Sustained correction of glycogen storage disease type II using adeno-associated virus serotype 1 vectors.

Glycogen storage disease type II (GSDII) is caused by a lack of functional lysosomal acid alpha-glucosidase (GAA). Affected individuals store glycogen in lysosomes beginning during gestation, ultimately resulting in fatal hypertrophic cardiomyopathy and respiratory failure. We have assessed the utility of recombinant adeno-associated virus (rAAV) vectors to restore GAA activity in vivo in a mouse model of GSDII (Gaa(-/-)). A single systemic administration of a rAAV serotype 1 (rAAV1) vector to neonate animals resulted in restored cardiac GAA activity to 6.4 times the normal level (mean=641+/-190% of normal (Gaa(+/+)) levels with concomitant glycogen clearance) at 11 months postinjection. Greater than 20% of normal levels of GAA activity were also observed in the diaphragm and quadriceps muscles. Furthermore, functional correction of the soleus skeletal muscle was also observed compared to age-matched untreated Gaa(-/-) control animals. These results demonstrate that rAAV1 vectors can mediate sustained therapeutic levels of correction of both skeletal and cardiac muscles in a model of fatal cardiomyopathy and muscular dystrophy.

Intercellular transfer of the virally derived precursor form of acid alpha-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.

Effect of altering filling pattern on diastolic pressure-volume curve.

BACKGROUND: The early-to-late ventricular filling ratio (E:A) is widely used to index diastolic function. While filling patterns reflect diastolic properties, they can also modulate chamber pressures due to myocardial viscoelasticity. We hypothesized that such feedback can potentially temper effects of delayed relaxation and/or volume loading on diastolic pressures. METHODS AND RESULTS: Six isolated blood-perfused canine left ventricles were studied with ejection and filling controlled by an intracavitary volume servo-pump. Diastolic filling was determined by a simulated atrial pressure source that was either constant or varied to yield dual-phase filling at a specified E:A ratio. E:A ratio was randomly set to 3:1, 1:3, or 1:1, and data were recorded at each ratio at three different preloads. With principally early filling (E:A=3:1), diastolic pressure rise from viscosity increased in proportion with the relaxation time constant (r=.91, P<.0001). However, this dependence was lost as E:A ratio declined (eg, P=.63 for E:A 1:3). Furthermore, E:A=3:1 yielded 37% to 50% lower end-diastolic pressures at similar volumes (versus E:A=1:3) as initial viscous forces decayed. Offsetting early and late filling effects led to little net change in mean diastolic pressure independent of E:A ratio or preload. CONCLUSIONS: Diastolic filling pattern itself influences chamber pressures early and late in diastole due to viscoelasticity, with larger net effects on end-diastolic pressure. Since E:A ratio normally falls with delayed relaxation but rises with higher preload or reduced compliance, the present results suggest that changes in filling pattern may modulate direct effects of such factors on elevating diastolic pressure.