Figure 3 Time course of rAAV2/8-HMBS-mediated hepatic HMB-synthase expression. AIP mice were sacrificed 1, 2, 6, 12, 24, and 36 weeks after vector administration and the hepatic HMB-synthase activities were determined. Results are shown as mean �� SD. Of … rAAV2/8-HMBS- and saline-treated different AIP mice were challenged with intraperitoneal injections of phenobarbital at 2, 10, 22, and 34 weeks after treatment to evaluate whether the expressed HMB-synthase enzyme prevented the biochemical induction of porphyrin precursors. Consistent with previous studies, urinary ALA and PBG increased markedly in the saline-treated AIP control mice, reaching levels of six- and tenfold greater, respectively, than the mean baseline values.

10,11 In contrast, phenobarbital induction in the rAAV2/8-HMBS-treated AIP mice did not increase their urinary ALA or PBG concentrations, which remained at levels similar to those in the wild-type mice (Figures 4a,b). This was consistent in all four experiments in which the mice were induced with phenobarbital. Figure 4 Urinary porphyrin precursor levels following phenobarbital injections. After collection of two baseline urine samples (days 1 and 2), increasing doses of phenobarbital (110, 120, 125, 130 mg/kg/day) were administered to wild-type (triangles) and … To investigate whether the AAV8-mediated HMB-synthase activity altered the response of hepatic ALAS1 expression to phenobarbital induction, relative ALAS1 transcript levels were determined at baseline and 9 hours after the fourth and final phenobarbital injection (130 mg/kg) at 34 weeks after rAAV2/8-HMBS treatment.

Real-time PCR analysis showed that baseline mean ALAS1 expression levels in the saline-treated AIP mice were approximately threefold higher than those of wild-type mice (relative ALAS1 transcript levels: 47.9 and 17.8, respectively), consistent with previous findings.10 Nine hours following the final phenobarbital injection, mean hepatic ALAS1 expression levels in the saline-treated AIP mice were markedly increased, whereas only slight increases were detected in the wild-type mice (230 versus 26.6, respectively). Not only did AAV8 treatment of the AIP mice normalize baseline hepatic ALAS1 levels (47.9 to 11.6), but it also decreased the phenobarbital-induced ALAS1 expression by approximately threefold (230 to 82.0). rAAV2/8-HMBS therapy improves neuromotor function of AIP mice Previously, it was shown that the AIP mice develop chronic progressive neuromotor impairment by 6 months of age.8 Therefore, the impact of rAAV2/8-HMBS treatment on motor coordination and balance skills was assessed by rotarod testing at 24 weeks Carfilzomib after vector administration, when the mice were 7 months of age.