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

Abstract

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