Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral-mediated gene transfer.
BACKGROUND
Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig.


METHODS
Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR).


RESULTS
The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues.


CONCLUSIONS
Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs.
Yan
Xing
X
Salivary Gland Disease Center and the Molecular Laboratory for Gene Therapy, School of Stomatology, Capital Medical University, Beijing, PR China.
Voutetakis
Antonis
A
Zheng
Changyu
C
Hai
Bo
B
Zhang
Chunmei
C
Baum
Bruce J
BJ
Wang
Songlin
S
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D000906:Antibodies / Q000097:blood; D000917:Antibody Formation; D004921:Erythropoietin / Q000008:administration & dosage / Q000097:blood / Q000378:metabolism*; D018014:Gene Transfer Techniques; D019382:Human Growth Hormone / Q000008:administration & dosage / Q000097:blood / Q000378:metabolism*; D006801:Humans; D008297:Male; D009928:Organ Specificity; D010306:Parotid Gland / Q000378:metabolism* / Q000473:pathology; D021381:Protein Transport; D013552:Swine; D013556:Swine, Miniature / Q000378:metabolism*; D014018:Tissue Distribution; D019076:Transgenes
D000906:Antibodies; D004921:Erythropoietin; D019382:Human Growth Hormone