and B-mode/Doppler US imaging in ex vivo porcine kidneys to mechanistically study renal localization of the carrier, and optimize conditions for synchronous guidance and acoustic activation. Biophysical insights gained from these studies will be used to refine nanoemulsion formulation to achieve compartment-specific renal localization, and to optimize acoustic activation in kidneys using clinically relevant US pressures. In aim 3, pilot in vivo studies will quantitatively assess the efficiency of US-programmed gene editing in kidney tissue using an RFP-reporter murine model. Parallel delivery assays using Cas9 base editors will test specificity and efficacy of single-nucleotide polymorphism repair in PKD1 genes, and resultant modulation of cystogenesis in phenotypic models of ADPKD. Results will be benchmarked against current liposomal RNP delivery systems to evaluate performance. Success of these high-risk/high- reward studies will provide the foundation for a clinically-relevant, imaging-guided and quantitative gene-editing tool for human kidney disease that leverages portable and non-invasive diagnostic US. We also expect to gain mechanistic insights that may allow for future development of therapies against other genetic kidney disorders, as well as novel diagnostic modalities and molecular biosensing technologies to probe renal function.