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.

项目摘要超过60种遗传疾病与刺穿肾功能和单基因肾脏疾病有关占终末期肾脏疾病的约15％。虽然CRISPR机械具有巨大的热潜能为了纠正致病性肾突变，目前缺乏可用的临床相关技术有效地将CRISPR构造提供给肾脏，并在复杂的三维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脏验证和超声（US）敏感氟纳米材料能够成像引导基因编辑核核蛋白（RNP）递送到肾脏中小管。这项技术将为整个领域建立强大的工具。这项策略的基础是我们的发现氟化学调节器或“ ftags”家族，与蛋白质可逆地接口以使其能够从Acoustico响应性易流性纳米乳液中加载和美国可编程的交付损害蛋白质的结构或生物活性。来自我们小组的发表研究表明，ftagged蛋白质可以使用临床诊断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空间控制递送。利用这一进步，并受到最近发现的可变形纳米颗粒的启发我们将在肾管中积聚，我们将设计这些Fluoro Nanovectors以将基础编辑RNP提供给肾小管影响单核苷酸多态性的基因修复；专注于常染色体主导多囊肾脏疾病（ADPKD）作为典范应用。为了实现这一目标，在目标1中，我们执行严格蛋白质-FTAG相互作用的生化分析在其对CAS9的方法学优化的途径中：SGRNA RNP 基本编辑。 AIM 2将对整个组织荧光和B型/多普勒US成像在离体猪中成像肾脏可以机械研究载体的肾脏定位，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用于完善纳米乳液公式以实现隔室特异性肾定位，并优化声学激活在使用临床相关的美国压力的肾脏中。在AIM 3中，试点体内研究将定量评估使用RFP-REPORTER鼠模型，在肾脏组织中使用USPROGMED基因编辑的效率。并行交付使用CAS9基础编辑器的测定将测试PKD1中单核苷酸多态性修复的特异性和效率基因，以及ADPKD表型模型中囊形的调节。结果将以基准为基准针对当前的脂质体RNP输送系统来评估性能。这些高风险/高风险的成功奖励研究将为临床上的，成像引导和定量基因编辑提供基础利用便携式和非侵入性诊断的人类肾脏疾病工具。我们也期望获得机械洞察力可能允许对其他遗传肾脏疾病的未来疗法开发，以及新型的诊断方式和分子生物传感技术探测肾功能。