Ultrasound-programmable gene editing in kidneys

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.