A Potential Therapy Using Antisense Oligonucleotides to Treat Autosomal Recessive Polycystic Kidney Disease.

(1) Background: Autosomal recessive polycystic kidney disease (ARPKD) is a rare ciliopathy characterized by progressively enlarged kidneys with fusiform dilatation of the collecting ducts. Loss-of-function mutations in the PKHD1 gene, which encodes fibrocystin/polyductin, cause ARPKD; however, an efficient treatment method and drug for ARPKD have yet to be found. Antisense oligonucleotides (ASOs) are short special oligonucleotides which function to regulate gene expression and alter mRNA splicing. Several ASOs have been approved by the FDA for the treatment of genetic disorders, and many are progressing at present. We designed ASOs to verify whether ASOs mediate the correction of splicing further to treat ARPKD arising from splicing defects and explored them as a potential treatment option. (2) Methods: We screened 38 children with polycystic kidney disease for gene detection using whole-exome sequencing (WES) and targeted next-generation sequencing. Their clinical information was investigated and followed up. The PKHD1 variants were summarized and analyzed, and association analysis was carried out to analyze the relationship between genotype and phenotype. Various bioinformatics tools were used to predict pathogenicity. Hybrid minigene analysis was performed as part of the functional splicing analysis. Moreover, the de novo protein synthesis inhibitor cycloheximide was selected to verify the degraded pathway of abnormal pre-mRNAs. ASOs were designed to rescue aberrant splicing, and this was verified. (3) Results: Of the 11 patients with PKHD1 variants, all of them exhibited variable levels of complications of the liver and kidneys. We found that patients with truncating variants and variants in certain regions had a more severe phenotype. Two splicing variants of the PKHD1 genotypes were studied via the hybrid minigene assay: variants c.2141-3T>C and c.11174+5G>A. These cause aberrant splicing, and their strong pathogenicity was confirmed. We demonstrated that the abnormal pre-mRNAs produced from the variants escaped from the NMD pathway with the use of the de novo protein synthesis inhibitor cycloheximide. Moreover, we found that the splicing defects were rescued by using ASOs, which efficiently induced the exclusion of pseudoexons. (4) Conclusion: Patients with truncating variants and variants in certain regions had a more severe phenotype. ASOs are a potential drug for treating ARPKD patients harboring splicing mutations of the PKHD1 gene by correcting the splicing defects and increasing the expression of the normal PKHD1 gene.