Ultrasound-programmable gene editing in kidneys

肾脏超声可编程基因编辑

基本信息

批准号：
10370610
负责人：
Scott Hammond Medina
金额：
$ 17.81万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-24 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10370610
关键词：
3-Dimensional Acoustics Adjuvant Adopted Affect Autosomal Dominant Polycystic Kidney Benchmarking Biochemical Biological Biological Assay Biophysics Biosensing Techniques CRISPR/Cas technology Cell Line Cells Chemicals Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complex Contrast Media Cyst DNA Development Doppler Ultrasound Emulsions End stage renal failure Engineering Family Family suidae Fluorescence Fluorine Fluorocarbons Follow-Up Studies Formulation Foundations Future Gene Delivery Genes Genetic Genetic Diseases Genetic Polymorphism Genetic Recombination Genome Hematological Disease Human Image In Situ In Vitro Investigation Kidney Kidney Diseases Laboratories Link Liposomes Mediating Messenger RNA Methodology Methods Modality Modeling Modification Molecular Monitor Multimodal Imaging Mutation Nanotechnology Nephrons Nucleotides Other Genetics PKD1 gene Particle Size Pathogenicity Pathologic Performance Phenotype Precision therapeutics Property Protein Analysis Proteins Publishing Recombinants Renal Tissue Renal function Renal tubule structure Reporter Reporting Ribonucleoproteins Route Sepharose Single Nucleotide Polymorphism Specificity Structure Structure-Activity Relationship Surface Tension System Techniques Technology Testing Therapeutic Time Tissues Toxic effect Transfection Transgenic Mice Ultrasonography Urologic Diseases Viral Visualization Work base cell type clinical diagnostics clinically relevant disease-causing mutation ex vivo imaging gene repair gene therapy high reward high risk high throughput screening image guided immunogenicity in vitro Assay in vivo insight molecular phenotype mouse model mutant nanocarrier nanoemulsion nanomaterials nanoparticle nanovector noninvasive diagnosis novel diagnostics particle portability pressure programs protein structure repaired response spatiotemporal success technology development therapy development tool tool development vaporization vector

项目摘要

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.

项目概要超过 60 种遗传病与肾功能受损和单基因肾病有关约占终末期肾病的 15%，而 CRISPR 机制具有巨大的治疗潜力。为了纠正致病性肾脏突变，目前缺乏临床相关技术高效地将 CRISPR 构建体递送至肾脏并在复杂的三维空间中精确控制基因编辑该催化工具开发项目将建立肾脏许可性和超声（美国）敏感性。氟纳米材料能够通过成像引导将基因编辑核糖核蛋白（RNP）输送到肾脏中这项技术将为整个领域建立一个强大的工具，这是我们这一战略的基础。发现了一系列含氟化合物佐剂或“FTag”，它们与蛋白质可逆地相互作用，使其能够加载到声响应氟纳米乳液中并进行美国可编程输送，无需我们小组发表的研究表明，FT 标记的蛋白质会损害蛋白质的结构或生物活性。可以使用临床诊断 US 在组织中进行外部引导和激活，以提供按需和体外和体内三维组织中功能蛋白的时空控制递送。利用这一进步，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 传递到肾小管影响单核苷酸多态性的基因修复；多囊肾病 (ADPKD) 作为示例性应用为了实现这一目标，我们在目标 1 中执行严格的要求。蛋白质-FTag 相互作用的生化分析，以优化 Cas9:sgRNA RNP 的方法 Aim 2 将在离体猪中配对全组织荧光和 B 模式/多普勒超声成像。肾脏机械地研究载体的肾脏定位，并优化同步条件从这些研究中获得的指导和声学激活将用于改进。纳米乳剂配方可实现隔室特异性肾脏定位，并优化声激活在目标 3 中，试点体内研究将使用临床相关的美国压力对肾脏进行定量评估。使用 RFP 报告小鼠模型进行美国程序基因编辑在肾组织中的效率。使用 Cas9 碱基编辑器的检测将测试 PKD1 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 表型模型中囊肿发生的调节结果将进行基准测试。对照当前的脂质体 RNP 递送系统来评估这些高风险/高-的性能。奖励研究将为临床相关、影像引导和定量基因编辑奠定基础我们还期望获得利用便携式和非侵入性超声诊断的人类肾脏疾病工具。机制见解可能有助于未来开发针对其他遗传性肾脏疾病的疗法，以及用于探测肾功能的新型诊断方式和分子生物传感技术。