Neonatal Hepatic Gene Therapy for mucopolysaccharidosis I mice

Sarah Chung¹, Lingfei Xu, Bin Wang, Yuli Liu, Annabel Fu², Biology Department, Washington University, St. Louis, MO¹ Washington University School of Medicine, Department of Hematology²

Mucopolysaccharidosis I (MPS I) is a lysosomal storage disease caused by deficient activity of α-L-iduronidase (IDUA), a lysosomal hydrolase of 65 kD that degrades heparan sulfate and dermatan sulfate. Heparan and dermatan sulfates are glycosaminoglycans (GAGS), or long chains of polysaccharides of dissacharide units, containing N-aceytlgalatosamine or N-acetylglucosamine with a uronic acid. The low activity of IDUA enzyme leads to a build up of storage within the lysosome, which increases the size of the lysosome and also disrupts many functions within the cell. Clinical effects of the more severe form of MPS I result in hepatosplenomegaly, skeletal abnormalities, corneal clouding, acute mental retardation with implication of early death. The milder form of MPS I produces joint stiffness, aortic valve diseases, mild hepatosplenomegaly, mild corneal clouding with a relatively normal lifespan.

Available animal models to test therapies for MPS I disease are mice, dogs, and cats. So far, we have tested enzyme activity in organs of MPS I mice. Our prediction was that RV-treated MPS I mice that had a minimum of 2% of normal IDUA enzyme activity would exhibit marked differences in clinical traits of the MPS I untreated and treated mice. Previous trials of gene therapy in humans have shown that patients with 2%-5% of normal IDUA enzyme activity have essentially no clinical signs of MPS I.

Since lysosomes are in all eukaryotic cells the most effective method of gene therapy would facilitate the movement of the enzyme throughout the body. During processing in the Golgi, lysosomal enzymes gain mannose 6-phosphate (M6P), which binds to the mannose 6-phosphate receptor (M6PR) on cells, for transportation of the enzyme to the lysosome within the cell. Human IDUA contains 6 N-linked glycosylation sites, of which 2 possess mannose 6-phosphate (M6P). Our RV vector was liver specific so the virus targeted the replicating hepatocytes in the neonates. Using a Moloney murine leukemia virus (MLV) based retroviral vector, we inserted a liver specific alpha-antitrypsin promoter and canine IDUA cDNA into the vector (hAAT-cIDUA-WPRE. Once the liver produced enzyme, the cell secreted approximately 10% of the enzyme. IDUA enzyme then moved to other organs that had the M6PR receptor, which allowed them to take up the enzyme.

Project objectives were to demonstrate that a minimum of 2% of normal enzyme activity in RV-treated mice would result in comparably normal physical traits. We also wanted to study the different levels of IDUA activity in each organ to establish the efficacy of hepatic gene therapy.

Injecting this retrovirus into two day old mice, we recorded the serum activity levels and sacrificed the 13 mice at six weeks. After homogenizing the organ, we determined the enzyme activity in units (nmoles of substrate metabolized per hour) per mg of protein. Our results for the IDUA activity in RV-treated mice were: 114 U/mg in liver (28500% of normal), 137 U/mg in spleen (1877% of normal), 57 U/mg in kidney (1403% of normal), 68 U/mg in spleen (327% of normal), 14 U/mg in kidney (175% of normal), .459 U/mg in brain (26% of normal), 45 U/mg in heart (2111% of normal), 16 U/mg in small intestines (238% of normal), 22 U/mg in large intestines (187% of normal), and 2.799 U/mg in muscles (507% of normal).

We used the enzyme HexB as our negative control, to show that low levels of IDUA results in high activity of other lysosomal enzymes. This is based on the postulation that the lysosome will compensate low IDUA enzyme activity with higher activity in HexB. Our results for the HexB activities in RV-treated mice were: 1729 U/mg in liver (118% of normal), 5335 U/mg in spleen (117% of normal), 1471 U/mg in kidney (85.47% of normal), 2500 U/mg in thymus (86% of normal), 1251 U/mg in lung (194% of normal), 286 U/mg in brain (8.42% of normal), 297 U/mg in heart (48% of normal), 811 U/mg in small instestines (72% of normal), 364 U/mg in large intestines (98% of normal), 190 U/mg in muscles (152% of normal).

Thus, our results proved that neonatal hepatic gene therapy shows dramatic change in enzyme activity of IDUA. The IDUA enzyme activity level in all organs, except for the brain, was higher than normal enzyme activity; however, the brain has greater than 2% of normal IDUA enzyme activity. In addition, our negative control shows us that the RV-treated have near normal levels of HexB.

The next step in our project is to assay 10 month old untreated, heterozygous, and RV-treated MPS I mice. Untreated MPS I mice usually die from complications of the disease at 11 months; in order for us to compare normal and RV-treated mice to the MPS I mice at later stages of the disease, we must sacrifice them before the average MPS I date of death. We will use the same protocol as the six-week old mice assays, and compare the 10 month period of therapy to the six week old mice results.