AAV2-mediated transfer of the human aquaporin-1 cDNA restores fluid secretion from irradiated miniature pig parotid glands

Abstract

Previously (Shan et al, 2005), we reported that adenoviral vector-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to minipig parotid glands following irradiation (IR) transiently restored salivary flow to near normal levels. This study evaluated a serotype 2, adeno-associated viral (AAV2) vector for extended correction of IR (single dose; 20 Gy)-induced, parotid salivary hypofunction in minipigs. At 16 weeks following the IR parotid salivary flow decreased by 85-90%. AAV2hAQP1 administration at week 17 transduced only duct cells and resulted in a dose-dependent increase in salivary flow to approximately 35% of pre-IR levels (to approximately 1 ml per 10 min) after 8 weeks (peak response). Administration of a control AAV2 vector or saline was without effect. Little change was observed in clinical chemistry and hematology values after AAV2hAQP1 delivery. Vector-treated animals generated high anti-AAV2 neutralizing antibody titers by week 4 (approximately 1:1600) and significant elevations in salivary (approximately 15%), but not serum, granulocyte macrophage colony-stimulating factor levels. Following vector administration, salivary [Na(+)] was dramatically increased, from approximately 10 to approximately 55 mM (at 4 weeks) and finally to 39 mM (8 weeks). The findings demonstrate that localized delivery of AAV2hAQP1 to IR-damaged parotid glands leads to increased fluid secretion from surviving duct cells, and may be useful in providing extended relief of salivary hypofunction in previously irradiated patients.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1

Effect of irradiation on parotid saliva flow rate in minipigs. One parotid gland of animals (n=9) was irradiated with 20 Gy, as described in the Materials and Methods. Parotid saliva flow rates were thereafter measured at weeks 4 and 16. Data shown are the mean±SD of salivary flow rates for the targeted glands (open bars; note that for the 16 week value the SD is too small to be seen) and the contralateral glands (black bars). Data were analyzed using a one-way analysis of variance (F=72.69, p

Figure 2

Effect on saliva flow rates of AAV2hAQP1 administration to minipig parotid glands after irradiation. At week 17 following 20 Gy irradiation, either AAV2hAQP1 (A, B), AAV2LacZ (B) or saline buffer (B) were administered to a single parotid gland. Panel A shows the effect of different doses of AAV2hAQP1 on parotid saliva flow rates in irradiated glands (n=2/group; data are mean values ±SD obtained 12 weeks following vector administration; note that for the 3×1011 vp dose the SD is too small to be seen). The data in Panel A was not analyzed for statistical significance, because there were only two animals/group. Panel B shows salivary flow rates at different times following administration of AAV2hAQP1, AAV2LacZ or saline buffer to irradiated parotid glands (n=3/group; data are mean values ±SD). Time zero corresponds to week 17 following irradiation. Note that for the 8-week time-point with buffer and AAV2LacZ treatment the SDs are too small to be seen. Data were analyzed using a one-way analysis of variance (F=27.88, p<0.001), followed by the Tukey test. All of the parotid flow rates for the AAV2hAQP1-treated minipigs are significantly different from those of the buffer- and AAV2LacZ-treated animals at weeks 4 (p<0.01 vs buffer; p<0.05 vs AAV2LacZ), 6 (p<0.001) and 8 (p<0.001). There were no significant differences in parotid flow rates between the buffer- and AAV2LacZ-treated animals at every time-point.

Figure 3

AAV2-mediated transgene expression in minipig parotid glands. Representative results of Western blot analyses are shown. 10 ug of crude membrane protein was subjected to SDS–gel electrophoresis and Western blotting, as described in Materials and Methods. Lane 1, positive control membranes from rat kidney; lane 2, membranes from an AAV2hAQP1-transduced, irradiated minipig parotid gland (8 weeks after 1×1011 vp was delivered) showing AQP1 expression; lane 3, membranes from an AAV2LacZ-transduced, irradiated minipig parotid gland (8 weeks after 1×1011 vp was delivered) showing little AQP1 expression; lane 4, membranes from saline-infused, irradiated minipig parotid gland, also showing little AQP1expression; lane 5, membranes from the non-irradiated, contralateral side, showing AQP1 expression normally present in minipig parotid glands, which is derived from microvascular endothelial cells,,. The migration position of all immunopositive protein bands represents the nonglycosylated AQP1 monomer (28 kDa).