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Abstract: FR-PO406

Self-Complementary Adeno-Associated Viral Gene Delivery in the Metanephric Organ Culture Identifies Pvt1 as Modulator of Cystogenesis

Session Information

Category: Genetic Diseases of the Kidneys

  • 1101 Genetic Diseases of the Kidneys: Cystic

Authors

  • Aboudehen, Karam S., University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
  • Eckberg, Kara, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
  • Weisser, Ivan D., University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
  • Buttram, Daniel Joseph, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
  • Somia, Nikunj, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
  • Igarashi, Peter, University of Minnesota Twin Cities, Minneapolis, Minnesota, United States
Background

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder characterized by the formation of kidney cysts, which originate from the epithelial tubules of the nephron. The metanephric organ culture (MOC) is an ex vivo system wherein explanted embryonic kidneys undergo tubular differentiation and renal development. The MOC has been previously used to study PKD; however, difficulty in manipulating gene expression has impeded its effectiveness in identifying genes and pathways that are involved in cystogenesis.

Methods

We tested the ability of different viruses as a gene-delivery tool by comparing the transduction efficiency of three serotypes scAAV vectors. We then utilized scAAV serotype DJ to deliver shRNA to knockdown Pvt1, a long non-coding RNA (lncRNA), which was upregulated in cystic MOCs, in Pkd1 and Pd2 mutant mice, and humans with ADPKD. E14.5 metanephroi from wild type CD-1 and Pkd1-null embryos were incubated with scAAV-DJ/shRNA for 4 hours and cultured on a transwell dish between 24 and 96 hours. Semi quantitative qRT-PCR, western blot, and cyst index analysis were performed at 96 hours.

Results

scAAV/DJ displayed robust transduction efficiency of the epithelial cells of the MOC by 67%. shRNA delivery by scAAV/DJ downregulated the expression of Pvt1 by 45% and redcued cyst index by more than 50% in explanted wild-type and Pkd1-mutant MOCs treated with cAMP. Mechanistically, knockdown of Pvt1 decreased the expression of c-MYC protein by up to 60% without affecting Myc mRNA.

Conclusion

Data from this study suggest that Pvt1 modulates cystogenesis and targeting Pvt1 could provide therapeutic benefits for patients with ADPKD. scAAV/DJ could be utilized to rapidly and inexpensively screen and identify genes and pathways that are involved in cystogenesis.

Funding

  • NIDDK Support