Thomas J. Fraites

Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml.

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease.

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid α-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.

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.

Intercellular Transfer of the Virally Derived Precursor Form of Acid α-Glucosidase Corrects the Enzyme Deficiency in Inherited Cardioskeletal Myopathy Pompe Disease

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.

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.