Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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 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. GSD-Ia is a pediatric genetic disorder. The rAAV-G6PC 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 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 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 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- protein. The editing reagents formulated in lipid nanoparticles (LNP) 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 4% of normal hepatic G6Pase- 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 only therapies currently available to treat 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/-catenin signaling regulates the expression of a variety of downstream mediators implicated in renal fibrosis, including multiple genes in the RAS. Sustained activation of Wnt/-catenin signaling is associated with the development and progression of renal fibrotic lesions 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, in turn, activate Wnt/-catenin signaling. We show that GSD-Ia mice display AKI that leads to activation of the Wnt/-catenin/RAS axis. Renal fibrosis was demonstrated by increased renal levels of Snail1, -smooth muscle actin (-SMA), and extracellular matrix proteins, including collagen-I1 and collagen-IV. Treating GSD-Ia mice with a CBP/-catenin inhibitor, ICG-001, significantly decreased nuclear translocated active -catenin and reduced renal levels of renin, Snail1, -SMA, and collagen-IV. The results suggest that inhibition of Wnt/-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 AT to GC 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, 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 (LNP) 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