Intraperitoneal injection of rAAV2/8-HMBS to the AIP mice demonstrated increased levels of hepatic HMB-synthase activity that effectively and continuously prevented the phenobarbital induction of ALA and PBG for over 36 weeks. Ganetespib cancer Results rAAV2/8-HMBS-mediated expression is dose-responsive and tissue specific AIP mice were intraperitoneally administered 3.8 �� 1011, 5.7 �� 1011, or 7.6 �� 1011 DNase-resistant particles of rAAV2/8-HMBS and hepatic HMB-synthase levels were determined 1 week later. Although the saline-treated AIP mice had baseline activity of ~30% of wild-type, a dose-dependent increase of hepatic HMB-synthase activity was seen in the rAAV2/8-HMBS-treated mice (Figure 1). The highest vector dose (7.

6 �� 1011 DNase-resistant particles) achieved HMB-synthase levels slightly greater than mean wild-type levels (Figure 1), thus, this dose was used for all subsequent studies. Figure 1 Dose-response of hepatic HMB-synthase expression. AIP mice were administered 3.8 �� 1011, 5.7 �� 1011, or 7.6 �� 1011 drp of rAAV2/8-HMBS and the hepatic HMB-synthase activities were determined 1 week later. Black bars represent HMB-synthase … To investigate the tissue specificity of rAAV2/8-HMBS-mediated expression, various tissues including liver, kidney, heart, and brain were isolated 1 week after vector administration and evaluated for AAV vector copy number and HMB-synthase activities. Quantitative PCR analysis detected the highest amounts of rAAV2/8-HMBS vector DNA in the liver, with ~3.6 �� 104 copies/500 ng total DNA, followed by the kidney and heart, which had ~1.

6 �� 103 and ~500 copies/500 ng total DNA, respectively (Figure 2). Not unexpectedly, negligible amounts were detected in the brain. The HMB-synthase activity was increased ~3.5-fold in the liver, whereas the activity in the kidney, which had considerable amounts of the rAAV2/8-HMBS vector, remained at baseline levels (Figure 2). These results indicated that expression of HMB-synthase from the rAAV2/8-HMBS vector, driven by the ��1-microglobulin enhancer and ��1-antityrpsin promoter, was primarily restricted to the liver. Figure 2 Tissue specificity of rAAV2/8-HMBS-mediated HMB-synthase expression. Various tissues from saline- and rAAV2/8-HMBS-treated AIP mice were assayed for HMB-synthase activity (right) and vector copy number (left). The data presented are means + SD …

Sustained rAAV2/8-HMBS-mediated hepatic expression of HMB-synthase protects against phenobarbital-induced urinary ALA and PBG accumulation Hepatic HMB-synthase activity was determined in the rAAV2/8-HMBS-treated mice at 1, 2, 6, 12, 24, and 36 weeks after vector administration. Mean hepatic HMB-synthase activity increased to levels slightly greater than wild-type levels Brefeldin_A by 1 week and continually increased up to 6 weeks, when it achieved maximal mean activity of ~1.3-fold over mean wild-type levels (Figure 3).