Adenoviral-mediated Transfer Of The Human Aquaporin-1 CDNA To Irradiated Minipig Parotid Glands

Patients experience a loss of salivary function as a result of radiation damage to salivary glands during and following radiotherapy of malignancies in the head and neck. Currently, there is no satisfactory treatment for irradiation-damaged glands. However, aquaporin -1 cDNA transferring to salivary glands offers a novel way to treat this condition. A previous work showed that it was possible to increase salivary fluid flow from irradiation-damaged submandibular glands in a rat model through an adenoviral-mediated transfer of human aquaporin-1 (hAQP1) cDNA to surviving epithelial cells (Delporte, et al, PANAS, 1997). However, the results of study weren't tested and verified in large animals. Morphology and function of Minipig's parotid gland are similar to those of humans. Hence, minipig is an ideal animal in study of salivary gland (Wang, et al. Dentomaxillofac Radiol, 1998). We firstly set up a convenient large animal model of radiation damage to parotid glands, then in this model study the effect of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated minipig parotid glands on the parotid flow rate, saliva chemistry serum chemistry.In this study, at first, we developed a convenient large animal model of irradiation damage to parotid gland for further evaluating the effect of adenovirus-mediated transfer of the human aquaporin-1 cDNA to irradiated minipig parotid glands using this model. Materials and methods: A single dose of irradiation (IR 0Gy, 5Gy, 10Gy, 15Gy and 20Gy) was delivered via a linear acceletator to one side of parotid glands of sixteen mini pigs. In pre–IR, post-IR 4 weeks and 16 weeks, parotid saliva flow rate, saliva chemistry, hematological, serum chemistry and histological assessment were performed. Parotid tissue was removed for quantitative histopathologic assessment at 16 weeks. Results: 20Gy IR treatment of the targeted gland resulted in >80% (P<0.01) reduction in parotid saliva output at 16 wk (3074±381μl/10 min pre-IR vs 558±117μl/10 min post-IR). Saliva output in the contralateral gland was reduced >50% (P<0.05). 15Gy IR resulted in >60% (P<0.05) reduction at 16wk. There were several transient alterations in clinical laboratory parameters after irradiation-damage parotid glands of minipigs, including K+ significant increase in post-IR saliva, WBC decrease in post-IR blood, AMY increase in serum chemistry. Histological findings showed a dose-related inflammatory response, and atrophy of the gland parenchyma. Conclusions: This study indicates that 20Gy IR treatment of the parotid gland can induce significant saliva secretion and set up a large animal model for irradiation damage to parotid gland in minipig.In the second part, adenoviral-mediated transfer of human aquaporin-1 cDNA was delivered to irradiated parotid glands in this animal model to evaluate the effect of this treatment on parotid flow rate and chemistry of the irradiated parotid glands. Materials and methods: Nineteen minipigs models were subjected to 20Gy IR directed at one parotid gland. Thereafter, randomly assigned minipigs were administered either infusate buffer (2 minipigs), or infusate buffer containing either the Ad5 vectors AdAQP1 (109 pfu 7 minipigs, 108 pfu 2 minipigs) or AdLuc (encoding luciferase; Li et al, J Gene Med, 2004, 109 pfu 6 minipigs, 108 pfu 2 minipigs). Parotid flow rate was measured, collected saliva chemistry, hematological, serum chemistry were determined pre-IR, 4 weeks, 16 weeks post-IR, 3, 7, 14days after virus transfer. Immunohistological, QPCR and Western blot assessment were performed in day 3, 7 and 14 after vector delivery. Results: Three days after administration of 108 pfu of either vector (i.e., 2.5 x 104 pfu/ul infusate), no significant change in parotid saliva output was observed from IR'd glands, compared to levels measured before vector delivery. However, three days following administration of 109 pfu of AdAQP1 (i.e., 2.5 x 105 pfu/ul infusate), a highly significant increase in parotid saliva output was observed (p = 0.024), averaging 1367±257μl/10 min. On average, the level of saliva output achieved following AdAQP1 was 81±18% of pre-IR values. Conversely, three days following administration of 109 pfu of AdLuc there was no significant change in saliva output. By day 7 following AdAQP1 delivery, parotid saliva output had decreased to 69±20% of pre-IR values (p = 0.058), and by day 14 was further reduced. These results demonstrate in minipigs that administration of the AdAQP1 vector to an IR-damaged salivary gland leads to significant recovery of secretory function, as has been observed to occur in IR rats, correspondingly K+ significant decreasing in 109 pfu AdAQP1 delivering parotid glands of saliva. Furthermore, the dose of AdAQP1 shown to be effective here (2.5 x 105 pfu/ul infusate) is a dose that results in comparable transgene expression levels in both mice and minipigs (Li et al, ibid), and is markedly lower than the doses of AdAQP1 used in our previous IR rat studies (~107-108 pfu/ul infusate). Immunohistochemical detection showed that at 3 days following administration of 109 pfu AdAQP1, aquaporin-1 protein was expressed mainly in ductal and partially in some acinar cells. Pathologicial observations of infected glands showed a rapidly developing inflammatory response, which was characterized by a predominantly lymphocyte infiltrate, and atrophy of the gland parenchyma. Conclusion: in aggregate, these findings suggest that localized delivery of AdAQP1 to IR-damaged salivary glands can significantly increase parotid flow rate and may be clinically used for patients with IR-induced salivary hypofunction.