Melissa A. Lewis

Recombinant Adeno-Associated Virus Serotype 9 Leads to Preferential Cardiac Transduction In Vivo

Heart disease is often the end result of inherited genetic defects, which may potentially be treatable using a gene-transfer approach. Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a realistic method for the treatment of such disorders. Here, we demonstrate and compare the natural affinity of specific AAV serotype capsids for transduction of cardiac tissue. We compared the previously accepted optimal rAAV serotype for transduction of skeletal muscle, rAAV2/1, with rAAV2/8 and the newer rAAV2/9 vectors carrying the CMV-lacZ construct in their respective abilities to transcend vasculature and transduce myocardium following intravenous delivery of 1×1011 vector genomes in neonatal mice. We found that both rAAV2/8 and rAAV2/9 are able to transduce myocardium at ≈20- and 200-fold (respectively) higher levels than rAAV2/1. Biodistribution analysis revealed that rAAV2/9 and rAAV2/8 demonstrate similar behavior in extracardiac tissue. Vector genome quantification showed an increase in genome copy numbers in cardiac tissue for several weeks following administration, which corresponds to expression data. In addition, we intravenously administered 1×1011 vector genomes of rAAV2/9-CMV-lacZ into adult mice and achieved an expression biodistribution profile similar to that found following delivery to newborns. Although higher doses of virus will be necessary to approach those levels observed following neonatal injections, adult myocardium is also readily transduced by rAAV2/9. Finally, we have demonstrated physiological disease correction by AAV9 gene transfer in a mouse model of Pompe disease via ECG tracings and that intravenous delivery of the same vector preferentially transduces cardiac tissue in nonhuman primates.

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.

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.