Banon-Maneus, E. | Search bioRxiv

bioRxiv2026

Minimally Invasive in Situ Perfusion Method for Targeted AAV Delivery in Native Kidney: Proof of Concept in Pigs

Currently, there is not any method for efficient and targeted delivery of gene therapies using viral vectors to the native kidneys. Adeno-associated viruses (AAV) are limited by hepatotoxicity and poor kidney tropism. This study aimed to develop a minimally invasive approach that overcomes both challenges. We evaluated in pigs a technique leveraging external perfusion systems to transiently isolate native kidney vascularization. Using a percutaneous femoral approach, fluoroscopy-guided catheterization of the kidney artery and vein enabled the establishment of a temporary isolated kidney perfusion circuit permitting native kidney in-situ perfusion. AAV delivery was then assessed by detecting construct encoding Td-Tomato protein, its mRNA and fluorescence 6 days after the perfusion. The method was achieved with two different external perfusion systems and was not associated with procedure related mortality or any major complication. Td-Tomato was detected in the perfused kidney without a significant detection in the liver which showed no inflammatory infiltrates. We developed, in pigs, a minimally invasive method permitting in-situ native kidney perfusion that enables kidney-targeted AAV delivery. Besides, it limits liver off-target transduction, potentially reducing its main side effect, hepatotoxicity, when AAV is systemically injected. It offers fast-track translational possibilities for developing kidney-targeted gene therapies. Finally, it may permit additional non-AAV therapeutics for patients with kidney diseases.

genetics↗

bioRxiv2025

Enhanced Kidney Targeting and Distribution of Tubuloids During Normothermic Perfusion

Tubuloids have become a promising tool for kidney disease model and regeneration, although their ability for integration and regeneration in vivo is not well documented. Here we establish, characterize and compare human tubuloids using two optimized protocols, which added a prior isolation of tubular cells (Crude tubuloids) and proximal tubular cells (F4 tubuloids). Secondly, using this protocol, healthy rat-derived tubuloids were established. Finally, we compared two strategies to deliver GFP tubuloids in a kidney host: 1) subcapsular/intracortical injection and 2) tubuloid infusion during normothermic preservation in a rat transplantation model and a human discarded kidney. F4 tubuloids achieved a more differentiation state compared to Crude tubuloids. When analyzing tubuloid delivery to the kidney, normothermic perfusion was more efficient compared to in vivo injection, observing fully-developed tubules in the host parenchyma after 1 week and 1 month. Normothermic perfusion also successfully delivered kidney tubuloids to a discarded human kidney for transplantation. These results suggest that tubuloids administered during normothermic perfusion represent a potential strategy to enhance the translatability of kidney regenerative therapies to the clinical practice to condition kidney grafts and to treat kidney diseases.

bioengineering↗