Abstract: SA-PO301

Characterization of a Synthetic Adeno-Associated Virus (AAV-2/Anc80) That Targets Kidney Stroma and Validation through Gli2 Deletion in Fibrosis

Session Information

Category: Cell Biology

  • 204 Extracellular Matrix Biology, Fibrosis, Cell Adhesion

Authors

  • Ikeda, Yoichiro, Washington University, School of Medicine, St. Louis, Missouri, United States
  • Xiao, Ru, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Vandenberghe, Luk, Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, United States
  • Sun, Zhao, Washington University in St Louis, Saint Louis, Missouri, United States
  • Humphreys, Benjamin D., Washington University School of Medicine, Clayton, Missouri, United States
Background

AAV is a non-integrating virus currently in human clinical trials for gene therapy. There are no reports of AAV with tropism to kidney, limiting our ability to deliver genetic material to that organ. We characterized the kidney tropism for a panel of AAV serotypes to set the stage for future use in human clinical trials.

Methods

Pseudotyped AAV with various capsid proteins and promoters were prepared and tested to validate the efficacy of transduction in mouse kidney. Initial studies utilized GFP reporters and later studies used AAV viruses that express Cre recombinase.
To establish whether an AAV-based approach could efficiently delete floxed genes in kidney pericytes, we injected AAV-2/Anc80-CASI-Cre (3x10^11 GC/mouse) into Gli2(f/f);R26(tdTomato) mice or control. After three weeks, we performed UUO surgery, then kidneys were analyzed. Human iPSC kidney organoids and primary human kidney fibroblasts from patients were exposed to AAV-GFP and analyzed with fluorescent microscopy.

Results

Of all serotypes of AAV analyzed, AAV-2/Anc80-CASI most efficiently transduced kidney cell types. When AAV-2/Anc80-CASI-Cre was injected into R26(tdTomato) reporter mice, 58.9±3.5% (mean±SEM) of all pericytes became TdTomato positive. Other reporter positive cells were mesangial cells and juxtaglomerular cells. AAV-Cre injected Gli2(f/f); R26(tdTomato) mice had reduced fibrosis including 30 – 60% reduction in aSMA, fibronectin, collagen1a1, collagen3a1 mRNA and protein levels after UUO compared to control, which is consistent to immunofluorescent stainings of aSMA and collagen1. There were 40% fewer myofibroblasts in the Gli2flox compared to control kidneys after UUO. There was no toxicity to extrarenal organs with this dose, though higher dose (10^12 GC/mouse) of AAV-2/8-CASI did cause hepatitis. AAV-2/Anc80-CASI-GFP drove GFP specifically in stromal cells of human iPSC kidney organoids (30% of total Meis1 positive cells) and primary human kidney fibroblasts.

Conclusion

We demonstrate that AAV-Cre targets kidney pericytes and efficiently deleted Gli2 leading to an antifibrotic effect. AAV strategies will be useful both in preclinical models but also in human clinical trials.

Funding

  • NIDDK Support