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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Detection of HSPG and AAV2 transduction of miniature pig parotid glands. The immunodetection of HSPG is shown in (A) and (B). (A) Photomicrograph of parotid gland section used as a control for HSPG immunostaining (×200). (B) Photomicrograph of parotid gland section stained for HSPG. For details of immunostaining procedures, see Materials and methods. (B) Arrowheads point to the luminal membranes of duct cells. Note also staining for HSPG in extracellular matrix (×200). AAV2 transduction of parotid duct cells is shown in (C) to (E). AAV2LacZ was administered, or not, to miniature pig parotid glands and β-galactosidase activity was revealed by staining with X-Gal and nuclear fast red, as described in the Materials and methods. (C) Photomicrograph of a parotid gland section of a control animal showing an absence of β-galactosidase-positive staining (×400) (D) Photomicrograph of a parotid gland from a miniature pig 6 weeks after administration of 10¹¹ pu of AAV2LacZ. The blue color indicates β-galactosidase-positive staining in duct cells (×200). (E) Higher magnification of duct from boxed area of (D) showing positive β-galactosidase activity (×400)

Figure 2

Time course of human erythropoietin (hEpo) detection and hematocrit values in miniature pigs after parotid gene transfer. (A) Average salivary hEpo levels over the 32-week experimental time for animals transduced with AAV2hEpo (n = 3) or not (controls, n = 3). (B) Average serum hEpo levels over the 32-week experimental time for animals transduced with AAV2hEpo or not (controls). (C) Average hematocrit values for transduced and control animals over the experimental time course. Data shown are the mean ± SD. At some control time-points, the SD values are smaller than the size of the symbol used

Figure 3

Detection of hEpo in saliva and serum, as well as hematocrit levels, of miniature pigs in the last experimental cohort. Data shown are the mean ± SEM for samples prior to vector administration (Pre-vector; time ‘0’), 6 weeks after vector administration, and 12 weeks after vector administration

Figure 4

Scattergram of serum anti-hEpo antibody levels in miniature pigs from the last two experimental cohorts. Data shown are from individual animals at three times after vector administration; weeks 2, 12 and 32 (only for one cohort). Each symbol represents a separate animal. Miniature pigs treated with AAV2hEpo are indicated by filled symbols and control animals (glands infused with buffer or AAV2LacZ) are indicated by open symbols. Unilateral refers to animals receiving vector in only one parotid gland, whereas animals designated as bilateral were administered vector to both parotid glands