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. 2023 Nov;46(6):1147-1158.
doi: 10.1002/jimd.12660. Epub 2023 Aug 18.

CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia

Ananya Samanta  1 , Nelson George  1 , Irina Arnaoutova  1 , Hung-Dar Chen  1 , Brian C Mansfield  1 , Christopher Hart, Troy Carlo, Janice Y Chou  1
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CRISPR/Cas9-based double-strand oligonucleotide insertion strategy corrects metabolic abnormalities in murine glycogen storage disease type-Ia

Ananya Samanta et al. J Inherit Metab Dis. 2023 Nov.

Abstract

Glycogen storage disease type-Ia (GSD-Ia), characterized by impaired blood glucose homeostasis, is caused by a deficiency in glucose-6-phosphatase-α (G6Pase-α or G6PC). Using the G6pc-R83C mouse model of GSD-Ia, we explored a CRISPR/Cas9-based double-strand DNA oligonucleotide (dsODN) insertional strategy that uses the nonhomologous end-joining repair mechanism to correct the pathogenic p.R83C variant in G6pc exon-2. The strategy is based on the insertion of a short dsODN into G6pc exon-2 to disrupt the native exon and to introduce an additional splice acceptor site and the correcting sequence. When transcribed and spliced, the edited gene would generate a wild-type mRNA encoding the native G6Pase-α protein. The editing reagents formulated in lipid nanoparticles (LNPs) were delivered to the liver. Mice were treated either with one dose of LNP-dsODN at age 4 weeks or with two doses of LNP-dsODN at age 2 and 4 weeks. The G6pc-R83C mice receiving successful editing expressed ~4% of normal hepatic G6Pase-α activity, maintained glucose homeostasis, lacked hypoglycemic seizures, and displayed normalized blood metabolite profile. The outcomes are consistent with preclinical studies supporting previous gene augmentation therapy which is currently in clinical trials. This editing strategy may offer the basis for a therapeutic approach with an earlier clinical intervention than gene augmentation, with the additional benefit of a potentially permanent correction of the GSD-Ia phenotype.

Keywords: genome editing; glucose-6-phosphatase-α; glycogen storage disease type-Ia mouse model; lipid nanoparticles; nonhomologous end-joining.

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Conflict of interest statement

CONFLICT OF INTEREST

Ananya Samanta, Nelson George, Irina Arnaoutova, Hung-Dar Chen, Brian C. Mansfield, and Janice Y. Chou declare that they have no conflicts of interest.

Christopher Hart and Troy Carlo are former employees of CRISPR Therapeutics, Inc., and own shares of CRISPR Therapeutics stock.

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