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Adeno-Associated Virus Vector Retrograde Transduction of Transected Murine Facial Motor Neurons
Matthew Q Miller, MD1,2; Iván Coto Hernández, PhD1; Steven Minderler, Lab technician1; Josette Nammour, PhD3; Carrie Ng, Lab technician3; Casey A Maguire, PhD3; Nate Jowett, MD1 1Surgical Photonics & Engineering Laboratory, Mass Eye & Ear, Boston, MA; 2Department of Otolaryngology-Head and Neck Surgery, University of North Carolina, Chapel Hill, NC; 3The Maguire Laboratory, Massachusetts General Hospital, Boston, MA

Introduction: Peripheral nerve injuries yield devastating consequences and nerve repair outcomes remain suboptimal. There is a critical need for novel therapeutic strategies to improve peripheral nerve regeneration. Gene therapy carries potential for enhancing nerve regeneration following injury. Though adeno-associated virus (AAV) vectors have been employed for transduction of intact mammalian peripheral neurons, transduction of axotomized neurons relevant to management of peripheral nerve transection injuries has not been reported.

Materials & Methods: Proximal stumps of transected facial nerve branches in C57Bl/6J mice were immersed in low-dose AAV solutions (5.9E11 viral genomes/mL) followed by retrograde labelling with Fluoro-Gold. Facial motor nuclei in brainstem were later volume-imaged via whole-mount two-photon excitation microscopy, and machine-learning based image segmentation employed to quantify the proportion of transgene-expressing neurons.

Results: Transduction efficiencies were 33.28% (SD 5.71%) for AAV-PHP.S, 18.59% (SD 6.93%) for AAV-F, 6.55% (SD 3.73%) for AAV-9, and 2.81% (SD 2.20%) for AAV-6.

Conclusions: This study confirms successful in vivo retrograde transgene delivery to transected peripheral neurons, which carries potential as a research tool and future therapeutic strategy.

Figure Legends:
Fig. 1: AAV indicates location of transected buccal branch immersion in AAV solution. FG indicates Fluoro-Gold delivery site 3 weeks later.
Fig. 2: Neuron cell body segmentation of whole mount mouse facial buccal subnuclei transduced with pAAV-CAG-tdTomato packaged in AAV6 (a, 27 cell bodies), AAV9 (b, 38 cell bodies), AAV-F (c, 170 cell bodies), and AAV-PHP.S (d, 289 cell bodies). Scale bar 50 µm.
Fig. 3: Efficient section-free quantification of AAV-PHP.S facial motoneuron transduction in buccal subnucleus using 3D reconstructions of two photon microscopy x-y-z tile scans (a, b, and c). Fluoro-Gold and tdTomato separated based on excitation wavelength. Neuronal cell body segmentations (d and e) revealed 368 cell bodies labeled by Fluoro-Gold and 289 also expressing tdTomato. Scale bar 50 µm.

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