2006 Grants: First year reviews Dr. Maria Pia Cosma "AAV-mediated gene therapy of Hunter syndrome in the MPS II mouse model" TIGEM, Naples, Italy Mucopolysaccharidosis type II (MPSII; Hunter syndrome) is a lysosomal storage disorder that arises due to the deficiency of iduronate 2-sulfatase (IDS) enzyme activity. We recently characterized the ids knockout, which shows skeleton deformations and an elevated accumulation of glycosaminoglycans in the urine and in many of organs. In addition, the performance of the knockout mice in the open field and walking pattern tests were severely compromised. We designed an efficient gene therapy approach to treat these MPSII mice using adeno-associated viral (AAV) vectors. AAV2/8TBG-IDS viral particles were administrated intravenously in adult animals. The plasma and tissue IDS activities were completely restored in all of the treated mice, up to nine months after treatment. This rescue of IDS activity resulted in the full clearance of glycosaminoglycan accumulation in the urine and in all of the tissues analyzed, with correction of the skeleton malformations and normalization of the performance in the locomotor tests (Cardone et al. Hum Mol Genet 2006). It is gratefully acknowledged that these studies were made possible through funding provided by the National MPS Society Award 2004. With regard to the current MPS Award 2006, to date, our progress towards the aims proposed is as follows: Aim 1: Characterization of the brain defects in MPSII mice. We have performed an initial characterization of the brain defects in the MPSII mouse model and seen a loss of Purkinje cells and cellular vacuolization in different regions of the brain: the hippocampus, thalamus, cerebellum and brainstem. We also noted GAG accumulation within the choroids plexus of the ventricular region. To further characterize the brain abnormalities in MPSII mice, we planned to analyze the morphology and numbers of neurons, astrocytes and oligodendrocyte, using different neural markers. To begin this analysis, control wild-type adult mice and MPSII adult mice, all at 12 months of age, were perfused with PFA and the brains postfixed and embedded in paraffin. Sections were analyzed by immunohistochemical analysis using NeuN as the neuronal cell marker, calbindin as the Purkinje cell marker and GFAP as the astrocyte marker. We detected a decreased number of NeuN-positive neurons in the cortex and almost no positive calbindin signal in the cerebellum of the MPSII mice, as compared to the wild-type animals. In contrast, we detected an increased positive GFAP signal in the astrocytes of the MPSII brain sections. These preliminary data suggest that neurodegeneration and gliosis occur in the brains of adult MPSII mice. Aim 2: Development of a gene therapy approach for the treatment of the MPSII pups to anticipate disease manifestation. Preliminary short-term experiments have indicated that when the idsy/- pups are injected with the AAV2/5CMV-IDS and AAV2/8TBG-IDS vectors into the temporal vein, they show a rescue of IDS activity and a clearance of GAG accumulation in all of the organs analyzed, including the brain. To see whether it is possible to prevent the MPSII phenotype, newborn (2 days after birth) idsy/- pups received 1x1011 total particles of AAV2/5CMV-IDS, of AAV2/8TBG-IDS or of AAV2/5CMV-EGFP in the temporal vein. We plan to follow the efficacy of the therapy throughout their lifetime. We are measuring IDS activity in the plasma and GAG accumulation in the urine each month. The IDS-injected mice are now 13 months old and show very high levels of circulating enzyme in the plasma, and GAG clearance in the urine, with respect to the control group. In addition, we planned to test the efficacy of the intra-muscular therapy. For this, we have constructed the AAV2/1MCK-IDS vector. The AAV2/1 serotype in combination with the muscle creatinine kinase promoter (MCK) allows efficient muscle transduction and robust expression of the transgene. We tested the prepared vector in wild type mice. Thus, a group of wild-type animals have been injected in the anterior tibialis with 2x1010 total particles of AAV2/1MCK-IDS. The injected animals were sacrificed one month after the injection and the transduced muscles were harvested. The measured IDS activity was extremely high in the injected muscle, with respect to the activities measured in the control (non-injected) group. Aim 3: Development of a gene-therapy approach to correct the CNS defects of the MPSII mice. To treat the neuropathological features via systemic delivery of the viral particles, we constructed the AAV2/4CMV-IDS vector and disseminated it into the cerebro-spinal fluid through injection into ventricular region IV. The goal of this approach is to transduce CNS ependymal cells and test if these cells, once transduced, can serve as a source for enzyme secretion into the surrounding brain parenchyma and CSF. A group of with 1x1010 particles of AAV2/4CMV-IDS or with 1x1010 of AAV2/5CMV-GFP viral particles as control. The mice were sacrificed one month after treatment and the brains analyzed. An increased IDS activity was seen in the brain homogenates of IDS-injected mice, with respect to the controls. In addition clearance of GAG accumulation was the brain sections of the treated mice. Dr. Shunji Tomatsu "Development of a therapeutic bone-targeting system for MPS" Department of Pediatrics, Saint Louis University, Pediatric Research Institute We have finished the untagged enzyme experiment on adult and newborn MPS IVA mice. The tagged enzyme is now under investigation. We summarized the results as follows. In the second year, we will finish the bone-tagged enzymes as well. Summary of the results: 1) Adult MPS IVA tolerant mouse: The pharmacokinetics and biodistributions were determined for two recombinant human GALNSs produced in CHO cell lines: native-GALNS and sulfatase-modifier-factor 1 (SUMF1) modified GALNS. Preclinical studies of enzyme replacement therapy (ERT) by using two GALNS enzymes were performed on MPS IVA mice. The half-lives in blood circulation of two phosphorylated GALNS enzymes were similar (native, 2.4 min; SUMF1, 3.3 min). After intravenous doses of 250 units/g body weight were administered, each enzyme was primarily recovered in liver and spleen, with detectable activity in other tissues including bone and bone marrow but not in the brain. At 4 h postinjection, enzyme activity was retained in the liver, spleen, bone, and bone marrow at levels that were 20% - 850% of enzyme activity in the wild-type mice. After intravenous doses of 250 units/g of native GALNS, 250, 600, or 1,000 units/g of SUMF1-GALNS were administered weekly for 12 weeks, MPS IVA mice showed marked reduction of storage in visceral organs, bone marrow cells, osteoblasts, osteocytes, ligaments, and periosteum. A dose-dependent clearance of storage material was observed including brain and cartilage cells although the heart valves were refractory and variable. The blood KS level assayed by tandem mass spectrometry was reduced nearly to normal level. These preclinical studies demonstrate the storage clearance of tissue and blood KS by administered GALNS, thereby providing the in vivo rationale for the design of ERT trials in MPS IVA. Preliminary results by using the bone-targeting enzymes showed that intravenous doses of 250 units/g of the bone targeting GALNS was the same effective as 1,000 units/g of SUMF1-GALNS was used. 2) Newborn MPS IVA knockout mice: We have used knock-out MPS IVA mice to assess the effects of long-term enzyme-replacement therapy initiated at birth. MPS IVA mice received weekly i.v. injections of 250 units/g body weight recombinant human native or SUMF1-GALNS until 14 wk of age. Either GALNS is able to reach brain and bone until the blood-brain barrier completely closes at 10â€"14 d of age and avascular region in cartilage cell layer appears. MPS IVA mice that were treated from birth demonstrated near normalization or complete reversal of lysosomal storage in most tissues including bone marrow, bone (osteocytes, osteoblasts, periosteum, and cartilage), ligaments, and heart valves. Nearly absence in storage vacuoles in cells of the CNS in MPS IVA mice treated from birth was also observed. MPS IVA mice treated from birth kept normal level of serum KS significantly lower than untreated MPS IVA mice cells. Preliminary results by using the bone-targeting enzymes showed that intravenous doses of 250 units/g of the bone targeting GALNS was the same effective as native or SUMF1-GALNS was used. These data suggest that enzyme that enters the brain and the cartilage in the first few weeks of life, before the blood-brain barrier and cartilage cell layers mature, is able to protect against accumulation of storage material in MPS IVA mice. Dr. Doug Brooks "Evaluation of Enzyme Enhancement Therapy for Mucopolysaccharidosis Type IIIA" Women’s and Children’s Hospital North Adelaide, South Australia Australia Genetic diseases are a fundamental cause of human suffering, depriving many children (up to 2.5% of births) and some adults of a normal existence. Lysosomal storage disorders are a group of over 45 different genetic disorders, each involving a defect in lysosomal function. Mucopolysaccharidosis IIIA is a lysosomal storage disorder that affects mainly children, but also some adults and can result in very severe brain disease. Mucopolysaccharidosis IIIA is caused by a defect in an enzyme called sulphamidase. We plan to develop a new therapy, which will involve stabilising the patient’s mutant sulphamidase protein, to increase the level of enzyme activity and thus treat the disease. To increase the stability of the mutant sulphamidase protein we will evaluate small molecule compounds called chemical chaperones that either interact with the outside of the protein or help stabilise the protein from within, by interaction with either the core of the molecule or the active site of the protein. In October 2006 we commenced this project by appointing an experienced research officer for this research project. Because of the drive to develop a new therapeutic strategy for mucopolysaccharidosis IIIA, we first set about validating all of the assay systems that would be required to convince a regulatory authority of the soundness of any data generated. We also cultured a number of cell lines expressing point mutations in CHO-K1 cells and selected fibroblast cell lines that would be appropriate for evaluating the action of chemical chaperones. These cell lines will be used to show increased protein stability and amount of protein/activity in response to chemical chaperone treatment. These initial objectives were completed early in 2007 and showed that the sulphamidase assays were both accurate and not subject to significant variation. We have now commenced a preliminary screen of the compounds proposed as chemical chaperones. In previous work, we showed that protein stabilisers like glycerol and trehalose (that act on the outside of a protein) can improve the yield of mutant protein and activity, but for sulphamidase we have seen no affect in expression cell lines that had different point mutations. We then evaluated several compounds that would be expected to bind to the inner core of the molecule, to help stabilise the mutant sulphamidase protein. We evaluated glucosamine (a low affinity inhibitor of sulphamidase) and glucosamine N-sulphate and showed some variable increases in the sulphamidase protein. This may indicate a partial effect, or that the conditions that we used were not optimal and we are currently pursuing this line of investigation. Finally, we have just evaluated another different type of inhibitor and have some indications of a very promising result. Thus, we believe that we are on target to achieve the following two scientific aims later in 2007: Identification of a chemical chaperone that will enhance the level of mutant sulphamidase protein and enzyme activity in cultured cells. Demonstrate that chemical chaperone treatment can correct the MPS IIIA biochemical storage defect, in vitro.