Targeting phrenic motoneurons using adeno‐associated viral vectors

Adeno‐associated viruses (AAV) have become promising vectors for gene transfer due to their wide range of tissue specificity, long‐term expression, and low immunogenicity. Our laboratory developed an intrapleural approach for retrograde targeting of phrenic motoneurons. This delivery technique, combined with the transduction efficiency of AAV, creates a novel method of increasing gene expression in a targeted manner. Our goal was to test six GFP‐expressing AAV serotypes (2, 6, 7, 8, 9, and 10) for transduction efficiency of phrenic motoneurons at 3 and 10 weeks after intrapleural injection. Cholera toxin subunit B was also injected intrapleurally to label phrenic motoneurons. The cervical spinal cord was fixed, excised, and cut longitudinally. GFP fluorescence was enhanced by immunostaining with a GFP antibody. We found no evidence of GFP expression in motoneurons after injection with serotypes 2, 6, and 10 (1 × 10 11 genome copies). In contrast, serotypes 8 and 9 (2 × 10 11 genome copies) resulted in GFP expression in dendrites of motoneurons after 3 or 10 weeks. At 10 weeks after injection with serotype 7 (1 × 10 11 genome copies), GFP expression was evident primarily in dendrites but also in soma of motoneurons. Future use of AAV serotype 7, 8, or 9 has the potential for targeted delivery of a neurotrophic factor receptor to phrenic motoneurons. Supported by NIH grant HL96750 and a grant from Paralyzed Veterans of America.