Reviews of 1st Year Funding awarded in 2003 Elena Aronovich, PhD Pediatrics and Institute of Human Genetics University of Minnesota, Minneapolis, MN "SB (Sleeping Beauty) Transposon-mediated Gene Therapy for MPS I". The Sleeping Beauty (SB) transposon system created at the University of Minnesota is one of the few non-viral gene therapy systems that are able to integrate genes into human chromosomes. This is especially relevant for treating genetic disorders, such as mucopolysaccharidoses, that require life-long expression of the therapeutic gene. The purpose of this funded project is to see if the SB transposon system can efficiently deliver the ?-L-iduronidase (IDUA) gene to chromosomes in MPS I mice for long-term correction of the disease. The SB transposon system consists of two parts: the transposon that carries the therapeutic gene and the source of transposase enzyme that cuts the gene out of the plasmid and pastes it into the chromosomal DNA. Without the transposase, the therapeutic gene will still be expressed, but only short-term, since it is unlikely that it will be integrated into the chromosome. We constructed a series of SB transposon vectors that all provided high IDUA activity but differed in the level of the transposase enzyme. The constructs were tested for transposition efficiency in cell culture. The most efficient construct was then injected into MPS I mice that were completely deficient in IDUA activity. The control group of MPS I mice did not get the transposase. The mice were immune-suppressed with the drug cyclophosphamide to allow detection of IDUA, which is recognized as a foreign protein in the MPS I mice. Two other groups of MPS I mice were treated with the therapeutic transposon vectors, but without immune suppression. Blood samples were collected one day after treatment and once every two weeks thereafter. Delivery of the therapeutic transposon was considered technically successful by the observation of plasma IDUA activity >100-fold of wild type levels the day after treatment. In treated MPS I mice that did not receive cyclophosphamide, IDUA activity was undetectable in all mice six weeks after delivery of the therapeutic gene. No IDUA activity was detected in the livers of these mice three months after plasmid administration. In all immune-suppressed mice, the initial plasma activity levels dropped approximately 150-fold over the first two weeks after delivery, but then remained stable in transposase-positive mice (up to five times higher than in the wild-type mice). But in transposase-negative (control) MPS I mice the IDUA levels continued to decline gradually over three months. In the liver, at three months, IDUA activity was detected in both transposase-positive and transposase-negative groups. However, in the former group, these levels were on average four fold higher. The obtained levels of IDUA activity were sufficient to reduce dramatically the number and size of pathologic inclusions in liver, up to their elimination, which was demonstrated by staining with toluidine blue of liver sections. Thus, in a long-term experiment (three month experiment) a single dose of SB transposon system resulted in partial to complete correction of IDUA activity in livers of treated adult MPS I mice. Our future efforts will be to: conduct a longer-term (6-12 month) study of IDUA expression in treated mice, define a pattern of biodistribution of the circulating IDUA enzyme in the treated MPS I mouse organs, and find a way to prevent more efficiently the inhibitory antibody response to the therapeutic IDUA enzyme. The results of this year's work have been reported at the annual WORLD Lysosomal Disease Research Network Symposium, Minneapolis, Minnesota, May 13-15, 2004: Applications of the Sleeping Beauty Transposon To Lysosomal Storage Diseases Perry B. Hackett, Elena L. Aronovich, Jason B. Bell, Betsy T. Kren, Brenda Koniar, Roland Gunther, Scott McIvor, Chester B. Whitley (platform presentation) Long-term Expression of Sleeping Beauty Transposon in the Murine Models of Mucopolysaccharidosis (MPS) Type VII and Type I Elena L. Aronovich, Jason B. Bell, Lalitha R. Belur, Joel L. Frandsen, Roland Gunther, Brenda Koniar, David C.C. Erickson, Beth Larson-Debruzzi, John R. Ohlfest, R. Scott McIvor, Perry B. Hackett, and Chester B. Whitley Dr. Judith Mosinger Ogilvie Ophthalmology & Visual Sciences Washington University School of Medicine, St. Louis, MO "Intravitreal gene therapy in MPS IIIB knockout mice" We have made considerable progress on two goals. First, we have successfully built a breeding colony of MPS IIIB knockout mice. These animals are now available in sufficient numbers for experimental testing. Secondly, we are making the gene transfer vector. Although Fu et al (Mol Ther 5:42-49, 2002) have constructed rAAV vectors encoding NAGLU cDNAs, we decided to prepare our own vector in order to be able to compare results from this study to those previously reported for AAV-GUSB in MPS VII mice (Hennig et al, J Neurosci, 23:3302, 2003; Hennig et al, Mol Ther, in press). We started with the same backbone previously used for a ?-glucuronidase vector. We then inserted human NAGLU cDNA from pCMV-hNAGLU (a kind gift from Elizabeth Neufeld), excised with EcoRI and ligated into pTR-CAGG?530 in place of GUSB (excised by partial digest). The insert orientation was confirmed by restriction digest as follows: ENZYME PREDICTED SIZES BANDS SEEN EcoRI 4829, 2481, 1590 bp 5.0, 2.3, 1.6 kB Sma I 4540, (3221+1390) or (3686+1189) 4.5, 3, ~1.5 kB Bgl II 4940, (3586+370) or (2286+1515) 5.0, 3.5 Sma I+Not I 4540, (2888+1385) or (3514+760), 172 These results indicate no recombination of inverted terminal repeats and that the NAGLU cDNA is in the correct orientation to produce protein. In order to determine whether the construct produces active NAGLU enzyme, we transfected four fibroblast cell lines prepared from NAGLU -/- and +/+ littermate tissues according to the Mark Sands' Lab protocol. We tested the following conditions: transfection with 3 dilutions of plasmid containing the AAV genomic construct, control plasmid DNA, medium with no DNA, and not transfected. NAGLU -/- cell cultures showed a slight but clear, dose-dependent increase in positively stained cells in wells that had been transfected with AAV-NAGLU construct. NAGLU enzyme activity levels in medium from transfected cultures were slightly higher than untreated or sham-transfected culture medium for NAGLU-deficient flat cell cultures. These results indicate that the construct does produce active enzyme and we are now proceeding to make the virus for gene therapy in the MPS IIIB knockout mouse. Philippe Moullier, M.D., Ph.D., and N. Matthew Ellinwood, D.V.M., Ph.D. Inserm U 0649-Laboratoire de Th?rapie G?nique, CHU H?tel-Dieu. Nantes, France "Evaluation of Gene Therapy in the Canine Model of MPS IIIB". The focus of this grant is the further development of the canine model of MPS IIIB with an emphasis on gene therapy. Broadly speaking there have been two main goals in this grant. The first group of aims involves the construction and analysis of gene therapy vectors to be used in vivo in the canine model that are designed to deliver normal copies of the canine N-acetyl-?-D-glucosaminidase (NaGlu) cDNA. The second group of aims involves further characterization of the canine model so that therapies can be evaluated in a timely manner, obviating the need to assess their impact on clinical signs, which are adult onset in this canine model. In pursuit of the first set of aims, work is underway on the construction of a gene therapy vector containing the canine NaGlu cDNA in the context of a recombinant adeno-associated viral (rAAV) vector derived from the AAV serotype 2. This vector will be assessed by intracerebral injections in the canine model. In support of the second aim of the grant, work has progressed on a better understanding of the natural history of the disease in the canine model. The canine model of MPS IIIB is unlike other models in two respects. First, combined with the canine models of MPS IIIA, it is among the only large animal models of MPS disorders to show overt neurological clinical signs. Secondly, unlike the clinical signs of MPS seen in other large animal models, these clinical signs are of early adult onset. This latter fact makes it critical that biochemical markers and/or pathological findings which differ between normal and affected dog be identified at a young age. To this end we have pursued analysis of affected dogs looking at histopathological signs of disease, as well as at biochemical changes in the brains of affected dogs. Histological, lesions associated with lysosomal storage can be distinguished in the liver and kidney of affected dogs as early as 3 months of age. These findings were also seen in affected dogs at six months of age. No findings of lysosomal storage has been seen in the central nervous system of affected dogs at these ages using convention histopathological techniques, however semi-thin section analysis will be pursued. In an effort to find central nervous system biochemical markers associated with disease in the canine model, we have conducted ganglioside analysis of the cerebral gray matter of affected dogs, ages three months to six years of age. This work was done in at the Lyon-Sud Medical School in Lyon, France, in collaboration with Dr Marie T. Vanier and with the assistance of her graduate student Lucie Verot. Gangliosides, some types of which have long been known to accumulate in the brains of patients with some forms of the MPS disorders, were found to be elevated in dogs as early as 3 months of age. The accumulated gangliosides (GM2 and GM3), remain elevated relative to age matched normal controls from 3 months of age onward, and this elevation increases until the end stages of the disease. In the course of this year of the grant, Dr. N. Matthew Ellinwood, who was funded as a post-doctoral fellow by this grant, was selected to begin an assistant professorship in the Animal Genetics group within the Animal Science Department at Iowa State University, in Ames Iowa. This is primarily a research appointment, and Dr. Ellinwood, whose position begins October 1, 2004, will continue to work on canine MPS IIIB as the major focus of his research. The canine breeding colony, the establishment of which was supported by the National MPS Society, has been housed at the University of Pennsylvania, and is in the process of being transferred to Iowa State University, a process which will be completed this fall. In consideration of Dr. Ellinwood's change of status, it has been proposed to the National MPS Society that the second year of this grant be transferred to Dr. Ellinwood at Iowa State University, where it will serve as the source of a stipend for a post-doctoral fellow in Dr. Ellinwood's research laboratory. Findings presented above have been presented in abstract form at an NIH symposium on the Glycoproteinoses and Related Disorders (April 2004), and are submitted to the American Society of Human Genetics meeting (October, 2004). All work present has acknowledged the funding support of the National MPS Society. Dr. S. Byers Department of Genetic Medicine, Women's and Children's Hospital, North. Adelaide, South Australia "Inhibition of glycosaminoglycan synthesis as a therapy for mucopolysaccharidosis type IVA and VI" The goal of this proposal is to develop and evaluate substrate deprivation therapy for MPS IVA and MPS VI, using small molecular weight inhibitors of glycosaminoglycan synthesis. In the first instance therapy for MPS IVA has been prioritized. One of the obstacles to evaluation of any type of therapy for MPS IVA is the availability of a convenient in vitro system to monitor correction of storage. The most widely used cell type, the skin fibroblast, does not synthesize or store significant amounts of keratan sulphate and is therefore not suitable for testing therapy regimens. Our first aim was to develop an in vitro assay system. To achieve this, the keratan sulphate containing domain (G1-G2) of the large cartilage proteoglycan, aggrecan, was isolated and cloned into 2 different expression vectors; an HIV-1 based lentivirus (pHIVmpsvG1-G2) and pCDNA3.1v8HisTOPO (pTOPOG1-G2). Normal cells infected with pHIVmpsvG1-G2 expressed low levels of the keratan sulphate domain as determined by Western blot. Work is in progress to optimize expression from both constructs and assess both skin fibroblast and bone osteoblast cells as mediators of expression. The concept of substrate deprivation therapy and its application to keratan sulphate synthesis has been demonstrated in normal bovine articular cartilage cell cultures. Cartilage chondrocytes produce large amounts of keratan sulphate containing proteoglycans. The addition of either a general inhibitor of glycosaminoglycan synthesis or a small molecular weight inhibitor of keratan sulphate synthesis to cell culture medium resulted in a decrease in the level of keratan sulphate produced. Based on other experiments with different glycosaminoglycan types, we have shown that inhibition of glycosaminoglycan synthesis results in decreased storage of gag degradation products in the appropriate MPS skin fibroblast cells. Similar experiments will be performed with the keratan sulphate inhibitors once we have fully developed our in vitro assay for keratan sulphate storage. Large scale synthesis of the small molecular weight inhibitor of keratan sulphate synthesis has been initiated. Dr. Calogera M. Simonaro Department of Human Genetics, Mount Sinai School of Medicine, New York, NY "Joint & Bone Disease in the Mucopolysaccharidoses: Identification of New Therapeutic Targets & BioMarkers Using Animal Models" The mucopolysaccharidoses (MPS) are inherited metabolic disorders resulting from the defective catabolism of glycosaminoglycans (GAGs). We previously used MPS animal models to investigate the pathophysiology of the joints and bones, major sites of pathology in these disorders, and found enhanced chondrocyte apoptosis and nitric oxide production associated with TNF-? and Il-1. We now report that the stimulation of MPS connective tissue cells by these inflammatory cytokines causes enhanced secretion of several matrix-degrading metalloproteinases (MMPs). In addition, expression of tissue inhibitor of metalloproteinase (TIMP)-1 was elevated, consistent with the enhanced MMP activity. These findings were not restricted to one particular MPS disorder or species, and are consistent with previous observations in animal models with chemically induced arthritis. BrdU incorporation studies also revealed that MPS chondrocytes proliferated up to five-fold faster than normal chondrocytes, and released elevated levels of TGF to counteract the marked chondrocyte apoptosis and matrix degradation associated with MMP expression. However, despite this compensatory mechanism, studies of endochondral ossification revealed a reduction in chondrodifferentiation in the growth plates. Thus, although MPS chondrocytes grew faster, most of the newly formed cells were immature and could not mineralize into bone. Our studies suggest that altered MMP expression, most likely stimulated by inflammatory cytokines and nitric oxide, is an important feature of the MPS disorders. These data also identify several proinflammatory cytokines, nitric oxide, and MMPs as novel therapeutic targets and/or biomarkers of MPS joint and bone disease. This information should aid in the evaluation of existing therapies for these disorders, such as enzyme replacement therapy (ERT) and bone marrow transplantation (BMT), and may lead to the development of new therapeutic approaches. Pathological and Molecular Characterization of Feline Mucolipidosis II: Urs Giger, PD Dr. med. vet. FVH Section of Medical Genetics, University of Pennsylvania Philadelphia, PA "Pathological and Molecular Characterization of Feline Mucolipidosis II: The First Model of Human I-Cell Disease" Mucolipidosis II (ML II), also called I-cell disease, is a unique cellular storage disease leading to severe skeletal malformations, growth and mental retardation, and death within the first decade of life. Although ML II is caused by faulty trafficking of enzymes to reach cellular organelles (lysosomes), it shares many clinical features of the more common forms of mucopolysaccharidoses (MPS). We have established a colony of domestic shorthair cats with naturally-occurring ML II, the first model in which to study this rare storage disease. Recently we have documented the clinical features in cats and the autosomal recessive mode of inheritance (Mazrier et al J Heredity 2003) and documented the close homology and minor differences between the disorder in humans and cats. As the pathology is hardly described in humans with ML-II, we were keen to characterize the pathology of feline ML II in tissues from affected cats and compare the results to the scant information from human patients. We have prepared histological preparations from autopsied animals and are analyzing each tissue by Jessica Caverly VMD PhD, a veterinary pathologist who received a Reentry Fellowship from NIH for these studies. Furthermore, we are extracting the various tissues to identify the specific storage material including mucopolysaccharides and gangliosides in collaborations with others. Similarly we have cultured fibroblast from affected cats to further characterize the specific inclusions so classic for I-cell disease. Finally, tissues with specific pathology will also be assessed by electron microscopy. Although the deficient enzyme has recently been identified in humans, the molecular basis remains unknown in affected cats as well as humans. Through William Canfield at Genzyme we were able to gain access to the sequence of the human enzyme N-acetylglucosamine-1-phosphotransferase, compared that sequence to the recently released canine transferase sequence and have developed primers to amplify the exons from normal and affected cats. We have thus far about 1kb amplified and sequenced and there seems to be close homology between species. We are also using fibroblast cultures for RT-PCR of the feline cDNA and thereby should be able to get the entire sequence shortly, therefore we are in a good position to characterize feline I-cell disease at the molecular genetic level. Some of our initial findings were presented at the National MPS Society meeting in Mainz and further collaborations for storage pathology and biochemistry were established. Thereby, the knowledge gained in feline ML II will likely further our understanding of this disease in humans and provides the necessary characterization for this animal model to become useful in the development and assessment of the safety and efficacy of novel therapeutic interventions. Philippe Moullier, M.D., Ph.D., and N. Matthew Ellinwood, D.V.M., Ph.D. Inserm U 0649-Laboratoire de Th?rapie G?nique, CHU H?tel-Dieu. Nantes, France "Evaluation of Gene Therapy in the Canine Model of MPS IIIB". The focus of this grant is the further development of the canine model of MPS IIIB with an emphasis on gene therapy. Broadly speaking there have been two main goals in this grant. The first group of aims involves the construction and analysis of gene therapy vectors to be used in vivo in the canine model that are designed to deliver normal copies of the canine N-acetyl-?-D-glucosaminidase (NaGlu) cDNA. The second group of aims involves further characterization of the canine model so that therapies can be evaluated in a timely manner, obviating the need to assess their impact on clinical signs, which are adult onset in this canine model. Dr. John Hopwood Lysosomal Diseases Research Unit, Women's and Children's Hospital North Adelaide, South Australia, Australia The Sleeping Beauty transgene vehicle ? a potential new therapy for MPS-IIIA The present study is investigating a potential new treatment for lysosomal storage disorders (LSD) that affect the brain, the 'Sleeping Beauty transgene vehicle'. Sleeping Beauty (SB) is able to transport genetic material into cells. We believe that we will be able to use Sleeping Beauty to transport the genetic material into cells that is required to make lysosomal enzymes. In LSD patients, this would mean that cells treated with SB would make lysosomal enzyme 'normally', and these disorders could thereby be treated. In particular, we are excited about the ability of Sleeping Beauty to enter the cells in the brain. The MPS IIIA mouse model is being used in these studies. Our preliminary studies have focused on the use of SB that has a red fluorescent tag (so that it can be located within the brain) (SB-dsRed), which has been constructed by Professor Clifford Steer and Dr Betsy Kren and colleagues. SB-dsRed has been used in the first instance to obtain some understanding of how SB moves around the brain from the injection site and how many cells it treats ? we can determine this by looking to see how many red cells there are in a section of mouse brain. Ideally, we would like SB to treat every cell within the brain. In the next phase of the study, we will utilise the SB vector that is capable of transporting the genetic material into cells that is required to make the lysosomal enzyme sulphamidase. SB-sulphamidase is being constructed by Dr Kren in Minnesota at present. We have used the SB-dsRed vector and have undertaken: (1) preliminary studies investigating the potential of Sleeping Beauty as a future long-term treatment for LSD that affect the brain. The first step in this process has been to determine how long SB-treated cells are capable of producing the red fluorescent tag (or in the future, sulphamidase). We would like SB to enable cells to produce lysosomal enzymes on a long-term (years) basis, so that this treatment does not have to be given too frequently. These experiments have been carried out in human unaffected and MPS-IIIA cells grown in culture in the laboratory; and (2) subsequent studies where we injected SB-dsRed directly into the newborn and adult mouse brain to determine where the fluorescent tag is seen within the brain. The findings from this work are discussed below. Our work with cultured unaffected and MPS-IIIA cells indicates that SB is able to enter these cells and result in long-term production of dsRed (or in the future, lysosomal enzymes). We observed the presence of red fluorescent cells for at least five-months in culture (the longest time we have studied thus far). Approximately 1% of either unaffected or MPS-IIIA cells were treated by SB, a figure consistent with that achieved by other researchers in other cell types. Both unaffected and MPS-IIIA cells were used in this study as it was important to establish that SB treats unaffected and MPS-IIIA cells in the same manner. Adult MPS IIIA mice received direct injections of SB-dsRed into the brain, and the location of cells treated by SB was determined by observing sections of brain under a fluorescence microscope. We detected red fluorescent cells for up to six-weeks post-injection near the injection site, as expected, but also in a nearby region of the brain that contains cells capable of dividing throughout the life of the animal. This is an exciting observation as it suggests that SB is able to enter dividing cells, thus increasing the number of 'treated' cells within the brain. Injection of SB-dsRed into the adult mouse brain did not result in any complications, nor was there observable tissue damage and all mice recovered uneventfully from the injection procedure, as indicated by steady weight gain and behavioural observations. In more recent studies, newborn MPS IIIA mice received injections of SB-dsRed into the ventricles (fluid filled spaces) in the brain and the location of SB-treated cells (as determined by the presence of the red fluorescent protein) was determined two-weeks later. Treated cells were observed in even more widespread areas of brain. These experiments are continuing. In summary, these preliminary studies pave the way for planned experiments using the SB-sulphamidase vector in MPS-IIIA mice (as a precursor to MPS-IIIA dog and then MPS-IIIA human studies). We are hopeful that cells in widespread areas of the brain will be treated with SB, thus alleviating the deleterious effects of reduced lysosomal enzyme activity in this disorder.