Adeno-associated virus-mediated correction of a canine model of glycogen storage disease type Ia

Abstract

Glycogen storage disease type Ia (GSDIa; von Gierke disease; MIM 232200) is caused by a deficiency in glucose-6-phosphatase-alpha. Patients with GSDIa are unable to maintain glucose homeostasis and suffer from severe hypoglycemia, hepatomegaly, hyperlipidemia, hyperuricemia, and lactic acidosis. The canine model of GSDIa is naturally occurring and recapitulates almost all aspects of the human form of disease. We investigated the potential of recombinant adeno-associated virus (rAAV) vector-based therapy to treat the canine model of GSDIa. After delivery of a therapeutic rAAV2/8 vector to a 1-day-old GSDIa dog, improvement was noted as early as 2 weeks posttreatment. Correction was transient, however, and by 2 months posttreatment the rAAV2/8-treated dog could no longer sustain normal blood glucose levels after 1 hr of fasting. The same animal was then dosed with a therapeutic rAAV2/1 vector delivered via the portal vein. Two months after rAAV2/1 dosing, both blood glucose and lactate levels were normal at 4 hr postfasting. With more prolonged fasting, the dog still maintained near-normal glucose concentrations, but lactate levels were elevated by 9 hr, indicating that partial correction was achieved. Dietary glucose supplementation was discontinued starting 1 month after rAAV2/1 delivery and the dog continues to thrive with minimal laboratory abnormalities at 23 months of age (18 months after rAAV2/1 treatment). These results demonstrate that delivery of rAAV vectors can mediate significant correction of the GSDIa phenotype and that gene transfer may be a promising alternative therapy for this disease and other genetic diseases of the liver.

FIG. 1.

Neonatal delivery of rAAV2/8 results in short-term improvement of fasting glucose homeostasis and blood lactate levels. (A) Fasting blood glucose levels of untreated (no gene therapy; GSDIa) and heterozygous littermate control (HET) and the rAAV2/8-treated GSDIa dog at 2, 4, and 8 weeks postinjection. (B) Blood lactate levels of untreated (GSDIa) and rAAV2/8-treated GSDIa dogs at 2 and 4 weeks postinjection after 3 hr of fasting. *GSDIa, dog blood lactate level taken at 1 hr of fasting, after which fasting was discontinued to avoid complications.

FIG. 2.

Redosing with rAAV2/1 results in improved fasting glucose homeostasis and blood lactate levels. (A) Fasting blood glucose levels of the rAAV2/8 plus rAAV2/1-treated GSDIa dog, 8 weeks after rAAV2/1 delivery (open circles), wild-type (WT) control (solid circles), and an untreated (no gene therapy) GSDIa dog (solid triangle). (B) Fasting blood lactate levels of an untreated GSDIa dog (GSDIa); the gene therapy-treated GSDIa dog (rAAV2/1 plus rAAV2/8) at 2, 4, and 9 hr of fasting; and control heterozygous (HET; n = 5) and wild-type (n = 4) dogs after 12 hr of fasting.

FIG. 3.

Hepatic G6Pase enzyme activity and liver histology are improved in the gene therapy-treated dog. (A) Liver G6Pase activity in wild-type (WT; n = 3), heterozygous (HET; n = 3), and gene therapy-treated dogs 20 weeks after rAAV2/8 delivery (AAV8), a gene therapy-treated dog 6 months after redosing with rAAV1 (AAV8 + 1), and an untreated dog, which did not receive gene therapy (GSDIa). (B) Histochemical analysis of G6Pase activity in the liver of an untreated dog (22 weeks old, no gene therapy; GSDIa), a wild-type dog (WT, 20 weeks old), and an rAAV2/8-treated dog (AAV8) 20 weeks postinjection. Brown staining is indicative of lead trapping of phosphate generated by G6P hydrolysis by active G6Pase. (C) Periodic acid–Schiff (PAS) and hematoxylin and eosin (H&E) staining of liver tissue from a gene therapy-treated dog 20 weeks after rAAV2/8 delivery (AAV8) and 6 months after rAAV2/1 redosing (AAV8 + 1). (D) ¹H-MRS spectra of hepatic glycogen in a heterozygous (Het) dog, a wild-type (WT) dog, an untreated (G6Pase^–/–) dog, and a gene therapy-treated dog 20 weeks after rAAV2/8 delivery (rAAV2/8) and 6 months after rAAV2/1 redosing (rAAV2/8 + 2/1).

FIG. 3.

Hepatic G6Pase enzyme activity and liver histology are improved in the gene therapy-treated dog. (A) Liver G6Pase activity in wild-type (WT; n = 3), heterozygous (HET; n = 3), and gene therapy-treated dogs 20 weeks after rAAV2/8 delivery (AAV8), a gene therapy-treated dog 6 months after redosing with rAAV1 (AAV8 + 1), and an untreated dog, which did not receive gene therapy (GSDIa). (B) Histochemical analysis of G6Pase activity in the liver of an untreated dog (22 weeks old, no gene therapy; GSDIa), a wild-type dog (WT, 20 weeks old), and an rAAV2/8-treated dog (AAV8) 20 weeks postinjection. Brown staining is indicative of lead trapping of phosphate generated by G6P hydrolysis by active G6Pase. (C) Periodic acid–Schiff (PAS) and hematoxylin and eosin (H&E) staining of liver tissue from a gene therapy-treated dog 20 weeks after rAAV2/8 delivery (AAV8) and 6 months after rAAV2/1 redosing (AAV8 + 1). (D) ¹H-MRS spectra of hepatic glycogen in a heterozygous (Het) dog, a wild-type (WT) dog, an untreated (G6Pase^–/–) dog, and a gene therapy-treated dog 20 weeks after rAAV2/8 delivery (rAAV2/8) and 6 months after rAAV2/1 redosing (rAAV2/8 + 2/1).

FIG. 4.

Heterozygous dogs have increased hepatic glycogen as compared with wild-type dogs. PAS staining of liver from two wild-type animals (top row; 1.4 and 3.9 years old, respectively) and two heterozygous animals (bottom row; 1.4 [littermate to WT animal directly above] and 4.7 years old).