Previously published experiments in rodents, miniature pigs, and nonhuman primates using Ad5 and adeno-associated viral (AAV) vectors have demonstrated the potential clinical utility of SGs for gene therapeutics applications (e.g., Hoque et al.
; Baum et al.
; Voutetakis et al.
; Yan et al.
). SGs can secrete transgene products both into saliva and the bloodstream (Baum et al.
), which can be applied to treat both oral and upper gastrointestinal tract disorders, as well as systemic monopeptide deficiencies. SGs use two general secretory pathways: a predominant RSP leading to exocrine (apical) protein secretion into saliva via zymogen granules and a CSP, leading mainly to endocrine (basolateral) secretion into the interstitium and bloodstream (Castle and Castle, 1998
; Baum et al.
; Isenman et al.
; Gorr et al.
). Previously, we have extensively studied transgenic secretory protein sorting in SGs of male mice and minipigs, using Ad5 vectors encoding hGH and hEPO cDNAs. Whereas in both species hGH continues to follow the same general secretory pathway (RSP) as in their primary site of production, hEPO (a CSP protein) is handled in a dramatically different manner by male mouse (see herein; Voutetakis et al.
) and minipig SGs (Yan et al.
). The present study was conducted in part to address this remarkable dissimilarity.
Average Erythropoietin Levels in Serum and Saliva of Male Mice, Rats, Miniature Pigs, and Rhesus Macaques after AdhEPO Gene Transfera
Male macaques received either 1010
particles per gland of either AdhGH or AdhEPO via intraoral cannulation of both parotid glands. All animals tolerated vector administration well. Saliva was of normal consistency with absence of purulence. Food consumption and weight gain were normal, suggesting parotid gland transduction by the Ad5 vectors caused little to no ill effects. There were no significant changes in hematological values, but there were a few minor, clinically insignificant and dose-independent changes in serum chemistry values, generally similar for both vectors (e.g., amylase, creatine phosphokinase, and lactate dehydrogenase levels transiently increased and serum Fe2+
decreased; data not shown). Because no experimental animals were killed, no tissue biodistribution data are available. However, we performed qPCR studies to assess vector presence in blood samples 10
hr, and 1 day after administration. Importantly, the absence of vector in all of these samples suggests that little if any dissemination occurred beyond the targeted glands (data not shown; and Zheng et al.
; Yan et al.
). Furthermore, previously published detailed functional and safety studies with administration of similar doses of an Ad5 vector encoding human aquaporin-1 (AdhAQP1) to macaque parotid glands (O'Connell et al.
) showed no untoward local or systemic effects.
The pattern of transgene production herein (i.e., the rapid elevation followed by rapid decline within 2 weeks; and ) is identical to what we have previously seen in rodent (Kagami et al.
), miniature pig (Li et al.
), and macaque (O'Connell et al.
) parotid glands, that is, it is kinetically reproducible in all species. Further, it seems to be Ad5 vector specific because long-term SG secretion of hEPO and rhesus EPO has been observed with AAV2 vectors (Voutetakis et al.
). As observed in our previous mouse, rat, and minipig experiments, macaque serum hGH levels were below the detection limit at almost all time points for both dose groups. Accordingly, when we measured IGF-I levels in the AdhGH-treated macaques no significant changes were observed (data not shown).
FIG. 1. Serum and salivary human growth hormone (hGH) levels in macaques. Two groups (n=2 per dose group) of male macaques received either 1011 (high dose; A) or 1010 (low dose; B) particles of AdhGH via intraoral cannulation of the parotid (more ...)
FIG. 2. Serum and salivary human erythropoietin (hEPO) levels in macaques. Two groups (n=2 per dose group) of male macaques received either 1011 (high dose; A) or 1010 (low dose; B) particles of AdhEPO via intraoral cannulation of the parotid (more ...)
After AdhEPO administration to the high-dose group, day 3 salivary hEPO concentrations were ~7-fold higher than those in serum, but the total amount of secreted hEPO was ~15-fold higher in serum (not shown; see also low dose group in ). These hEPO sorting results in macaques after Ad5 vector-mediated gene transfer are intermediate to those observed in mice and minipigs and somewhat similar to those found for rats (). Furthermore, the pattern of hEPO secretion is similar to that previously observed with rAAV2RhEPO administration to nonhuman primates (Voutetakis et al.
), suggesting the sorting results are independent of the vector type used for transduction (Ad5 vs. AAV2), or of the cell types targeted (Ad5 targets both acinar and duct cells, whereas AAV2 targets primarily duct cells; O'Connell et al.
; Voutetakis et al.
Interestingly, the transient rise in serum hEPO levels of these nonanemic animals did not result in an increase in relevant hematologic parameters (e.g., red blood cell count, hemoglobin, and hematocrit percent; Kendall, 2001
). We know that the transgene product, hEPO protein, is biologically active, as in minipigs treated with the same AdhEPO vector much lower serum hEPO levels led to significant elevations in hematocrit (Yan et al.
). The reason for the lack of demonstrable biological function in the macaques herein is unclear, but may be attributable to tighter physiological controls on red cell production in this species compared with minipigs.
Gene transfer to SGs for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. However, as pointed out by the results presented herein, (1) clear differences in the sorting of the model transgenic CSP protein hEPO exist between the four animal models studied (mouse, rat, minipig, and macaque) and (2) it is difficult to predict exactly how transgenic hEPO protein would be sorted by human SGs. Although the RSP is considered a more complex and tightly controlled secretion pathway than the CSP (Loh et al.
), our current and previous results clearly show that in all studied species, the model RSP protein hGH is rigorously sorted via an exocrine pathway into saliva. In contrast, transgenic hEPO can be found both in saliva and serum in various proportions depending on the species studied.
The mechanism(s) responsible for the differences in hEPO-sorting patterns in SGs shown in are not yet clear. We hypothesize that our hEPO results reflect important differences in the expression and/or metabolic fate of key molecules essential to the recognition and sorting of the protein within SGs of mice, rats, minipigs, and macaques. Since the description of the SNARE (soluble N
-ethylmaleimide-sensitive factor attachment protein receptor) hypothesis by Rothman (1994
), it is clear that the intracellular pathways followed by membrane and secretory proteins in epithelial cells, including SGs (e.g., Fölsch et al.
; Gaisano, 2000
), are directed by a huge repertoire of finely regulated molecules (e.g., Nakatsu and Ohno, 2003
; Rodriguez-Boulan et al.
). Thus, there are many intracellular steps at which a metabolic or genetic change can lead to a dramatic difference in the final destination of a membrane or secretory protein.
In conclusion, for SGs to be generally useful for systemic gene therapeutics sorting differences such as presented herein must be understood so that human applications can be performed efficiently and safely. On the basis of our studies, we hypothesize that mouse and rat submandibular glands, which are in general structurally and physiologically similar, have significant inherent differences in one or more key intracellular molecule(s) used in sorting hEPO for secretion. Identification of those differences may lead to useful strategies to employ for potential clinical applications of SG gene transfer.