Impaired clearance of accumulated lysosomal glycogen in advanced Pompe disease despite high-level vector-mediated transgene expression

Abstract

Background: Infantile-onset glycogen storage disease type II (GSD-II; Pompe disease; MIM 232300) causes death early in childhood from cardiorespiratory failure in the absence of effective treatment, whereas late-onset Pompe disease causes a progressive skeletal myopathy. The limitations of enzyme replacement therapy could potentially be addressed with adeno-associated virus (AAV) vector-mediated gene therapy.

Methods: AAV vectors containing tissue-specific regulatory cassettes, either liver-specific or muscle-specific, were administered to 12- and 17-month-old Pompe disease mice to evaluate the efficacy of gene therapy in advanced Pompe disease. Biochemical correction was evaluated through acid alpha-glucosidase (GAA) activity and glycogen content analyses of the heart and skeletal muscle. Western blotting, urinary biomarker, and Rotarod performance were evaluated after vector administration.

Results: The AAV vector containing the liver-specific regulatory cassette secreted high-level human GAA into the blood and corrected glycogen storage in the heart and diaphragm. The biochemical correction of the heart and diaphragm was associated with efficacy, as reflected by increased Rotarod performance; however, the clearance of glycogen from skeletal muscles was relatively impaired compared to in younger Pompe disease mice. An alternative vector containing a muscle-specific regulatory cassette transduced skeletal muscle with high efficiency, but also failed to achieve complete clearance of accumulated glycogen. Decreased transduction of the heart and liver in older mice, especially in females, was implicated as a cause for reduced efficacy in advanced Pompe disease.

Conclusions: The impaired efficacy of AAV vector-mediated gene therapy in old Pompe disease mice emphasizes the need for early treatment to achieve full efficacy.

Fig. 1. Endpoint analysis following AAV vector administration

Twelve-month old male GAA-KO mice were injected intravenously with AAV-SPhGAApA (n=5, except for the last interval n=4) or PBS (n=3). A single AAV vector-treated mouse died in the sixth month of the study of an undetermined cause, at 18 months old when GAA-KO mice are reaching the end of their lifespan [35]. Mean +/− s.d shown, and significant differences indicated (*) based upon P < 0.05 calculated with an unpaired T-test with Welch's correction (A) Rotarod testing. The change in Rotarod time was calculated for each mouse during the indicated time intervals. (B) Weight. The change is weight was calculated for each mouse during the indicated time intervals. Each line represents an individual mouse. (C) Urinary biomarker, [Glc₄], was analyzed for each mouse (n=4 in each group).

Fig. 2. Biochemical correction with chimeric hGAA in GAA-KO mice

Mean +/−s.d. is shown. Control mice were age-matched, PBS-injected GAA-KO mice (n=4). Significant differences indicated (*), based upon P < 0.05 calculated with a two-tailed homoscedastic Student's t-test. (A) Plasma GAA was assayed for GAA-KO mice following administration of the AAV vector (n=5, except for the last time point n=4). (B) Tissue GAA was assayed for GAA-KO mice 24 weeks following administration of the AAV vector (n=4). (C) Glycogen content in tissues for mice in (B).

Fig. 3. Western blot detection of hGAA in tissues

Tissues analyzed 24 weeks following administration of the AAV vector to 12 month-old (18 months) or 3 month-old (9 months) GAA-KO mice. Three different proteins were detected, hGAA, lysosomal associated membrane protein 2 (LAMP-2), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH served as a control to indicate equal loading of each lane. Heart from GAA-KO mice, following administration of the AAV2/8 vector (AAV) or mock treatment (PBS). Each lane represents an individual mouse. Controls were a PBS-injected, age-matched GAA-KO mice, and a C57BL/6 mouse (wildtype).

Fig. 4. Tissue analysis following AAV vector administration

Twelve-month old male GAA-KO mice were injected intravenously with AAV-MHCK7hGAApA (n=5), or PBS (n=3). Two AAV vector-treated mouse died in the second month of the study of an undetermined cause, at an age when GAA-KO mice are reaching the end of their lifespan [35]. Mean +/− s.d shown, and significant differences indicated (*) based upon P < 0.05 calculated with an unpaired T-test with Welch's correction. (A) GAA activity in the indicated tissues. (B) Glycogen content for the mice shown in (A).

Fig. 5. Tissue analysis following dual vector administration to 17 month-old GAA-KO mice

Seventeen-month old female GAA-KO mice were injected intravenously with AAV-LSPhGAApA and AAV-MHCK7hGAApA (n=4). (A) GAA activity in the indicated tissues. (B) Glycogen content for the mice shown in (A). (C) Vector genome quantification by Realtime PCR. Detection of the hGAA cDNA in GAA-KO mice following AAV-SPhGAApA administration at 3 months old (Young, n=4) or 12 months old (Old, n=4 for liver, n=2 for heart), or AAV-LSPhGAApA and AAV-MHCK7hGAApA co-administration at 17 months old (Very Old, n=4). Mean +/− s.d shown, and significant differences indicated by (*) indicating p < 0.05, (**) indicating p<0.01, and (***) indicating p<0.001 as calculated with an unpaired T-test.