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Kidney Week

Abstract: SA-PO637

Targeted Delivery of Small Interfering RNA (siRNA) to Proximal Tubule Cells in the Kidneys

Session Information

Category: Genetic Diseases of the Kidneys

  • 1202 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Ding, Shiying, Judo Bio, Cambridge, Massachusetts, United States
  • Long, Kimberly R., University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States
  • Cunniff, Jeremy, Judo Bio, Cambridge, Massachusetts, United States
  • Lucas, Johnny, Judo Bio, Cambridge, Massachusetts, United States
  • Rong, Haojing, Judo Bio, Cambridge, Massachusetts, United States
  • Belanger, Adam, Judo Bio, Cambridge, Massachusetts, United States
  • Zhang, Hongmei, Judo Bio, Cambridge, Massachusetts, United States
  • Lawrence, Jonathan F., Judo Bio, Cambridge, Massachusetts, United States
  • Rbaibi, Youssef, University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States
  • Weisz, Ora A., University of Pittsburgh Department of Medicine, Pittsburgh, Pennsylvania, United States
  • Sehgal, Alfica, Judo Bio, Cambridge, Massachusetts, United States
Background

Small interfering RNA (siRNA) is a clinically validated therapeutic modality, which silences gene expression via RNA interference (RNAi). Although siRNAs are secreted through the kidney, siRNA mediated knock-down in the kidney is still limited, largely due to challenges with optimal delivery. Proximal tubule epithelial cells (PTECs) within the nephron are attractive targets for utilizing RNAi, where the primary mode of entry is likely endocytic uptake. Here we describe a targeted approach to deliver conjugated siRNAs exploiting the PTECs’ internalizing receptors. Understanding the mechanisms of siRNA uptake in kidney PTECs is pivotal for developing efficient and safe delivery platforms targeting renal diseases.

Methods

In the present study, we investigated siRNA uptake in vitro and in vivo using conjugated siRNAs. A well-differentiated, opossum kidney (OK) PTECs cell line was used as a model system to mimic proximal tubule biology both morphologically and functionally, for studying siRNA uptake. Fluorescently tagged siRNAs were employed to observe uptake and intracellular trafficking. Additionally, siRNAs conjugated with target ligands were administered in mice to assess exposure in vivo, measured by mass spectrometry and microscopy. We further measured siRNA mediated knock-down across multiple genes in mouse kidneys.

Results

The siRNAs were taken up by OK cells in both time and concentration dependent manners, predominantly from the apical surface. We further studied the mechanism of conjugated siRNA uptake using specific inhibitors and cell specific knockouts of various PTEC endocytic receptors. Conjugation with selected ligands of megalin significantly enhanced siRNA uptake both in OK cells and in mouse kidneys. In addition, these conjugations led to significant gene knock-down across multiple targets.

Conclusion

The work suggests that PTECs employ megalin to facilitate siRNA uptake via endocytosis and can be used for gene silencing. Elucidating the mechanism(s) for siRNA uptake in the proximal tubule aids in the development of RNA-based therapeutics. Future work will focus on ligand conjugation optimization and chemical modification to improve kidney specific silencing activity.

Funding

  • Commercial Support – Judo Bio