Molecular Genetics Of Heritable Human Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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. GSD-Ia and GSD-Ib patients manifest a common phenotype of impaired glucose homeostasis and long-term risk of HCA/HCC. The etiology of HCA/HCC in GSD-I is unknown. Autophagy is 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. Studies have shown that a deficiency in hepatic autophagy can contribute 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 which is also a tumor suppressor. We have shown that G6Pase- deficiency impairs hepatic autophagy through downregulation of SIRT1/FoxO3a signaling. We hypothesized that in GSD-Ib, G6PT deficiency will lead to hepatic autophagy deficiency. Using mouse models of GSD-Ib, we showed that G6PT deficiency leads to impaired hepatic autophagy evident from attenuated expression of many components of the autophagy network, impaired 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 p-AMPK. Hepatic overexpression of either SIRT1 or LKB1 in G6PT-deficient liver restored autophagy and SIRT1/FoxO3a/LKB1/AMPK signaling. The hepatosteatosis in GSD-Ib mice reduced SIRT1 expression. LKB1 overexpression reduced hepatic triglycerides levels, linking LKB1/AMPK signaling to SIRT1 expression. Taken together, our data show that G6PT deficiency leads to impaired autophagy in GSD-Ib primarily by downregulation of SIRT1/FoxO3a/LKB1/AMPK signaling. We have generated 4 efficacious G6PC gene transfer rAAV vectors for GSD-Ia gene therapy. They are rAAV-G6PC that expresses the wild-type G6PC, rAAV-co-G6PC that expresses a codon-optimized (co) G6PC, rAAV-G6PC-S298C that expresses a G6PC-S298C variant with increased efficacy, and rAAV-co-G6PC-S298C. Using the rAAV-G6PC and rAAV-co-G6PC vectors, we have shown that rAAV-treated G6pc-/- mice expressing 3% of normal hepatic G6Pase- activity maintain glucose homeostasis and show no evidence of HCA/HCC. Our rAAV-G6PC/rAAV-co-G6PC vector technology (US patent # 9,644,216) was commercially licensed to Ultragenyx Pharmaceutical who has launched a phase I/II clinical trial for GSD-Ia (NCT03517085) using the rAAV-co-G6PC vector. The rAAV-co-G6PC vector contains a 20% change in the native G6PC coding sequence. While routinely used in clinical therapies, codon-optimized vectors may not always be optimal. To develop alternative approaches to increase the potency of the G6PC gene transfer vector, we generated the rAAV-G6PC-S298C and the rAAV-co-G6PC-S298C vectors (US patent #10,415,044), the latter combines codon optimization with the S298C variant in the same construct. In a short-term (4 weeks) study using G6pc-/- mice, we showed that the efficacy of the rAAV-G6PC-S298C and rAAV-co-G6PC-S298C vectors were 3- and 5-fold higher than that of the rAAV-G6PC vector. We then undertook a 65-76-week study to examine the longer-term efficacy of these vectors. We infused 4 separate groups of G6pc-/- mice, each with one of the rAAV vectors at 3 x1012 vp/kg and examined phenotypic correction of the mice at age 65-76 weeks. We showed that hepatic G6Pase- activities in rAAV-co-G6PC, rAAV-G6PC-S298C, and rAAV-co-G6PC-S298C-treated G6pc-/- mice were 2.2-, 2.3-, and 6.2-fold higher, respectively than that in rAAV-G6PC-treated mice. The rAAV-G6PC-S298C vector that contains a 2% change in the native G6PC coding sequence provides equal or greater efficacy to the codon optimization approach, offering a valuable alternative vector for clinical translation in human GSD-Ia. The rAAV-co-G6PC-S298C vector that is 3-fold more efficient than that of the rAAV-co-G6PC vector currently used in the clinical trials offers a vector of choice for clinical translation in human GSD-Ia. 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, and can lead 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 huR83C mouse strain by inserting the entire coding sequence of the human G6PC-p.R83C cDNA along with human G6PC 3-UTR into exon 1 of the mouse G6pc gene at the ATG start codon. This insertion places the human cDNA 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 first 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 treated newborn huR83C 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 huR83C mice expressed significant levels of hepatic G6Pase- activity with an editing efficiency up to 60% and displayed normalized blood metabolite profiles. Taken together, our data demonstrate the potential of base-editing to correct the G6PC-p.R83C mutation and the abnormal GSD-Ia phenotype.