296. Transgene Expression and Neutralizing Antibody Production after Submandibular Gland Administration of AdhAQP1
Authors: Corinne M. Goldsmith, Changyu Zheng, Molly Vallant, Richard D. Irwin, Bruce J. Baum
Journal: Molecular Therapy (2006)

The treatment of patients with head and neck cancers typically includes ionizing radiation (IR). A significant consequence of this is the irreversible damage to salivary glands in the IR field. Without saliva, these patients suffer considerable morbidity, including oral infections, mucositis, dysphagia, as well as frank discomfort. Currently, there is no adequate treatment for this condition. Previously (Delporte et al, PNAS, 1997; Shan et al, Mol Ther, 2005), we showed that serotype 5 adenoviral (Ad5) -mediated transfer of the human aquaporin-1 (hAQP1) cDNA, via AdhAQP1, resulted in a restoration of near normal salivary flow to both rat submandibular and minipig parotid glands four months following IR. In preparation for clinical studies testing this strategy, we conducted a toxicology study with AdhAQP1 in Fischer 344 rats over a 13-week period. Herein, we describe the assessment of transgenic hAQP1 expression in targeted salivary glands, as well as the neutralizing antibody responses to vector delivery. A total of 100 animals per gender were studied. On day zero, animals received either vehicle (diluent buffer), or AdhAQP1 (2 × 10e8, 8 × 10e9 or 2 × 10e11 particle units; pu) via retroductal submandibular gland delivery. Five animals per gender and dosage group were sacrificed on days 3, 15, 29, 57 and 92. Protein from submandibular gland aqueous extracts (1000×g supernatants) was slot blotted onto nitrocellulose filters, and hAQP1 detected with a polyclonal antibody. Neutralizing antibodies were measured as the dilution of serum that resulted in 50% inhibition of the transduction of 293 cells with an Ad5 vector encoding luciferase, AdRSVLuc (MOI = 10). All vector-treated animals survived until scheduled sacrifice, and in general toxicological observations were comparable to findings previously reported with similar Ad5 vectors expressing different transgenes (O'Connell et al, J Oral Pathol Med, 2003; Zheng et al, Oral Dis, 2006). Transgene expression was readily seen in day 3 samples and was detectable in most high dose samples through the day 15-time point. Thereafter (days 29, 57, 92), slot blot results from vector-treated animals were not different from those of vehicle-treated animals. Additionally, on day 3, the expression of transgene was vector dose-dependent. None of the vehicle-treated rats showed the presence of neutralizing antibodies to Ad5 vectors in their sera. However, by day 29, four animals in the 2 × 10e8 pu vector dosage group exhibited low levels of serum neutralizing antibodies (2/gender; 1:4, 1:8, 1:8, 1:16), while all animals receiving 8×10e9 pu AdhAQP1 were positive for neutralizing antibodies (1:64 – 1:256; median 1:128). Similarly, all animals receiving 2×10e11 pu AdhAQP1 were positive for neutralizing antibodies on day 29, but with much higher titers (1:256 – 1:2048; median 1:1024). These results show that following AdhAQP1 administration to rat submandibular glands, hAQP1 protein is expressed, and anti-Ad5 neutralizing antibodies are produced, in a dose and time-dependent manner.