Neonatal gene therapy of glycogen storage disease type Ia using a feline immunodeficiency virus-based vector

Abstract

Glycogen storage disease type Ia (GSD-Ia), also known as von Gierke disease, is caused by a deficiency of glucose-6-phosphatase-alpha (G6Pase), a key enzyme in glucose homeostasis. From birth, affected individuals cannot maintain normal blood glucose levels and suffer from a variety of metabolic disorders, leading to life-threatening complications. Gene therapy has been proposed as a possible option for treatment of this illness. Vectors have been constructed from feline immunodeficiency virus (FIV), a nonprimate lentivirus, because the wild-type virus does not cause disease in humans. Previously, we have shown that these vectors are capable of integrating stably into hepatocyte cell lines and adult murine livers and lead to long-term transgene expression. In the current work, we have assessed the ability to attenuate disease symptoms in a murine model of GSD-Ia. Single administration of FIV vectors containing the human G6Pase gene to G6Pase-alpha(-/-) mice did not change the biochemical and pathological phenotype. However, a double neonatal administration protocol led to normalized blood glucose levels, significantly extended survival, improved body weight, and decreased accumulation of liver glycogen associated with the disease. This approach shows a promising paradigm for treating GSD-Ia patients early in life thereby avoiding long-term consequences.

Figure 1

FIV vectors produce stable and prolonged transgene expression following neonatal administration. One-day-old BALB/c mice were administered 3 × 10⁷ FIV viral particles carrying the gene encoding luciferase or EGFP driven by the liver-specific hAAT promoter or the CMV promoter. (a) At various times after treatment, luciferase expression was measure by CCCD camera imaging; mean ± SD; P < 0.0001. (b) Representative bioluminescence photographs of mice taken 6 months after transduction with the viral vectors, FIV-hAAT-Luc (upper panel) or FIV-CMV-Luc (lower panel). (c) Immunohistochemical detection of EGFP⁺ cells in liver sections analyzed at the indicated time points following transduction. Bar = 100 µm. (d) Computerized quantification of EGFP⁺ cells as represented in c using the Ariol automated image analysis system. Data are shown as mean ± SD. EGFP, enhanced green fluorescent protein; FIV, feline immunodeficiency virus.

Figure 2

Double administration of FIV-hAAT-G6Pase to knockout neonatal mice leads to prolonged survival and improvement in disease symptoms. G6Pase-α^−/− mice were administered a double injection of viral particles or plasmid DNA at 1 and 6 days after birth. (a) Survival curves of mice receiving FIV viral particles carrying the G6Pase-α gene (n = 18), the EGFP reporter gene (n = 2), or naked plasmid DNA containing the G6Pase-α gene (n = 3) (log-rank test; P < 0.0001). (b) Blood glucose levels were measured biweekly, after transduction with the FIV-hAAT-G6Pase vector. Between 4 and 16 mice were sampled at each time point in the treated KO mice group, and between 1 and 8 mice in the healthy age-matched control group (mean ± SD). No significant difference (P < 0.05) was observed between the two groups after the 8th week of surveillance. (c) Mice were weighed at birth, and at 4, 8, 16, and 24 weeks of age; virally transduced G6Pase-α^−/− mice (treated KO) n = 31, 6, 3, 5, 5; untreated healthy age-matched siblings (control) n = 45, 3, 2, 4, 2, respectively. Mean ± SD is shown. (d) Proviral vector copy number (VCN) (columns) was measured in livers of FIV-transduced G6Pase-α^−/− mice at 4 and 6 months after treatment and compared to serum glucose levels; C, untransduced control. FIV, feline immunodeficiency virus; GFP, green fluorescent protein; KO, knockout.

Figure 3

Restoration of hepatic G6Pase-α activity and reduction in hepatic glycogen accumulation in knockout mice following FIV-hAAT-G6Pase transduction. (a) G6Pase-α enzyme activity in microsomes extracted from liver samples was measured at various times after FIV treatment. Healthy siblings (control), n = 2; untreated KO mice at 12 days, n = 3; treated KO mice at 3, 8, and 24 weeks of age, n = 6, 4, 4, respectively. Data are shown as % activity relative to healthy controls ± SD. Values of all treated mice were significantly higher than untreated KO mice (P < 0.05). (b) Liver glycogen content of FIV-transduced mice (n = 5 at 4 and 6 months) relative to healthy controls (n = 3 at 4 months and n = 2 at 6 months); mean fold increase ± SD is shown (P < 0.05 between all groups). (c) The level of ALT and AST liver enzymes was measured in the serum of treated G6Pase-α^−/− mice (treated KO) (n ≥ 3) and healthy age-matched controls (n ≥ 2) at 4 (4m) and 6 (6m) months after transduction. *P < 0.05. ALT, alanine transaminase; AST, aspartate transaminase.

Figure 4

Reduced lipid accumulation in the livers of G6Pase-α^−/− mice following feline immunodeficiency virus vector administration. Representative photomicrographs of liver hematoxylin and eosin–stained sections from (a–c) normal, (d–f) untreated 2-week-old G6Pase-α KO pups, and treated G6Pase-α KO mice (g–i) 4 months and (j–l) 6 months after transduction. Original magnification ×40 in a,d, and g (bar = 100 µm); ×100 in b,e, and h (bar = 50 µm); ×200 in c, f, and i (bar = 20 µm). KO, knockout.