Elizabeth D. Brooks

Induction of autophagy improves hepatic lipid metabolism in glucose-6-phosphatase deficiency.

BACKGROUND & AIMS Glucose-6-phosphatase (G6Pase α, G6PC) deficiency, also known as von Gierkes disease or GSDIa, is the most common glycogen storage disorder. It is characterized by a decreased ability of the liver to convert glucose-6-phosphate (G6P) to glucose leading to glycogen and lipid over-accumulation progressing to liver failure and/or hepatomas and carcinomas. Autophagy of intracellular lipid stores (lipophagy) has been shown to stimulate fatty acid β-oxidation in hepatic cells. Thus, we examined autophagy and its effects on reducing hepatic lipid over-accumulation in several cell culture and animal models of GSDIa. METHODS Autophagy in G6PC-deficient hepatic cell lines, mice, and dogs was measured by Western blotting for key autophagy markers. Pro-autophagic Unc51-like kinase 1 (ULK1/ATG1) was overexpressed in G6PC-deficient hepatic cells, and lipid clearance and oxidative phosphorylation measured. G6PC(-/-) mice and GSDIa dogs were treated with rapamycin and assessed for liver function. RESULTS Autophagy was impaired in the cell culture, mouse, and canine models of GSDIa. Stimulation of the anti-autophagic mTOR, and inhibition of the pro-autophagic AMPK pathways occurred both in vitro and in vivo. Induction of autophagy by ULK1/ATG1 overexpression decreased lipid accumulation and increased oxidative phosphorylation in G6PC-deficient hepatic cells. Rapamycin treatment induced autophagy and decreased hepatic triglyceride and glycogen content in G6PC(-/-) mice, as well as reduced liver size and improved circulating markers of liver damage in GSDIa dogs. CONCLUSIONS Autophagy is impaired in GSDIa. Pharmacological induction of autophagy corrects hepatic lipid over-accumulation and may represent a new therapeutic strategy for GSDIa.

In vivo zinc finger nuclease-mediated targeted integration of a glucose-6-phosphatase transgene promotes survival in mice with glycogen storage disease Type IA

Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (P < 0.04). Furthermore, transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Targeted alleles were 4.6-fold more common in livers of mice with GSD Ia, as compared with normal littermates, at 8 months following vector administration (P < 0.02). This suggests a selective advantage for vector-transduced hepatocytes following ZFN-mediated integration of the G6Pase vector. A short-term experiment also showed that 3-month-old mice receiving the ZFN had significantly-improved biochemical correction, in comparison with mice that received the donor vector alone. These data suggest that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve vector persistence and efficacy, and lower mortality in GSD Ia.

Pathogenesis of growth failure and partial reversal with gene therapy in murine and canine Glycogen Storage Disease type Ia

Glycogen Storage Disease type Ia (GSD-Ia) in humans frequently causes delayed bone maturation, decrease in final adult height, and decreased growth velocity. This study evaluates the pathogenesis of growth failure and the effect of gene therapy on growth in GSD-Ia affected dogs and mice. Here we found that homozygous G6pase (-/-) mice with GSD-Ia have normal growth hormone (GH) levels in response to hypoglycemia, decreased insulin-like growth factor (IGF) 1 levels, and attenuated weight gain following administration of GH. Expression of hepatic GH receptor and IGF 1 mRNAs and hepatic STAT5 (phospho Y694) protein levels are reduced prior to and after GH administration, indicating GH resistance. However, restoration of G6Pase expression in the liver by treatment with adeno-associated virus 8 pseudotyped vector expressing G6Pase (AAV2/8-G6Pase) corrected body weight, but failed to normalize plasma IGF 1 in G6pase (-/-) mice. Untreated G6pase (-/-) mice also demonstrated severe delay of growth plate ossification at 12 days of age; those treated with AAV2/8-G6Pase at 14 days of age demonstrated skeletal dysplasia and limb shortening when analyzed radiographically at 6 months of age, in spite of apparent metabolic correction. Moreover, gene therapy with AAV2/9-G6Pase only partially corrected growth in GSD-Ia affected dogs as detected by weight and bone measurements and serum IGF 1 concentrations were persistently low in treated dogs. We also found that heterozygous GSD-Ia carrier dogs had decreased serum IGF 1, adult body weights and bone dimensions compared to wild-type littermates. In sum, these findings suggest that growth failure in GSD-Ia results, at least in part, from hepatic GH resistance. In addition, gene therapy improved growth in addition to promoting long-term survival in dogs and mice with GSD-Ia.

Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy.

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. This study evaluates 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 5 × 1011 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, 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 the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the 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 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1ys/ys mice.

Large animal models and new therapies for glycogen storage 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.

Letter to the Editors: Concerning “Long-term safety and efficacy of AAV gene therapy in the canine model of glycogen storage disease type Ia” by Lee et al.

Dear Editors, Recently, Lee et al. reported seven dogs with glycogen storage disease (GSD) type Ia treated with adenoassociated virus (AAV) vector-mediated gene therapy that apparently prevented long-term hepatic or renal complications (Lee et al. 2018). However, there are problems with this conclusion. Notably, Lee et al. describe how dogs underwent 24-h care, receiving glucose feeds every 30–60 min. Furthermore, Lee et al. describe two GSD Ia dogs on this same rigorous dietary therapy that lived to > 5 years of age with mostly normal laboratory values, implying that gene therapy had few additional benefits over nutritional therapy. Our group recently published a very similar gene therapy study, but the GSD Ia dogs were maintained on a more typical diet of only three feedings per day (Brooks et al. 2018). However, 4/5 AAV vector-treated dogs developed hepatocellular adenomas and carcinoma and 3/5 developed chronic kidney disease. The key difference between the two studies was the degree of nutritional support and not the gene therapy. Both studies demonstrated low hepatic glucose 6-phosphatase (G6Pase) activity in dogs at the endpoint, as well as marked hepatic glycogen accumulation. Figure 1 in Lee et al. also demonstrates a decrease in blood glucose over time in GSD Ia dogs, without specifying if the dogs were fed or fasted. Brooks et al. fasted dogs to demonstrate prevention of hypoglycemia. Both studies reported very small amounts of vector DNA at the end of life in most of the GSD Ia dogs. Two dogs in Lee et al. had very low vector DNA present in the liver at the end of the study (0.01 to 0.02 copies/cell) and correspondingly low G6Pase activity from 2.2 to 3.8% of activity in the liver of normal carriers. One dog in Lee et al. had elevated vector DNA (Fig. 2; dog GE) in association with low G6Pase activity (Fig. 3). In contrast, Brooks et al. reported higher vector DNA (0.04– 0.16 copies/cell; Supplementary Fig. 2) and G6Pase activity (28% of carrier activity; Fig. 1) in the liver. Thus, the effect of gene therapy was similar in both studies. Lee et al. reported no renal complications, other than proteinuria in one dog and mild, intermittent increases in bloody urea nitrogen (BUN) and/or creatinine in two different dogs. Importantly, recombinant AAV8 vectors highly like the one used by Lee et al. had no effect on kidney involvement in G6pc mice with GSD Ia (see references therein), and, therefore, gene therapy would not be protective of the kidneys in dogs. The kidney sparing in Lee et al. can be attributed to intensive dietary therapy. Brooks et al. reported tubule-interstitial lesions and glomerular changes in the older GSD Ia dog cohort treated with AAV vectors. In conclusion, both papers demonstrate that gene therapy has greatly prolonged life in GSD Ia dogs and led to low levels of G6Pase activity persistence. However, evidence is lacking that gene therapy in the absence of a strict dietary regimen can prevent hepatocellular tumors and renal disease. Communicating Editor: Terry Derks

Bezafibrate induces autophagy and improves hepatic lipid metabolism in glycogen storage disease type Ia

Abstract Glucose‐6‐phosphatase &agr; (G6Pase) deficiency, also known as von Gierkes Disease or Glycogen storage disease type Ia (GSD Ia), is characterized by decreased ability of the liver to convert glucose‐6‐phosphate to glucose leading to glycogen accumulation and hepatosteatosis. Long‐term complications of GSD Ia include hepatic adenomas and carcinomas, in association with the suppression of autophagy in the liver. The G6pc−/− mouse and canine models for GSD Ia were treated with the pan‐peroxisomal proliferator‐activated receptor agonist, bezafibrate, to determine the drugs effect on liver metabolism and function. Hepatic glycogen and triglyceride concentrations were measured and western blotting was performed to investigate pathways affected by the treatment. Bezafibrate decreased liver triglyceride and glycogen concentrations and partially reversed the autophagy defect previously demonstrated in GSD Ia models. Changes in medium‐chain acyl‐CoA dehydrogenase expression and acylcarnintine flux suggested that fatty acid oxidation was increased and fatty acid synthase expression associated with lipogenesis was decreased in G6pc−/− mice treated with bezafibrate. In summary, bezafibrate induced autophagy in the liver while increasing fatty acid oxidation and decreasing lipogenesis in G6pc−/− mice. It represents a potential therapy for glycogen overload and hepatosteatosis associated with GSD Ia, with beneficial effects that have implications for non‐alcoholic fatty liver disease.

311. In Vivo Zinc Finger Nuclease-Mediated Targeted Integration of a Glucose-6-Phosphatase Transgene Enhances Biochemical Correction in Mice with Glycogen Storage Disease Type IA

Glycogen storage disease type Ia (GSD Ia) is caused by glucose-6-phosphatase (G6Pase) deficiency in association with severe, life-threatening hypoglycemia that necessitates lifelong dietary therapy. Here we show that use of a zinc-finger nuclease (ZFN) targeted to the ROSA26 safe harbor locus and a ROSA26-targeting vector containing a G6PC donor transgene, both delivered with adeno-associated virus (AAV) vectors, markedly improved survival of G6Pase knockout (G6Pase-KO) mice compared with mice receiving the donor vector alone (p<0.04) out to 8 months of age. Transgene integration has been confirmed by sequencing in the majority of the mice treated with both vectors. Surviving G6Pase-KO mice at 8 months of age had decreased glycogen content compared with young untreated G6Pase-KO controls, which correlated with the long-term survival of these mice that otherwise perish before weaning. A short-term experiment resolved difficulty observing biochemical differences between the dual-vector and single-vector groups, caused by a selective advantage for strongly-responding mice: 3-month-old mice receiving the ZFN had significantly reduced hepatic glycogen accumulation and improved G6Pase activity, compared with mice that received the donor vector alone. These data demonstrate that the use of ZFNs to drive integration of G6Pase at a safe harbor locus might improve transgene persistence and efficacy, and lower mortality in GSD Ia.

701. Long-Term Complications of Glycogen Storage Disease Type IA in the Dog Model Treated With Gene Replacement Therapy

Glycogen storage disease type Ia (GSD-Ia) in dogs closely resembles human GSD-Ia where untreated patients develop complications associated with glucose-6-phosphatase (G6Pase) deficiency (hypoglycemia, hyperlipidemia, growth retardation, and early death). For the past 3 decades survival of human patients that are placed under intensive nutritional management with uncooked corn starch has improved; however, long-term complications persist including renal failure, nephrolithiasis, hepatic adenomas, and a high risk for hepatocellular carcinoma. Affected dogs fail to thrive with dietary therapy alone, but treatment with gene replacement therapy using adeno-associated viral vectors (AAV) expressing G6Pase has greatly prolonged life and prevented hypoglycemia. In this study, 7 GSD-Ia affected dogs treated with AAV-G6Pase were followed up to 8 years of life to describe long-term complications. All required readministration of AAV vector(s) pseudotyped as a new serotype to avoid anti-AAV antibodies, due to decreased ability to maintain normoglycemia during fasting. Two dogs developed chronic renal disease as denoted by polyuria, polydipsia, and azotemia at 6 and 8 years of age. Calcium oxalate and phosphate urolithiasis was detected in 2 dogs; 1 dog had evidence of polycystic ovarian disease. Four of the 7 dogs were euthanized due to reaching humane endpoints related to liver and/or kidney disease, at 3 to 8 years of life, and 1 dog succumbed to acute hypoglycemia. Necropsy revealed several focal hepatic lesions in all deceased dogs; 2 dogs had lesions confirmed histologically as hepatocellular carcinoma, the largest tumor reached 8 cm in diameter (Fig. 1). One dog demonstrated lesions consistent with hepatocellular adenoma and in the other 2, there was hepatocellular hyperplasia. There was no significant difference in amount of vector DNA in hepatic tumors compared with normal tissue (n=4 dogs), suggesting that insertional mutagenesis by the AAV vector was not the mechanism for tumor formation (Fig. 2). View Large Image | Download PowerPoint Slide View Large Image | Download PowerPoint SlideGlomerular sclerosis, fibrosis of Bowmans capsules and focal interstitial nephritis were found in the 2 dogs with clinical renal disease. Two dogs, both female, remain in good health with no evidence of liver masses appreciable on ultrasound examination; they are 4 and 5 years of age. Hepatocellular carcinoma, kidney disease, polycystic ovaries and urolithiasis are common findings among GSD-Ia patients. Here we show that the canine GSD-Ia model demonstrates similar long-term complications in spite of AAV-mediated gene replacement therapy. Further development of gene therapy is needed to prevent long-term complications of GSD-Ia.