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Kidney Week

Abstract: PO1597

Biobank of Urinary Cells and Human Kidney Organoids Reveals Nephropathic Cystinosis Phenotypes and Gene Therapy Strategy

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Helms, Louisa, University of Washington, Seattle, Washington, United States
  • Gomez, Ivan G., University of Washington, Seattle, Washington, United States
  • David, Dries, Katholieke Universiteit Leuven Groep Biomedische Wetenschappen, Leuven, Flanders, Belgium
  • Cruz, Nelly M., University of Washington, Seattle, Washington, United States
  • Levtchenko, Elena N., Katholieke Universiteit Leuven Groep Biomedische Wetenschappen, Leuven, Flanders, Belgium
  • Gijsbers, Rik, Katholieke Universiteit Leuven Groep Biomedische Wetenschappen, Leuven, Flanders, Belgium
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States
Background

Cystinosis is a rare disorder caused by mutations in CTNS encoding a cystine transporter, leading to renal Fanconi syndrome and kidney failure. Cysteamine treatment slows, but does not prevent these outcomes and animal models fail to exhibit Fanconi syndrome. Stem cell derived kidney organoids exhibit structures with segmented, nephron-like segments, providing an in vitro platform to study nephropathic cystinosis. As a monogenic disorder, gene therapy is an attractive therapeutic approach which can be optimized in kidney organoids. However, cystinosis patients are rare, and iPS cells representing this population are needed.

Methods

Cells from the urine of 16 patients with cystinosis and control subjects were reprogrammed into iPS cells via Sendai virus. CRISPR gene editing was applied to non-cystinotic iPS cells generating 8 CTNS-/- lines with isogenic controls. Patient-derived and CRISPR-derived CTNS mutant and control iPS cell cohorts were differentiated into kidney organoids and propagated in suspension culture to assess cystinotic phenotype. Organoids were transduced at different stages of differentiation with lenti and adeno-associated viruses with fluorescent reporters to assess efficacy of gene transfer.

Results

Patient-derived and CRISPR-derived stem cells exhibited >100 fold increased intracellular cystine content and vacuole-like structures, compared to controls. Both patient and CRISPR iPSCs differentiated into kidney organoids with proximal and distal tubules and podocyte segments. However, cystinotic organoids developed lobular cyst-like structures in suspension culture over multiple weeks, which were reduced with cysteamine treatment. Lentiviral and AAV transduction successfully entered kidney organoid structures and co-localized with nephron markers when transduced at early stages of differentiation.

Conclusion

We have established a biobank of urinary and iPS cells representing over 20 cystinotic genotypes, with phenotypes in both patient- and CRISPR-derived CTNS mutant lines that show improvement with cysteamine. Viral transduction of kidney organoids can be timed to produce high levels of entry. This biobank provides a comprehensive resource for patient-specific development of more efficacious therapeutics for cystinotic nephropathy, including gene therapy.

Funding

  • Other NIH Support