Efficient Targeted Transgene Delivery to Injured Lower Motor Neurons

Abstract

Peripheral nerve injuries yield devastating consequences, and surgical repair outcomes remain suboptimal. Novel therapeutic strategies such as gene therapy could improve peripheral nerve regeneration. Though adeno-associated virus (AAV) vectors have delivered transgenes to intact peripheral neurons, transduction of transected neurons relevant to management of peripheral nerve injuries has not been reported. Herein, in vivo transduction efficiency of axotomized murine facial neurons using four AAV capsids packaging a fluorescent reporter transgene, tdTomato, is characterized. Proximal stumps of transected facial nerve branches in C57Bl/6J mice were immersed in AAV solutions. Four weeks later, facial motor nuclei were volume-imaged via whole-mount two-photon excitation microscopy, and machine learning-based image segmentation quantified the proportion of transgene expressing neurons. We observed remarkable retrograde transduction efficiency with AAV-PHP.S and AAV-F, with expression levels sufficient to detect intrinsic tdTomato fluorescence. This study confirms successful in vivo retrograde transgene delivery to transected peripheral neurons, an approach that carries potential as a research tool and future therapeutic strategy.