An evolutionary approach to optimizing glucose-6-phosphatase-α enzymatic activity for gene therapy of glycogen storage disease type Ia
Lisa Zhang, Jun-Ho Cho, Irina Arnaoutova, Brian C Mansfield, Janice Y Chou, Lisa Zhang, Jun-Ho Cho, Irina Arnaoutova, Brian C Mansfield, Janice Y Chou
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
Source: PubMed