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Principal Investigator: JANICE CHOU
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2024
Award: $2,208,533
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
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, contributes to hepatocarcinogenesis. Autophagy can be regulated positively by SIRT1, 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 LKB1. To understand the pathways contributing to hepatocarcinogenesis in GSD-Ib, we hypothesized that impaired hepatic autophagy is a significant contributor. In this study, we show 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 show that overexpression of either SIRT1 or LKB1 in G6PT-deficient liver restored autophagy and SIRT1/FoxO3a and LKB1/AMPK signaling. The hepatosteatosis in G6PT-deficient liver decreased SIRT1 expression. LKB1 overexpression reduced hepatic triglycerides 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.
GSD-Ia is a pediatric genetic disorder. The rAAV-G6PC1 vector used in Phase III clinical trial for GSD-Ia (NCT05139316) is episomally expressed. Currently, there is insufficient clinical data to understand if 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 G6pc1-R83C mouse strain carrying the prevalent pathogenic G6PC1-p.R83C variant and showed that the G6pc1-R83C mice exhibit the GSD-Ia phenotype. 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 G6pc1-R83C mice. Using the G6pc1-R83C mice, we now 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 G6pc1 exon-2. The strategy is based on the insertion of a short dsODN into G6pc1 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 were delivered to the liver. Mice were treated either with one dose of LNP-dsODN or 2 doses of LNP-dsODN. The G6pc1-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed 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.
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 current therapies for GSD-Ia are dietary therapies which have significantly alleviated 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 have previously shown that one mechanism underlies GSD-Ia nephropathy is fibrosis mediated by activation of the renin-angiotensin system (RAS). The Wnt/β-catenin signaling regulates the expression of vauious downstream mediators implicated in renal fibrosis, including RAS genes. Sustained activation of Wnt/β-catenin signaling leads to renal fibrosis that can lead to chronic kidney disease. In this study, we examined the molecular mechanism underlying GSD-Ia nephropathy. Damage to the kidney proximal tubules is known to trigger acute kidney injury (AKI) that can activate Wnt/β-catenin signaling. We show that GSD-Ia mice display AKI and Wnt/β-catenin/RAS axis activation. Renal fibrosis was demonstrated by increased renal levels of Snail1, α-SMA, and extracellular matrix proteins. Treating GSD-Ia mice with a CBP/β-catenin inhibitor, ICG-001, significantly decreased nuclear translocated active β-catenin and reduced 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.
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 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 G6PC1-c.247C>T/p.R83C, carrying a single G>A transition in the G6PC1 gene, is the most prevalent pathogenic mutation identified in Caucasian GSD-Ia patients that can be targeted by ABEs. We first generated a humanized knock-in G6PC1-R83C mouse strain (huR83C) and showed that the huR83C mice lacked hepatic and renal G6Pase-α activity and manifested a human GSD-Ia pathophysiology.
We then evaluated the efficacy of ABE to correct the G6PC1-p.R83C variant in the huR83C mice following systemic administration of editing reagents formulated in lipid nanoparticles, hereafter referred to as BEAM-301 and 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 ~60% could be restored in the edited huR83C mice. The edited mice maintained glucose homeostasis, survived long-term, and lacked hepatic tumors. Significantly, no decline in hepatic G6Pase-α activity was observed over the 53-week study. In summary, we have developed a base-editing strategy for in vivo correction of a pathogenic G6PC1 variant in the native genetic locus, that may offer a durable and highly efficient therapeutic for patients with GSD-Ia.
Terms: <1H-Purin-6-amine><3' Splice Site><AAV vector><AAV-based vector><Active Sites><Acute Renal Failure with Renal Papillary Necrosis><Adenine><Age><Albumins><Angiotensin-Forming Enzyme><Angiotensinogenase><Animal Model><Animal Models and Related Studies><Attenuated><Autophagocytosis><Autophagosome><Autoregulation><Basal Transcription Factor><Basal transcription factor genes><Benign Hepatoma><Beta Cadherin-Associated Protein><Beta-1 Catenin><Blood><Blood Glucose><Blood Reticuloendothelial System><Blood Sugar><CRADA><CRISPR><CRISPR approach><CRISPR based approach><CRISPR based therapeutics><CRISPR based treatment><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR therapeutics><CRISPR tools><CRISPR treatment><CRISPR-CAS-9><CRISPR-Cas based therapeutics><CRISPR-based disease therapeutics><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based therapy><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas therapeutics><CRISPR/Cas9><CRISPR/Cas9 technology><CRISPR/Cas9 therapeutics><CRISPR/Cas9 therapy><CRISPR/Cas9 treatment><CRISPR/Cas9-based therapy><CUL-2><Carcinoma><Cas nuclease technology><Cas9 based therapeutics><Caucasian><Caucasian Race><Caucasians><Caucasoid><Caucasoid Race><Causality><Cell Communication and Signaling><Cell Signaling><Childhood><Chronic Kidney Failure><Chronic Renal Disease><Chronic Renal Failure><Clinical><Clinical Data><Clinical Trials><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats based therapeutics><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Clustered Regularly Interspaced Short Palindromic Repeats therapeutics><Collaborations><Collagen IV><Collagen Type IV><Compensation><Complex><Complication><Cooperative Research and Development Agreement><Couples><Cytoplasm><D-Glucose><D-Glucose-6-phosphate phosphohydrolase><DNA Therapy><Development><Dextrose><Diet therapy><Disease><Disorder><Dose><Double-Stranded DNA><Down-Regulation><Dysfunction><Endoplasmic Reticulum><Epithelial cancer><Ergastoplasm><Etiology><Excretory function><Exhibits><Exons><Extracellular Matrix Proteins><Face><Family member><Fatty Liver><Fibrosis><Functional disorder><Future><Gene Transcription><Gene Transfer Clinical><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Alteration><Genetic Change><Genetic Diseases><Genetic Intervention><Genetic Transcription><Genetic defect><Genomic DNA><Gierke's Disease><Gluconeogenesis><Glucose><Glucose-6-Phosphatase Deficiency><Glucose-6-Phosphate><Glucose-6-Phosphate Phosphohydrolase><Glucosephosphatase Deficiency><Glycogen><Glycogen Storage Disease Type I><Glycogen storage disease type Ia><Glycogenosis 1><Goals><Hepatic><Hepatic Neoplasms><Hepatocarcinogenesis><Hepatocellular Adenoma><Hepatorenal Glycogen Storage Disease><Heritability><Homeostasis><Human><Hydrolysis><Hypoglycemia><Impairment><Inorganic Phosphate Transporter><Integral Membrane Protein><Intestinal><Intestines><Intracellular Communication and Signaling><Intrinsic Membrane Protein><Kidney><Kidney Diseases><Kidney Urinary System><Knock-in><LKB1><LKB1/STK11 Gene><Laboratories><Legal patent><Licensing><Link><Liver><Liver Carcinogenesis><Liver Cell Adenoma><Liver Steatosis><Liver neoplasms><Malignant Epithelial Neoplasms><Malignant Epithelial Tumors><Mediating><Mediator><Messenger RNA><Metabolic><Mice><Mice Mammals><Modern Man><Molecular><Molecular Genetics><Monitor><Mouse Strains><Murine><Mus><Mutation><NHEJ><Neonatal><Nephropathy><Non-Homologous End Joining><Non-homologous DNA End Joining><Nonhomologous DNA End Joining><Nonhomologous End Joining><Nuclear Translocation><Occidental><Oligo><Oligonucleotides><Outcome><PRO2286><Patents><Pathogenicity><Pathologic Processes><Pathological Processes><Pathway interactions><Patients><Pharmaceutical Agent><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Phase><Phase 3 Clinical Trials><Phase III Clinical Trials><Phenotype><Phosphate Transport Proteins><Phosphate Transporters><Phosphates><Phosphorylation><Physiologic><Physiological><Physiological Homeostasis><Physiopathology><Protein Phosphorylation><Proteins><Proximal Kidney Tubules><RNA Expression><RNA Splicing><Reagent><Recombinant adeno-associated virus><Recombinant adeno-associated virus (rAAV)><Renal Disease><Renin><Renin-Angiotensin System><Risk><SIRT1><SIRT1 gene><STK11><STK11 gene><Seizures><Signal Transduction><Signal Transduction Systems><Signaling><Sirtuin 1><Snails><Splice Acceptor Sites><Splicing><System><Technology><Therapeutic><Transcription><Transcription Factor Proto-Oncogene><Transcription factor genes><Transmembrane Protein><Transmembrane Protein Gene><Triacylglycerol><Triglycerides><Type IV (Basement Membrane) Collagen><Up-Regulation><Upregulation><Variant><Variation><Vitamin B4><Von Gierke's Disease><acute kidney injury><adeno-associated viral vector><adeno-associated virus vector><ages><attenuate><attenuates><autophagy><base editing><base editor><beta catenin><biological signal transduction><blood glucose regulation><bowel><carcinogenesis in the liver><causation><chronic kidney disease><damage to kidney><determine efficacy><developmental><dietary therapy><disease causation><disease causing variant><disease-causing mutation><ds-DNA><dsDNA><efficacy analysis><efficacy assessment><efficacy determination><efficacy evaluation><efficacy examination><epithelial carcinoma><evaluate efficacy><examine efficacy><excretion><faces><facial><gDNA><gene augmentation intervention><gene augmentation therapy><gene corrected><gene correction><gene editing platform><gene editing system><gene editing technology><gene editing tools><gene locus><gene repair therapy><gene therapy><gene-based therapy><gene-editing toolkit><genetic condition><genetic disorder><genetic locus><genetic technology><genetic therapy><genome mutation><genomic correction><genomic location><genomic locus><genomic therapy><glucose RA><glucose biosynthesis><glucose control><glucose homeostasis><glucose production><glucose rate of appearance><glucose regulation><glucose-6-phosphatase><glycogenosis type I><hepatic body system><hepatic carcinogenesis><hepatic neoplasia><hepatic neoplasm><hepatic organ system><hepatic steatosis><hepatic tumor><hepatocellular carcinogenesis><hepatorenal glycogenosis><hepatosteatosis><hypercalcinuria><hypercalciuria><hypercalciuric><hypoglycemic><hypoglycemic episodes><in vivo><inhibitor><inorganic phosphate><kidney damage><kidney disorder><kidney fibrosis><knockin><lipid based nanoparticle><lipid nanoparticle><liver cancer pathogenesis><liver kinase B1><liver tumor><liver tumorigenesis><mRNA><metabolic phenotype><metabotype><model of animal><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><new treatment approach><new treatment strategy><next generation therapeutics><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutics><novel therapy><novel therapy approach><oligos><overexpress><overexpression><pathogenic variant><pathophysiology><pathway><pediatric><pharmaceutical><phase III protocol><pre-clinical study><preclinical study><rAAV><recombinant AAV><renal><renal damage><renal disorder><renal fibrosis><renal proximal tubule><repair><repair strategy><repaired><therapeutically effective><transcription factor><transgene expression><urinary><vector><white race><β-catenin>