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Principal Investigator: JANICE CHOU
Organization: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
Fiscal Year: 2022
Award: $1,602,501
Funding agency: Eunice Kennedy Shriver National Institute of Child Health and Human Development
The rAAV-co-G6PC vector used in the current phase I/II clinical trial is episomally expressed and the long-term durability of expression in humans is currently being established. We therefore sought to explore the use of the CRISPR/Cas9 technology to correct a pathogenic GSD-Ia variant in its native genetic locus. The most prevalent pathogenic mutation identified in Caucasian GSD-Ia patients is G6PC-p.R83C, representing 32% of diseased alleles. Using the CRISPR/Cas9-based gene editing technology, we generated a GSD-Ia mouse disease model, the G6pc-R83C mouse homozygous for the G6PC-p.R83C mutation and showed that the G6pc-R83C mice manifest impaired glucose homeostasis mimicking that of human GSD-Ia. We then used a CRISPR/Cas9-based gene editing system to treat newborn G6pc-R83C mice and showed that the treated mice grew normally to age 16 weeks without hypoglycemia seizures. The treated G6pc-R83C mice, expressing 3% of normal hepatic G6Pase- activity, maintained glucose homeostasis, displayed normalized blood metabolites, and could sustain 24 hours of fasting. Taken together, we have developed a second-generation therapy in which in vivo correction of a pathogenic G6PC-p.R83C variant in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia disorder.
Clinically, GSD-Ib patients manifest a metabolic phenotype of impaired blood glucose homeostasis and 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 evolutionary 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 -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 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.
We have generated 4 efficacious G6PC gene transfer rAAV vectors for GSD-Ia gene therapy; rAAV-G6PC expressing the wild-type (WT) G6PC, rAAV-coG6PC expressing a codon-optimized (co) G6PC, rAAV-G6PC-S298C expressing a G6PC-S298C variant with increased efficacy, and rAAV-coG6PC-S298C. Our rAAV-G6PC/rAAV-coG6PC vector (US patent #9,644,216) technology was licensed to Ultragenyx Pharmaceutical Inc who has launched a phase I/II clinical trial (NCT03517085) in 2018 and followed by a phase III clinical trial (NCT05139316) in 2022 using the rAAV-GPE-coG6PC vector. To examine the long-term efficacy of these rAAV vectors, we conducted a long-term (66-76 week) gene transfer study in G6pc-/- mice using these rAAV vectors. All treated G6pc-/- mice survived to age 66-76 weeks, and the outcomes were additive. Hepatic G6Pase- activities in rAAV-G6PC-S298C-, rAAV-coG6PC-, and rAAV-coG6PC-S298C-treated G6pc-/- mice were 1.7-, 1.7-, and 4.4-fold higher, respectively than that in rAAV-G6PC-WT-treated mice. The efficacy of the rAAV-coG6PC-S298C vector is 2.6-fold higher than the rAAV-coG6PC vector currently used in phase III clinical trial (NCT05139316). Taken together, the rAAV-G6PC-S298C and rAAV-coG6PC-S298C vectors offer attractive clinical alternatives.
We explore the Adenine base editor (ABE)-based technologies that enable a programmable conversion of AT to GC in genomic DNA for GSD-Ia therapies. 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, 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 that contains a single G>A transition in the G6PC gene. We first generated a homozygous humanized R83C/R83C mouse strain, the G6PC-R83C 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. This 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 G6PC-R83C 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 treated newborn G6PC-R83C mice with lipid nanoparticles encompassing the guide RNA and mRNA encoding ABE (LNP-ABE) and showed that the treated mice grew normally to age 8 weeks without hypoglycemia seizures. The LNP-ABE-treated G6PC-R83C mice expressed significant levels of hepatic G6Pase- activity with an editing efficiency up to 60% and displayed normalized blood metabolite profiles and could tolerate 24 hours of fasting. Taken together, our data demonstrate the potential of base-editing to correct the G6PC-p.R83C mutation in its native genetic locus could lead to potentially permanent, durable, long-term correction of the GSD-Ia disorder.
GSD-Ia patients manifest nephromegaly caused by marked glycogen accumulation and nephropathy. The current dietary therapies have significantly alleviated metabolic abnormalities and delayed chronic renal disease and renal insufficiency in GSD-Ia patients. However, the underlying pathological processes remain uncorrected, glomerular hyperfiltration, hypercalciuria, hypocitraturia, and urinary albumin excretion still occur in metabolically compensated GSD-Ia patients. We have shown that one mechanism that underlies GSD-Ia nephropathy is fibrosis mediated by activation of the renin-angiotensin system (RAS). The Wnt/-catenin signaling that promotes fibrosis controls the expression of RAS genes. We hypothesized that elevated renal glycogen could elicit acute kidney injury (AKI) that activates Wnt/-catenin signaling and promotes fibrosis. Here we show that G6pc-/- mice displayed impaired renal glucose homeostasis and AKI. Renal levels of -catenin increased markedly in G6pc-/- mice during postnatal development, along with elevated renal levels of renin, angiotensinogen, and snail1. Renal fibrosis was evident by increased renal levels of -smooth muscle actin (-SMA) and extracellular matrix (ECM) proteins. ICG-001, a -catenin inhibitor, reduced renal levels of renin, snail1, -SMA, and ECM proteins, indicating that targeting the Wnt/-catenin s
Terms: <(hydroxymethylglutaryl-CoA reductase (NADPH)) kinase><0-4 weeks old><1H-Purin-6-amine><5'-AMP-activated protein kinase><AAV vector><AMP-activated kinase><AMP-activated protein kinase><AMPK enzyme><ATP-protein phosphotransferase><Active Sites><Acute Renal Failure with Renal Papillary Necrosis><Adenine><Age><Albumins><Alleles><Allelomorphs><Angiotensin-Forming Enzyme><Angiotensinogen><Angiotensinogenase><Animal Model><Animal Models and Related Studies><Attenuated><Autophagocytosis><Autophagosome><Basal Transcription Factor><Basal transcription factor genes><Benign Hepatoma><Blood><Blood Glucose><Blood Reticuloendothelial System><Blood Sugar><CAP102 protein><CRADA><CRISPR approach><CRISPR based approach><CRISPR based therapeutics><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR therapeutics><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas therapeutics><CRISPR/Cas9><CRISPR/Cas9 technology><CRISPR/Cas9 therapeutics><CRISPR/Cas9 therapy><Carcinoma><Cas nuclease technology><Cas9 based therapeutics><Caucasian><Caucasian Race><Caucasians><Caucasoid><Caucasoid Race><Causality><Cell Communication and Signaling><Cell Signaling><Chronic Kidney Failure><Chronic Renal Disease><Chronic Renal Failure><Clinical><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><Code><Coding System><Codon><Codon Nucleotides><Collaborations><Complex><Cooperative Research and Development Agreement><Couples><Cytoplasm><D-Glucose-6-phosphate phosphohydrolase><DNA Therapy><Data><Development><Diet therapy><Disease><Disease model><Disorder><Down-Regulation><Downregulation><Dysfunction><Endoplasmic Reticulum><Enhancers><Enlarged Liver><Epithelial cancer><Ergastoplasm><Etiology><Excretory function><Exons><Extracellular Matrix Proteins><Face><Family member><Fasting><Fatty Liver><Fibrosis><Functional disorder><Future><G(s), alpha Subunit><G(s), α Subunit><G(s)alpha><G(s)α><GTP-Binding Protein alpha Subunits, Gs><GTP-Binding Protein α Subunits, Gs><Gene Transfer><Gene Transfer Clinical><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Generations><Genes><Genetic Alteration><Genetic Change><Genetic Intervention><Genetic defect><Genome><Genomic DNA><Gierke's Disease><Glucose-6-Phosphatase Deficiency><Glucose-6-Phosphate><Glucose-6-Phosphate Phosphohydrolase><Glucosephosphatase Deficiency><Glucosephosphates><Glycogen><Glycogen Storage Disease Type I><Glycogen storage disease type Ia><Glycogenosis 1><Goals><Growth><Gs alpha Family G-Protein><Gsα><Guide RNA><Gαs><HMG CoA reductase (NADPH) kinase><HMG CoA reductase kinase><HMG coenzyme A reductase (NADPH) kinase><Hepatic><Hepatocarcinogenesis><Hepatocellular Adenoma><Hepatomegaly><Hepatorenal Glycogen Storage Disease><Heritability><Hour><Human><Hydrolysis><Hyperlipemia><Hyperlipidemia><Hypertensinogen><Hyperuricemia><Hypoglycemia><Impairment><Initiation Codon><Initiator Codon><Inorganic Phosphate Transporter><Integral Membrane Protein><Interphase Cell><Intestinal><Intestines><Intracellular Communication and Signaling><Intrinsic Membrane Protein><Kidney><Kidney Diseases><Kidney Failure><Kidney Insufficiency><Kidney Urinary System><Kinase Family Gene><Kinases><Laboratories><Lead><Legal patent><Link><Liver><Liver Carcinogenesis><Liver Cell Adenoma><Liver Steatosis><Malignant Epithelial Neoplasms><Malignant Epithelial Tumors><Mediating><Messenger RNA><Metabolic><Mice><Mice Mammals><Modern Man><Molecular Genetics><Mouse Strains><Murine><Mus><Mutation><Nephropathy><Newborn Infant><Newborns><Non-dividing Cell><Nondividing Cell><Nonsense Codon><Occidental><Organelles><Outcome><Patents><Pathogenicity><Pathologic Processes><Pathological Processes><Pathway interactions><Patients><Pb element><Pharmaceutical Agent><Pharmaceuticals><Pharmacologic Substance><Pharmacological Substance><Phase 1/2 Clinical Trial><Phase 3 Clinical Trials><Phase I/II Clinical Trial><Phase III Clinical Trials><Phosphate Transport Proteins><Phosphate Transporters><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Physiopathology><Premature Stop Codon><Proangiotensin><Process><Protein Kinase><Protein Phosphorylation><Protein-Serine Kinase><Protein-Serine-Threonine Kinases><Protein-Threonine Kinase><Proteins><Recombinant adeno-associated virus><Recombinant adeno-associated virus (rAAV)><Regulatory Ns Protein><Renal Disease><Renal Failure><Renal Insufficiency><Renin><Renin-Angiotensin System><Renin-Substrate><Reporting><Resting Cell><Risk><SIRT1><SIRT1 gene><Seizures><Serine Kinase><Serine-Threonine Kinases><Serine/Threonine Protein Kinase Gene><Signal Transduction><Signal Transduction Systems><Signaling><Sirtuin 1><Smooth Muscle Actin><Smooth Muscle Actin Staining Method><Start Codon><Stimulatory Gs G-Protein><System><Technology><Therapeutic><Threonine Kinase><Time><Tissue Growth><Transcript><Transcription Factor Proto-Oncogene><Transcription factor genes><Transmembrane Protein><Transmembrane Protein Gene><Transphosphorylases><Triacylglycerol><Triglycerides><UTRs><Untranslated Regions><Up-Regulation><Upregulation><Variant><Variation><Vitamin B4><Von Gierke's Disease><Work><acute kidney injury><adeno-associated viral vector><adeno-associated virus vector><ages><alpha E-catenin><alpha Subunit Stimulatory GTP-Binding Protein><alpha catenin><alpha-1 catenin><alpha-Gs><alphaE catenin><autophagy><base><base editing><base editor><biological signal transduction><blood glucose regulation><bowel><cadherin-associated protein 102 kDa><carcinogenesis in the liver><causation><chronic kidney disease><developmental><dietary therapy><disease causation><disorder model><efficacy analysis><efficacy assessment><efficacy evaluation><efficacy examination><environmental stresses><environmental stressor><epithelial carcinoma><evaluate efficacy><examine efficacy><excretion><faces><facial><fasted><fasts><gDNA><gRNA><gene locus><gene repair therapy><gene therapy><gene-based therapy><genetic locus><genetic technology><genetic therapy><genome mutation><genomic locus><genomic therapy><glucose RA><glucose control><glucose homeostasis><glucose phosphate><glucose production><glucose rate of appearance><glucose regulation><glucose-6-phosphatase><glycogen synthase a kinase><glycogenosis type I><heavy metal Pb><heavy metal lead><hepatic body system><hepatic carcinogenesis><hepatic organ system><hepatic steatosis><hepatocellular carcinogenesis><hepatorenal glycogenosis><hepatosteatosis><hydroxyalkyl protein kinase><hydroxymethylglutaryl-CoA-reductase kinase><hypercalcinuria><hypercalciuria><hypercalciuric><hypoglycemic><hypoglycemic episodes><in vivo><indel><inhibitor><insertion-deletion><insertion-deletion mutation><insertion/deletion><insertion/deletion mutation><kidney disorder><kidney fibrosis><lipid based nanoparticle><lipid nanoparticle><liver cancer pathogenesis><liver tumorigenesis><mRNA><metabolic phenotype><metabotype><model of animal><model organism><new drug treatments><new drugs><new therapeutics><new therapy><newborn child><newborn children><next generation therapeutics><novel drug treatments><novel drugs><novel therapeutics><novel therapy><nutrient deprivation><nutritional deprivation><ontogeny><overexpress><overexpression><pathophysiology><pathway><phase III protocol><phosphorylase b kinase kinase><post-natal development><postnatal development><programs><promoter><promotor><rAAV><recombinant AAV><renal><renal disorder><renal fibrosis><therapeutically effective><transcription factor><uricacidemia><urinary><vector><virtual><white race><α-Gs><α-catenin><αE-catenin>