Late responses to adenoviral-mediated transfer of the aquaporin-1 gene for radiation-induced salivary hypofunction

Abstract

We evaluated late effects of AdhAQP1 administration in five subjects in a clinical trial for radiation-induced salivary hypofunction (http://www.clinicaltrials.gov/ct/show/NCT00372320?order=). All were identified as initially responding to human aquaporin-1 (hAQP1) gene transfer. They were followed for 3-4 years after AdhAQP1 delivery to one parotid gland. At intervals we examined salivary flow, xerostomic symptoms, saliva composition, vector presence and efficacy in the targeted gland, clinical laboratory data and adverse events. All displayed marked increases (71-500% above baseline) in parotid flow 3-4.7 years after treatment, with improved symptoms for ~2-3 years. There were some changes in [Na⁺] and [Cl^-] consistent with elevated salivary flow, but no uniform changes in secretion of key parotid proteins. There were no clinically significant adverse events, nor consistent negative changes in laboratory parameters. One subject underwent a core needle biopsy of the targeted parotid gland 3.1 years post treatment and displayed evidence of hAQP1 protein in acinar, but not duct, cell membranes. All subjects responding to hAQP1 gene transfer initially had benefits for much longer times. First-generation adenoviral vectors typically yield transit effects, but these data show beneficial effects can continue years after parotid gland delivery.

Figure 1

Stimulated parotid salivary flow rates of the targeted gland at key time points from each subject responding positively to AdhAQP1 treatment. The color-coding for subjects in this figure are the same as in Figure 3, and as those published in Baum et al . The Y-axis shows salivary flow in mL/min from the targeted parotid gland. The X-axis shows five key time points in this study, with the specific days for each indicated above data points in the figure. Baseline represents each subject’s initial visit to the NIH Clinical Center prior to any procedures being performed. The initial peak increase in salivary flow represents the time point (varied from day 7-42) following AdhAQP1 administration when the subject’s parotid salivary flow was maximal. Both the Baseline and initial peak data were presented previously in Baum et al . The endpoint of the originally approved study was on day 360 visit after vector delivery for each subject. Thereafter, the original clinical protocol was amended and we received permission to evaluate the effects of AdhAQP1 administration to all five responder-subjects for an additional two years of follow-up. The exact days following vector administration for the two follow-up visits are different for each subject (shown also in Table 1). They are shown in the figure and are as follows: #19 (days 1124 and 1708, respectively), #50 (1132 and 1531), #99 (721 and 1141), #103 (797 and 1119), and #118 (721 and 1086).

Figure 2

Depiction of the changes from baseline for stimulated parotid salivary flow rates at each time point in this clinical trial from the AdhAQP1-treated gland (A) and the contralateral untreated gland (B). Statistical significance is indicated using the dark (p<0.05) and light blue (p>0.05) shading of each bar, and the 95% confidence intervals are depicted. Bars above the line represent an increase in salivary flow rate, while those below indicate a decrease in salivary flow rate. Statistical analysis used GEE modeling as presented in Tables 2 and 3. Visit numbers represent the following time points: 1 (baseline), 2 (6 hours), 3 (day 1), 4 (day 2), 5 (day 3), 6 (day 7), 8 (day 28), 9 (day 42), 10 (day 90), 11 (day 120), 12 (day 150), 13 (day 180, 14 (day 360), 15 (follow-up visit 1), 16 (follow-up visit 2). The exact times for the two follow-up visits are presented in Figure 1 and its legend.

Figure 3

Visual analog scale (VAS) measurements of two key xerostomic symptoms at key time points in responder-subjects. A. Rate the dryness in your mouth; B. Rate how much saliva is in your mouth. A lower score indicates an improvement in the symptom. The color-coding for subjects in this figure are the same as in Figure 1, and as those published in Baum et al . Baseline and initial peak data were presented previously in Baum et al . As noted in the legend to Figure 1, the endpoint of the originally approved study was on day 360 visit after vector delivery for each subject. The two follow-up visits are different for each subject (presented in Figure 1). They are as follows: #19 (days 1124 and 1708, respectively), #50 (1132 and 1531), #99 (721 and 1141), #103 (797 and 1119), and #118 (721 and 1086). The Y-axis represents the visual analogue scale numerical value [10 cm scale, with 10 being the driest mouth (A) or the lowest amount of saliva (B) imaginable]. The form used for the visual analogue scale measurements can be found in Supplemental Figure 1 and was derived from Pai et al .

Figure 3

Visual analog scale (VAS) measurements of two key xerostomic symptoms at key time points in responder-subjects. A. Rate the dryness in your mouth; B. Rate how much saliva is in your mouth. A lower score indicates an improvement in the symptom. The color-coding for subjects in this figure are the same as in Figure 1, and as those published in Baum et al . Baseline and initial peak data were presented previously in Baum et al . As noted in the legend to Figure 1, the endpoint of the originally approved study was on day 360 visit after vector delivery for each subject. The two follow-up visits are different for each subject (presented in Figure 1). They are as follows: #19 (days 1124 and 1708, respectively), #50 (1132 and 1531), #99 (721 and 1141), #103 (797 and 1119), and #118 (721 and 1086). The Y-axis represents the visual analogue scale numerical value [10 cm scale, with 10 being the driest mouth (A) or the lowest amount of saliva (B) imaginable]. The form used for the visual analogue scale measurements can be found in Supplemental Figure 1 and was derived from Pai et al .

Figure 4

Anti-Ad5 serum neutralizing antibody titers in responder-subjects. Anti-Ad5 neutralizing serum antibodies employed a previously described assay ^,. The assay tests the ability of serum dilutions to block the transduction of 293 cells by an Ad5 vector, AdCMVLuc encoding luciferase. The titers (Y-axis) represent serum dilutions resulting in a 50% inhibition of transduction. The X-axis represents time points in the study. B = baseline, while all other time points shown are days following AdhAQP1 administration. Individual subject symbols are indicated. No sample was available to perform this assay at the second follow-up visit of subject 19. The exact days of each follow-up visit are listed in the legends of Figures 1 and 3, and Table 1.Note that serum neutralizing antibody titers found in all time points through day 42 have recently been reported and were generally similar to those shown in the figure .

Figure 5

Assessment of protein composition in patient parotid saliva samples by BisTris PAGE (A), cationic PAGE (B) and anionic PAGE (C). Left panels: samples from subject 19; middle panels: samples from subject 50; right panel, samples from subject 99. Volumes loaded in A, B and C were 10 ul, 25 ul and 50 ul, respectively. In B, samples indicated with * and ** indicate 50 ul and 75 ul volumes were loaded, respectively. Numbers above the lanes refer to the day post treatment, with B in that position indicating baseline. The migration positions and molecular weight (MW) of the major proteins in each of the three PAGE types are indicated: Amylase in A; histatins (His) 1, 3 and 5 in B; and proline-rich proteins 1 and 2 (PRP1, PRP2) in C. The MW standards used in A, B and C were 10 ul BioRad MW standard, 8 ug histatin 1, 3, and 5 each, and 20 ug PRP1, respectively. Note low histatin levels in the subject samples, but increasing the saliva amounts analyzed revealed their presence. Note furthermore that all subjects expressed acidic PRPs and that different isoforms were expressed by the three subjects.

Figure 6

Results of conventional PCR assay with DNA extracted from tissue obtained with a modified sialoendoscopic biopsy of subject # 99. See SUBJECTS AND METHODS for details of the PCR reaction conditions and primers. A. Schematic diagram of AdhAQP1 with the region of the PCR amplicon shown as a red line. The Ad5 genome is shown in black. The human cytomegalovirus promoter (CMV) is shown in orange. The human aquaporin-1 transgene is shown in green, followed by the SV40 polyadenylation signal shown in blue. B. PCR Result. Lane M contains DNA standard markers (sizes shown to left). In the left panel, lane P is the positive control sample and shows the 565 bp amplicon as obtained from an extract of AdhAQP1. Lane #99 shows the same 565 bp amplicon, which was obtained from DNA extracted from the targeted parotid gland tissue of subject 99 at follow-up visit 1 (day 721 after AdhAQP1 administration). In the right panel, lanes M and P are the same, but the sample in the lane labeled NPG was obtained from the normal parotid gland of a male volunteer who had not been administered AdhAQP1.

Figure 7

Images from core needle biopsy specimen obtained from subject #19 at follow-up visit 1 (day 1124 after AdhAQP1 administration). A. H&E staining of the parotid gland tissue sample, showing the presence of acini and ducts. B. Control for immunofluorescence staining using normal rabbit IgG as the primary antibody. The nuclei are stained using DAPI and have a blue color. C. Tissue stained with an antibody to human AQP5. The immunofluorescence staining observed is localized only to the luminal membrane of acinar cells (a) and the closely adjacent intercalated duct region. Larger ducts (d) do not express AQP5 and are unstained. D. Tissue stained with an antibody to human AQP1. The immunofluorescence staining is found in three cell types: myoepithelial, vascular endothelial and acinar. Normally, AQP1 is only present in myoepithelial and vascular endothelial cells (Gresz et al ). Acinar cells that can be seen expressing AQP1 (right central and bottom portion of panel) were transduced with AdhAQP1 administered to subject # 19 1124 days previously. E. An enlarged region of Panel D showing the presence of AQP1 in myoepithelial (yellow arrows) and vascular endothelial cells (red, smaller arrows) and the negative staining of a non-transduced acinus. F. An enlarged region of Panel D showing the abundant presence of AQP1 in the basolateral and luminal membranes of a transduced acinus. G. AQP1 localization in a biopsy specimen from a normal, male human volunteer’s parotid gland, i.e., without AdhAQP1 transduction. There is no immunofluorescence staining in acinar cells. H. An enlarged region of Panel G clearly showing the absence of AQP1 staining in acinar (a) and duct (d) cells, but its presence in myoepithelial cells (yellow arrows) and vascular endothelial cells (smaller red arrows). See SUBJECTS AND METHODS for details on the staining procedures and antibodies used.