Targeting Podocytes: Optimized Adeno-Associated Virus Delivery for Gene Therapy in a Neonatal Mouse

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Background: Pathogenic variants in NPHS1 cause the most severe form of infantile-onset nephrotic syndrome that remains unresponsive to current treatments. While adeno-associated virus (AAV)-mediated gene replacement therapy is effective in other organs, targeted gene delivery to the kidney presents unique challenges due to limited accessibility. This study aims to identify an effective neonatal injection route for AAV that achieves efficient podocytes transduction in a mouse model, establishing a basis for gene therapy in glomerular diseases. Methods: B6;129 neonatal mice (postnatal day 1–5) received >1.0×1013 vg/kg of AAV-KP1-CAG-tdTomato (TdT) via three routes: (i) retro-orbital (RO, 10–15 µL, 31 G BD Insulin syringe, manual), (ii) intrarenal subcapsular (IR, 10 µL, 33 G syringe pump, over 2 minutes), and (iii) percutaneous intracardiac (IC, 10 µL, 33 G Hamilton syringe, slow manual injection). Kidneys and liver were harvested 10–20 days post injection, paraffin-embedded, sectioned, and stained for TdT and WT1. Whole-slide images were acquired using a Nikon microscope, TdT signals were quantified manually or using QuPath software. Podocyte transduction was quantified in ≥30 glomeruli per animal. Results: Both RO and IR routes led to detectable podocyte transduction, reaching up to 18 % (RO: 18%, IR: median 16%, [14–18%], n=3) of the WT1-positive cells. IR also transduced glomeruli in the contralateral kidney, indicating systemic distribution. In contrast, IC injection resulted in robust TdT expression in both kidneys, with up to 48% ([30–48%], n=3) of podocytes co-expressing TdT, approximately 2.5-fold higher than in RO or IR. Conclusion: Percutaneous IC delivery of the podocyte-tropic AAV-KP1 capsid in neonatal mice achieved ∼50 % podocyte transduction, surpassing RO and IR routes. This enhanced efficiency of the IC route is likely due to direct arterial perfusion from the left ventricle, facilitating effective delivery to the renal vasculature. These findings establish proof-of-principle for systemic AAV-mediated gene delivery to podocytes in neonatal mice and support its potential application for future gene therapy for glomerular diseases.

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