Systemic correction of the muscle disorder glycogen storage disease type II after hepatic targeting of a modified adenovirus vector encoding human acid-alpha-glucosidase

Abstract

This report demonstrates that a single intravenous administration of a gene therapy vector can potentially result in the correction of all affected muscles in a mouse model of a human genetic muscle disease. These results were achieved by capitalizing both on the positive attributes of modified adenovirus-based vectoring systems and receptor-mediated lysosomal targeting of enzymes. The muscle disease treated, glycogen storage disease type II, is a lysosomal storage disorder that manifests as a progressive myopathy, secondary to massive glycogen accumulations in the skeletal and/or cardiac muscles of affected individuals. We demonstrated that a single intravenous administration of a modified Ad vector encoding human acid alpha-glucosidase (GAA) resulted in efficient hepatic transduction and secretion of high levels of the precursor GAA proenzyme into the plasma of treated animals. Subsequently, systemic distribution and uptake of the proenzyme into the skeletal and cardiac muscles of the GAA-knockout mouse was confirmed. As a result, systemic decreases (and correction) of the glycogen accumulations in a variety of muscle tissues was demonstrated. This model can potentially be expanded to include the treatment of other lysosomal enzyme disorders. Lessons learned from systemic genetic therapy of muscle disorders also should have implications for other muscle diseases, such as the muscular dystrophies.

Figure 1

Construction of AdhGAAΔpol. Schematic representation.

Figure 2

Human GAA secretion after infection of human cells. Human 293 cells were infected with the respective vectors, and medium was removed and assayed for GAA activity at the indicated time points.

Figure 3

Detection of hGAA in the plasma of treated mice. 1 × 10⁹ plaque-forming units of AdhGAAΔpol were intravenously injected into the respective strains of mice, and GAA activities in the plasma was measured at the respective time points. n = 3 for Ad treated mice, and n = 5–6 for uninfected mice. dpi, days post-infection.

Figure 4

Detection of hGAA protein in plasma of treated animals. Equivalent amounts of plasma were probed for the presence of hGAA proteins, as described in Materials and Methods. The 110-kDa precursor protein represents unprocessed hGAA whereas the processed form of hGAA has a molecular mass of 76 kDa.

Figure 5

Detection of hGAA activity in the tissues of treated mice. Respective tissues were harvested 12 days after AdhGAAΔpol infection and were analyzed for GAA activity, as described in Materials and Methods. n = 3 for Ad treated animals, and n = 4–6 for uninfected control animals.

Figure 6

Lack of detection of hGAA mRNA in quadriceps and heart tissues of AdhGAAΔpol-treated GAA-KO mice. Respective tissues from a C57BL/6 mouse or tissues derived from GAA-KO mouse that had been given AdhGAAΔpol 12 days prior were harvested, and total RNA was analyzed for the presence of hGAA, as described in Materials and Methods. The Ad-treated mouse had GAA activities (nmol/hr/mg) of 44.3 in the heart, 214.7 in the quadriceps muscle, and 21,790 in the liver whereas C57BL/6 mice had average GAA activities of 22.15 in the heart, 10.2 in the quadriceps, and 98.7 in the liver. The amount of 28s rRNA present in each sample before probing is presented as well.

Figure 7

Periodic acid-Schiff detection of glycogen in situ. Cryosections from 2.5-month-old mouse tissues were periodic acid-Schiff stained for glycogen as described in Materials and Methods. Source of the tissue sections are as follows: (a) GAA-KO, gastrocnemius, untreated. (b) GAA-KO, gastrocnemius, 12 days post-AdhGAAΔpol. (c) GAA-KO, quadriceps, untreated. (d) GAA-KO, quadriceps, 12 days post-AdhGAAΔpol. (e) GAA-KO, diaphragm, untreated. (f) GAA-KO, diaphragm, 12 days post-AdhGAAΔpol. (g) GAA-KO, heart, untreated. (h) GAA-KO, heart, 4 days post-AdhGAAΔpol. (i) GAA-KO, heart, 12 days post-AdhGAAΔpol. (j) Wild-type, heart, untreated.