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National Institutes of Health

Eunice Kennedy Shriver National Institute of Child Health and Human Development

2023 Annual Report of the Division of Intramural Research

Molecular Genetics of Heritable Human Disorders

Janice Chou
  • Janice Y. Chou, PhD, Head, Section on Cellular Differentiation
  • Irina Arnaoutova, PhD, Staff Scientist
  • Lisa Zhang, PhD, Staff Scientist
  • Hung-Dar Chen, PhD, Contract Technician
  • Cheol Lee, PhD, Contract Technician
  • Kunal Pratap, PhD, Visiting Fellow
  • Ananya Samanta, PhD, Visiting Fellow
  • Brian C. Mansfield, PhD, Special Volunteer

We conduct research to delineate the pathophysiology of and develop novel therapies for type I glycogen storage disease (GSD-I) subtypes GSD-Ia and GSD-Ib. GSD-Ia is caused by a deficiency in the liver/kidney/intestine–restricted glucose-6-phosphatase-α (G6Pase-α or G6PC), and GSD-Ib is caused by a deficiency in the ubiquitously expressed glucose-6-phosphate transporter (G6PT or SLC37A4). G6Pase-α is an endoplasmic reticulum (ER) transmembrane protein that regulates intracellular glucose production by catalyzing the hydrolysis of G6P to glucose and phosphate. The active site of G6Pase-α faces into the ER lumen and depends on G6PT, another ER transmembrane protein, to translocate G6P from the cytoplasm into the ER lumen. To function, G6Pase-α couples with G6PT to form a G6Pase-α/G6PT complex, which maintains interprandial glucose homeostasis. GSD-Ia and GSD-Ib patients manifest a common metabolic phenotype of impaired glucose homeostasis and the long-term complications of hepatocellular adenoma/carcinoma (HCA/HCC) and renal disease. There is no cure for either GSD-Ia or GSD-Ib. The current dietary therapies have enabled GSD-I patients to maintain a normalized metabolic phenotype if strictly adhered to. However, the underlying pathological processes remain uncorrected, and HCA/HCC and renal disease still occur in metabolically compensated GSD-I patients. We generated animal models of GSD-Ia and GSD-Ib, which are being exploited to both delineate the disease more precisely and to develop new treatment approaches, including gene therapy. We also generated G6PC– and G6PT–expressing recombinant adeno-associated virus (rAAV) vectors and showed that rAAV vector–mediated gene argumentation therapies for GSD-Ia and GSD-Ib are safe and efficacious. Our rAAV–G6PC vector (US patent #9,644,216) technology was licensed to Ultragenyx Pharmaceutical Inc (Novato, CA), who launched a phase I/II clinical trial (NCT03517085) in 2018, now in phase III (NCT05139316). To explore alternative genetic technologies for GSD-I therapies, we have established formal collaborations under the CRADA (cooperative research and development agreement) with CRISPR Therapeutics (Cambridge, MA) and Beam Therapeutics (Cambridge, MA) to evaluate the efficacy of CRISPR/Cas9–based and adenine base editor (ABE)–based gene-editing systems, respectively, to correct gene-specific G6PC mutations in animal models of GSD-Ia.

Molecular mechanism underlying hepatic autophagy impairment in GSD-Ib

Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and a long-term risk of hepatocellular adenoma/carcinoma (HCA/HCC). The etiology of HCA/HCC in GSD-Ib is unknown. Studies have shown that deficiency in autophagy, an evolutionarily conserved, degradative process that produces energy and building blocks through lysosomal degradation of intracellular proteins and organelles in times of nutrient deprivation and environmental stresses contributes to hepatocarcinogenesis. Autophagy can be regulated positively by sirtuin 1 (SIRT1), AMP–activated protein kinase (AMPK), and forkhead box O (FoxO) transcription factor family members. In the liver, AMPK is activated via phosphorylation of the AMPK α-subunit at residue T172 by the liver kinase B-1 (LKB1), a serine/threonine kinase.

To understand the pathways contributing to hepatocarcinogenesis in GSD-Ib, we hypothesized that impaired hepatic autophagy is a significant contributor. In this study, we showed that G6PT deficiency leads to impaired hepatic autophagy evident from attenuated expression of many components of the autophagy network, decreased autophagosome formation, and reduced autophagy flux. The G6PT–deficient liver displayed impaired SIRT1 and AMPK signaling, along with reduced expression of SIRT1, FoxO3a, LKB1, and the active p-AMPK. Importantly, we showed that overexpression of either SIRT1 or LKB1 in the G6PT–deficient liver restored autophagy and SIRT1/FoxO3a and LKB1/AMPK signaling. The hepatosteatosis in the G6PT–deficient liver lowered SIRT1 expression. LKB1 overexpression reduced hepatic triglyceride levels, providing a potential link between LKB1/AMPK signaling upregulation and the increase in SIRT1 expression. In conclusion, downregulation of SIRT1/FoxO3a and LKB1/AMPK signaling underlies impaired hepatic autophagy, which may contribute to HCA/HCC development in GSD-Ib. Understanding this mechanism may guide future therapies [Reference 1].

CRISPR/Cas9–based double strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type Ia.

GSD-Ia is a pediatric genetic disorder. The rAAV-G6PC vector used in the Phase III clinical trial for GSD-Ia (NCT05139316) is episomally expressed. Currently, there are insufficient clinical data to understand whether multi-decade episomal transgene expression can be maintained in the human liver at a therapeutic level. We therefore explored alternative genetic technologies for GSD-Ia therapy, such as CRISPR/Cas9–based gene editing. We previously generated a G6pc–R83C mouse strain carrying the prevalent pathogenic G6PC-p.R83C variant and showed that the G6pc–R83C mice exhibit the pathophysiology of impaired glucose homeostasis mimicking human GSD-Ia. In an initial exploration of CRISP/Cas-9–based editing using AAV to deliver the CRISPR reagents, we showed that a homology-directed repair strategy could correct the abnormal metabolic phenotype of neonatal G6pc–R83C mice.

Using the G6pc–R83C mice, we explored a CRISPR/Cas9–based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the non-homologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon, and to introduce an additional splice-acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents, formulated in lipid nanoparticles (LNPs), were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with 2 doses of LNP-dsODN at age 2 and 4 weeks. The G6pc–R83C mice receiving successful editing expressed about 4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed a normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene-augmentation therapy, which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype [Reference 3].

Figure 1.

Figure 1

Click image to view.

CRISPR/Cas9–based double-strand oligonucleotide (dsODN) insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type Ia.

Base editing corrects metabolic abnormalities and prevents hepatocarcinogenesis in murine GSD-Ia.

We explore the adenine base editor (ABE)–based technologies that enable a programmable conversion of A•T to G•C in genomic DNA for GSD-Ia therapy. The ABE system works in both dividing and non-dividing cells, is reported to produce virtually no indels or off-target editing in the genome, and can correct a pathogenic variant in its native genetic locus, leading to permanent, therapeutically effective long-term expression. This is a collaborative study with Beam Therapeutics, Cambridge, MA, under a CRADA.

The G6PC-p.R83C is the most prevalent pathogenic mutation identified in Caucasian GSD-Ia patients, which contains a single G→A transition in the G6PC gene. We first generated a homozygous humanized (hu) R83C/R83C mouse strain, the huR83C mouse, by inserting the entire coding sequence of the human G6PC-p.R83C along with human G6PC 3′-UTR into exon 1 of the mouse G6pc gene at the ATG start codon. The insertion places the human transcript under the control of the native mouse G6pc promoter/enhancer. The mouse G6pc gene is disrupted by a premature STOP codon created in the mouse G6pc exon 1. We showed that the huR83C mice manifest impaired glucose homeostasis characterized by growth retardation, hypoglycemia, hyperlipidemia, hyperuricemia, hepatomegaly, and nephromegaly mimicking the abnormal metabolic phenotype of human GSD-Ia. We then explored the efficacy of ABE to correct the G6PC-p.R83C variant in the huR83C mice following systemic administration of editing reagents formulated in LNPs, and we monitored phenotypic correction up to 53 weeks of age. We showed that physiological levels of hepatic G6Pase-α activity with an editing efficiency up to about 60% could be restored in the edited huR83C mice. The edited mice maintained glucose homeostasis, survived long-term, and lacked hepatic tumors. While LNP–ABE failed to transduce the kidney, nephromegaly was improved in the edited mice. In summary, the ABE–mediated gene editing corrected a pathogenic G6PC variant in the native genetic locus, offering a permanent, non-inheritable, correction for GSD-Ia.

Inhibition of Wnt/β-catenin signaling reduces renal fibrosis in murine glycogen storage disease type Ia.

Renal disease is a serious long-term complication for GSD-Ia. The early kidney manifestations of GSD-Ia are impaired renal gluconeogenesis, and nephromegaly caused by increased glycogen accumulation. The only therapies currently available to treat GSD-Ia are dietary therapies, which significantly alleviate metabolic abnormalities but only delay the onset of chronic kidney disease. The underlying pathological processes remain uncorrected, and glomerular hyperfiltration, hypercalciuria, hypocitraturia, and urinary albumin excretion still occur in metabolically compensated GSD-Ia patients. We previously showed that one mechanism underlying GSD-Ia nephropathy is fibrosis mediated by activation of the renin-angiotensin system (RAS).

Wnt/β-catenin (catenins are components of adherens junctions) signaling regulates the expression of a variety of downstream mediators implicated in renal fibrosis, including several genes in the RAS. Sustained activation of Wnt/β-catenin signaling is associated with the development and progression of renal fibrotic lesions, which can lead to chronic kidney disease. We examined the molecular mechanism underlying GSD-Ia nephropathy. Damage to kidney proximal tubules is known to trigger acute kidney injury (AKI) which can, in turn, activate Wnt/β-catenin signaling. We showed that GSD-Ia mice display AKI, which leads to activation of the Wnt/β-catenin/RAS axis. Renal fibrosis was demonstrated by increased renal levels of Snail1 (zinc finger transcriptional repressor), α-smooth muscle actin (α-SMA), and extracellular matrix proteins, including collagen-Iα1 and collagen-IV. Treating GSD-Ia mice with the CBP/β-catenin (CBP is a CREB-binding protein, where CREB is a cAMP response element–binding protein) inhibitor ICG-001 significantly reduced nuclear translocated active β-catenin and renal levels of renin, Snail1, α-SMA, and collagen-IV. The results suggest that inhibition of Wnt/β-catenin signaling may be a promising therapeutic strategy for GSD-Ia nephropathy [Reference 4].

Additional Funding

  • The Children’s Fund for Glycogen Storage Disease Research
  • CRISPR Therapeutics (Cambridge, MA), under a cooperative research and development agreement (CRADA)
  • Beam Therapeutics, Inc (Cambridge, MA), under a CRADA


  1. Gautam S, Zhang L, Lee C, Arnaoutova I, Chen HD, Resaz R, Eva A, Mansfield BC, Chou JY. Molecular mechanism underlying hepatic autophagy impairment in glycogen storage disease type Ib. Hum Mol Genet 2023 32:262–275.
  2. Chou JY, Mansfield BC. Gene therapy and genome editing for type I glycogen storage diseases. Frontiers Mol Med 2023 3:1167091.
  3. Samanta A, George N, Arnaoutova I, Chen HD, Mansfield BC, Hart C, Carlo T, Chou JY. CRISPR/Cas9-based double strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type Ia. J Inherit Metab Dis 2023 46(6):1147–1158.
  4. Lee C, Pratap K, Zhang L, Chen HD, Gautam S, Arnaoutova I, Raghavankutty M, Starost MF, Kahn M, Mansfield BC, Chou JY. Inhibition of Wnt/β-catenin signaling reduces renal fibrosis in murine glycogen storage disease type Ia. Biochim Biophys Acta Mol Basis Dis 2023 1870(1):166874.


  • Troy Carlo, PhD, Prime Medicine Inc, Cambridge, MA
  • Alessandra Eva, PhD, Istituto Giannina Gaslini, Genoa, Italy
  • Christopher Hart, PhD, Prime Medicine Inc, Cambridge, MA
  • Michael Kahn, PhD, Beckmann Research Institute, City of Hope, Duarte, CA
  • Matthew F. Starost, PhD, Diagnostic & Research Services Branch, Division of Veterinary Resources, NIH, Bethesda, MD


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