Intrathoracic injection of Adeno‐associated virus serotype‐1 (AAV1) is transported to the spinal cord

Abstract

Our goal is to develop a clinically relevant method for transgene delivery to both the diaphragm and phrenic motoneuron pool (located in cervical spinal segments C3-C5). An AAV1 vector containing the ubiquitous cytomegalovirus immediate early gene promoter and the control gene LacZ (AAV1-CMV-LacZ) was diluted in sterile saline and mixed with a gel solution to obtain a total volume of 100μ L (4.0 x 1011 particles). Mice were anesthetized, placed supine, and a 26G needle was inserted between the ribs to a depth of 3 mm. Following the 100μ L injection, mice were held upright for 10 minutes to allow the gel media (containing AAV1-CMV-LacZ) to stay in contact with the diaphragm. Four months post-injection, diaphragm and spinal segments C3-C5 were harvested. PCR was performed on isolated DNA using primers specific to the LacZ gene. Mice injected with AAV1-CMV-LacZ were positive for LacZ compared to negative controls in both tissues. The protein product of LacZ (beta-galactosidase; rlu/μ g protein above sham injected control tissue) was present in both the diaphragm (18914 ± 9346) and cervical spinal segments C3-C5 (2380 ± 792). We conclude that AAV1-CMV-LacZ is transported to the spinal cord after an intrathoracic injection. Ongoing experiments are aimed at co-localizing AAV1 with retrogradely labled phrenic motoneurons. NIH DK58327 LRD supported by: T32 HD043730/UF Alumni Assoc. Fellowship