# Intravenous rAAV9 Produces Time-Resolved Parenchymal Labeling Downstream of the Vasculature in Adult Mice

## TL;DR

This study maps which cells outside blood vessels in adult mice become labeled after an intravenous gene therapy injection, helping improve understanding of how genetic material reaches tissues.

## Contribution

The study provides a time-resolved map of parenchymal labeling after intravenous rAAV9 delivery in adult mice.

## Key findings

- Durable labeling was observed in several parenchymal cell populations across multiple organs and the nervous system.
- Reporter activation was consistent in nonvascular cells relevant to systemic gene delivery.
- The findings suggest potential mechanistic routes like viral transcytosis or extracellular vesicle transfer.

## Abstract

Systemic gene therapy is often limited by uncertainty about which cells outside blood vessels actually receive and express delivered genetic cargo after injection into the bloodstream. In this study, we injected a single dose of a gene-delivery virus into healthy adult mice and used a fluorescent reporter system that permanently marks cells that receive a genetic “switch” carried by the virus. By examining many organs over time, we created a time-resolved map of which cell types outside the vasculature become labeled after an intravenous dose. We found durable labeling in several parenchymal cell populations across peripheral organs and the nervous system, identifying specific tissues that warrant mechanistic follow-up to determine how labeling occurs after vascular exposure. These data provide a reference resource for researchers optimizing minimally invasive delivery strategies and for interpreting downstream labeling events after systemic administration.

Intravenous delivery of recombinant adeno-associated virus serotype 9 can lead to reporter activation in cell types beyond the vasculature, but the routes enabling downstream parenchymal labeling remain unclear. Here, we provide a systematic, time-resolved map of parenchymal labeling after a single intravenous dose of rAAV9 encoding Cre recombinase under a ubiquitous promoter in healthy adult Ai9 reporter mice. Following retro-orbital administration, we quantified tdTomato-positive labeling across 25 targets at multiple time points over six months and observed durable reporter activation in several nonvascular parenchymal populations relevant to systemic gene-delivery applications. We also identify a set of parenchymal cell types that are consistently labeled in both this vascularly initiated reporter system and our prior adult VE-cadherin-driven reporter paradigm, supporting a connection to vascular exposure without asserting lineage relationships. These results nominate mechanistic routes for future disambiguation, including viral transcytosis across endothelium, endothelial cell transdifferentiation and extracellular-vesicle-mediated transfer. The dataset and methods provide a reference framework for investigators optimizing systemic delivery and interpreting downstream labeling in vivo.