Glycogen storage disease type I: Genetic etiology, clinical manifestations, and conventional and gene therapies

Abstract

Glycogen storage disease type I (GSDI) is an inherited metabolic disorder characterized by a deficiency of enzymes or proteins involved in glycogenolysis and gluconeogenesis, resulting in excessive intracellular glycogen accumulation. While GSDI is classified into four different subtypes based on molecular genetic variants, GSDIa accounts for approximately 80%. GSDIa and GSDIb are autosomal recessive disorders caused by deficiencies in glucose‐6‐phosphatase (G6Pase‐α) and glucose‐6‐phosphate‐transporter (G6PT), respectively. For the past 50 years, the care of patients with GSDI has been improved following elaborate dietary managements. GSDI patients currently receive dietary therapies that enable patients to improve hypoglycemia and alleviate early symptomatic signs of the disease. However, dietary therapies have many limitations with a risk of calcium, vitamin D, and iron deficiency and cannot prevent long‐term complications, such as progressive liver and renal failure. With the deepening understanding of the pathogenesis of GSDI and the development of gene therapy technology, there is great progress in the treatment of GSDI. Here, we review the underlying molecular genetics and the current clinical management strategies of GSDI patients with an emphasis on promising experimental gene therapies.

1. INTRODUCTION

Glycogen storage disease type I (GSDI) was first described by von Gierke in 1929, so it is also known as von Gierke disease. Glycogen storage disease type I is an inherited metabolic disorder caused by abnormalities of enzymes or proteins that are involved in glycogenolysis and gluconeogenesis. ¹ GSDI can be classified into four different subtypes based on molecular genetic variants: GSDIa, GSDIb, GSDIc, and GSDId. GSDIc and GSDId cases have been reported rarely, and GSDIa and GSDIb subtypes are the most clinically recognized. ² The probability of GSDI is estimated to be 1 in 100,000 live births, and GSDIa accounts for approximately 80%. ³ GSDIa and GSDIb are autosomal recessive disorders caused by glucose‐6‐phosphatase (G6Pase‐a) and glucose‐6‐phosphate‐transporter (G6PT) deficiency, respectively. ⁴ Both G6Pase‐a and G6PT are embedded in the membrane of the endoplasmic reticulum and are functionally coupled to maintain interprandial glucose homeostasis. Functionally, G6PT transports glucose‐6‐phosphate (G6P) from the cytoplasm to the endoplasmic reticulum lumen, where G6Pase‐a hydrolyzes G6P into glucose and inorganic phosphate. ⁵

The primary metabolic abnormality in GSDIa and GSDIb is fasting hypoglycemia and lactic acidosis in the neonatal period, as G6Pase‐α and G6PT deficiencies result in the inability to hydrolyze G6P into glucose and phosphate. However, most newborns are free of symptoms as soon as they are fed foods containing enough glucose to prevent hypoglycemia, which usually only occurs at increased intervals between feedings. More commonly, the first symptom of GSDI is a protruding abdomen at about 3–6 months of age due to marked hepatomegaly, followed by progressive enlargement of the liver. Clinical features include doll‐like facies, growth retardation, short stature, and a distended abdomen owing to apparent hepatomegaly and nephromegaly. The high G6P level is shunted to alternative metabolic pathways, especially de novo lipogenesis, causing abnormal biochemical manifestations, including hypoglycemia, hyperlipidemia, hyperuricemia, hyperlactatemia, and hypertriglyceridemia. ⁵ , ⁶ In most of the patients with GSDI, liver enlargement decreases with age; however, 75% of patients older than 25 years are at risk for developing hepatocellular adenomas (HCA), of which 10% of HCA patients undergo malignant transformation into hepatocellular carcinoma (HCC). ⁷ Moreover, patients with GSDI can also develop long‐term nephropathy due to excessive accumulation of glycogen and lipids in the kidney. ⁸ Liver transplantation and kidney transplantation are the ultimate curative therapy for patients with GSDI. ⁹ Neutropenia and myeloid dysfunction are unique to patients with GSDIb, which are associated with recurrent bacterial infections and enterocolitis. ¹⁰ In addition to the presence of symptoms and a wide spectrum of characteristic biochemical abnormalities mentioned above, molecular genetic testing, and/or enzymatic testing of liver biopsy tissue provide clues to the diagnosis of GSDI. In patients with GSDI, the liver, kidney, and small intestinal mucosa are impaired due to excessive glycogen storage, and the cardiovascular system is typically unaffected; however, there were several early case reports showing pulmonary hypertension occurred in patients with GSDI. ¹¹ , ¹² Pulmonary hypertension is a very rarely seen complication, which has a very bad prognosis and its physiopathology is poorly understood.

Both GSDIa and GSDIb are fatal in childhood, if left untreated. The primary treatment for patients with GSDI focuses on dietary therapy, including continuous nocturnal nasogastric/intragastric infusion of glucose or oral administration of uncooked cornstarch to control glycemia and associated metabolic abnormalities. ⁴ , ¹³ Proper dietary management is helpful to prevent hypoglycemia and alleviate complications. Uncooked cornstarch (UCS) is an option starch choice to manage hypoglycemia for patients because of its slow‐release carbohydrate properties. ¹⁴ However, enzyme replacement therapy is not appropriate for patients with GSDI because both G6Pase‐α and G6PT are highly hydrophobic transmembrane proteins. Gene therapy offers therapeutic advantages for diseases caused by defective and abnormal genes, especially monogenic diseases. ¹⁵ Gene therapy involves the use of vectors to deliver the target gene to the relevant tissues or organs. There are two gene therapy strategies according to different delivery systems: viral vector‐based delivery and non‐viral vector‐based delivery. The viral vector‐based delivery includes lentiviral vectors (LV), retroviral vectors, adenoviral (Ad) vectors, adeno‐associated virus (AAV) vectors, and the like. The non‐viral vector‐based delivery can include cationic polymer vectors, liposome vectors, and nanoparticle vectors. ¹⁶ There are numerous preclinical trials to investigate the viral vectors, which have been reported to account for 70% of gene therapy programs. ¹⁷ An effective viral vector should be capable of delivering the transgene stably and efficiently to the tissue of interest. AAV is the most promising gene transfer vector for the treatment of glycogen storage disease type I due to its excellent safety profile, low immunogenicity, and durable and efficient transgene expression. ¹⁸ The AAV vectors encoding different species G6Pase/SLC37A4 directed by the specific promoter/enhancer are administered to GSDI dogs or mice models that closely mimic severe GSDI in humans.

In this review, we will introduce the current understanding of the molecular genetics underlying GSDIa and GSDIb. We then summarize the clinical manifestations and pathologic phenotypes of GSDI on hepatic and renal metabolism as well as immune system. Lastly, we overview the current therapeutic strategies for GSDI including: (1) continuous dietary/nutritional therapies for GSDI patients, (2) conventional drug treatment for GSDI patients, and (3) viral vector‐mediated gene delivery with an emphasis on AAV‐based gene delivery systems.

2. MOLECULAR GENETICS OF GLYCOGEN STORAGE DISEASE TYPE I DISORDERS

2.1. Molecular pathogenesis of glycogen storage disease type Ia

Glycogen storage disease type Ia (GSDIa; OMIM#232200) is an autosomal recessive disorder that causes abnormal carbohydrate metabolism due to deleterious mutations in the glucose‐6‐phosphatase (G6Pase‐a or G6PC, OMIM*613742). ¹⁹ , ²⁰ Human G6PC gene is a single‐copy gene composed of five exons, localized on 17q21.31, encoding a highly hydrophobic, 357 amino‐acid glycoprotein. ²¹ To date, 118 G6PC mutation types have been identified in GSDIa patients, including 89 missense/nonsense, 5 splicing, 1 regulatory, 19 small insertions/deletions, 3 indels, and 1 gross deletions (via The Human Gene Mutation Database http://www.hgmd.cf.ac.uk/ac/gene.php?gene=G6PC). G6Pase‐α is expressed primarily in liver, kidney, and intestine and contributes to maintaining interprandial blood glucose homeostasis by reversibly hydrolyzing G6P into glucose and phosphate in the final common reaction of glycogenolysis and gluconeogenesis. ²¹ In gluconeogenic organs, G6Pase is embedded in the membrane of the endoplasmic reticulum (ER) by nine transmembrane helices and has the amino‐terminus (NH2) located in the lumen, while the carboxyl‐terminus located in the cytoplasm ²² (Figure 1A). Based on the crystal structure of the vanadium haloperoxidase, the active site residues of G6Pase‐a are all located on the lumen side of endoplasmic reticulum (ER) comprising Lys‐76, Arg‐83, His‐119, Arg‐170, and His‐176. ²² , ²³ , ²⁴ The current paradigm for the G6Pase‐α reaction mechanism is that His‐176 acts as a nucleophile that covalently bound the phosphate of G6P forming a phosphohistidine‐enzyme intermediate. ²²

FIGURE 1.

Potential targets of disrupted glucose homeostasis. Glucose‐6‐phosphatase‐α (G6Pase‐α) and glucose‐6‐phosphate transporter (G6PT) are shown embedded within the membrane of the endoplasmic reticulum (ER). In gluconeogenic organs, such as liver, kidney, and intestine, G6PT couples with G6Pase‐α to maintain interprandial blood glucose homeostasis. G6PT transports glucose‐6‐phosphate (G6P) from the cytoplasm to the lumen of the ER, where G6Pase‐α hydrolyzes G6P into glucose and inorganic phosphate. (A) Glycogen storage disease type Ia (GSDIa) is a glycogen metabolic disorder and manifests hypoglycemia and secondary metabolic symptoms. (B) Glycogen storage disease type Ib (GSDIb) is a metabolic and immune disorder. GSDIb manifests overlapping and distinct phenotypes compared with GSDIa.

2.2. Molecular pathogenesis of glycogen storage disease type Ib

Glycogen storage disease type Ib (GSDIb, OMIM#232220) is an autosomal recessive disorder caused by a defect in the glucose‐6‐phosphate transporter (G6PT1) encoded by the solute carrier family 37 members 4 (SLC37A4) gene. ¹⁹ Human SLC37A4 gene is a single‐copy gene composed of nine exons, localized on 11q23.3. G6PT encodes 429 amino‐acid protein consisting of 10 transmembrane helices on the endoplasmic reticulum membrane with their amino‐terminus and carboxyl‐terminus located in the cytoplasm. ²⁵ , ²⁶ To date, 115 SLC37A4 gene mutation types have been identified, including 65 missense/nonsense, 18 splicing, 1 regulatory, 27 small insertions/deletions, 2 indels, and 2 gross deletions (via The Human Gene Mutation Database, www.hgmd.cf.ac.uk/ac/gene.php?gene=SLC37A4). G6PT is ubiquitously expressed in all tissues and acts as a phosphate‐linked transporter, transporting the translocation of cytoplasmic G6P into the lumen of the endoplasmic reticulum, exchanging inorganic phosphate stored in the lumen. ²⁶ In the endoplasmic reticulum membrane, G6Pase‐α and G6PT are functionally coupled, and G6PT facilitates G6Pase‐α to catalyze the hydrolysis of the intracellular G6P into glucose to maintain interprandial glucose homeostasis (Figure 1B). Table 1 summarizes the similarities and differences between GSDIa and GSDIb characteristics.

TABLE 1.

Similarities and differences of Glycogen storage disease type Ia (GSDIa) versus Glycogen storage disease type Ib (GSDIb).

Characteristics	GSDIa (von Gierke disease)	GSDIb
Affected gene	G6PC	SLC37A4
Enzyme deficiency (protein)	Glucose‐6‐phosphatase (G6Pase‐α)	Glucose‐6‐phosphate‐transporter (G6PT)
Chromosome location	17 (17q21.31)	11 (11q23.3)
Genome components	Five exons	Nine exons
Inheritance	AR	AR
Probability	80%	20%
Located	Liver, Kidney, Small intestine	Similarities with GSDIa + Neutrophils
Clinical description	Doll‐like facies, Growth retardation, Hepatomegaly, Nephromegaly, Hypoglycaemia, Hyperlipidemia, Hyperlactacidemia, Hyperuricemia, Hypertriglyceridemia	Symptoms of GSDIa + Neutropenia, Myeloid dysfunction
Long‐term complications	HCA HCC Renal disease, Pulmonary hypertension	Complications of GSDIa + recurrent bacterial infections, Inflammatory bowel disease

Abbreviations: AR, Autosomal recessive; HCA, Hepatocellular adenomas; HCC, Hepatocellular carcinoma.

3. CLINICAL MANIFESTATIONS AND PATHOLOGIC PHENOTYPES OF GLYCOGEN STORAGE DISEASE TYPE I DISORDERS

3.1. Liver phenotypes of Glycogen storage disease type I disorders

The liver is a pivotal organ for regulating glucose homeostasis, keeping the blood glucose concentration at a relatively constant level, which is extremely important to ensure the utilization of various tissues and organs. Glucose is metabolized through a variety of direct competing pathways, glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), glycogenesis, glycogenolysis, gluconeogenesis, and other hexose metabolism ²⁵ (Figure 2). Glycogen storage disease type I is a genetic metabolic disorder in which G6Pase‐a or G6PT deficiency leads to extensive glycogen deposition. Both GSDIa and GSDIb are characterized by the aberrant utilization of glycogen, resulting in excessive glycogen accumulation in the cytoplasm of gluconeogenic organs (liver, kidney, and small intestine). Consequently, glucose production is insufficient and excessive G6P is shunted to alternative metabolic pathways, resulting in hypoglycemia and typical secondary metabolic abnormalities (e.g., hyperlactatemia, hypertriglyceridemia, hyperlipidemia, and hyperuricemia). ² , ²⁷ These findings are consistent with an observational study among 14 patients (11 GSDIa and 3 GSDIb), in which their plasma metabolomic profiles revealed alterations in metabolic flux mainly involving fuel and energy metabolism, lipids and fatty acids metabolism, amino acid and methyl‐group metabolism, urea cycle, and purine/pyrimidine metabolism compared to a cohort of age‐matched healthy controls. ²⁸

FIGURE 2.

The essential role of G6P metabolism in gluconeogenesis. G6P can be hydrolyzed into glucose and inorganic phosphate in the terminal rate‐limiting step of gluconeogensis and glycogenolysis by G6Pase‐α. They are responsible for maintaining glucose homeostasis. Furthermore, G6P can be hydrolyzed to produce energy through glycolysis and TCA cycle. G6P can also provide ribose 5‐phosphate via the pentose phosphate pathway for nucleotide and nucleic acid biosynthesis. Conversely, G6P can be synthesized into glycogen via glycogenesis for energy storage.

Excessive fat and glycogen accumulations in the liver and kidney contribute to progressive hepatomegaly and nephromegaly, respectively. ²⁰ Hepatomegaly tends to be prevalent in the younger children, resulting in abdominal protrusion; however, the size of the liver gradually decreases with age. Thus, dysregulated hepatic glycogen metabolic disorder can further trigger the development and/or progression of HCA, HCC, and eventually liver failure. ²⁹ HCA and HCC are severe long‐term complications in patients with GSDI. ³⁰ The severity of biochemical abnormalities and long‐term complications are correlated with the extent of hepatic G6PC activity deficiency in preclinical in vivo models. Several studies support this hypothesis: In a 70–90 weeks study, Lee al. showed that rAAV‐G6PC‐mediated gene therapy expressed 3%–63% of wild‐type hepatic G6Pase‐a activity in G6pc ^−/− mice, maintaining glucose hemostasis and preventing chronic HCA formation. ⁷ , ³¹ After that, they further showed that the rAAV‐vector‐treated G6pc ^−/− mice expressing 0.2% of normal hepatic G6Pase‐α activity experienced hypoglycemia, while expressing 0.5%–1.3% of normal hepatic G6Pase‐α activity showed no evidence of HCA formation. The authors concluded that 0.5%–1.3% of normal hepatic G6Pase‐α activity is the threshold of hepatic G6Pase‐α activity required to block HCA formation in GSDIa mice. ³² However, Kwon al. showed G6pt ^−/− mice expressing <6% of normal hepatic G6PT activity developed HCAs after treating with rAAV‐GPE‐G6PT vectors. ³³ More recently, Rutten al. Demonstrated that affected mice with residual G6PC activity <60% showed a markedly increase in hepatomegaly, fasting hypoglycemia, hyperlactatemia, hypertriglyceridemia, glycogen contents, as well as oleate synthesis. These symptoms were more prominent in affected mice with G6PC activity <25%. ³⁴

3.2. Kidney phenotypes of Glycogen storage disease type I disorders

Kidney is also recognized as a major affected organ in patients with GSDI. ³⁵ The high level of G6P results in the activation of glycogenesis and de novo lipogenesis metabolic pathway, which is responsible for accumulation of renal glycogen and lipid. This phenomenon leads to a progressive decline in kidney function in patients with GSDI, most of whom are at risk for developing chronic kidney disease (CKD). ³⁶ One study showed that 14 patients with GSDI had a significant increase in glomerular filtration rate (GFR). ³⁷ This characteristic abnormality of glomerular hyperfiltration is firstly identified in patients with GSDI, followed by microalbuminuria, hypercalciuria, hypocitraturia, and later proteinuria. ⁸ , ³⁸ About 70% of patients involved progress to proteinuria, and probably 30% develop end‐stage renal disease demanding dialysis. In some patients, renal function has deteriorated and gradually developed to nephrolithiasis, nephrocalcinosis, and eventually renal failure. ³⁹ The underlying mechanism of kidney disease in patients with GSDI and its molecular pathways involved needs to be further elucidated.

A mouse model of kidney‐specific G6Pase deficiency (K. G6pc ^−/− mice) was generated to characterize the development of nephropathy in GSDIa by Clar et al. ⁴⁰ The study showed that renal G6PC knockout led to excessive glycogen accumulation in proximal tubules, resulting in tubular dilation and nephromegaly, as well as microalbuminuria after 6 months of G6pc deletion. ⁴⁰ Several studies have shown that the activation of renin‐angiotensin system (RAS), enhance of renal oxidative stress, increased expression of transforming growth factors, and insufficient energy in renal tubular epithelial cells are associated with glomerular damage and nephropathy in the GSDI mice. ⁶ , ⁴¹ , ⁴²

3.3. Immune dysfunction in Glycogen storage disease type I disorders

Both GSDIa and GSDIb patients present generally similar clinical symptoms, whereas GSDIb patients have an impact in immune disorders, including neutropenia and myeloid dysfunction, causing recurrent bacterial infections, inflammatory bowel disease, and mucosal lesions. ¹ , ³³ , ⁴³ , ⁴⁴ The therapeutic approach for patients with GSDIb is the administration of granulocyte‐colony stimulating factor (G‐CSF) in order to increase the number of neutrophils and mitigate the incidence of infections. ²⁵ A study investigated the hypothalamus‐pituitary‐thyroid axis in patients with GSDI demonstrated that 57% patients with GSDIb were observed to have an increased prevalence of thyroid autoimmunity and hypothyroidism. ⁴⁵ The underlying mechanism between neutropenia and/or neutrophil dysfunction in GSDIb and its relationship to G6P homeostasis is unclear.

In 2003, Kuijpers et al. observed a significantly higher level of apoptosis in circulating neutrophils in patients with GSDIb, which may be associated with granulocyte dysfunction. ⁴⁶ A mass spectrometric glycomic profiling of neutrophils in GSDIb suggested a novel explanation that hypoglycosylation of the electron transporting subunit of NADPH oxidase, gp91phox, was responsible for the correlation of neutrophil dysfunction and neutropenia in GSDIb patients. ⁴⁷ Most recently, Veiga‐da‐Cunhaet al. found 1,5‐anhydroglucitol‐6‐phosphate (1,5‐AG6P), a structural analog of G6P, accumulated in patients with G6PT or G6PC3 deficiency. ⁴⁸ In patients with GSDIb, 1,5‐AG6P cannot be transported from the cytosol into the ER by G6PT, resulting in the excessive accumulation of 1,5‐AG6P in neutrophils. ⁴⁴ The exact relationship of 1,5‐AG6P accumulation and G6PT mutations was confirmed by Veiga‐da‐Cunhaet al. As they found that cytosolic accumulating noncanonical metabolite 1,5‐AG6P leads to neutropenia and neutrophil dysfunction in G6PT‐deficient patients. ⁴⁸ It is well known that 1,5‐AG6P is an inhibitor of hexokinases, and mature neutrophils primarily depend on glycolysis for energy. The improvement in neutrophil function is mainly due to the decrease of intracellular 1,5‐AG6P concentration.

4. CLINICAL MANAGEMENT AND THERAPIES OF GLYCOGEN STORAGE DISEASE TYPE I DISORDERS

4.1. Dietary/nutritional therapies for Glycogen storage disease type I disorders

Before gene therapy technology had been developed, most of the early investigations focused on using dietary management and/or conventional drugs to treat GSDI. Currently, there are no targeted pharmaceutical interventions for patients with GSDI. Regimented dietary therapy is taken to treat this metabolic disruption disease. In the 1960s, portacaval shunts were used to correct patients with congenital metabolic errors. This surgical strategy shunts blood from the portal venous system into the vena cava system, which is an excellent way to favorably prevent dietary carbohydrates from passing through the liver and allow glucose more readily available to peripheral tissues. ¹³ , ⁴⁹ Following this procedure, portacaval shunts coincidentally relieve hypoglycemia and avoid glycogen deposition in the liver; however, hypohepatia, jaundice, ascites, and even hepatic encephalopathy will occur after portacaval shunting.

In the 1970s, continuous nutrition therapy, including total parenteral nutrition (TPN), nocturnal nasogastric infusion, and nighttime intragastric feeding, was introduced to well prevent universally fatal hypoglycemia. ¹³ These clinic trials demonstrated that patients with type 1 disease receiving intragastric feeding with a high‐glucose formula combined with frequent daytime feeding can prevent hypoglycemia and reverse metabolic anomalies associated with GSDI ⁵⁰ ; however, continuous feeding is possibly interrupted especially during the night. Hence, parents of affected infants must take some measures to avoid interruption in nighttime feeding because recurrent hypoglycemia can lead to seizures and even death from pump failures. ⁶ The rate administration of carbohydrates is not a constant formula, and plasma glucose concentration needs to be considered to minimize organic acidemia in children with GSDI. The carbohydrate infusions rates are calculated based on the body weight and age: the rate of administration for children under 6 years is 7–9 mg per kg per minute, for older children such as schoolchildren or adolescents is 5–6 mg per kg per minute for 10 h, and for adults is 3–4 mg per kg per minute for 8–10 hours. ⁵¹ , ⁵² , ⁵³ Interestedly, the study's mathematical formula showed that estimated glucose requirements were linearly correlated with brain size, rather than to weight and age. ¹³

Subsequent, investigations tested numerous starches with the intention to find a slow‐release carbohydrate source. In particular, uncooked cornstarch (UCS) is an ideal starch to manage hypoglycemia for children and adults with GSDs. ¹⁴ Compared to different starchy foods, cornstarch has several characteristics in preventing hypoglycemia. First, cornstarch digests and absorbs slowly, which can avoid a rapid increase in blood glucose in a short period of time and maintain glucose concentrations for a longer time. Consequently, cornstarch results in lower insulin concentration, which is beneficial because high insulin concentration increases the risk of hypoglycemia and decreases the production of lactate and ketone bodies. ¹³ , ¹⁴ , ⁵¹ A smaller amount of cornstarch was used to maintain normal glucose concentrations, and the recommended dosage for children is 1.75–2.5 g/kg every 6 h. ⁵¹ Over the past 50 years, strict dietary therapies have transformed GSDs from a fatal metabolic disorder to one with a good prognosis, extending the lifespan and improving the quality of life of GSDs patients. ¹³

4.2. Adjunct drug therapies for Glycogen storage disease type I disorders

Adjunct drug therapies have improved GSDI patient's management (Table 2). Clinically, renin‐angiotensin system (RAS) blockers, such as angiotensin‐converting inhibitors (ACEi) or angiotensin receptor blockers (ARBs), have been reported to effectively reverse hypertension and delay progression of chronic renal failure in patients with GSDI. ³⁸ , ⁵⁴ , ⁵⁵ , ⁵⁶ Patients with GSDIb are often treated with G‐CSF to improve symptoms of inflammatory bowel disease (IBD) and prevent severe neutropenia. ⁵⁷ , ⁵⁸ , ⁵⁹ A study reviewed medical records of 103 patients with GSDIb subjected to drug therapy with G‐CSF, and data indicated that G‐CSF can stimulate peripheral blood neutrophil counts and ameliorate the occurrence of infections. ⁶⁰ Other studies have shown that vitamin E is equally useful for alleviating disease manifestation associated with neutropenia. ⁶¹ , ⁶² However, there has been increasing evidence that long‐term G‐CSF administration can lead to the occurrence of malignancies (e.g., MDS or AML). ⁵⁷ , ⁶³ Thus, more effective and safe medications are required to treat patients with GSDIb. Empagliflozin, an inhibitor of the renal glucose cotransporter SGLT2, has emerged as a new and appropriate treatment option for neutropenia and neutrophil dysfunction in patients with GSDIb. ⁶⁴ A report of the clinical data from 112 pediatric and adult individuals with GSDIb clearly showed that empagliflozin markedly improves neutrophil counts and has a positive effect for neutrophil dysfunction‐related symptoms. ⁶⁵ , ⁶⁶ Empagliflozin has also been used to treat GSDIb patients, resulting in decreased serum 1,5‐AG and neutrophil 1,5‐AG6P levels. ⁴³ , ⁴⁴ , ⁶⁷ , ⁶⁸

TABLE 2.

Current therapeutics used in Glycogen storage disease type I (GSDI) models or GSD patients.

Agents	Targets	Subject	Results	References
Bezafibrate	PPAR	G6pc−/− mice	Decreased hepatic glycogen and triglyceride partially reversed hepatic autophagy defect Increased AAV vector‐mediated genome editing	71
		GSD‐Ia dog
Fenofibrate	PPAR‐α	L.G6pc −/− mice	Decreased hepatic glycogen and triglyceride and renal triglyceride Prevented liver or kidney damages	70, 72, 73
		K.G6pc −/− mice
		G6pc −/− mice
VK2809	THR β	G6pc −/− mice	Decreased hepatic triglyceride Stimulated hepatic autophagic flux Promote mitochondrial β‐oxidation	69
G‐CSF	Neutrophils	GSDIb patients	Raised blood neutrophil counts Ameliorates inflammatory bowel symptoms Increased risk of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML)	57, 58, 59, 60, 63, 63
Empagliflozin	SGLT2	GSDIb patients	Reduced plasma 1,5AG levels Reduced intracellular 1,5AG6P Improved bowel health, growth, and laboratory parameters	43, 44, 64, 65, 66, 67, 68
ACEi	ACE	GSDI patients	Decreased glomerular filtration rate Delayed progression of chronic renal failure	37, 54, 55, 56
Vitamin E	Antioxidant	GSDIb patients	Raised neutrophil counts Reduced frequency and severity of infections	61, 62

Abbreviations: ACEi, angiotension converting enzyme inhibitors; G6pc, glucose 6 phosphatase‐α; G6pt, glucose 6 phosphate transporter; G‐CSF, granulocyte‐colony stimulating factor; GSDI, glycogen storage disease type I; K.G6pc −/− mice, Kidney‐specific G6pc deficient mice; L.G6pc −/− mice, Liver‐specific G6pc deficient mice; PPAR, peroxisome proliferator‐activated receptor; PPAR‐α, peroxisome proliferator‐activated receptor‐alpha; SGLT2, sodium‐dependent glucose transporters 2; THR β, thyroid hormone receptor β.

In addition, there are several novel pharmacological agents developed for the treatment of GSDIa patients. VK2809, a thyroid hormone receptor agonist selective for liver tissues, was used to treat GSDIa mice. VK2809 stimulation decreased hepatic triglyceride levels by inducing autophagy and mitochondrial biogenesis promoting fatty acid β‐oxidation. ⁶⁹ Similarly, fenofibrate and bezafibrate are used to improve hepatic and/or renal lipid metabolism and reduce glycogen storage in neonatal G6pc ^−/− mice through the induction of autophagy to promote β‐oxidation of fatty acids. ⁷⁰ , ⁷¹ Furthermore, fenofibrate, via the promotion of fatty acid β‐oxidation, significantly decreased the hepatic and renal glycogen and triglyceride accumulations, and prevented liver injury and nephropathy in L.G6pc ^−/− and K.G6pc ^−/− mice. ⁷² These findings suggested that the reduction of hepatic lipid by pharmacologically induction autophagy may be a novel therapeutic strategy for GSDIa. ⁷³

4.3. Gene therapies for Glycogen storage disease type I disorders

4.3.1. Adenovirus vector‐mediated gene therapies for Glycogen storage disease type I disorders

Somatic gene therapy is a promising treatment for inherited diseases caused by mutations in genes, such as GSDIa and GSDIb ¹⁸ (Table 3). Gene therapy studies have demonstrated the first and early generation adenovirus vectors expressing G6Pase‐a or G6PT effectively normalized the metabolic abnormalities in GSDI mice and canine models. ²⁶ In 2000, Zingone al. infused an adenovirus vector carrying murine G6Pase gene into G6pc ^−/− mice (Ad‐mG6Pase) via the retro‐orbital vein and restored hepatic G6Pase activity to 19% of that of G6Pase ^+/+ mice, thereby improving pathological manifestations and increasing the survival rate after weaning to 100%. However, the kidneys of adenovirus‐infused G6pc ^−/− mice showed little or no detectable expression of the G6Pase. ⁷⁴ , ⁷⁵ To compensate for the limited efficiency of adenovirus alone, Sun al. proposed a strategy of co‐administration with adenovirus and adeno‐associated virus type 2 vectors (Ad/AAV‐mG6Pase) to maintain sustained G6Pase expression in the liver and kidney of neonatal G6pc ^−/− mice. ⁷⁶ In addition, neonatal G6pt ^−/− mice were infused intravenously with adenoviral vector containing human G6PT (Ad‐hG6PT) effectively delivered G6pt mRNA to the liver, bone marrow, and spleen and corrected neutropenia as well as myeloid abnormalities. ⁵ Nevertheless, it is known that Ad‐mediated transient transgene expression is not suitable for maintaining long‐term G6Pase activity and can cause serious immunogenicity. To achieve sustained transgene expression and alleviate viral toxicity, a helper‐dependent adenovirus vector encoding G6Pase (HDAd‐G6Pase) was administered to the GSDIa murine ⁷⁷ and canine model, ⁷⁸ directed by the human apolipoprotein AI promoter/enhancer. As a result, HDAd‐mediated delivery of G6Pase effectively corrected physiological and biochemical abnormalities in G6Pase‐KO mice and GSDIa dogs, markedly prolonging median survival time to 7 months compared to 5–6 weeks in Ad‐treated mice. ⁷⁶ , ⁷⁷

TABLE 3.

List of experimental gene therapies in Glycogen storage disease type I (GSDI) models.

Author/year	Gene	Treatment vector	Promoter enhancer	Species models	Infusion‐method‐age	Results
Zingone et al. 2000	Murine G6PC	Adenoviral	RSV	G6pc −/− mice	Retro‐orbital vein (14‐day‐old)	Restored hepatic G6Pase activity to 19% of G6Pase+/+
Beaty et al. 2002	Canine G6PC	AAV	Mouse albumin promoter/enhancer	GSD Ia dogs	Intravenous (3‐ or 4‐day‐old)	Resulted in sustained hepatic G6Pase activity; improved liver histology and biochemical parameters
Mao‐Sen et al. 2002	Murine G6PC	Co‐infused adenoviral; AAV	RSV; CBA promoter/CMV enhancer	G6pc −/− mice	Temporal vein (1‐day‐old); intravenous (2‐week‐old)	Resulted in sustained G6Pase expression in both the liver and the kidney; corrected the murine GSD‐Ia disease
Koeberl et al. 2006	Canine G6PC	AAV8	Nucleotides −1372 to −11 of the canine G6PC 5′‐fanking sequence	G6pc −/− mice	Intravenous (2‐week‐old)	Prolonged median survival to 7 months; restored hepatic G6Pase activity to 25% of normal level at 7 mouths of age
Ghosh et al. 2006	Murine G6PC	AAV1; AAV8	CBA promoter/CMV enhancer	G6pc −/− mice	Temporal vein (1‐ or 2‐day‐old); retro‐orbital vein (1‐week‐old)	Increased hepatic G6Pase activity; improved survival; corrected the metabolic abnormalities for the 57 weeks
Koeberl et al. 2007	Canine G6PC	HDAd	Human apolipoprotein AI	G6pc −/− mice	Intravenous (2‐week‐old)	Increased hepatic G6Pase activity over tenfold between 3 days and 28 weeks; prolonged median survival to 7 months; reduced liver glycogen storage; reversed the physiological and biochemical abnormalities
Yiu et al. 2007	Human SLC37A4	Adenoviral	CMV	G6pt −/− mice	Temporal vein (1 or 2‐day‐old); retro‐orbital vein (2‐week‐old)	Restored G6PT mRNA level in the liver, bone marrow, and spleen; corrected metabolic and myeloid abnormalities
Koeberl et al. 2008	Human G6PC	AAV2/8	Nucleotides −298 to +128 of the human G6PC 5′‐fanking sequence	G6pc −/− mice; GSD Ia dogs	Retro‐orbital sinus (12 ± 1 day of age mice); Venipuncture of the jugular vein (3 days of age dogs)	Prevented hypoglycemia, corrected liver G6Pase deficiency; reduced liver glycogen storage in GSD‐Ia mice and dogs; prolonged survival
Yiu et al. 2009	Human SLC37A4	AAV8	CBA promoter/CMV enhancer	G6pt −/− mice	Temporal vein (1‐day‐old)	Hepatic G6PT activity was 50% of wild‐type at 2 weeks post‐infusion but declined to 3% of wild‐type levels by age 6–72 weeks
Grinshpun et al. 2010	Human G6PC	FIV	EF‐1α, CMV, or hAAT	G6pc −/− mice	Temporal vein (1‐day‐old); retro‐orbital sinus (7 days of age)	Normalized blood glucose levels; improved body weight, and decreased accumulation of liver glycogen
Yiu et al. 2010	Human G6PC	AAV8	Nucleotides −2864 to −1 of the human G6PC 5′‐fanking sequence	G6pc −/− mice	Temporal vein (2‐day‐old); retro‐orbital sinus (2‐ or 4‐week‐old)	Normalized blood glucose levels, blood metabolites, hepatic glycogen, and hepatic fat
Weinstein et al. 2010	Murine G6PC	rAAV2/8; rAAV2/1	CBA promoter	GSD Ia dogs	Jugular vein (1 day old); portal vein injection (20‐week‐old)	Corrected of the GSD Ia phenotype
Luo et al. 2011	Human G6PC	AAV2/7	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (12 ± 1 day‐old)	Prevented hypoglycemia; reduced glycogen content in the liver and renal; reduced Albuminuria and renal fibrosis
Crane et al. 2011	Human G6PC	HDAd	Human apolipoprotein AI	GSD Ia dogs	Jugular venipuncture (3‐day‐old dogs)	Normalized blood glucose levels between 6 and 22 months elevated hepatic G6Pase activity and reduced glycogen content; prolonged survival
Demaster et al. 2012	Human G6PC	AAV2/7; AAV2/8; AAV2/9	Data not available	GSD Ia dogs	Jugular venipuncture (2‐ or 3‐day‐old)	Normalized glycogen content; restored hepatic G6Pase activity to 40% of normal in female dogs at 7 mouths of age
Lee et al. 2012	Human G6PC	AAV2/8	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐ or 4‐week‐old)	Prevented chronic HCA formation; restored hepatic G6Pase activity more than 3%–100% of wild‐type
Brooks et al. 2013	Human G6PC	AAV2/8; AAV2/9	Human G6PC 5′‐fanking sequence	G6pc −/− mice; GSD Ia dogs	(2‐week‐old) (1–3‐day‐old)	Improved growth; promoted long‐term survival
Lee et al. 2015	Human G6PC	AAV2/8	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐week‐old)	Prevented HCA formation and hepatic steatosis; expressed 0.5%–1.3% of wild‐type hepatic G6Pase‐α activity grow normally to age 75–90 weeks
Kim et al. 2015	Human G6PC	AAV2/8	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐week‐old)	Activated signaling by hepatic carbohydrate response element binding protein
Landau et al. 2016	Human G6PC	AVV2/8‐ZFN; AVV2/9‐ZFN; AVV2/8‐RoG6P; AVV2/9‐RoG6P	Thyroid hormone binding globulin liver‐specific promoter; human G6PC minimal promoter	G6pc −/− mice	Retro‐orbital (12 ± 1‐day‐old)	Improved survival; improved biochemical correction; improved vector persistence and efficacy, and lower mortality
Kwon et al. 2017	Human SLC37A4	rAAV	2.8 kb human G6PC; 1.62 kb human G6PT; 610‐bp kb human G6PT	G6pt−/− mice	Temporal vein (1‐day‐old); Retroorbital sinus (4‐week‐old)	Established the threshold of hepatic G6PT activity required to prevent tumor formation; protected against age‐related obesity and insulin resistance
Kim et al. 2017	Human G6PC; human co‐G6PC	rAAV	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐week‐old)	rAAV‐co‐G6PC vector is more effective than the rAAV‐G6PC vector; establish the threshold of hepatic G6Pase‐α activity required to prevent HCA/HCC
Lee et al. 2018	Human G6PC	rAAV1	2864‐bp human G6PC	GSD Ia dogs	Jugular venipuncture (1‐day‐old); portal vein (2–6‐month‐old)	Improved survival; prevented focal hepatic lesions or renal abnormalities
Brooks et al. 2018	Human G6PC	AAV2/7; AAV2/8; AAV2/9	Human G6PC minimal promoter	GSD Ia dogs	Data not available	Developed renal failure; exhibited progressive kidney disease histologically
Kang et al. 2019	Human G6PC	AVV2/9‐RoG6P; AVV2/9‐ZFN	Human G6PC minimal promoter	Liver G6pc −/− mice	Intravenous (7–8weeks‐old)	Corrected hepatic G6Pase deficiency; suppressed HCA and HCC
Cho et al. 2019	Human G6PC	rAAV	Human G6PC 5′‐fanking sequence	L‐ G6pc −/− mice	Retroorbital sinus (at 53 weeks post G6pc gene deletion)	Restored hepatic G6Pase‐α expression; prevented de novo HCA/HCC development
Zhang et al. 2019	Human co‐G6PC‐S298 C	rAAV	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐week‐old)	Efficacy of the rAAV‐hG6PC‐S298 C vector was3‐fold higher than that of the rAAV‐hG6PC‐WT vector
Zhang et al. 2020	Human G6PC‐S298 C	rAAV	Human G6PC 5′‐fanking sequence	G6pc −/− mice	Retro‐orbital sinus (2‐week‐old)	Treated with a lower dose to survive long term; expressing ≥3% of normal hepatic G6Pase‐a activity do not develop hepatic tumors;
Arnaoutova et al. 2021		rAAV8	U6 promoter	G6pc‐R83/R83 C mice	Remporal vein (newborn)	Displayed normalized blood metabolites expressing ≥3% of normal hepatic G6Pase‐a activity; increased 16‐week survival rate from 0% to 100%

Abbreviations: AAV, adeno‐associated virus; CBA, chicken β‐actin; CMV, cytomegalovirus; FIV, Feline immunodeficiency virus; G6PC, glucose 6 phosphatase‐α; G6PT, glucose 6 phosphate transporter; GSDI, glycogen, storage disease type I; RSV, rous sarcoma virus.

4.3.2. Adeno‐associated virus vector‐mediated gene therapies for Glycogen storage disease type I disorders

In contrast to first‐generation adenovirus vector‐mediated gene therapy, recombinant adeno‐associated virus (rAAV) has several unique characteristics as a gene delivery vector. There is no direct evidence that rAAV can cause vector genome‐mediated host genotoxicity in humans, making rAAV become the primary vector for gene transfer in vivo. ¹⁸ Investigators have identified 12 human AAV serotypes (AAV1 to AAV12), of which the most commonly used rAAV serotypes include AAV2, 5, 8, and 9. In 2002, Beaty et al. firstly developed an AAV vector (AAV‐AlbcG6PGH) containing mouse albumin promoter/enhancer driving the expression of canine G6Pase. The administration of this agent to GSDIa canines resulted in sustained G6Pase expression and improvement in hepatic histology and relevant biochemical parameters. ⁷⁹ Different serotypes of AAV exhibit different tropisms, which are mainly dependent on the interaction of the amino acid sequence and structure of AAV capsid with host cytokines, including cell surface receptors, signaling molecules, and co‐receptors. ⁸⁰ One study found that AAV serotype 8 (AAV8) has become one of the preferred vector serotypes for liver‐targeted gene therapy because it is a highly liver tropic and efficient vector with low preexisting immunity. ⁸¹ Koeberl et al. used AAV serotype 8‐based vectors expressing G6Pase to administrate to G6pc ^−/− mice or GSDIa dogs and that showed they could prolong median survival and restore hepatic G6Pase activity in affected models. ³⁹ , ⁸² In subsequent years, a large body of research has systemically shown that the administration of rAAV pseudotype 2/8 or 2/7 vectors expressing the wild‐type human G6Pase‐α (rAAV‐G6PC) achieve high efficiency of transduction and correct metabolic abnormalities in the infused G6pc ^−/− mice or GSDIa dogs, directed by the human G6PC promoter/enhancer (GPE). ⁷ , ⁹ , ³¹ , ³² , ³⁹ , ⁸³ , ⁸⁴ , ⁸⁵ , ⁸⁶ More recently, Kim al. found that the G6pc ^−/− mice treated with rAAV‐G6PC and rAAV‐co(codon‐optimized)‐G6PC expressed more than 3% of normal hepatic G6Pase‐α activity and prevented the development of age‐related obesity and insulin resistance. ³¹ , ⁸⁷

These evolutionary approaches of single amino acid variant have been widely used to improve the efficacy and potency of rAAV‐mediated gene transfer and therapy for GSD and reduce the adaptive immune response to the rAAV. ⁸⁸ , ⁸⁹ , ⁹⁰ Initially, Kim et al designed a rAAV vector expressing a codon‐optimized (co) G6Pase‐α (rAAV‐co‐G6PC) and found that rAAV‐co‐G6PC mediated more efficient hepatic G6Pase‐α expression than rAAV‐G6PC. ⁸⁷ Later, Zhang et al. constructed a rAAV‐G6PC‐S298 C vector with a single amino acid variant in the native human G6PC sequence. This rAAV‐G6PC‐S298 C vector mediated a threefold higher expression of G6Pase‐α activity than the native rAAV‐G6PC vector in the short‐term, which simultaneously enhanced and prolonged survival of the young mice under low dose of the vector. ⁸⁸ An alternative vector enabled researchers to further enhance initial G6Pase‐α enzymatic activity by simultaneously combining codon‐optimization with the S298 C variant (designated as rAAV‐co‐S298 C). ⁹⁰ Similarly, Cao et al. showed that serine (S) to cysteine (C) substitution at position 298 (S298 C) had elevated G6Pase‐α activity among 20 G6Pase‐α protein variants, while combining protein variant S298 C and codon optimization can maximize enzymatic activity. ⁸⁹

rAAV vector genomes are predominantly persisted in the nucleus in an extrachromosomal form (circularized dsDNA). AAV genome can mediate long‐term gene expression, relying solely on cellular proteins to convert the single‐stranded genome into stable circular and concatemeric episomal forms. ⁹¹ However, AAV vectors can transduce cells by integrating nonhomologous chromosomal locations, which have been developed for the treatment of GSDIa. ⁹² , ⁹³ The mouse ROSA26 locus is a ‘‘safe harbor’’ and is frequently used as a safe site for gene targeting by homologous recombination (HR). Mouse ROSA26 locus is a preferred site for gene targeting as it can achieve stable and efficient transgene expression without affecting the expression and function of other nearby endogenous genes. ⁹⁴ The engineered zinc finger nucleases (ZFNs) have been proposed as an optimal means of precise genome modification, which can generate site‐specific DNA double‐strand breaks in target genomic sequences. Studies have demonstrated the feasibility of gene targeting to the mouse ROSA26 locus directed by engineered ZFNs. ⁹⁴ , ⁹⁵ Studies have developed dual AAV vector‐mediated genome editing in G6Pase knockout mice, one vector (AAV‐ZFN) containing a ZFNs generating DNA double‐strand breaks in the ROSA26 gene and one vector (AAV‐G6Pase) containing G6PC donor transgene targeting ROSA26 locus. ZFN‐mediated targeted gene therapy integrates G6Pase gene cassette into the murine ROSA26 safe harbor locus, significantly prolonging and enhancing G6Pase expression in treated G6pc ^−/− mice. ⁹³ , ⁹⁶

5. DISCUSSION AND FUTURE DIRECTIONS

Glycogen storage disease type I is a rare inherited disease characterized by the overaccumulation of G6P, since G6Pase‐a and G6PT deficiency resulted in the failure to hydrolyze G6P into glucose and phosphate in the final step of glycogenolysis and gluconeogenesis. ⁹⁷ The patients with GSDI suffer from severe hypoglycemia and metabolic disorder associated complications. Currently, there are no specific drugs for this disease, and the focus is on preventing hypoglycemia and mitigating progression of related complications. Over the past 50 years, the care of patients with GSDI has improved following elaborate dietary regimens, including continuous nutrition therapy, restriction of non‐utilizable sugars, and frequent cornstarch feeding. Patients with GSDI currently receive dietary therapies that have enabled patients to improve hypoglycemia and remove early symptoms and signs of the disease. However, dietary therapies have many limitations with a risk of calcium, vitamin D, and iron deficiency and cannot prevent long‐term complications, such as progressive liver and renal failure. ⁹⁸

The high levels of G6P accumulate in the liver and are shunted to alternative metabolic pathways, especially glycogen synthesis and de novo lipogenesis. These findings suggested that reducing hepatic lipid may be a novel therapeutic strategy for patients with GSDI. Furthermore, studies have shown that glycogen metabolism has been linked to autophagy, such as Pompe disease and Lafora disease. ⁹⁹ , ¹⁰⁰ Activation of autophagy is critical for energy utilization and clearance of intracellular lipid, which can preferentially hydrolyze intrahepatic lipid droplets to promote mitochondrial fatty acids β‐oxidation. ¹⁰¹ A number of previous studies have demonstrated the use of drugs to promote hepatic autophagy in the treatment of GSDIa mice, such as VK2809, fenofibrate as well as bezafibrate. ⁶⁹ , ⁷¹ , ¹⁰² Recently, mediator complex subunit 1 (MED1) has play an important role in regulating hepatic autophagy, β‐oxidation of fatty acids, and mitochondrial function. ¹⁰³ The effect of MED1 on glycogen storage disease has not been studied and it may be an alternative drug for glycogen storage disease.

ACEi is currently one of the most effectual drugs for the treatment of hypertension and glomerular diseases. However, ACEi is unable to improve microalbuminuria and proteinuria. ³⁸ It may be the reason that G6P is overaccumulated in early patients with GSDI resulting in activating protein kinase C and regulating renal angiotensinogen. ¹⁰⁴ New agents are being developed for the treatment of renal damage in patients with GSDI. Transforming growth factor beta cytokine (TGF‐β) has been widely recognized a central participant of chronic renal disease. TGF‐β is upregulated and mediates chronic renal disease by promoting fibroblast proliferation, epithelial‐to‐mesenchymal transition, production of tubular and fibroblast extracellular matrix proteins (ECM), and podocyte injury. ¹⁰⁵ Thus, inhibiting TGF‐β axis and its crosstalk pathways can be a promising therapeutic strategy for renal disease. ¹⁰⁶

Considering that both G6Pase‐a and G6PT are highly hydrophobic transmembrane proteins, enzyme‐replacement therapy is not suitable for patients with GSDI. Up to now, gene therapy studies using GSDIa and GSDIb models of mice or dogs have shown that AAV vectors encoding G6Pase‐α deliver transgene to the liver or kidney can be directed by the chicken β‐actin promoter/CMV enhancer, ⁷⁶ , ¹⁰⁷ , ¹⁰⁸ the mouse albumin promoter/enhancer, ⁷⁹ the canine G6PC promoter, ⁸² or the human G6PC promoter. ⁷ , ³¹ , ³² , ⁸⁵ , ⁸⁸ , ⁹⁰ Unfortunately, these animal models have severe hypoglycemia and a short life expectancy, which make them unsuitable for studying the long‐term complications of gene therapy on GSDI. These studies have well‐demonstrated that the numbers of vector genomes could decline over time following rapid division of hepatocyte cell in young GSDIa mice; thus, the rAAV‐mediated gene therapy cannot be durably and efficiently expressed in transgenic tissues. ⁸⁸ , ⁹⁰ , ⁹³ , ⁹⁶ One study found that the significant increase in hepatic CD8⁺ lymphocyte counts was observed in G6pc ^−/− mice infused with AAV‐CBA at age 2 weeks; in contrast, low levels of hepatic CD8+ lymphocyte counts were observed with AAV8‐GPE under the same conditions. ⁸³ The results suggest the rapid decline and low efficacy of transgene expression are associated with the CBA promoter/CMV enhancer and there may be an inflammatory immune response against the AAV‐CBA vector. Previous gene therapy studies have used two different vectors, rAAV8‐GPE and rAAV8‐miGPE, encoding two human G6Pase‐α to treat the mouse model of GSDIa, directed by 2864‐bp of the human G6PC promote/enhancer and 328‐bp of minimal G6PC promote/enhancer, respectively. However, the results showed that the rAAV8‐GPE was more effective for hepatic G6Pase‐α expression than the rAAV8‐miGPE vector. ¹⁰⁹ The molecular mechanisms of promoters affecting vector genomes decline and transgene expression efficacy remain to be explored.

As one of the most promising gene therapy vectors, AAV vectors cannot be integrated into the host‐cell genomes and predominantly act as an episome in the host nucleus. ¹¹⁰ However, AAV‐mediated gene therapy has some limitations, including (1) immune response to the viral vector due to preexisting neutralizing antibodies in the body, (2) transgene expression in progressively lost over time, especially in the presence of underlying hepatocyte growth and regeneration, (3) high dose vector targeting of non‐permissive tissue is required. (4) AAV vectors package single‐stranded DNA (ssDNA) and therefore require time‐consuming steps of complementary‐strand synthesis or recruitment for transduction. Repeated administration of AVV‐based gene therapy is not ideal due to the aforementioned application limitations. In addition to Ad and AAV‐targeted gene therapy for GSDI, one study used a lentiviral vector based on feline immunodeficiency virus (FIV). ¹¹¹ Compared to adeno‐associated viruses, lentiviral vectors are clinically attractive, including: (1) lentiviruses integrate transgene‐carrying viruses into cellular genes, providing long‐term expression even in actively dividing cells; (2) immune response to the vector components is not elicited because FIV long‐termina repeat activity is negligible. However, the copy number of the viral genome integrated into the cellular genome may vary considerably between experiments and the potential genotoxicity of integrated genomes; thus, the full potential of lentiviral vectors for GSDI gene therapy remains to be determined.

New therapeutic vehicles are currently being developed for an increasing number of indications. A new therapeutic concept that has recently been proposed with particular applicability to metabolic diseases is the combination of gene therapy and other therapeutic strategies. Prior to gene therapy, dietary regimens and pharmacological interventions can be very effective in preventing hypoglycemia and alleviating liver damage. The combination of dietary regimens or pharmacological treatment with gene therapy has the potential to enhance therapeutic outcomes through early treatment of complications and prolonged vector persistence. Significant challenges remain before gene therapy moves from proof‐of‐concept to preclinical studies and clinical trials; we need to further innovate technology in order to generate safer and more effective programmable vectors to promote clinical applications and ultimately improve child health.

AUTHOR CONTRIBUTIONS

Jiamin Zhon, Jiaming Fan, Russell R. Reid, and Tong‐Chuan He conceived and designed the study. Jiamin Zhon, Yannian Gou, Piao Zhao, Xiangyu Dong, Meichun Guo, Aohua Li, Ailing Hao, Hue H. Luu, Tong‐Chuan He, Russell R. Reid, and Jiaming Fan drafted and revised the manuscript. All authors reviewed, edited, and approved the manuscript.

CONFLICT OF INTEREST STATEMENT

Tong‐Chuan He, Hue H. Luu and Russell R. Reid are the members of Pediatric Discovery Editorial Board. They were excluded from all editorial decision‐making related to the acceptance of this article for publication. The remaining authors declare no conflict of interests.

ETHICS STATEMENT

Not applicable.

Zhong J, Gou Y, Zhao P, et al. Glycogen storage disease type I: Genetic etiology, clinical manifestations, and conventional and gene therapies. Pediatr Discov. 2023;1(2):e3. 10.1002/pdi3.3

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.