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Abstract: FR-PO352

Development of Molecular Targeting Agents Enables Specific Gene Editing of Human Podocytes in Kidney Organoids

Session Information

Category: Development‚ Stem Cells‚ and Regenerative Medicine

  • 500 Development‚ Stem Cells‚ and Regenerative Medicine

Authors

  • Vo, Nicole, University of Washington, Seattle, Washington, United States
  • Freedman, Benjamin S., University of Washington, Seattle, Washington, United States

Group or Team Name

  • Dr. Benjamin Freedman lab
Background

Gene therapy offers many opportunities to treat kidney diseases. Kidney organoids are complex structures that resemble nephrons and can be used to develop gene therapy approaches. Commonly used gene transfer techniques, such as lentivirus and adeno-associated virus, are size limited, transient, or introduce DNA non-specifically into the genome. As in tissues, however, gene editing in organoids remains inefficient and non-specific. There is an unmet need for reporter systems that enable tracking of editing in different cell types. We sought to improve upon these challenges, using kidney organoids as a representative lineage.

Methods

To enable detection of genome editing events in different cell types, we developed a fluorescence-on (mCherry) reporter system in human iPS cells. Intact kidney organoids were transfected with CRISPR ribonucleoprotein (RNP) complexes. To improve delivery, we tested candidate surface markers to identify molecular targeting agents (MTAs) specifically recognizing podocytes, proximal tubules, or endothelial cells in live imaging assays. An MTA labeling podocytes (PodoTracker) was tethered to Cas9. Genome editing events were detected by changes in fluorescence in specific cell types, and next generation sequencing at the target locus.

Results

Confocal microscopy showed that mCherry+ cells were detected in kidney organoids treated with gRNA targeting fluorescence-on reporter, but not with a scrambled guide. Co-staining of gene edited cultures with nephron markers revealed editing in both proximal tubule cells and podocytes. By imaging analysis, the fluorescence-on editing efficiency in organoids was around 5%. MTAs successfully live-labeled podocytes within kidney organoids. Tethering of Cas9 to podocyte-targeting MTA produced increased rates of podocyte genome editing (mCherry+), relative to non-tethered Cas9.

Conclusion

Using fluorescence-on organoids, genome editing events can be monitored using in individual cell types, showing editing rates consistent with sequencing. Coupling of Cas9 to MTAs is a promising approach to enhance delivery and edit specific cell types of interest. Because the cultures are derived from pluripotent stem cells, the platform may also be readily adapted to detect genome editing in other organ lineages. In the future, we will also test whether our tubule MTAs can selectively enable editing in tubules.

Funding

  • NIDDK Support