Ultrasound-programmable gene editing in kidneys

项目总结 60多种遗传性疾病与肾功能受损和单基因肾脏疾病有关 约占终末期肾病的15%。虽然CRISPR机器拥有巨大的治疗潜力 为了纠正致病肾脏突变，目前缺乏与临床相关的技术来 高效地将CRISPR构建物输送到肾脏，并在复杂的三维空间中精确控制基因编辑 肾组织。该催化工具开发项目将建立肾许可和超声(US)敏感 能够在成像引导下将基因编辑核糖核蛋白(RNPs)输送到肾脏的氟纳米材料 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 发现一种氟化学佐剂家族，或称“FTags”，它可与蛋白质可逆地相互作用，使其能够 加载到声响应型氟纳米乳液中并从其进行美国可编程递送，没有 破坏蛋白质的结构或生物活性。我们小组发表的研究表明，FTTag蛋白 可以使用临床诊断超声在组织中进行外部引导和激活，以提供按需和 体内和体外三维组织中功能蛋白的时空控制传递。 利用这一进展，并受到最近的发现的启发，可变形的纳米颗粒可以被动地 在肾小管中积聚，我们将对这些氟纳米载体进行改造，将碱基编辑RNP输送到 肾小管影响单核苷酸多态的基因修复；关注常染色体显性 多囊肾病(ADPKD)是一个典型的应用。为了实现这一点，在目标1中，我们严格执行 对Cas9：sgRNA RNP进行方法学优化的蛋白质-FTag相互作用的生化分析 基础编辑。AIM 2将在体外配对猪的全组织荧光和B型/多普勒超声成像 从力学角度研究肾脏载体的肾脏定位，并优化同步条件 引导和声学激活。从这些研究中获得的生物物理见解将被用来改进 纳米乳剂配方可实现特定隔室的肾脏定位，并优化声学激活 在肾脏中使用临床相关的美国压力。在目标3中，体内试验研究将定量评估 使用RFP报告鼠模型的肾脏组织中美国程序化基因编辑的效率。并行交付 使用Cas9碱基编辑的分析将测试PKD1中单核苷酸多态修复的特异性和有效性 基因，以及在ADPKD表型模型中对囊变的结果调控。将对结果进行基准测试 对照目前的脂质体RNP递送系统来评估性能。这些高风险/高风险项目的成功 奖励研究将为临床相关、影像指导和定量基因编辑提供基础 人类肾脏疾病的工具，利用便携式和非侵入性诊断US。我们还预计将获得 机械性的洞察力，可能会允许未来开发针对其他遗传性肾脏疾病的治疗方法， 以及新的诊断模式和分子生物传感技术来探测肾功能。

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.