Reviews of the second year of funding for grants awarded in 2003 Intravitreal Gene Therapy in MPS IIIB Knockout Mice Dr. Judith Mosinger Ogilvie, Department of Biology, St. Louis University The objectives of this project were to fully characterize the functional and pathological degeneration of the MPS IIIB mouse model of Sanfilippo Syndrome type B, to determine whether visual function could be restored through intravitreal gene therapy, and to lay the groundwork for rational design of studies to assess function in the central nervous system. Preliminary studies in MPS VII mice with virus-mediated gene therapy resulted in correction of lysosomal storage in treated eyes and in several areas of the brain. However, because of the severity of the disease, we could not test the long-term functional consequences of treatment in these mice. We have succeeded in achieving the first objective with full characterization of the functional and pathological degeneration of the MPS IIIB mouse. Functional evaluation was performed with electroretinograms (ERGs) of retinas from MPS IIIB mice at 4, 8, 12, 16, 20, 30, 34, and 45 weeks of age. ERG testing of light- and dark-adapted mice produces a characteristic wave-form that allows for identification of deficits in different cell types within the retina. The dark-adapted retinal ERG response is depressed by 5 weeks and becomes progressively less sensitive with increasing age. The diminished sensitivity reflects a loss of rod photoreceptor function that achieves persistent significance after 15 weeks. No significant differences were observed at any age in retinal function after light adaptation indicating a normal level of cone photoreceptor function. We have performed histological analysis on retinas from MPS IIIB mice at the same time points. At 4 weeks of age, the mutant retina appeared grossly normal, although localized abnormalities were seen in retinal pigment epithelium (RPE) cell shape consistent with loss of cell polarization and/or delamination. By 8 weeks of age, lysosomal storage could be seen in vascular cells and microglia in the inner retina. Occasional pyknotic nuclei could be seen in the outer nuclear layer, which is comprised of photoreceptor cells, and further disruption in the RPE was seen. Photoreceptor degeneration, with shortening of the outer segments and cell loss in the outer nuclear layer, became apparent around 16 weeks with a decrease of 2-4 rows of nuclei by 20 weeks of age. Macrophage-like cells were apparent in the subretinal space between the RPE and photoreceptor outer segments by 16 weeks. Nearly half of the photoreceptor cells were gone by 30 – 34 weeks with only 3-4 rows of nuclei remaining at 45 weeks. Outer segments were further shortened and appeared swollen. Large, round, dense melanosome-like structures were seen in the RPE of mutant retinas, first becoming noticeable in the periphery of the mutant eye as early as 4 weeks, distributed throughout the RPE by 16 weeks, and becoming larger and more prominent by 30 weeks. In mutant retinas, unlike wild type controls, these structures were prominent in the mid to basal cytoplasm and were distinguished by their round shape. This thorough characterization of the retina of the MPS IIIB mouse model of Sanfilippo Syndrome type B is an important step forward in investigating potential therapeutic interventions for this disease. These results have been presented in abstract form at the Association for Research in Vision and Ophthalmology meeting (Hennig, et al., 2006) and are currently in preparation for publication with additional characterization performed by Dr. Mark Sands and collaborators (Heldermon, et al). Considerable effort was placed in producing a gene transfer vector. Initial results indicated that our construct produced active enzyme. MPS IIIB knockout mice and wild type controls were injected intravitreally with the vector and functional tests were performed at three time points. Tissue was harvested and processed. Unfortunately, the viral construct did not produce sufficient expression of the NAGLU enzyme to determine whether visual function could be restored through intravitreal gene therapy. Although we were disappointed with this result, other experiments performed concurrently with this work have enabled progress on the third objective. We collaborated with Drs. Mark Sands and Megan Griffey on the ppt1 mouse model of Batten's Disease. Those studies (Griffey, et al., 2005) were successful in establishing the groundwork for the future design of studies to assess the ability of intravitreal gene therapy to improve CNS function in lysosomal storage diseases. Publications: Griffey, M., S.L. Macauley, J.M. Ogilvie, M.S. Sands. (2005) AAV2-mediated ocular gene therapy for infantile neuronal ceroid lipofuscinosis. Mol. Ther. 12:413-21. Heldermon, C.D., Hennig, A., Vogler, C., Ohlemiller, K., Ogilvie, J.M., Breidenbach, A., Herzog, E.D., Sands, M.S. "Characterization of the murine model of Sanfilippo syndrome type B." In preparation. Hennig, A.K., M. Griffey, M.S. Sands, R.L. Gunkel, M.K. Murphy, J.M. Ogilvie. (2006) Electroretinogram Changes and Retinal Degeneration in Knockout Mouse Models of Four Lysosomal Storage Diseases. ARVO Abstract #5780 accessed at www.arvo.org. presented May 4, 2006 at the annual conference of the Annual Conference of the Association for Research in Vision and Ophthalmology. SB (Sleeping Beauty) Transposon-mediated Gene Therapy for MPS I Elena Aronovich, PhD, Pediatrics and Institute of Human Genetics University of Minnesota 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 to provide life-long expression of a therapeutic gene. The purpose of this funded project has been to see if the SB transposon system can efficiently deliver the a-L-iduronidase (IDUA) gene to chromosomes of liver in MPS I mice for long-term expression and 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, which cuts the gene out of the plasmid and pastes it into chromosomal DNA. Without the transposase, the therapeutic gene can be expressed, but generally only short-term, presumably because as an unintegrated episome, it is either lost or recognized as foreign DNA and inactivated. For this study we chose the very efficient, high-pressure "hydrodynamics-based" DNA injection, which targets the liver. We constructed SB transposon plasmid-based vectors and injected them into MPS I mice that were completely deficient of IDUA activity. Because our goal was in part to determine the efficacy of transposition as a way of providing long-term expression of IDUA protein, control groups of MPS I mice did not receive the transposase. Blood samples for plasma isolation were collected 1 day after treatment and once every 2 weeks thereafter. Plasma IDUA activity reached >100-fold of wild type levels on day 1 following treatment, but was essentially gone in all mice by 4-weeks. IDUA activity was not detected in the liver of mice 3 months after plasmid administration. We examined the duration of the transposon-delivered IDUA gene by PCR and IDUA expression in liver of unaffected mice over 6 months. As in the MPS I mice, plasma and liver IDUA activity reached supra-normal levels on day 1 and remained at this level for the first week, but reduced dramatically by the 2-week time point and by 4 weeks were indistinguishable from background. Notably, the presence of the IDUA transgene mirrored the IDUA activity time-line, i.e., the PCR product from the transgene was not detectable after 2 weeks post-injection. However, transposition was confirmed by an "excision assay" (which detects the PCR product of the plasmid that delivered the IDUA gene to the liver). The excision product was detectable for the first two weeks, but was undetectable thereafter. This suggests that cells that express the IDUA gene are cleared from the liver of treated mice by 4 weeks following injection. Our data suggest induction of an immune response either to the therapeutic protein or/and the cells that express the therapeutic gene. This conclusion was supported by our observations that in all cyclophosphamide immune-suppressed MPS I mice, the initial 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice. IDUA levels were stable in transposase-positive mice (up to 5 times higher than in the wild-type mice) whereas in transposase-negative (control) MPS I mice IDUA levels declined over 3 months. In the liver, at 3 months, IDUA activity was detected in both transposase-positive and transposase-negative groups. However, in the former group, these levels were on average 4-fold higher. The obtained levels of IDUA activity were sufficient to dramatically reduce the number and size of pathologic inclusions in the liver as demonstrated by toluidine blue staining of liver sections. Thus, with immune suppression, a single dose of the SB transposon system resulted in partial to complete biochemical correction (cure) of IDUA activity in the liver of treated adult MPS I mice. Our future effort will be directed at achieving systemic delivery of the therapeutic gene and finding a way to prevent immune responses to the therapeutic gene in MPS mice. The results of this work have been reported in three platform presentations and one poster at international meetings: American Society of Gene Therapy, 2005 Annual Meeting, St. Louis, MO, June 1-5, Duration of Expression of Sleeping Beauty Transposase by Hydrodynamic Injection of C57/BL6 Mice. Jason B. Bell, Elena L. Aronovich, Brenda Koniar Roland Gunther, Beth Larson-Debruzzi, Chester B. Whitley, R. Scott McIvor and Perry B. Hackett Mol. Therapy, 2005, v.11: S423 Third Annual International Conference on Transposition and Animal Biotechnology, Minneapolis, MN, June 23-24, 2005 : 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, John R. Ohlfest, R. Scott McIvor, Chester B. Whitley, and Perry B. Hackett WORLD Lysosomal Disease Clinical Research Network Annual Symposium 2004, May 13-15, Minneapolis, MN Application 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, R. Scott McIvor and Chester B. Whitley Long-term Expression of Sleeping Beauty Transposon in the Murine Models of Mucopolysaccharidosis (MPS) Type VII and Type I (poster presentation) Elena L. Aronovich, Jason B. Bell, Lalitha R. Belur, Joel L. Frandsen, Roland Gunther, Brenda Koniar, David C.C. Erickson, John R. Ohlfest, R. Scott McIvor, Perry B. Hackett, and Chester B. Whitley Evaluation of Gene Therapy in the Canine Model of MPS IIIB N. Matthew Ellinwood, D.V.M., Ph.D., Iowa State University, Ames, Iowa. 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-a-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 the course of this grant, originally awarded to the Laboratoire de Therapie Genique (Dr. Philippe Moullier), Dr. N. Matthew Ellinwood, who was funded from this grant as a post-doctoral fellow in Nantes, France, 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 primary research appointment began October 1, 2004, and Dr. Ellinwood's work will continue to focus on canine MPS IIIB. In consideration of Dr. Ellinwood's change of status, it has been agreed that the second year of this grant be transferred to Dr. Ellinwood at Iowa State University. Furthermore, to ensure that the fellowship be used optimally, Dr. Ellinwood has requested an extension of the second year of the award, so that the fellowship is used for primary research after a suitable candidate can be identified and after Dr. Ellinwood's laboratory and canine colony are settled at Iowa State University. The canine MPS IIIB breeding and research colony was fully transferred from the University of Pennsylvania in December of 2004, and our first ISU litter was whelped in May 2005. Research collaborations are underway with both a veterinary clinical neurologist and radiologist at the School of Veterinary Medicine at ISU. These efforts will allow for gene therapy experiments and further characterization of the model. In addition the laboratory is now poised to begin gene therapy evaluations, and a post-doctoral fellow, Dr. Chun Coa, has been identified, and will begin work in August 1, 2005. Work supported by this grant was presented at the American Society of Human Genetics meeting (October, 2004). All work present has acknowledged the funding support of the National MPS Society. Inhibition of Glycosaminoglycan Synthesis as a Therapy for Mucopolysaccharidosis Type IVA and VI Dr. S. Byers, Women's and Children's Hospital, North Adelaide, South Australia The goal of this proposal is to develop and evaluate substrate deprivation therapy (SDT) for MPS IVA and MPS VI. These MPS disorders arise from the deficiency of a lysosomal enzyme required for the degradation of keratan sulphate (KS) or dermatan sulphate (DS) glycosaminoglycan (gag) chains respectively. To be effective, SDT must therefore target the synthesis of these gag chains. By decreasing KS or DS synthesis, the balance between gag production and removal can be redressed in MPS IVA and MPS VI. Thus any remaining patient enzyme activity can more effectively degrade the reduced amount of gag arriving in the lysosome in the presence of inhibitor. We have synthesised a small molecular weight analogue of glucose and assessed its ability to inhibit the synthesis of KS and DS gags in cell culture and compared its effect with a non-specific gag synthesis inhibitor. Both inhibitors decrease gag synthesis in a dose dependent manner when added to the culture medium of normal bovine cartilage cells. Using a combination of size exclusion chromatography and enzyme digestion to identify individual gags, a >50% decrease in the synthesis of the KS-gag containing fraction but only a 15% decrease in the DS-gag containing fraction is observed, implying a differential effect on the two gag populations. Work is currently underway to characterize the size and structure of KS gags synthesised in the presence of inhibitor. These results offer "proof of concept" that SDT targeting gag synthesis as a treatment for MPS disorders has the potential to be a feasible therapy option. Joint and Bone Disease in Mucopolysaccharidosis Type VI Dr. Calogera Simonaro, Human Genetics, Mount Sinai School of Medicine The overall goal of our research is to fill the void in our understanding of MPS bone and joint disease and to develop new and improved therapies that might benefit MPS patients. We specifically study two animal models with MPS VI, but anticipate that the results obtained can be applicable to the general class of MPS disorders and benefit a wide range of patients. Our studies carried out over the past five years (funded in part by the MPS Society and published in two peer-reviewed papers) have revealed that glycosaminoglycan GAG accumulation is a direct cause of chondrocyte death in the articular cartilage and growth plates of MPS animals, leading to abnormal matrix homeostasis. This enhanced cell death also triggers a series of signaling events that lead to marked inflammatory disease. Together, these two factors (enhanced cell death and inflammation), lead to the characteristic bone and joint disease in the MPS disorders. In addition, cellular defects associated with the maturation of MPS growth plates are likely contributing to abnormal bone growth. Enzyme replacement studies in MPS animals and human patients have revealed that chondrocytes in joints and bones are difficult to reach following injection due to the poor vascular supply to these tissues and the fact that the target cells are embedded in a dense, negatively charged matrix. With the availability of enzyme replacement it is important to continue to investigate new approaches for improving enzyme delivery to these critical target tissues. We have modified the charge on these enzymes to make them less negatively charged, so that they might penetrate the cartilage matrix more efficiently and enter chondrocytes. Our data supports the notion that altering the charge might enhance its therapeutic usefulness for cartilage and bone. Our findings have important implications for the treatment of MPS individuals, as well as for the identification of novel biomarkers to monitor disease progression and therapeutic efficacy. Pathological and Molecular Characterization of Feline Mucolipidosis II: The First Model of Human I-Cell Disease Urs Giger, University of Pennsylvania Research Services 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. Clinical signs seem to be rapidly progressive with leg deformities evident from birth; furthermore these kittens also develop retinal and corneal changes that are being further defined. 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 analyzed histological preparations of various tissues from autopsied animals. Interestingly the storage lesions seem to develop slower and be restricted to specific tissues (Caverly et al in preparation). These tissues will be further assessed by electron microscopy. Similarly, specific storage material including mucopolysaccharides and gangliosides, extracted in collaborations with others, could only be found in a limited number of tissues. Finally, we have cultured fibroblast from affected cats to further characterize the specific inclusions so classic for I-cell disease. 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 (GNTPA), compared that sequence to the partial shotgun feline sequence of the transferase. We have completed the sequence of the entire feline GNTPA and characterized the close homology to the human GNTPA sequence and gene structure. Comparing the sequence of affected and normal healthy kittens we have identified a putative disease-causing missense mutation Tcherneva, Seng, Caverly, unpublished). Further studies are in progress to establish a screening test and to characterize the effect on the protein. Some of our initial findings were presented at the National MPS Society meeting in Mainz and further collaborations for future collaborations have been established internationally. 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.