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. 2021 Apr 7;29(4):1602-1610.
doi: 10.1016/j.ymthe.2020.12.027. Epub 2020 Dec 23.

Correction of metabolic abnormalities in a mouse model of glycogen storage disease type Ia by CRISPR/Cas9-based gene editing

Irina Arnaoutova  1 , Lisa Zhang  1 , Hung-Dar Chen  1 , Brian C Mansfield  2 , Janice Y Chou  3
Affiliations

Correction of metabolic abnormalities in a mouse model of glycogen storage disease type Ia by CRISPR/Cas9-based gene editing

Irina Arnaoutova et al. Mol Ther. .

Abstract

Glycogen storage disease type Ia (GSD-Ia), deficient in glucose-6-phosphatase-α (G6PC), is characterized by impaired glucose homeostasis and a hallmark of fasting hypoglycemia. We have developed a recombinant adeno-associated virus (rAAV) vector-mediated gene therapy for GSD-Ia that is currently in a phase I/II clinical trial. While therapeutic expression of the episomal rAAV-G6PC clinical vector is stable in mice, the long-term durability of expression in humans is currently being established. Here we evaluated CRISPR/Cas9-based in vivo genome editing technology to correct a prevalent pathogenic human variant, G6PC-p.R83C. We have generated a homozygous G6pc-R83C mouse strain and shown that the G6pc-R83C mice manifest impaired glucose homeostasis and frequent hypoglycemic seizures, mimicking the pathophysiology of GSD-Ia patients. 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-α activity, maintained glucose homeostasis, displayed normalized blood metabolites, and could sustain 24 h 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 phenotype.

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Figures

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Graphical abstract
Figure 1
Figure 1
The G6pc-R83C mice manifest impaired glucose homeostasis The G6pc-R83, G6pc-R83/R83C, and G6pc-R83C mice were designated as R83, R83/R83C, and R83C, respectively. The R83 and R83/R83C mice displaying a similar phenotype were collectively named as the control mice. (A) Liver and kidney microsomal G6Pase-α activity in 3-week-old R83 (n = 5), R83/R83C (n = 7), R83C (n = 12), G6pc+/+ (n = 6), G6pc+/− (n = 6), and G6pc−/− (n = 12) mice. (B) Histochemical analysis of G6Pase-α activity. Each image represents an individual mouse. The arrow indicates kidney cortex. Scale bars, 100 μm. (C) Western blot analysis of liver microsomal G6Pase-α using a monoclonal antibody against G6Pase-α. (D) Postnatal growth of control (○) and R83C (●) mice. Each point represents 10 animals. (E) Size comparison of R83 and R83C mice at age 3 weeks, showing growth retardation of the G6pc-R83C mice. (F) Liver and kidney weight of 3-week-old control (n = 10) and R83C (n = 10) mice. Data represent the mean ± SEM. ∗∗p < 0.005.
Figure 2
Figure 2
Phenotype analysis of the G6pc-R83C mice The G6pc-R83, G6pc-R83/R83C, and G6pc-R83C mice were designated as R83, R83/R83C, and R83C, respectively. The R83 and R83/R83C mice displaying a similar phenotype were collectively named as the control mice. (A) Serum glucose, triglyceride, cholesterol, lactate, and uric acid levels in 3-week-old control (n = 12) and R83C (n = 12) mice. (B) Liver glucose, triglyceride, glycogen, lactate, and G6P levels in 3-week-old control (n = 12) and R83C (n = 12) mice. (C) H&E-stained liver and kidney sections in 3-week-old R83 and R83C mice. Each plate represents an individual mouse. Scale bar, 20 μm. (D) Oil red O staining of liver and kidney from 3-week-old R83 and R83C mice. Scale bar, 20 μm. Data represent the mean ± SEM. , ∗∗p < 0.005.
Figure 3
Figure 3
In vivo correction of the human G6PC-p.R83C variant in the G6pc-R83C mice by a dual AAV-CRISPR-Cas9 strategy (A) Schematic diagram of the two AAV vectors, AAV8-mG6pc-gRNA and AAV8-SaCas9-gRNA. (B) HDR and indels in AAV-H liver cells analyzed at age 8 weeks.
Figure 4
Figure 4
Phenotypic analysis of the AAV-H and AAV-L mice at age 8 weeks The G6pc-R83 and G6pc-R83/R83C mice displaying a similar phenotype were collectively named as the control mice. Biochemical analysis was conducted in 8-week-old control (n = 28), AAV-H (n = 20), and AAV-L (n = 7) mice. (A) Liver microsomal G6Pase-α activity. (B) Histochemical analysis of hepatic G6Pase-α activity. Each image represents an individual mouse. Scale bars, 200 μm (50×) and 100 μm (100×). The numbers represent hepatic G6Pase-α activity expressed in the mice. (C) Fasting blood glucose levels in control (n = 16) and AAV-H (n = 12) mice. (D) Serum glucose, triglyceride, cholesterol, lactate, and uric acid levels in mice not subjected to fasting. (E) BW values. (F) BMI values. Data represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.005.
Figure 5
Figure 5
Biochemical analysis of AAV-H and AAV-L mice at age 8 weeks The G6pc-R83 and G6pc-R83/R83C mice displaying a similar phenotype were collectively named as the control mice. The analysis was conducted in 8-week-old control (n = 28), AAV-H (n = 20), and AAV-L (n = 7) mice. (A) Liver weight (LW)/BW values. (B) H&E and Oil Red O-stained liver sections in 8-week-old control, AAV-H, and AAV-L mice. The numbers represent hepatic G6Pase-α activity expressed in the mice. Each plate represents an individual mouse. Scale bar, 20 μm. (C) Hepatic levels of glucose, triglyceride, glycogen, lactate, and G6P. (D) Hepatic levels of G6pt transcript. (E) Serum insulin levels. Data represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.005.
Figure 6
Figure 6
Biochemical analysis of AAV-H mice at age 16 weeks The G6pc-R83 and G6pc-R83/R83C mice displaying a similar phenotype were collectively named as the control mice. The analysis was conducted in 16-week-old control (n = 10) and AAV-H (n = 10) mice. (A) Liver microsomal G6Pase-α activity. (B) Fasting blood glucose levels in 16-week-old control (n = 5) and AAV-H (n = 5) mice. (C) Serum levels of glucose, triglyceride, cholesterol, lactate, and uric acid in mice not subjected to fasting. (D) BW and LW/BW values. (E) BMI values. (F) Serum insulin levels. Data represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.005.
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