Identification of Cellular Determinants Impacting Mesangial Cells in IgA Nephropathy

Background: IgA nephropathy is a prevalent glomerulonephritis that frequently develop into end-stage renal disease, impacting over a million patients worldwide. Presently there is no disease-specific therapy, necessitating the development of targeted therapeutic intervention strategies and approaches for early diagnosis. One of the characteristic hallmarks of the disease is the predominant autoimmune complex deposition in the glomerular mesangium. However, the genetic regulators of the preferential accumulation of these IgAN immune complexes on the mesangial cells are not defined. Methods: Using our magnetic ranking cytometry platform (termed LEAPFROG for large scale phenotypic functional genomics), together with the versatility of genome-scale CRISPR/Cas9 pooled screening across primary mesangial cells and other non-impacted/non-mesangial kidney cell types in parallel, we will identify the cellular surface component(s) driving the predominant accumulation of autoimmune complexes on the mesangial cells. Using our pooled surfaceome libraries, we will systematically profile the impact of individual surface proteins on the sensitivity of galactose-deficient IgA induced IgAN complex deposition. To enrich subpopulation of mesangial cells with either high or low levels of IgAN complex deposit phenotypes, we can functionalize the commercially available monoclonal anti-Gd-IgA1 antibody or the in vitro purified IgAN complexes to be useable on our platform. The systematic comparison of next-generation sequencing based guide RNA enrichment of regulators of IgAN complex deposition accumulation on mesangial cells versus the non-mesangial cells is anticipated to enable identification of cellular components regulating the IgAN complex induced proliferation and activation of mesangial cells. Results: We have completed the lentiviral CRISPR guide RNA library targeting the cellsurfacome. It encompasses 3000 unique surface proteins, with 12,450 unique guides in total. Gererated cosmc knock out DAKIKI cell line for production of galactose-deficient IgA. These reagents, together with the KM55 antibody are being used for phenotypic screening of primary mesangial cells to identify genetic determinants impacting IgA nephropathy. Conclusion: Use of LEAPFROG-powered CRISPR-cas9 based loss-of-fucnction phenotypic screening is anticipated to identify potentially druggable targets for IgAN. Funding: NIDDK Support