Research Updates In 2004 the National MPS Society awarded grants to Dr Maia Pia Cosma for her work, ?Gene Therapy for MPS II? and to Dr. Mark Sands for his work ?Characterization of the Systemic Inflammatory Response to Lysosomal Storage?. Below are the reviews they submitted, summarizing the first year of research. Dr. Maria Pia Cosma TIGEM, Naples, Italy ?Gene Therapy for MPS II? Mucopolysaccharidosis type II (Hunter syndrome) is due to the deficiency of the enzyme iduronate sulfatase (IDS). The IDS enzyme promotes the degradation of complex molecules named glycosoamminoglycans (GAGs). In particular IDS promotes the degradation of dermatan sulfate and heparan sulfate, which once degraded can be eliminated from the cells. In Hunter Syndrome these compounds accumulate as un-degraded molecules in the cells of the patients. The cells, overloaded with these molecules within particular organelles, the lysosomes, initiate a systemic degeneration of all of the tissues of the body. To date, there is no effective treatment for MPSII and gene therapy is an attractive approach to cure the MPSII syndrome. To this aim we used the Hunter mouse model that exhibits many of the characteristics of MPSII, including skeleton abnormalities such as coarse faces, macrodactlya, and elevated accumulation of GAGs in the urine and many organs. We generated a viral vector carrying the human IDS that produces the active enzyme exclusively in the liver. The viral particles were administrated intravenously (IV) to group of adult MPS II mice and mainly targeted the liver. When the IDS enzyme was produced from the liver it was secreted into the bloodstream and taken up by the other organs leading to the correction of the systemic defects. We analyzed the correction of the disease phenotype in treated mice after one and seven months. In both groups, upon the gene therapy treatment, the plasma levels of IDS enzyme were increased in all treated mice and the accumulation of GAGs in the urines was reduced. We also carried out behavioral tests on the treated animals that performed as well as the normal wild-type control mice. Furthermore, even the skeletal abnormalities, such as coarse faces and macrodactlya, resulted completely corrected. The tissues of treated mice were also analyzed and showed a rescue of the enzymatic activity of IDS and complete GAGs clearance. Finally, microscopically, there was a marked reduction in vacuolization in all of the organs examined. We also characterized the CNS defects of the MPSII mice and found degeneration of Purkinje cells in the cerebellum and GAGs accumulation in the choroid plexus. The described gene therapy approach has the caveat that it does not correct the central nervous system defects. The IDS secreted from the liver into the bloodstream does not cross the brain blood barrier and therefore does not reach the brain. To rescue the central nervous system phenotype, we are performing experiments based on the delivery of the IDS adeno-associated vector in the cerebrospinal fluid and direct injection in the cerebellum. Preliminary results showed a partial clearance of the accumulation of GAGs in the choroid plexus. The results achieved so far demonstrate possibility and the efficacy of gene therapy by AAV carrying the active IDS to treat mucopolysaccharidosis type II.? Dr. Mark S. Sands Washington University, St. Louis, MO ?Characterization of the Systemic Inflammatory Response to Lysosomal Storage? Specific Aim 1: The goal of this Specific Aim was to determine if the abnormal clinical signs observed in the MPS I mouse (adipose storage deficiency, anemia, muscle wasting) were common to other lysosomal storage diseases. As presented in the preliminary data section of the proposal every mouse model of lysosomal storage disease had a significant decrease in adiposity (15-57% decrease compared to normal control animals). With respect to anemia, the models of MPS (I, IIIB and VII) all had anemia as presented in the preliminary data. However, neither the Neimann-Pick AB (NPAB) nor the infantile neuronal ceroid lipofuscinosis (INCL) models had significantly decreased hematocrits. We are currently sectioning quadriceps muscles from all of the models to determine if there is significant muscle wasting. Specific Aim 2: The goal of this Specific Aim was to determine if the abnormal levels of pro-inflammatory molecules seen in the MPS I model were common to other models of lysosomal storage disease. Our preliminary data showed that both MPS I and NPAB mice had significantly elevated levels of several cytokines and chemokines. Interestingly, the biggest changes were in the levels of circulating chemokines and soluble VCAM. We have completed our analyses of the MPSIIIB, MPS VII and INCL models. The levels of pro-inflammatory molecules varied among the various models but there was evidence of systemic inflammation in all of the models. MPS IIIB mice had elevated levels of MCP-1, MCP-3, MIP-1a, VCAM and IL-1b. MPS VII mice had elevated levels of MCP-1, MCP-3 and VCAM. INCL mice had elevated levels of soluble VCAM. Therefore, inflammation appears to be a common clinical feature of lysosomal storage diseases. Interestingly, MCP-1 and/or VCAM are elevated in every model of lysosomal storage disease. Specific Aim 3: The goal of this Specific Aim was to test the hypothesis that systemic inflammation contributed to the disease progression in murine models of lysosomal storage diseases. We proposed several different approaches to test this hypothesis. Since MCP-1 was significantly elevated in nearly every model of lysosomal storage disease, we obtained the MCP-1-null mouse from the Jackson Laboratory and have moved both the MPS I and NPAB mutations onto that genetic background. We currently have colonies of MCP-1- and NPAB-deficient mice that carry both the disease mutation and the MCP-1 mutation and are generating double mutant animals to be tested for adiposity, anemia and muscle wasting. We have also created an HIV-based gene transfer vector encoding the M3 protein from g-Herpes virus. M3 is a secreted protein that binds most chemokines with high affinity and inactivates the pro-inflammatory function. In this way we can determine the effects of selectively inhibiting the function of the elevated chemokines present in the murine models. We currently have several MPS I and MPS IIIB mice that have been injected with the HIV-M3 virus and are generating additional animals to complete those experimental groups. In a similar fashion, we have obtained a transgenic mouse model that expresses M3 from an inducible promoter in the presence of the antibiotic doxycycline. We have moved the transgenes onto the MPS I background and currently have mice that are expressing high levels of M3 systemically. All of the animals will be analyzed at 5 or 7 months of age to determine the effects of inhibiting a host of different chemokines (HIV-M3 and transgenic M3) or just MCP-1's actions (MCP-1 knockout).