Characterization of Pulmonary Tropism, Expression Kinetics and Safety of the Novel Adeno-Associated Virus-LungM3 Vector.

Abstract

This study investigated the lung-targeting characteristics, dose- and time-dependent effects, and safety of a novel engineered adeno-associated virus (AAV) vector, AAV-LungM3. In vitro, mouse primary lung organoids were transduced with AAV-LungM3 at doses of 103, 104, and 105 vector genomes (vg)/cell, and transduction was evaluated by fluorescence detection. For in vivo validation, C57BL/6 mice received intratracheal injection of AAV-LungM3 at doses of 0.5 × 1011, 1.0 × 1011, and 2.0 × 1011 vg. Flow cytometry was performed to assess the dose-dependent transduction specificity and efficiency of AAV-LungM3 in type I alveolar epithelial cells (AT1 cells) and type II alveolar epithelial cells (AT2 cells). Using the favorable dose determined by the dose-response assay, the temporal changes in AT2 cell transduction specificity and efficiency were further monitored over a 4-week period. In addition, hematoxylin and eosin (H&E) staining, detection of peripheral blood inflammatory factors, and analysis of plasma biochemical indicators were used to evaluate early-phase safety. In vitro transduction increased in a dose-dependent manner. In vivo assays revealed that the AT2 cell transduction specificity of AAV-LungM3 decreased gradually with increasing viral dose, whereas its transduction specificity for AT1 cells increased. Nevertheless, no significant differences in the transduction efficiency of AT1 cells and AT2 cells were observed among all dose groups. Longitudinal monitoring indicated that week 2 was the preferred time point for AAV-LungM3 transduction. In conclusion, under the tested conditions, AAV-LungM3 exhibited targeting specificity to AT2 cells and favorable in vivo expression with no observed toxic effects, serving as a potential delivery platform for pulmonary gene therapy.