Rapamycin control of exocrine protein levels in saliva after adenoviral vector-mediated gene transfer

Abstract

Transgene-encoded therapeutic secretory proteins can be efficiently secreted from salivary glands into saliva or the bloodstream after adenoviral (Ad)-mediated gene transfer. Since transgene expression from conventional vectors is typically unregulated, we evaluated the rapamycin-based dimerizer regulation system for control of transgene expression in, and consequent exocrine protein secreted from, rat salivary glands. We used human growth hormone (hGH) as a surrogate exocrine secretory protein. Two Ad vectors, Ad C4ZF3, encoding activation and DNA binding domain fusion polypeptides, and Ad Z12-I-GH-2, encoding hGH, were constructed and shown useful in vitro. Thereafter, both vectors were delivered into submandibular glands by retroductal infusion. After 24 h, rapamycin (0, 1, 3 or 10 mg/kg) was administered, and 20 h later hGH levels in saliva were determined. Salivary hGH levels were rapamycin concentration dependent. At a rapamycin dose of 10 mg/kg, total salivary hGH was 6937197 ng and the hGH concentration in saliva was 4.671.3 mg/ml. Over a 16-day experimental period, three separate administrations of rapamycin (3 mg/kg) induced distinct elevations of salivary hGH (B100-200 ng total hGH) that were entirely rapamycin dependent. This study demonstrates for the first time pharmacological control of transgenic exocrine protein production and presence in saliva after salivary gland gene transfer, and the potential for its application to the management of oral, oropharyngeal and upper gastrointestinal tract disorders.

Rapamycin control of transgene expression from a single AAV vector in mouse salivary glands

“…6 In that study, regulated rhesus EPO production was observed for 420 months (length of study) and was completely dependent on dimerizer drug delivery. 6 The single AAV2 vector described herein seems to retain the desirable features of the earlier generation rapamycin regulation systems, employing two vectors, as previously reported in muscle 5,6 and eye, 12 as well as SG, 7 and therefore seems to be a practical gene delivery tool for long-term therapeutics.…”

“…After administration of the rAd5 vector, robust and completely reversible, transgene expression was observed in rats. 7 However, such rAd5 vectors are unsuitable for achieving long-term transgene expression since they elicit a potent immune response. 8,9 In the absence of immunosuppressive therapy, transgene expression from first generation rAd5 vectors in SGs is typically lost within 7-14 days.…”

“…Following administration of rapamycin (3 mg/kg; a dose previously shown useful in rats), 7 and measurement of serum hEPO levels, the optimal vector dose determined was 10 10 particles/mouse administered to a single submandibular gland. All subsequent experiments herein presented were conducted with this vector dose.…”

“…6 We have recently shown that the rapamycin-inducible system can be useful for regulating transgene expression in SGs. 7 In those studies, we employed a two-vector system, with first-generation serotype 5 adenoviral (rAd5) vectors. After administration of the rAd5 vector, robust and completely reversible, transgene expression was observed in rats.…”

“…Rapamycin was initially dissolved in N, N-dimethylacetamide at a concentration of 5 mg/ml and stored at À201C. 7 Shortly before use, an equal volume of rapamycin (freshly diluted to 3 mg/ml) was mixed with PEG400: Tween 80 (9:1) and vortexed until homogenized, resulting in a 1.5 mg/ml final concentration. Rapamycin was diluted as needed in the same solution, and administered on day zero at 2 ml/g body weight by intraperitoneal injection.…”