Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle

Abstract

Enzyme replacement therapy (ERT) with acid α-glucosidase has become available for Pompe disease; however, the response of skeletal muscle, as opposed to the heart, has been attenuated. The poor response of skeletal muscle has been attributed to the low abundance of the cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle compared to heart. To further understand the role of CI-MPR in Pompe disease, muscle-specific CI-MPR conditional knockout (KO) mice were crossed with GAA-KO (Pompe disease) mice. We evaluated the impact of CI-MPR-mediated uptake of GAA by evaluating ERT in CI-MPR-KO/GAA-KO (double KO) mice. The essential role of CI-MPR was emphasized by the lack of efficacy of ERT as demonstrated by markedly reduced biochemical correction of GAA deficiency and of glycogen accumulations in double KO mice, in comparison with the administration of the same therapeutic doses in GAA-KO mice. Clenbuterol, a selective β(2)-agonist, enhanced the CI-MPR expression in skeletal tissue and also increased efficacy from GAA therapy, thereby confirming the key role of CI-MPR with regard to enzyme replacement therapy in Pompe disease. Biochemical correction improved in both muscle and non-muscle tissues, indicating that therapy could be similarly enhanced in other lysosomal storage disorders. In summary, enhanced CI-MPR expression might improve the efficacy of enzyme replacement therapy in Pompe disease through enhancing receptor-mediated uptake of GAA.

Fig. 1. Impaired rhGAA uptake in DKO mice

The homozygous DKO mice (n=4) and GAA-KO mice (n=4) were administered four weekly doses of rhGAA and sacrificed three days after the last injection. (A) GAA enzyme levels and (B) glycogen content were evaluated in the target tissues. Mean +/− standard deviation are shown.

Fig. 2. Western blot analysis of CI-MPR expression and GAA content of heart and skeletal muscle

(A) Western blot detection of CI-MPR and human GAA in the tissues of DKO and GAA-KO mice is shown, with molecular weights indicated. Each lane represents an individual mouse. Equivalent quantities of tissue homogenate were loaded for each mouse. (B) Signal for CI-MPR as quantified by densitometry of Western blots. Mean +/− standard deviation are shown.

Fig. 3. Enhanced Rotarod performance and weight gain following ERT plus clenbuterol treatment

GAA-KO mice were administered four weekly doses of rhGAA (20 mg/kg), and treated with clenbuterol (n=7) or untreated (n=5). (A) Rotarod latency and (B) weight at indicated time points. Mean +/− standard deviation are shown. Statistically significant alterations associated with clenbuterol treatment indicated (*).

Fig. 4. Enhanced efficacy from ERT plus clenbuterol treatment

Male GAA-KO mice were administered four weekly doses of 20 mg/kg body weight of rhGAA and sacrificed one week following the last injection. Groups of mice were treated with clenbuterol (n=6) or untreated (n=5) during ERT. Mice were euthanized for tissue analysis 4 weeks after vector injection. (A) GAA enzyme levels and (B) glycogen content were evaluated in the target tissues, including the tibialis anterior (Tib. Ant.). Statistically significant alterations associated with clenbuterol treatment indicated (*).

Fig. 5. Decreased glycogen accumulation in skeletal muscle following clenbuterol administration

Periodic-acid Schiff staining for glycogen in paraffin-embedded sections. Original magnification 400X. (A) Diaphragm following clenbuterol treatment and ERT. (B) Diaphragm following ERT alone. (C) Quadriceps following clenbuterol treatment and ERT. (D) Quadriceps following ERT alone.

Fig. 6. Western blot analysis of CI-MPR expression and GAA content of skeletal muscle following clenbuterol treatment

(A) Western blot detection of CI-MPR and human GAA in the tibialis anterior of GAA-KO mice is shown, with molecular weights indicated. Each lane represents an individual mouse. Equivalent quantities of tissue homogenate were loaded for each mouse. (B) Signal for CI-MPR as quantified by densitometry of Western blot images. Mean +/− standard deviation are shown. Statistically significant alterations associated with CI-MPR absence indicated (*).