674. Administration of rAAV2 and rAAV5 to the Parotid Gland of Non-Human Primates
Authors: A. Voutetakis, C. Zheng, J.A. Chiorini, M. Schmidt, S. Afione, M. Metzger, C. Dunbar, R.E. Donahue, B.J. Baum
Journal: Molecular Therapy (2006)

Previous studies from our laboratory showed that therapeutic levels of transgene expression were obtained from mouse submandibular glands after retro-ductal administration of relatively low doses (109 particles/animal) of a recombinant serotype 2 adeno- associated viral vector (rAAV2) encoding human erythropoietin (hEPO). The recombinant hormone was preferentially secreted into the bloodstream rather than into saliva. Serum hEPO levels steadily increased for an 8–12 week period and remained relatively stable until the end of the experiment (54 weeks; Voutetakis et al, PNAS, 2004).In the present study, we have extended this approach to non- human primates. Our goal was to compare the efficacy of two widely used AAV serotypes (2 and 5) and study the potential clinical applicability of gene transfer to the salivary glands in a large animal model. Both the rAAV2 and rAAV5 vectors used expressed rhesus macaque erythropoietin (RhEPO) under the control of the Rous Sarcoma Virus promoter. Viral vectors were produced using conventional methods and titers were determined by Q-PCR. A total of four groups (n=3/dosage group) of macaques received either 1010 (low dose) or 3x1011 (high dose) particles of either rAAV2RhEPO or rAAV5RhEPO via intraoral cannulation of the right parotid gland. The higher dose used was comparable on a particle per kg basis to the dosing used in our previous murine experiments. Serum and saliva were obtained at multiple time points, both prior to and post vector administration. RhEPO levels were measured using a commercially available hEPO Elisa kit which cross- reacts with RhEPO.Prior to vector administration, serum RhEPO levels were minimal (0.41±0.2 mU/ml; 6 weeks prior to vector delivery for all 12 macaques used) with no animal measuring >1.6 mU/ml. Saliva RhEPO levels were below the lowest standard used in the assay (0.8 mU/ml). Serum RhEPO levels increased to 3.36±1.2 and 3.8±0.75 mU/ml in the high dose groups for both rAAV2 and rAAV5 administered animals, respectively at week 8 post-delivery of vector. RhEPO expression was vector dose-dependent. Salivary RhEPO levels increased similarly. rAAV2-mediated RhEPO expression increased quickly and remained stable. Our data show that the kinetics of transgene expression are different between the two vector serotypes in salivary glands of macaques, and are not predicted by the kinetics observed in murines. In agreement with our mouse studies, RhEPO produced in the targeted gland was preferentially secreted into the bloodstream. However, hematocrit (Hct) values did not increase appreciably (maximum 5% compared to pre-administration levels), probably due to the relatively low overall levels of RhEPO achieved. The immune response (as measured by the presence of circulating anti-AAV antibody titers) was higher after AAV5 than after AAV2 vector administration. The latter result is in accord with those in mice (Katano et al Gene Ther, 2006).In summary, this study shows that administration of low doses of AAV2 and AAV5 vectors administered to the salivary glands of non-human primates can result in the production and secretion of a recombinant protein into the bloodstream. Previous studies from our laboratory showed that therapeutic levels of transgene expression were obtained from mouse submandibular glands after retro-ductal administration of relatively low doses (109 particles/animal) of a recombinant serotype 2 adeno- associated viral vector (rAAV2) encoding human erythropoietin (hEPO). The recombinant hormone was preferentially secreted into the bloodstream rather than into saliva. Serum hEPO levels steadily increased for an 8–12 week period and remained relatively stable until the end of the experiment (54 weeks; Voutetakis et al, PNAS, 2004). In the present study, we have extended this approach to non- human primates. Our goal was to compare the efficacy of two widely used AAV serotypes (2 and 5) and study the potential clinical applicability of gene transfer to the salivary glands in a large animal model. Both the rAAV2 and rAAV5 vectors used expressed rhesus macaque erythropoietin (RhEPO) under the control of the Rous Sarcoma Virus promoter. Viral vectors were produced using conventional methods and titers were determined by Q-PCR. A total of four groups (n=3/dosage group) of macaques received either 1010 (low dose) or 3x1011 (high dose) particles of either rAAV2RhEPO or rAAV5RhEPO via intraoral cannulation of the right parotid gland. The higher dose used was comparable on a particle per kg basis to the dosing used in our previous murine experiments. Serum and saliva were obtained at multiple time points, both prior to and post vector administration. RhEPO levels were measured using a commercially available hEPO Elisa kit which cross- reacts with RhEPO. Prior to vector administration, serum RhEPO levels were minimal (0.41±0.2 mU/ml; 6 weeks prior to vector delivery for all 12 macaques used) with no animal measuring >1.6 mU/ml. Saliva RhEPO levels were below the lowest standard used in the assay (0.8 mU/ml). Serum RhEPO levels increased to 3.36±1.2 and 3.8±0.75 mU/ml in the high dose groups for both rAAV2 and rAAV5 administered animals, respectively at week 8 post-delivery of vector. RhEPO expression was vector dose-dependent. Salivary RhEPO levels increased similarly. rAAV2-mediated RhEPO expression increased quickly and remained stable. Our data show that the kinetics of transgene expression are different between the two vector serotypes in salivary glands of macaques, and are not predicted by the kinetics observed in murines. In agreement with our mouse studies, RhEPO produced in the targeted gland was preferentially secreted into the bloodstream. However, hematocrit (Hct) values did not increase appreciably (maximum 5% compared to pre-administration levels), probably due to the relatively low overall levels of RhEPO achieved. The immune response (as measured by the presence of circulating anti-AAV antibody titers) was higher after AAV5 than after AAV2 vector administration. The latter result is in accord with those in mice (Katano et al Gene Ther, 2006). In summary, this study shows that administration of low doses of AAV2 and AAV5 vectors administered to the salivary glands of non-human primates can result in the production and secretion of a recombinant protein into the bloodstream.