CRISPR-HITI gene therapy, delivered via mesoscale nanoparticles, to restore NPHP1 function in PTEC’s.

Abstract

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal disease in children and adolescents. Most cases of juvenile NPHP are caused by homozygous full gene deletions of the NPHP1 gene coding for nephrocystin-1, a protein that localizes to the primary cilia. Lack of nephrocystin-1 is thought to impair cilia function, likely disrupting important developmental signalling pathways. Current therapies for NPHP are merely supportive, resulting in a high need for curative treatments. Gene therapy can offer a solution. More specifically, we want to utilize a strategy called CRISPR/Cas9-mediated homology-independent targeted integration (HITI), to knock-in a functional copy of the NPHP1 gene. The genetic cargo of this CRISPR-HITI gene therapy (Cas9, gRNA, and NPHP1 donor DNA) must be delivered efficiently to the target proximal tubule epithelial cells (PTEC’s). Mesoscale nanoparticles seem to be a good candidate vector for this, as previous research showed that they selectively target the PTEC’s of mice. The pharmacology group of Utrecht University recently developed a biallelic NPHP1 knock-out model in induced pluripotent stem cell derived kidney organoids. Here, we propose to use this organoid model to develop an in vitro test system for NPHP that allows easy access to the PTEC’s. This test system will then be used to determine the potential of the CRISPR-HITI system, delivered via mesoscale nanoparticles, to knock-in the therapeutic NPHP1 gene, and thereby restore disease phenotype.