#3460 DEVELOPMENT OF A NEW CELLULAR MODEL TO EVALUATE THE CLINICAL IMPACT OF PKD1 VARIANTS EXPLOITING CRISPR/CAS9 SYSTEM

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common single-gene disorder frequent progressive kidney disease, which ultimately leads to failure renal replacement therapy. Almost 80% of cases ADPKD are attributed germline mutations in PKD1, even though at least one second somatic event such as inactivation remaining wild-type PKD1 allele required for disease's manifestation. A significant portion all variants identified classified “variant unknown significance” (VUS) understanding their functional impact may be diagnostic importance patients families. So, a proper experimental model study different genetic lesions needed readily confirm pathogenicity. Method The HEK293T cell line was modified express an inducible Cas9 afterwards transfected with sgRNA targeting exon 15 generate clones carrying homozygous or heterozygous nonsense variants, validated by Sanger sequencing. Transcript level protein expression were evaluated, then read-outs set-up validate model. Heterozygous used introduce variant, c.11614G>A p.(Glu3872Lys) 42, using CRISPR system called base editors, able operate single-base substitutions (Figure 1A). Results (+/−) (−/−) generated. These cells showed slight decrement mRNA levels, PC1 loss confirmed full knock-out clones. Functionally, immunofluorescence staining highlighted cytoskeletal rearrangement lacking PC1, did not present actin protrusions variance WT cells. In agreement these results, PKD1−/− reduced migration demonstrated wound healing assay. starving conditions, characterized increased viability, resistance death disrupted autophagic pathway, LC3BI-II conversion, diminished. Validation also supported RNA sequencing data indicating that double lines display upregulation genes involved proliferative pathways epithelial-mesenchymal transition, well down-regulation organization. We specific variant C4 according ACMG criteria. To do so, PKD1+/+ PKD1+/− guide correctly positioned Cytosine Base Editor. Using this approach we could on 42 “double hit” represents more likely real-life situation. Functional assessment variant's pathogenicity comparison knock-outs currently ongoing. Conclusion conclusion, generated new PKD cellular can easily exploited reproduce some VUS identified, clinical exome sequencing, our cohort patients. obtained provide proof-of-principle feasibility allowed identify selective 1B) rapid screening assess phenotypic variants.