1002-OR: Expanding the Target Space for DKD: A Multiplexed Characterization of AAV Cellular Tropism Targeting Tubular Epithelial Cells for High Throughput In Vivo Perturb-seq

Introduction and Objective: In vivo modeling is essential to capture the dysregulated multicellular circuits driving DKD, but is limited by low throughput discovery. To identify causal drivers of DKD, we developed a multiplexed, in vivo pooled perturbation approach targeting tubular epithelial cells combined with single cell/nucleus RNAseq to link genetic perturbations to disease pathology. Methods: 12 individual single stranded (ss) AAV serotypes were packaged with barcoded DNA pools under pol3 promoter expressing a fluorescent reporter (ITR-Promoter-mGreenLantern-Sv40(polyA)-Pol3-Barcode-ITR) and pooled equimolarly. This library was administered IV into hamsters with insulin resistance or a rat model of 5/6 nephrectomy. Two weeks post-injection, GFP-positive nuclei were isolated and analyzed via snRNA-seq to map viral uptake to specific kidney cell types. Results: We identified three serotypes, AAV-KP1, AAV-DJ, and AAV-DJ9 that preferentially transduced proximal tubule epithelial cells (PTEC), specifically the injury vulnerable S3 segment (PT-S3). Notably, AAV-DJ and AAVD-J9 demonstrated ~7-fold higher selectivity for PTECs compared to AAV-KP1, a commonly used serotype previously validated in literature to target PTECs using localized delivery. Conversely, AAV-KP1 barcodes were significantly enriched in endothelial cells, a tropism not observed with other serotypes. Conclusion: In conclusion, our multiplexed RNA-barcoded screening platform successfully identifies serotypes that preferentially target and express in epithelial and/or endothelial cells, active drivers of kidney disease progression. These findings provide a critical toolbox for future high-throughput perturbation screens, enabling the interrogation of causal relationships between genetic signatures and DKD progression in vivo at scale. Disclosure G. Pharaoh: None. J. Nonora: None. D. Shao: None. C. Le: None. A. Gupta: None. C. Carrico: None. J. Chen: None. S. Shambhu: None. I. Driver: Stock/Shareholder; Current; AbbVie Inc., Amgen Inc.