Kidney Week

Abstract: FR-PO0091

Viral Gene Therapy Rescues Cystic and Fibrotic Tubular Phenotypes in ARPKD Organoids

Session Information

• ADPKD and Cystic Kidney Disease - 2
  October 23, 2026 | Location: Exhibit Hall A, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic (Monogenic)

Authors

• Robichaud, Jielu Hao, Mayo Clinic Minnesota, Rochester, Minnesota, United States

Jielu Hao Robichaud, MD

• Afrin, Humayra, Mayo Clinic Minnesota, Rochester, Minnesota, United States

Humayra Afrin, MD, PhD

• Chebib, Fouad T., Mayo Clinic in Florida, Jacksonville, Florida, United States

Fouad T. Chebib, MD, FASN

• Harris, Peter C., Mayo Clinic Minnesota, Rochester, Minnesota, United States

Peter C. Harris, PhD

• Gupta, Navin R., Mayo Clinic Minnesota, Rochester, Minnesota, United States

Navin R. Gupta, MD, FASN

Background

ARPKD is an often embryonically-lethal disease of progressive kidney cysts and fibrosis due to biallelic loss of PKHD1, whose 67 exons encode fibrocystin (FPC). >750 pathogenic PKHD1 variants have identified its functional domains and correlated genotype with phenotype. Despite growing understanding of molecular underpinnings, there are no approved treatments. Given the severity and early-onset of disease, gene therapy is attractive. DNA correction is hindered by the # of mutations and need for precise editing, which gene transfer circumvents. PKHD1 delivery is hindered by its size, yet fragments of its C-terminus (FPC-CTD) translocate to the nucleus to suppress cysts. To overcome phenotypic limitations of mouse models, we employ an organoid platform to test whether tubular delivery of FPC-CTD to ARPKD tubules hinders cystogenesis and fibrosis.

Methods

Kidney organoids from PKHD1-/- and isogenic wild-type hPSCs were infected with a library of GFP-tagged AAV serotypes at fixed dosage and longitudinally monitored by brightfield and epifluorescent microscopy. After 1 week, immunostaining for GFP and cell-type specific markers determined AAV tropism. Using the optimal serotype for tubular tropism, viral dosage was modulated and transgene delivery and tubular injury assessed by confocal microscopy. FPC-CTD delivery was assessed for reversal of forskolin-induced cystogenic and profibrotic tubular phenotypes.

Results

AAV2, AAV6, AAV6.2, AAVDJ, and AAVPHP.S transduce organoids; AAV2 best for LTL⁺ and CDH1⁺ tubules, AAV6.2 for PODXL+ podocytes, and AAVPHP.S for PDGFR-β+ stroma. AAV2 manifests dose-dependent tubular DNA damage (yH2AX+) and injury (KIM1+) from MOI 10³, 10⁴, 10⁵ vg/cell without correlate reduction in transgene delivery. Following infection with “loaded and tagged” AAV2-GFP-(FPC-CTD-5XMyc), immunostaining for Myc tag confirmed nuclear delivery of FPC-CTD to LTL+ tubules, where it rescued primary cilia by α-acetylated tubulin. Low-dose infection with “loaded and tagged” AAV2 prevented forskolin-induced organoid hypertrophy, tubular DNA damage and senescence, and LTL+ proximal and CDH1+ distal tubular cysts, compared to an "unloaded" control.

Conclusion

Complementary assessments of profibrotic and cystic phenotypes at variable viral concentrations informs the therapeutic window for AAV-based gene therapy in ARPKD by defining the maximally tolerated, and then the minimally effective, AAV dosage.

Acknowledgment

NIDDK U24DK126110 (PKD-RRC subaward); NIDDK U54DK144863 (Mayo Clinic Polycystic Kidney Disease Research Core Center); NIDDK P30 DK090728 (Mayo Clinic Robert M. and Billie Kelley Pimie Translational PKD Research Center)

Funding

• NIDDK Support