250. Cross-Correction in Pompe Mice after Immunomodulation and Hepatic Delivery of Recombinant AAA5 and AAV8 Vectors

Abstract

Top of pageAbstract Pompe disease is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme acid α-glucosidase (GAA). The disorder causes cardiac and skeletal myopathy in infants, is fatal within two years of life, and protein therapeutic trials are currently underway. The ultimate goal of viral vector-mediated correction includes secretion/re-uptake of recombinant hGAA via the mannose 6-phosphate receptor (M6PR) pathway. Previous experience with rAAV serotype 2 vectors demonstrated moderate levels of transgene expression in the liver. We anticipate that significant hepatic overexpression of human GAA will be necessary for systemic correction of Pompe disease. Therefore, we tested the ability of rAAV serotype 5 and 8 (rAAV5 and rAAV8) vectors to direct GAA overexpression and secretion from the liver. We cross-packaged rAAV2-ITR containing genomes carrying the hGAA cDNA under the control of the duck hepatitis B viral (DHBV) promoter into AAV5 and AAV8 capsids, respectively. In a pilot study using the rAAV5 vector in Gaa-/- mice, we delivered 1012 vector genomes (vg) intraportally, which resulted in 4- to 16-fold overexpression of GAA; inhibitory anti-GAA antibody formation; no detectable enzyme activity in the heart or skeletal muscle; and significant levels of enzyme activity in the diaphragm that was dependent on both antibody titer and hepatic expression levels. In an effort to modulate this observed immune response, we developed a neonatal model of tolerization using subcutaneous, low dose delivery of recombinant hGAA to 1-day-old Gaa-/- mice. At 8 weeks post-tolerization, we intraportally delivered 1012 vg of rAAV5 and rAAV8 respectively. Hepatic expression of hGAA was 1.7-fold greater in rAAV8-treated mice compared to rAAV5 (26 ± 4.8-fold wild-type v. 15 ± 1.6-fold wild-type). Some mice from both groups exhibited antibody formation despite neonatal tolerization, and rAAV8 mice, as a group, had higher antibody titers (43-fold untreated v. 7-fold for rAAV5). Specific subjects in both rAAV5- and rAAV8-treated groups maintained tolerance, and in those subjects, significantly higher GAA activities were observed in the heart, diaphragm and skeletal muscle tissues, compared to non-tolerant, treated mice. Specifically, tolerant rAAV8-treated mice had hepatic GAA activities that were 34-fold wild-type, while tolerant rAAV5 mice had 10-fold hepatic overexpression. For both rAAV5 and rAAV8 GAA-tolerant mice, we observed diaphragmatic enzyme activities up to 3-fold of wild-type and cardiac activities reached 50% of wild-type, with concomitant histological reduction of stored glycogen. In summary, we have established that superphysiologic levels of liver GAA expression can be directed by both rAAV5 and rAAV8. These levels are sufficient to mediate cross-correction of target tissues, but can be adversely modulated by a humoral response. Clinical therapeutic strategies may require a combination of high levels of liver GAA expression and immunomodulation.