Long‐term transduction of miniature pig parotid glands using serotype 2 adeno‐associated viral vectors

Abstract

Previously, using an adenoviral vector, we showed that miniature pigs could provide a valuable and affordable large animal model for pre-clinical gene therapy studies to correct parotid gland radiation damage. However, adenoviral vectors lead to short-term transgene expression and, ideally, a more stable correction is required. In the present study, we examined the suitability of using a serotype 2 adeno-associated viral (AAV2) vector to mediate more stable gene transfer in the parotid glands of these animals. Heparan sulfate proteoglycan was detected by immunohistochemistry. beta-galactosidase expression was determined histochemically. An AAV2 vector encoding human erythropoietin (hEpo) was administered via Stensen's duct. Salivary and serum hEpo levels were measured using an enzyme-linked immunosorbent assay. Serum chemistry and hematological analyses were performed and serum antibodies to hEpo were measured throughout the study. Vector distribution was determined by a quantitative polymerase chain reaction. Transgene expression was vector dose-dependent, with high levels of hEpo being detected for up to 32 weeks (i.e. the longest time studied). hEpo reached maximal levels during weeks 4-8, but declined to approximately 25% of these values by week 32. Haematocrits were elevated from week 2. Transduced animals exhibited low serum anti-hEpo antibodies (1 : 8-1 : 16). Vector biodistribution at animal sacrifice revealed that most copies were in the targeted parotid gland, with few being detected elsewhere. No consistent adverse changes in serum chemistry or hematology parameters were seen. AAV2 vectors mediate extended gene transfer to miniature pig parotid glands and should be useful for testing pre-clinical gene therapy strategies aiming to correct salivary gland radiation damage.