Glycogen storage disease type I (GSD-I) was first described by von Gierke in 1929 based on autopsy reports of 2 children who had excessive glycogen in their enlarged liver and kidneys. Similar findings were reported by Cori and Cori in 6 patients in 1952.1 Two of the patients had almost total deficiency of hepatic glucose-6-phosphatase-α (G6Pase-α or G6PC), whereas the remaining 4 had healthy enzyme activity. The puzzle was eventually solved in 1978 when Narisawa et al. identified a defect in intracellular transport of the substrate, named as glucose-6-phosphate transporter (G6PT).2 G6Pase-α and G6PT, both embedded in the endoplasmic reticulum (ER) membrane, form a functional complex that maintains glucose homeostasis between meals: G6PT translocates glucose-6-phosphate (G6P) from the cytoplasm into the lumen of the ER, and G6Pase-α hydrolyzes G6P into glucose and phosphate. A deficiency in G6Pase-α causes GSD-Ia, and a deficiency in G6PT causes GSD-Ib and both are autosomal recessive disorders with an overall incidence of approximately 1 in 100,000.3 Both GSD-Ia and GSD-Ib patients fail to hydrolyze G6P to glucose and thus share symptoms, including life-threatening hypoglycemia, hepatomegaly, and seizures, within the first year of life. Long-term complications include growth failure, pulmonary hypertension, formation of hepatic adenomas, and, occasionally, hepatocellular carcinoma (HCC) and renal failure. Therapies are aimed at controlling glycemia by dietary supplementation or continuous parenteral or intragastric infusion of carbohydrates. In contrast to most other inborn errors of metabolism, enzyme-replacement therapy is not possible for von Gierke's disease because the deficient enzyme is a hydrophobic ER-associated transmembrane protein that cannot be expressed in a soluble form. Gene therapy, which utilizes a vector to deliver the therapeutic gene to the target tissues, provides an attractive alternative therapy.
A vector based on adeno-associated virus (AAV) has been chosen as the main vector platform for GSD-Ia gene therapy because of its safety profiles, high in vivo transduction efficiency, stable transgene expression, and modest immunogenicity. The availability of both small-4 and large-animal5 models that closely mimic severe GSD-Ia in humans makes a preclinical evaluation of the efficacy of gene therapy feasible. The main target tissue is the liver, based on the success with human patients after liver transplantation (LT). The kidney is also a target organ to prevent renal failure, which frequently presents as a late complication in GSD-Ia patients with or without LT. Initial studies using AAV serotype 2–based vectors expressing G6Pase-α to treat infant GSD-Ia in dogs or mice showed suboptimal improvement.6, 7 In subsequent years, several new advances in the AAV field, such as novel AAV serotypes from nonhuman primate or human tissues8 and the discovery of self-complementary (sc)AAV,9 enabled researchers to further improve the efficacy of gene therapy for GSD-Ia. AAV serotype 8 (AAV8), a highly liver-tropic and efficient vector with low preexisting immunity in human populations, has become one of the preferred vector serotypes, especially for liver-directed gene therapy. Using AAV8 or AAV1 vectors prolonged survival, and partial biochemical correction was demonstrated in G6pc−/− mice.10, 11 However, both studies required high vector doses in excess of 2 × 1014 vector genome/kg while still failing to fully correct biochemical parameters and restore G6Pase deficiency. Further improvements in efficacy were achieved by using a scAAV8 vector expressing human G6Pase-α from a minimal human G6Pase promoter. A complete normalization of biochemical parameters for up to 1 year postvector administration was achieved in G6pc−/− mice, despite the use of a 600-fold lower dose than in previous studies.12 Prolonged survival for up to 1 year and sustained correction of hypoglycemia subsequent to AAV8 gene transfer was also demonstrated in GSD-Ia dogs.12 Further comparison with AAV7 and AAV9 vectors in G6pc−/− mice showed that AAV9 is more efficient in transducing kidney because of its broad tropism, and partial correction of renal failure was achieved.13 The use of human G6Pase promoter regions regulates G6Pases-α expression in response to glucose, dexamethasone, and insulin levels, therefore preventing potential overexpression of the enzyme as observed in animals treated with high vector doses.12, 14 They also bypass the limitations of liver-specific promoters, which have limited or no expression in kidney, or the problems of ubiquitous promoters, which are associated with cytotoxic T-cell response and rapid clearance of vector in the liver of young GSD-Ia mice.14 G6pc−/− mice treated with an AAV8 vector expressing the human G6Pase-α driven by the human G6PC promoter/enhancer (GPE) showed improved G6Pase-α expression and complete normalization of G6Pase-α deficiency in the liver for 24 weeks.14 Another challenge faced by gene therapy for GSD-Ia and for many other metabolic diseases that manifest soon after birth is the loss of efficacy and persistence after neonatal gene transfer resulting from the loss of episomal vector genomes caused by hepatocyte proliferation and liver growth. In addition, ongoing liver damage related to glycogen storage and hypoglycemia might accelerate the loss of vector genomes in liver. Two strategies were attempted to overcome this problem. In G6pc−/− mice, delaying the injection age from 2 days to 2 weeks significantly improved long-term efficacy.14 In GSD-Ia dogs, readministration with vector of a different serotype after the initial neonatal vector treatment restored long-term efficacy (prevention of hypoglycemia and marked reduction of glycogen storage in liver) and prolonged survival for up to 5 years.15
The advances made through these preclinical studies significantly prolonged the life of GSD-Ia animals, therefore allowing one to address the long-term efficacy of gene therapy. In GSD-Ia patients, one of the most significant chronic risks is hepatocellular adenoma (HCA), which develops in 70%-80% of GSD-I patients over 25 years of age.16, 17 In 10% of GSD-Ia patients, HCAs undergo malignant transformation to HCC. It is hard to assess HCA in the existing GSD-Ia dogs and G6pc−/− mice because of their short lifespan. The recently generated liver-specific G6pc-null (L-G6pc−/−) mice, however, have a significantly higher survival rate, allowing long-term observation of liver pathogenesis.18 Hepatic nodules were detected in 30%-40% of L-G6pc−/− mice at 12 months. After 18 months, all L-G6pc−/− mice developed multiple HCAs.18 The Lee et al. study in this issue demonstrates the long-term effect of AAV8/GPE-mediated gene therapy in G6pc−/− mice.19 The data show that AAV8-treated G6pc−/− mice expressed 3%-128% of normal levels of hepatic G6Pase-α activity, correlating to the vector doses they received. These treated mice grew normally for 70-90 weeks and exhibited normalized blood-metabolite and glucose-tolerance profiles. Furthermore, the treated G6pc−/− mice did not develop hepatic steatosis and had normal levels of hepatic triglycerides. Most important, for the first time, the investigators show that AAV8/GPE-mediated gene transfer prevented hepatic G6Pase-α deficiency-induced chronic HCA, despite the fact that some mice in the low-dose group only expressed 3% of normal G6Pase-α activity levels. The investigators further elucidated the role played by G6PT and the feedback mechanism to compensate the reduced G6Pase-α activity. These data are encouraging for advancing GSD-Ia translational research to bring the preclinical success to the bedside, which has already shown promise with gene therapy for other genetic diseases, such as hemophilia B.19, 20