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 (NCT03517085). 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.

Keywords: clinical translation for human GSD-Ia; expression optimization; gene therapy; glucose-6-phosphatase-α variant; recombinant adeno-associated virus.

Conflict of interest statement

Conflict of Interest

Lisa Zhang, Jun-Ho Cho, Irina Arnaoutova, Brian C. Mansfield, and Janice Y. Chou declare that they have no conflicts of interest. The Eunice Kennedy Shriver National Institute of Child Health and Human Development has applied patents on the vectors described in this article as an employee invention of Janice Chou.

© 2019 SSIEM.

Figures

Fig. 1.

Alignment of the amino acid sequences of mammalian G6Pase-α. The non-conserved amino acids between human and canine G6Pase-α are bracketed. The G6Pase-α sequences are GENBANK accession numbers U01120 (human), U91844 (dog), U00445 (mouse), U07993 (rat), XP_003942781 (Saimiri boliviensis), XP_002748054 (Callithrix jacchus), XP_003786361 (Otolemur garnettii), and XP_008066427 (Carlito syrichta).

Fig. 2.

Characterization of pSVL-hG6Pase-α variants harboring the non-conserved amino acids in canine G6Pase-α. (a) The hG6Pase-α enzyme is anchored in the ER membrane by 9 helices (H1 to H9), creating 4 cytoplasmic (C1 to C4) and 4 luminal (L1 to L4) loops. The amino-terminus is located in the ER lumen and the carboxyl-terminus in the cellular cytoplasm. The non-conserved amino acids in hG6Pase-α differing from that in canine G6Pase-α are indicated and shown in black. The Cys residues are shown in grey and indicated by arrow heads. (b) Phosphohydrolase activity of WT and variant hG6Pase-α after transfecting of the pSVL constructs into COS-1 cells. Numbers in parenthesis represent fold increase in phosphohydrolase activity over the WT construct. Data represent the mean ± SEM. *p < 0.05, **p < 0.005.

Fig. 3.

Transient expression assays of G6Pase-α constructs and phenotype analyses of rAAV-treated G6pc−/− mice. (a) Phosphohydrolase activity after transfecting the pSVL-hG6Pase-α-WT, pSVL-hG6Pase-α-S298C, pSVL-co-hG6Pase-α, or pSVL-co-hG6Pase-α-S298C construct into COS-1 cells. (b) Western-blot analysis of hG6Pase-α and β-actin after transient expression of the pSVL-hG6Pase-α-WT, pSVL-hG6Pase-α-S298C, pSVL-co-hG6Pase-α, pSVL-co-hG6Pase-α-S298C construct into COS-1 cell along with quantification by densitometry (n = 3). (c-f) Phenotypic analyses of rAAV-treated G6pc−/− mice. Two-week-old G6pc−/− mice were treated with 1012 or 1013 vp/kg of rAAV-WT (n = 12), rAAV-S298C (n = 12), rAAV-co (n = 12), or rAAV-co-S298C (n = 12) vector, and phenotype analyzed at age 4 weeks (6 mice/treatment) and 12 weeks (6 mice/treatment). (c) Hepatic microsomal G6Pase-α activity in mice treated with the rAAV vectors at 1012 vp/kg. (d) Vector genome copy numbers in the livers of 12-week-old mice. (e) Hepatic microsomal G6Pase-α activity in mice treated with the rAAV vectors at 1013 vp/kg. (f) Fasting blood glucose tolerance profiles and blood glucose levels following a 24-hour of fast. Data represent the mean ± SEM. *p < 0.05, **p < 0.005.

Fig. 4.

Phenotypic analyses of rAAV-treated G6pc−/− mice at age 12 weeks. G6pc−/− mice were treated at age 2 weeks with either 1012 vp/kg or 1013 vp/kg of rAAV-WT, rAAV-S298C, or rAAV-co, and analyzed at age 12 weeks. The data were obtained from WT (+/+, n = 12) and G6pc−/− mice treated with either 1012 vp/kg (n = 6 per vector) or 1013 vp/kg (n = 6 per vector) of rAAV-WT, rAAV-S298C, or rAAV-co. (a) BW. (b) LW/BW ratios and LW. (c) Hepatic glucose levels. (d) Relative G6pt mRNA levels. (e) Hepatic levels of G6P, triglyceride, and lactate. (f) Western-blot analysis of serum antibodies against human G6Pase-α. The serum samples were obtained from 12 week-old G6pc−/− mice treated at age 2 weeks with 1013 vp/kg of rAAV-WT, rAAV-S298C, or rAAV-co vector. Lanes 1: anti-hG6Pase-α antiserum; lanes 2, 6, 10: serum samples (1: 50 dilution) from WT mice; lanes 3, 7, 11: serum samples (1: 50 dilution) from rAAV-WT-treated G6pc−/− mice; lanes 4, 8, 12: serum samples (1: 50 dilution) from rAAV-S298C-treated G6pc−/− mice; and lanes 5, 9, 13: serum samples (1: 50 dilution) from rAAV-co-treated G6pc−/− mice. Data represent the mean ± SEM. *p < 0.05, **p < 0.005.