Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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. While we have shown that CRISPR/Cas9-based homology directed repair (HDR) and non-homologous end joining (NHEJ) gene editing strategies can correct the GSD-Ia phenotype in G6pc-R83C mice, the efficacy of the HDR-mediated editing in non-dividing cells was low and the success of the NHEJ-mediated editing was dependent on the efficiency of inserting the editing DNA oligonucleotide in the correct orientation. We therefore 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, 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 prevalent human variant G6PC1-c.247C>T/p.R83C, carrying a single G>A transition in the G6PC1 gene is amenable to ABE editing. We first generated a humanized G6PC1-R83C mouse strain (huR83C) by inserting the entire human G6PC1-R83C (G6PC1-c.247C>T) coding sequence into exon 1 of the mouse G6pc gene, in a manner that knocked out the native mouse G6pc gene expression while placing the expression of the human G6PC1-R83C under the control of the mouse G6pc promoter/enhancer. We showed that the huR83C mice lacked hepatic and renal G6Pase-α activity and manifested a human GSD-Ia phenotype. We then evaluated the efficacy of ABE to correct the G6PC1-p.R83C variant in the huR83C mice following systemic administration of BEAM-301, the ABE editing reagent containing ABE mRNA/gRNA encapsulated in a lipid nanoparticles (LNP) formulation. Newborn (NB) or 3-week-old mice were infused with BEAM-301 and monitored for phenotypic correction up to age 1 year. We showed that the efficiency of correcting the G6PC1-R83C variant correlates with the activity of hepatic G6Pase-α restored in the edited mice. Notably, an editing efficiency up to ~60% could be obtained. All edited mice harboring ≥3 units of hepatic G6Pase-α activity (~5% editing) displayed an improved metabolic profile, sustained 24 hours of fasting, and survived through 1 year. Significantly, no decline in hepatic G6Pase-α activity was observed over the 1-year study. Conversely, the untreated huR83C mice exhibited fasting hypoglycemia, with none surviving longer than age 6 weeks. An off-target analysis of BEAM-301 identified only a single intronic, guide-dependent off-target site, highlighting the high precision of this base-editing approach. Integrated risk assessment determined that editing at this site is unlikely to affect the function of any associated genes, indicating a negligible overall risk. These findings supported Beam Therapeutics’ successful application for Investigational New Drug status for BEAM-301, enabling the initiation of a Phase I/II clinical trial for GSD-Ia in January 2025. We are aware of individuals with GSD-Ia carrying the G6PC1 p.R83C variant who previously declined gene augmentation therapy, holding out hope for a gene editing strategy tailored to their specific mutation. It is heartening to witness this development now becoming a reality. Liver dysfunction in GSD-Ia patients results in fasting hypoglycemia, which is neonatally fatal without dietary therapies, while kidney dysfunction results in nephropathy. Several liver-directed G6PC1 gene therapies have shown clinical promise, including a completed phase III trial for G6PC1 gene augmentation (NCT05139316) currently under Food and Drug Administration review, and an adenine base editor strategy targeting the most prevalent G6PC1 mutation has recently entered phase I/II clinical trials (NCT06735755). The rAAV8-G6PC1 vector used in gene augmentation selectively targets the liver and does not transduce the kidney. Similarly, lipid nanoparticles delivering the adenine base editor accumulate in the liver. Consequently, developing effective kidney-targeted therapies remains challenging. Previous studies suggest that strict dietary management can help delay the progression of renal disease. The G6pc-/- (GSD-Ia) mice mimic human GSD-Ia, but fewer than 30% G6pc-/- mice survive beyond weaning, and at 3-weeks of age early-stage renal dysfunction is not detectable via serum marker analysis, necessitating studies in older mice. To address this, we developed a liver G6Pase-α augmented (L-G6PC1), kidney G6Pase-α null (Knull) model for nephropathy studies. To study the impact of restoration of liver-specific G6Pase-α on GSD-Ia nephropathy, we generated two groups of liver augmented GSD-Ia mice: one expressing 86% of normal liver G6Pase-α activity (L-G6PC1-high) and another expressing 2% of normal liver G6Pase-α activity (L-G6PC1-low). Since the mouse background is a global G6PC1 knockout, the liver augmented mice lack kidney G6Pase-α expression. We evaluates kidney function in L-G6PC1-high and L-G6PC1-low mice at 12 weeks of age. Both groups exhibited impaired renal glucose homeostasis, altered renal glucose reabsorption, acute kidney injury, and early signs of renal fibrosis. However, mice with near-normal liver G6Pase-α activity had better renal glucose reabsorption and homeostasis with lower serum levels of cystatin C and blood urea nitrogen, key markers of kidney function. These findings highlight the potential of liver-directed G6PC1 gene therapy to enhance metabolic control and mitigate early kidney disease in GSD-Ia. Endogenous glucose production and interprandial blood glucose homeostasis rely on a functional G6PT/G6Pase complex. While a single glucose-6-phosphate transporter (G6PT) exists, there are two distinct G6Pase enzymes: G6Pase-α (G6PC1) and G6Pase-β (G6PC3). GSD-Ib results from a deficiency of the ubiquitously expressed G6PT. G6Pase-α is primarily expressed in the liver, kidney, and intestine, whereas G6Pase-β, though also ubiquitous, is about sixfold less active than G6Pase-α and highly expressed in the kidney but minimally in the liver. In the liver, glucose homeostasis is mainly regulated by the G6PT/G6Pase-α complex, while in the kidney, both G6PT/G6Pase-α and G6PT/G6Pase-β contribute. Clinically, patients with GSD-Ia and GSD-Ib exhibit impaired glucose homeostasis and are at long-term risk for renal disease. Renal glucose regulation is maintained by the coordinated activity of G6PT/G6Pase-α and G6PT/G6Pase-β. In GSD-Ia (G6pc1-/-) mice, G6PT/G6Pase-β activity remains in the kidney, whereas in GSD-Ib mice, neither G6PT/G6Pase complex is functional. Based on this distinction, we hypothesize that renal G6PT/G6Pase-β activity in GSD-Ia may help mitigate renal disease compared to GSD-Ib. Preclinical studies have shown that in GSD-Ia, impaired renal gluconeogenesis and excessive glycogen accumulation lead to acute kidney injury, which activates Wnt/β-catenin signaling, a key driver of fibrosis, resulting in progressive renal fibrosis. We now show that G6pt-/- (GSD-Ib) mice display similar pathological features, including Wnt/β-catenin–mediated fibrosis, but with significantly higher renal triglyceride levels than age-matched GSD-Ia mice during postnatal weeks 1 to 3. This lipid accumulation is a known contributor to kidney disease progression. In GSD-Ia, G6PT/G6Pase-α activity is absent in both liver and kidney, while G6PT/G6Pase-β remains active in the kidney. In contrast, GSD-Ib lacks both G6PT complexes in all tissues. These findings suggest that the G6PT/G6Pase-β activity in GSD-Ia exerts a protective effect on renal function, mitigating the severity of kidney disease compared to GSD-Ib.