Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands

Abstract

Objectives: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands.

Methods: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval.

Results: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal.

Conclusion: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

Figure 1

Schematic representation of the AAV-TF-RhEpo-2.3w vector. ITR, inverted terminal repeat. Pcmv, cytomegalovirus promoter. TF, transcription factor. IRES, internal ribosome entry site. pA, polyadenylation signal. PIL-2, interleukin-2 promoter. RhEpo, rhesus erythropoietin cDNA. See Materials and Methods for additional details.

Figure 2

Demonstration of rapamycin responsiveness of the AAV-TF-RhEpo-2.3w vector in vitro. Vector was added to 293 cells at the indicated multiplicities of infection. After 24 hours, medium was replaced with serum-free medium, and rapamycin added at the indicated concentrations. Following incubation for an additional 24 hours, medium was collected and assayed for rhesus erythropoietin (RhEpo). Data shown are the average of duplicate determinations. See Materials and Methods for additional details.

Figure 3

Effect of rapamycin administration on serum RhEpo (rhesus erythropoietin) levels in all six macaques across the entire time course of this study. A. Experimental timeline for this study. Upward facing arrow indicates time when vector was administered (day 0), while downward facing arrows indicate times (* symbol) when blood samples were collected for determination of RhEpo (rhesus erythropoietin) levels, CBC (complete blood counts) and serum chemistry values. The # and ^ symbols indicate times when rapamycin was administered. Note that only one animal was administered rapamycin on day 98 (# 6852), because of difficulty getting venous access on day 84. See text for additional details. B. Each panel represents data from an individual monkey. The “y-axis” indicates the relative level of RhEpo detected 24 hours after rapamycin administration with 1 (dashed line in each panel) equal to no difference in serum RhEpo levels from before rapamycin administration, i.e., Relative RhEpo level = serum RhEpo after rapamycin/serum RhEpo before rapamycin. Thus, values <1 indicate a reduction in serum RhEpo levels compared with before rapamycin administration, and values >1 indicate an increase in serum RhEpo levels compared with before rapamycin administration. The three left panels indicate low dose animals and the three right panels indicate high dose animals. Specific animal study numbers are shown in the bottom right corner of each panel. Animal #6851 is the macaque that experienced the severe adverse event and was euthanized. Note that the final data point shown for animal #6847 is approximate, as the before rapamycin administration RhEpo level on day 84 was undetectable and the after rapamycin administration RhEpo level was 0.2 mU/ml. Also, note that the last time point for animal #6852 was on day 98 after vector delivery, because of technical difficulties in obtaining blood on day 84. The last time point for the remaining four macaques that completed the study was on day 84. See text for additional details.