Long-term complications of glycogen storage disease type Ia in the canine model treated with gene replacement therapy

Abstract

Background: Glycogen storage disease type Ia (GSD Ia) in dogs closely resembles human GSD Ia. Untreated patients with GSD Ia develop complications associated with glucose-6-phosphatase (G6Pase) deficiency. Survival of human patients on intensive nutritional management has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatocellular adenomas (HCA), and a high risk for hepatocellular carcinoma (HCC). Affected dogs fail to thrive with dietary therapy alone. Treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia in affected dogs. However, long-term complications have not been described to date.

Methods: Five GSD Ia-affected dogs treated with AAV-G6Pase were evaluated. Dogs were euthanized due to reaching humane endpoints related to liver and/or kidney involvement, at 4 to 8 years of life. Necropsies were performed and tissues were analyzed.

Results: Four dogs had liver tumors consistent with HCA and HCC. Three dogs developed renal failure, but all dogs exhibited progressive kidney disease histologically. Urolithiasis was detected in two dogs; uroliths were composed of calcium oxalate and calcium phosphate. One affected and one carrier dog had polycystic ovarian disease. Bone mineral density was not significantly affected.

Conclusions: Here, we show that the canine GSD Ia model demonstrates similar long-term complications as GSD Ia patients in spite of gene replacement therapy. Further development of gene therapy is needed to develop a more effective treatment to prevent long-term complications of GSD Ia.

Fig. 1.

Liver G6Pase persists throughout lifetime of AAV-G6Pase treated GSD Ia dogs increasing survival and ability to maintain normoglycemia during fasting. a. Hepatic G6Pase staining of AAV-G6Pase treated GSD Ia, Dog R. G6Pase is shown with brown staining. 100x b. Hepatic G6Pase staining of a GSD Ia carrier shown for comparison. 100x c. Mean ±SD of G6Pase activity detected in livers of GSD Ia dogs at end of life, AAV-G6Pase treated dogs have significantly higher G6Pase activity than untreated GSD Ia dogs , but both treated and untreated GSD Ia dogs do not achieve G6Pase activity as high as that found in carrier/wild type animals as shown through Student’s t test. Untreated, affected puppies had a low residual G6Pase activity in liver as described (Kishnani et al 2001). d. Mean ±SD of glycogen detected in GSD Ia dogs at end of life. Both treated and untreated GSD Ia dogs have increased glycogen compared with carriers and wild type through student’s t test. e. AAV-G6Pase dogs have a greatly increased survival over untreated GSD Ia dogs; as shown using a Log-rank (Mantel- Cox) test, p=0.008. f. Using the Area Under Curve (AUC), glucose curves throughout the lifetime of AAV-G6Pase treated dogs demonstrate abilities to fast and maintain normoglycemia.

Fig. 2.

Liver and kidney disease in GSD Ia dogs. a. Hepatomegaly of Dog H noted on right lateral abdominal radiographs, liver far surpasses the border of the rib cage. b. Liver histology from Dog L with relatively normal parenchyma entrapped between two encroaching areas of focal nodular hyperplasia with marked hepatocyte enlargement and vacuolation (H&E 200x). c. Liver mass histology from dog De demonstrates nests and thickened trabeculae of neoplastic cells more prominent with absence of lobular architecture (H&E 200x). Inset box (400x) demonstrates a mitotic figure in center. d. Liver mass histology from dog L is composed of large, vacuolated neoplastic hepatocytes compresses and invades adjacent more normal hepatic parenchyma. Normal lobular architecture not present in neoplastic mass (adenoma, Arrows). There is a thin layer of compressed more normal hepatocytes above the adenoma. In upper most part of photomicrograph are nests and trabeculae thickened by neoplastic hepatocytes 2 or more cells wide (carcinoma, arrowheads). (PAS, 100x). e. Normal renal glomerulus (PAS, 200X). f. Glomerulus with changes of FSGS demonstrating thickened PAS positive glomerular basement membrane as well as thickened Bowman’s capsule (PAS 200x). g. Tubulo-interstitial changes as part of the spectrum of chronic renal lesions in dog H. Note interstitial mononuclear inflammatory infiltrate and thickened peritubular basement membrane. (PAS 200x) h. PAS positive droplets in proximal tubular epithelium (PAS 200x). i. Kidney histology from Dog R (100x) There is clustering of glomeruli with varying changes. In several glomeruli, there is moderate eosinophilic thickening of Bowman’s capsules (sclerosis). In other glomeruli, the sclerosis of the Bowman’s capsules is more severe and there is moderated to marked eosinophilic thickening of the mesangium of one or more segments of the glomeruli tufts. A sclerotic remnant of a glomerulus is also present. The proximal convoluted tubular epithelial cells are markedly swollen with clear, cytoplasmic vacuoles. j. Creatinine increased over time in dogs R, H and L.