Kerry O. Cresawn

Gel-mediated Delivery of AAV1 Vectors Corrects Ventilatory Function in Pompe Mice With Established Disease

Pompe disease is a muscular dystrophy that results in respiratory insufficiency. We characterized the outcomes of targeted delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragms of Pompe mice with varying stages of disease progression. We observed significant improvement in diaphragm contractile strength in mice treated at 3 months of age that is sustained at least for 1 year and enhanced contractile strength in mice treated at 9 and 21 months of age, measured 3 months post-treatment. Ventilatory parameters including tidal volume/inspiratory time ratio, minute ventilation/expired CO2 ratio, and peak inspiratory airflow were significantly improved in mice treated at 3 months and tested at 6 months. Despite early improvement, mice treated at 3 months and tested at 1 year had diminished normoxic ventilation, potentially due to attenuation of correction over time or progressive degeneration of nontargeted accessory tissues. However, for all rAAV2/1-treated mice (treated at 3, 9, and 21 months, assayed 3 months later; treated at 3 months, assayed at 1 year), minute ventilation and peak inspiratory flows were significantly improved during respiratory challenge. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors can significantly augment ventilatory function at initial and late phases of disease in a model of muscular dystrophy.

911. Comparison of rAAV Serotype, Promoter, and Treatment Age for the Correction of Glycogen Storage Disease Type II

Glycogen storage disease type II is a prototypical lysosomal storage disease caused by a deficiency in the enzyme acid -glucosidase (GAA). This disorder causes cardiac and skeletal myopathy and respiratory insufficiency. In the most severely affected individuals, GSDII is fatal within 2 years of life. In effort to achieve optimal levels of transgene expression in the affected cardiac and skeletal muscles, we evaluated 3 recombinant adeno-associated viral (rAAV) vectors expressing human GAA (hGAA); rAAV1- and rAAV8-hGAA under control of the CMV promoter and rAAV8-hGAA under control of a liver specific promoter (LSP). These vectors were delivered to both neonatal and adult GSDII mice. In general, treatment of neonatal mice was more successful than adult mice and serotype 8 vectors resulted in higher expression levels than serotype 1 vectors. At 10 wks post-injection (PI), rAAV8-CMV-hGAA- treated mice had 2.8-fold higher levels of GAA activity in the heart and 10.5-fold higher levels in the diaphragm compared to rAAV1-CMV-hGAA- treated mice (with averages of 45-fold normal and 8.7-fold normal in the respective tissues of rAAV8-treated mice). GAA activity in the hind-limb muscles was also significantly improved in rAAV8-treated mice with up to 83 30% of normal GAA activity in the tibialis anterior muscle. In neonatal mice treated with rAAV8-LSP-hGAA, GAA levels gradually dropped from 1 to 10 wks PI, which was attributed to the progressive loss of vector genomes in the transduced neonatal liver.

Gene therapy for lysosomal storage disorders

Publisher Summary This chapter discusses gene therapy for the treatment of lysosomal storage diseases (LRDs). Gene therapy is an attractive alternative to enzyme replacement therapy (ERT) for several reasons. Gene therapy offers the potential for a sustained therapeutic effect from a single administration of vector. In contrast to ERT, this single treatment decreases the probability of an immune response to the enzyme and would also decrease the cost to the patient. LSDs are ideal candidates for gene therapy treatment for many reasons. LSDs are generally well-characterized, single-gene disorders. While the amount of enzyme required correcting the disease varies depending on the enzyme deficiency and affected tissues, restoring enzyme levels to those observed in unaffected heterozygote patients or patients with a milder LSD phenotype should significantly improve survival and quality of life. The ability of cells to both secrete newly synthesized enzyme and endocytose circulating enzyme allows for gene therapy-mediated correction of multiple tissues either by systemic vector administration or direct vector administration to one tissue that will in turn synthesize and secrete functional enzyme for another.

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

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.