Elizabeth Drake Brooks

Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. We evaluate the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1ys/ys). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1ys/ys mice at a dose of 5x1011 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at age of 3 months, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in liver. Glycogen contents were reduced to wild-type levels in muscles and significantly reduced in liver and brain. At 9 months of age, though GBE activity was only significantly elevated in heart, glycogen levels were significantly reduced in liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at age of 9 months. This suggests an alleviation of damage and improvement of function in liver and muscle by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in the Gbe1ys/ys mice.

Glycogen storage disease type IIIa (GSD IIIa) is caused by a deficiency of glycogen debranching enzyme activity. Hepatomegaly, muscle degeneration, and hypoglycemia occur in human patients at an early age. Long-term complications include liver cirrhosis, hepatic adenomas, and generalized myopathy. A naturally occurring canine model of GSD IIIa that mimics the human disease has been described, with progressive liver disease and skeletal muscle damage likely due to excess glycogen deposition. In the current study, long-term follow-up of previously described GSD IIIa dogs until 32 mo of age (n = 4) and of family-owned GSD IIIa dogs until 11 to 12 y of age (n = 2) revealed that elevated concentrations of liver and muscle enzyme (AST, ALT, ALP, and creatine phosphokinase) decreased over time, consistent with hepatic cirrhosis and muscle fibrosis. Glycogen deposition in many skeletal muscles; the tongue, diaphragm, and heart; and the phrenic and sciatic nerves occurred also. Furthermore, the urinary biomarker Glc4, which has been described in many types of GSD, was first elevated and then decreased later in life. This urinary biomarker demonstrated a similar trend as AST and ALT in GSD IIIa dogs, indicating that Glc4 might be a less invasive biomarker of hepatocellular disease. Finally, the current study further demonstrates that the canine GSD IIIa model adheres to the clinical course in human patients with this disorder and is an appropriate model for developing novel therapies.

Glycogen storage disease (GSD) consists of more than 10 discrete conditions for which the biochemical and genetic bases have been determined, and new therapies have been under development for several of these conditions. Gene therapy research has generated proof-of-concept for GSD types I (von Gierke disease) and II (Pompe disease). Key features of these gene therapy strategies include the choice of vector and regulatory cassette, and recently adeno-associated virus (AAV) vectors containing tissue-specific promoters have achieved a high degree of efficacy. Efficacy of gene therapy for Pompe disease depend upon the induction of immune tolerance to the therapeutic enzyme. Efficacy of von Gierke disease is transient, waning gradually over the months following vector administration. Small molecule therapies have been evaluated with the goal of improving standard of care therapy or ameliorating the cellular abnormalities associated with specific GSDs. The receptor-mediated uptake of the therapeutic enzyme in Pompe disease was enhanced by administration of β2 agonists. Rapamycin reduced the liver fibrosis observed in GSD III. Further development of gene therapy could provide curative therapy for patients with GSD, if efficacy from preclinical research is observed in future clinical trials and these treatments become clinically available.

Enzyme replacement therapy (ERT) is the standard-of-care treatment of Pompe disease, a lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA). One limitation of ERT with recombinant human (rh) GAA is antibody formation against GAA. Similarly, in adeno-associated virus (AAV) vector-mediated gene transfer for Pompe disease, development of antibodies against the GAA transgene product and the AAV vector prevents therapeutic efficacy and vector readministration, respectively. Here a nondepleting anti-CD4 monoclonal antibody (mAb) was administrated intravenously prior to administration of an AAV2/9 vector encoding GAA to suppress anti-GAA responses, leading to a substantial reduction of anti-GAA immunoglobulins, including IgG1, IgG2a, IgG2b, IgG2c, and IgG3. Transduction efficiency in liver with a subsequent AAV2/8 vector was massively improved by the administration of anti-CD4 mAb with the initial AAV2/9 vector, indicating a spread of benefit derived from control of the immune response to the first AAV2/9 vector. Anti-CD4 mAb along with AAV2/9-CBhGAApA significantly increased GAA activity in heart and skeletal muscles along with a significant reduction of glycogen accumulation. Taken together, these data demonstrated that the addition of nondepleting anti-CD4 mAb with gene therapy controls humoral immune responses to both vector and transgene, resulting in clear therapeutic benefit in mice with Pompe disease.

Glycogen storage diseases (GSD), a unique category of inherited metabolic disorders, were first described early in the twentieth century. Since then, the biochemical and genetic bases of these disorders have been determined, and an increasing number of animal models for GSD have become available. At least seven large mammalian models have been developed for laboratory research on GSDs. These models have facilitated the development of new therapies, including gene therapy, which are undergoing clinical translation. For example, gene therapy prolonged survival and prevented hypoglycemia during fasting for greater than one year in dogs with GSD type Ia, and the need for periodic re‐administration to maintain efficacy was demonstrated in that dog model. The further development of gene therapy could provide curative therapy for patients with GSD and other inherited metabolic disorders.

Recently, we reported that progression of liver fibrosis and skeletal myopathy caused by extensive accumulation of cytoplasmic glycogen at advanced age is the major feature of a canine model of glycogen storage disease (GSD) IIIa. Here, we aim to investigate whether rapamycin, a specific inhibitor of mTOR, is an effective therapy for GSD III. Our data show that rapamycin significantly reduced glycogen content in primary muscle cells from human patients with GSD IIIa by suppressing the expression of glycogen synthase and glucose transporter 1. To test the treatment efficacy in vivo, rapamycin was daily administered to GSD IIIa dogs starting from age 2 (early-treatment group) or 8 months (late-treatment group), and liver and skeletal muscle biopsies were performed at age 12 and 16 months. In both treatment groups, muscle glycogen accumulation was not affected at age 12 months but significantly inhibited at 16 months. Liver glycogen content was reduced in the early-treatment group but not in the late-treatment group at age 12 months. Both treatments effectively reduced liver fibrosis at age 16 months, consistent with markedly inhibited transition of hepatic stellate cells into myofibroblasts, the central event in the process of liver fibrosis. Our results suggest a potential useful therapy for GSD III. Rapamycin inhibited glycogen accumulation in GSD IIIa patient muscle cells. Rapamycin reduced muscle glycogen content in GSD IIIa dogs at advanced age. Rapamycin effectively prevented progression of liver fibrosis in GSD IIIa dogs. Our results suggest rapamycin as potential useful therapy for patients with GSD III.