The upstream enhancer elements of the G6PC promoter are critical for optimal G6PC expression in murine glycogen storage disease type Ia

Abstract

Glycogen storage disease type-Ia (GSD-Ia) patients deficient in glucose-6-phosphatase-α (G6Pase-α or G6PC) manifest impaired glucose homeostasis characterized by fasting hypoglycemia, growth retardation, hepatomegaly, nephromegaly, hyperlipidemia, hyperuricemia, and lactic acidemia. Two efficacious recombinant adeno-associated virus pseudotype 2/8 (rAAV8) vectors expressing human G6Pase-α have been independently developed. One is a single-stranded vector containing a 2864-bp of the G6PC promoter/enhancer (rAAV8-GPE) and the other is a double-stranded vector containing a shorter 382-bp minimal G6PC promoter/enhancer (rAAV8-miGPE). To identify the best construct, a direct comparison of the rAAV8-GPE and the rAAV8-miGPE vectors was initiated to determine the best vector to take forward into clinical trials. We show that the rAAV8-GPE vector directed significantly higher levels of hepatic G6Pase-α expression, achieved greater reduction in hepatic glycogen accumulation, and led to a better toleration of fasting in GSD-Ia mice than the rAAV8-miGPE vector. Our results indicated that additional control elements in the rAAV8-GPE vector outweigh the gains from the double-stranded rAAV8-miGPE transduction efficiency, and that the rAAV8-GPE vector is the current choice for clinical translation in human GSD-Ia.

Keywords: AAV; Adeno-associated virus; G6P; G6PC promoter/enhancer; G6Pase; GPE; GSD-Ia; Gene therapy; Glucose-6-phosphatase; Glycogen storage disease type I; HCA; adeno-associated virus; glucose-6-phosphatase; glucose-6-phosphate; glycogen storage disease type Ia; hepatocellular adenoma.

Fig. 1

Biochemical analyses in 12-week-old wild type and rAAV8-treated G6pc−/− mice. (A) Hepatic microsomal G6Pase-α activity and its relationship to vector genome copy numbers. (B) Growth curve. (C) BMI values. (D) Blood glucose levels. GPE-high, high dose rAAV8-GPE-treated (○); miGPE-high, high dose rAAV8-miGPE-treated (●); GPE-low, low dose rAAV8-GPE-treated (□); miGPE-low, low dose rAAV8-miGPE-treated (∎) G6pc−/− mice; (+/+), wild type (▾) mice. Hepatic G6Pase activity represents the combined data from the two centers (n = 12 per treatment) and the other data are from the NIH (n = 6 per treatment). Data are mean ± SEM. *P < 0.05.

Fig. 2

Phenotypic analyses in 12-week-old wild type and rAAV8-treated G6pc−/− mice. (A) Liver weight. (B) Hepatic glycogen contents. (C) Hepatic triglyceride contents. GPE-high (n = 6), high dose rAAV8-GPE-treated; miGPE-high (n = 6), high dose rAAV8-miGPE-treated; GPE-low (n = 6), low dose rAAV8-GPE-treated; miGPE-low (n = 6), low dose rAAV8-miGPE-treated G6pc−/− mice; (+/+), wild type mice. Data are mean ± SEM. *P < 0.05.

Fig. 3

Fasting blood glucose and glucose tolerance profiles in 12-week-old wild type and rAAV8-treated G6pc−/− mice. (A) Fasting blood glucose profiles. (B) Blood glucose levels following a 24-hour fast. (C) Glucose tolerance profiles. GPE-high (n = 6), high dose rAAV8-GPE-treated (○); miGPE-high (n = 6), high dose rAAV8-miGPE-treated (●); GPE-low (n = 6), low dose rAAV8-GPE-treated (□); miGPE-low (n = 6), low dose rAAV8-miGPE-treated (∎) G6pc−/− mice; (+/+), wild type mice (n = 24) (▾). Data are mean ± SEM. *P < 0.05, **P < 0.005.