Therapeutic neonatal hepatic gene therapy in mucopolysaccharidosis VII dogs

Katherine Ponder

John Melniczek

Lingfei Xu

Margaret Weil

Hamutal Mazrier+8 authors

^{Departments of Internal Medicine and Biochemistry and Molecular Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110;}^{Departments of Pathobiology and Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104;}^{Baker Institute, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853; and}^{Scheie Eye Institute, 51 North 39th Street, University of Pennsylvania, Philadelphia, PA 19104}

^{To whom reprint requests should be addressed. E-mail:}ude.nnepu.tev@sniksahm.

Edited by Stuart A. Kornfeld, Washington University School of Medicine, St. Louis, MO, and approved July 17, 2002

Received 2002 Jun 12

Abstract

Dogs with mucopolysaccharidosis VII (MPS VII) were injected intravenously at 2–3 days of age with a retroviral vector (RV) expressing canine β-glucuronidase (cGUSB). Five animals received RV alone, and two dogs received hepatocyte growth factor (HGF) before RV in an attempt to increase transduction efficiency. Transduced hepatocytes expanded clonally during normal liver growth and secreted enzyme with mannose 6-phosphate. Serum GUSB activity was stable for up to 14 months at normal levels for the RV-treated dogs, and for 17 months at 67-fold normal for the HGF/RV-treated dog. GUSB activity in other organs was 1.5–60% of normal at 6 months for two RV-treated dogs, which was likely because of uptake of enzyme from blood by the mannose 6-phosphate receptor. The body weights of untreated MPS VII dogs are 50% of normal at 6 months. MPS VII dogs cannot walk or stand after 6 months, and progressively develop eye and heart disease. RV- and HGF/RV-treated MPS VII dogs achieved 87% and 84% of normal body weight, respectively. Treated animals could run at all times of evaluation for 6–17 months because of improvements in bone and joint abnormalities, and had little or no corneal clouding and no mitral valve thickening. Despite higher GUSB expression, the clinical improvements in the HGF/RV-treated dog were similar to those in the RV-treated animals. This is the first successful application of gene therapy in preventing the clinical manifestations of a lysosomal storage disease in a large animal.

Abstract

The mucopolysaccharidoses (MPS) are lysosomal storage diseases (LSD) that result from deficient activity of enzymes that degrade glycosaminoglycans (GAGs) (1, 2). The overall incidence of MPS is 1:27,000 live births (3). MPS VII (Sly syndrome) is due to deficient β-glucuronidase (GUSB; EC 3.2.1.31) activity and results in growth retardation, mobility problems, dysostosis multiplex, facial dysmorphia, hepatosplenomegaly, corneal clouding, cardiac valvular abnormalities, and mental retardation (4). MPS VII dogs are homozygous for an arginine to histidine substitution at amino acid 166 in the canine GUSB (cGUSB) protein (5, 6). Features in MPS VII dogs resemble those in humans, except mental retardation is difficult to assess, and hepatosplenomegaly is less severe.

The clinical manifestations of LSD can be reduced by enzyme replacement therapy (ERT), which involves i.v. injection of enzyme. Enzyme that contains mannose 6-phosphate (M6P) can be internalized via the M6P receptor (M6PR) present on the surface of most cells (7, 8). ERT has been therapeutic in mice with MPS VII (reviewed in ref. 9), large animals with MPS (10–12), and humans with LSD (13–15). The rationale for hepatic gene therapy is that the liver will continuously secrete GUSB with M6P into the blood, because most cells that produce functional GUSB secrete some enzyme that is phosphorylated.

We and others have demonstrated that Moloney murine leukemia virus (MLV)-based retroviral vectors (RV) can stably express proteins from the liver in rodents (reviewed in ref. 16). Although efficient transfer into hepatocytes of adults by using an MLV-based RV requires induction of replication with hepatocyte growth factor (HGF) (17) or other methods, the rapid liver growth allowed transduction to occur without any stimulus for hepatocyte replication in newborn mice (18) and dogs (19). We report the clinical improvements seen in six MPS VII dogs that were transduced with a cGUSB-expressing RV as neonates and followed for 6–17 months.

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Acknowledgments

We thank Paula Henthorn for the canine GUSB cDNA, Stuart Kornfeld and Walter Gregory for assistance with the mannose 6-phosphate receptor column experiments, William Sly, Stuart Kornfeld, and Mark Sands for discussions of the manuscript, James Hayden for radiograph illustrations, and veterinary students and the University Laboratory Animal Resources staff for assistance with animal care. This work was supported by National Institutes of Health Grants DK54061 and K02 DK02575 (to K.P.P.), DK54481 and RR02512 (to M.E.H.), and DK46637 and Training Grant RR07063 (to J.H.W.), and Washington University Digestive Diseases Research Core Center Grant P30 52574.

Acknowledgments

Abbreviations

GUSB	β-glucuronidase
cGUSB	canine GUSB
M6P	mannose 6-phosphate
MPS	mucopolysaccharidosis
LSD	lysosomal storage disease
ERT	enzyme replacement therapy
HGF	hepatocyte growth factor
RV	retroviral vector
MLV	Moloney murine leukemia virus
MR	mitral regurgitation

Abbreviations

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Footnotes

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