487. Liver-Targeted Gene Therapy in Glycogen Storage Disease Type Ia (GSD-Ia) Requires Widespread, Regulated Glucose-6-phosphatase

Abstract

The glucose-6-phosphatase (G6Pase) deficiency of GSD-Ia prevents the conversion of glycogen to glucose in the liver. G6Pase overexpression was associated with blood glucose elevations; therefore, unregulated G6Pase expression should be avoided when considering strategies for gene therapy for GSD-Ia. A minimal G6Pase promoter containing insulin-responsive sequence elements was placed upstream of the human G6Pase cDNA in AAV-GRPhG6PpA. This vector was pseudotyped as AAV2/8 and administered to two week-old affected GSD-Ia mice (3 × 1011 DNase-resistant vector particles; 6 × 1013 particles/kg). An AAV2/8 vector containing the universally active CB promoter, AAV-CBcG6PpA, was also administered to affected GSD-Ia mice; however, the CB promoter is NOT glucose-responsive. Importantly, G6Pase expression with the glucose-responsive promoter (GRP) was as effective as the CB promoter/enhancer at correcting hypoglycemia. AAV-GRPhG6PpA significantly corrected hypoglycemia in fasting, affected GSD-Ia mice at 4 weeks of age. Pseudotyping AAV-GRPhG6PpA as AAV2/8 or AAV2/1 corrected hypoglycemia equivalently in GSD-Ia mice. Resolution of hypoglycemia was associated with the correction of other biochemical abnormalities, including hypercholesterolemia and G6Pase deficiency/glycogen storage in the liver. Furthermore, AAV- GRPhG6pA promoted normal growth and prolonged the survival of GSD-Ia mice by greater than 6 months, in comparison to untreated, affected mice which failed to grow and did not survive weaning. AAV-GRPhG6PpA was modified by deleting the D sequence from the terminal repeat to package scAAV-GRPhG6PpA, thereby increasing the efficiency of liver transduction. When scAAV- GRPhG6PpA, pseudotyped as AAV2/8, was administered to 2 week- old affected G6Pase-KO mice (3 × 1011 vector particles), blood glucose was significantly increased (90 +/- 16 mg/dl versus 31 +/- 19 mg/dL for untreated, affected mice; p<0.005). Furthermore, the number of scAAV vector particles administered could be reduced 10-fold compared to the analogous non-scAAV vector, while still correcting hypoglycemia and prolonging survival. An AAV vector encoding a liver-specific promoter (AAV-LSPhG6PpA) to drive unregulated G6Pase expression was administered to unaffected (wild-type) G6Pase-KO mice at 2 weeks of age and a glucose tolerance test was performed at 1 month old. The glucose tolerance test detected elevated glucose, in comparison to untreated wild-type mice, which reflected glucose intolerance related to unregulated G6Pase expression driven by the LSP. In contrast, AAV-GRPhG6PpA administration did not cause glucose intolerance in affected or wild-type mice. Hence, an scAAV vector efficaciously delivered a regulated transgene to liver without causing glucose intolerance in GSD-Ia. The glucose-6-phosphatase (G6Pase) deficiency of GSD-Ia prevents the conversion of glycogen to glucose in the liver. G6Pase overexpression was associated with blood glucose elevations; therefore, unregulated G6Pase expression should be avoided when considering strategies for gene therapy for GSD-Ia. A minimal G6Pase promoter containing insulin-responsive sequence elements was placed upstream of the human G6Pase cDNA in AAV-GRPhG6PpA. This vector was pseudotyped as AAV2/8 and administered to two week-old affected GSD-Ia mice (3 × 1011 DNase-resistant vector particles; 6 × 1013 particles/kg). An AAV2/8 vector containing the universally active CB promoter, AAV-CBcG6PpA, was also administered to affected GSD-Ia mice; however, the CB promoter is NOT glucose-responsive. Importantly, G6Pase expression with the glucose-responsive promoter (GRP) was as effective as the CB promoter/enhancer at correcting hypoglycemia. AAV-GRPhG6PpA significantly corrected hypoglycemia in fasting, affected GSD-Ia mice at 4 weeks of age. Pseudotyping AAV-GRPhG6PpA as AAV2/8 or AAV2/1 corrected hypoglycemia equivalently in GSD-Ia mice. Resolution of hypoglycemia was associated with the correction of other biochemical abnormalities, including hypercholesterolemia and G6Pase deficiency/glycogen storage in the liver. Furthermore, AAV- GRPhG6pA promoted normal growth and prolonged the survival of GSD-Ia mice by greater than 6 months, in comparison to untreated, affected mice which failed to grow and did not survive weaning. AAV-GRPhG6PpA was modified by deleting the D sequence from the terminal repeat to package scAAV-GRPhG6PpA, thereby increasing the efficiency of liver transduction. When scAAV- GRPhG6PpA, pseudotyped as AAV2/8, was administered to 2 week- old affected G6Pase-KO mice (3 × 1011 vector particles), blood glucose was significantly increased (90 +/- 16 mg/dl versus 31 +/- 19 mg/dL for untreated, affected mice; p<0.005). Furthermore, the number of scAAV vector particles administered could be reduced 10-fold compared to the analogous non-scAAV vector, while still correcting hypoglycemia and prolonging survival. An AAV vector encoding a liver-specific promoter (AAV-LSPhG6PpA) to drive unregulated G6Pase expression was administered to unaffected (wild-type) G6Pase-KO mice at 2 weeks of age and a glucose tolerance test was performed at 1 month old. The glucose tolerance test detected elevated glucose, in comparison to untreated wild-type mice, which reflected glucose intolerance related to unregulated G6Pase expression driven by the LSP. In contrast, AAV-GRPhG6PpA administration did not cause glucose intolerance in affected or wild-type mice. Hence, an scAAV vector efficaciously delivered a regulated transgene to liver without causing glucose intolerance in GSD-Ia.