An evolutionary approach to optimizing glucose‐6‐phosphatase‐α enzymatic activity for gene therapy of glycogen storage disease type Ia

Abstract

Glycogen storage disease type-Ia (GSD-Ia), caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC), is characterized by impaired glucose homeostasis with a hallmark hypoglycemia, following a short fast. We have shown that G6pc-deficient (G6pc−/−) mice treated with recombinant adeno-associated virus (rAAV) vectors expressing either wild-type (WT) (rAAV-hG6PC-WT) or codon-optimized (co) (rAAV-co-hG6PC) human (h) G6Pase-α maintain glucose homeostasis if they restore ≥3% of normal hepatic G6Pase-α activity. The co vector, which has a higher potency, is currently being used in a phase I/II clinical trial for human GSD-Ia (NCT 03517085). While routinely used in clinical therapies, co vectors may not always be optimal. Codon-optimization can impact RNA secondary structure, change RNA/DNA protein-binding sites, affect protein conformation and function, and alter posttranscriptional modifications that may reduce potency or efficacy. We therefore sought to develop alternative approaches to increase the potency of the G6PC gene transfer vectors. Using an evolutionary sequence analysis, we identified a Ser-298 to Cys-298 substitution naturally found in canine, mouse, rat, and several primate G6Pase-α isozymes, that when incorporated into the WT hG6Pase-α sequence, markedly enhanced enzymatic activity. Using G6pc −/− mice, we show that the efficacy of the rAAV-hG6PC-S298C vector was 3-fold higher than that of the rAAV-hG6PC-WT vector. The rAAV-hG6PC-S298C vector with increased efficacy, that minimizes the potential problems associated with codon-optimization, offers a valuable vector for clinical translation in human GSD-Ia. K E Y W O R D S clinical translation for human GSD-Ia, expression optimization, gene therapy, glucose-6-phosphataseα variant, recombinant adeno-associated virus

mRNA therapy restores euglycemia and prevents liver tumors in murine model of glycogen storage disease

“…Therefore, we selected the S298C protein variant for further analysis. This finding is consistent with and supports previous studies by Zhang et al, which reported similar improvements in protein expression with the S298C variant 47 , 48 . Based on predicted topology analysis 49 , the S298C substitution falls within the eighth transmembrane domain of hG6Pase-α, downstream of residues R83, H119, and H176 50 , that are directly involved in hG6Pase-α activity (see predicted topology, Supplementary Fig.…”

Gene therapy and genome editing for type I glycogen storage diseases

“…Their synthetic mRNA, which contained complete N1-methylpseudouridine substitution, and included 5′and 3′untranslated regions along with the coding sequence and a poly(A) tail, was packaged in a LNP formulation and delivered intravenously. Supporting the study of Zhang et al (2019), Cao et al (2021) showed that the G6PC-S298C variant had a 2-fold higher specific activity than WT G6PC, when expressed in vitro, and the activity of the encoded enzyme was further increased by codon-optimization. As previously reported (Zhang et al, 2019), the increase in enzymatic activity of the G6PC-S298C variant correlated with a corresponding increase in the stability of the expressed protein, and notably, the overall clearance rate of G6PC-WT and G6PC-S298C proteins were similar.…”

“…While routinely used in clinical therapies, codon-optimized vectors may not always The effects of codon optimization and catalytic activity optimization are additive and stable. (A) G6pc _/_ mice were treated at age 2 weeks with 10 12 vp/kg rAAV-G6PC-WT, rAAV-G6PC-S298C, rAAV-coG6PC, or rAAV-coG6PC-S298C (n = 6 per group) and analyzed at age 4 weeks (Zhang et al, 2019). (B) G6pc _/_ mice were treated at age 2 weeks with 3 x10 12 vp/kg rAAV-G6PC-WT, rAAV-G6PC-S298C, rAAV-coG6PC, or rAAV-coG6PC-S298C (n = 6 per group) and analyzed at age 66-76 weeks.…”

Evolving AAV-delivered therapeutics towards ultimate cures

“…infusion n.a. NCT03517085; phase 1/2 2018 Ultragenyx [ 43 ] ABO-102 (scAAV9.U1a.hSGSH) MPS IIIA SGSH AAV9 0.5 × 10 13 1 × 10 13 3 × 10 13 ≥ 6 months, i.v. infusion Signs of efficacy NCT02716246; phase 1/2 NCT04360265 (follow-up) 2016; 2020 Abeona Therapeutics [ 44 ] AT132 X-linked myotubular myopathy hMTM1 AAV8 1 × 10 14 3 × 10 14 ≤ 5 years, males; i.v.…”