2nd Year Research Reviews – 2010


Mark J. Osborn, PhD (eight month extension awarded)
University of Minnesota, Minneapolis, MN
Gene therapy for the central nervous system pathology of MPS I


Brett E. Crawford, Ph.D.
Zacharon Pharmaceuticals Inc., La Jolla CA
Glycosaminoglycan inhibitors as substrate reduction therapies for MPS II

With the continued support from the National MPS Society, the National Institute of Neurological Disorder and Stroke and Pfizer, we have made significant progress toward developing a new therapeutic approach designed to treat both the neurological and non-neurological symptoms of MPS.  This approach is based on compounds that modify glycosaminoglycan synthesis so that certain lysosomal enzymes are not required to degrade them.

The funding from the National MPS Society has provided critical support that has helped us progress this drug development from an idea to an NIH grant supported program and recently to a partnership with Pfizer.  The following is a brief description of our progress over the last year:

Testing the In Vivo Efficacy of Lead Compounds. Through the first year of support, we identified a series of analogs with improved potency in cellular models of MPS.  Our most potent analogs are active in the 100 to 500 nM range, a 100-fold improvement from the original compound.  Over the last year, these compounds have been evaluated for their drug-like properties and pharmacological characteristics in a wide range of enzymatic, cellular, and rodent models.  Three of these potent compounds with acceptable pharmacological properties were recently tested in the MPS IIIA mouse model.  These in vivo studies demonstrated that all three compounds reduced the lysosomal accumulation of glycosaminoglycans n the brain.  Future studies will explore the minimal effective dose and dose required to achieve a phenotypic benefit in the mouse model.

Expanded Drug Discover Effort. With the additional support of Pfizer over the last year, we have also expanded our efforts to identify additional therapeutic approaches to MPS.  We have used our cellular model of MPS and the Sensi-Pro® assay to screen hundreds of compounds from Pfizer’s collections that are potent inhibitors of known drug targets.  Our goal is to identify new drug targets that could accelerate the clinical testing of active agents in MPS patients.  These studies have revealed a number of potentially active compounds that we are currently characterizing further.

In the nest year we will continue to optimize the drug-like properties of the most potent inhibitors, explore novel approaches to MPS, and test the improved compounds in the MPS mouse models.  We are optimistic that a novel therapy will emerge from these studies and move closer to clinical testing in the near future.  We sincerely appreciate the dire need for effective therapies for these devastating diseases and are committed to bring a new therapy to the clinic as soon as possible.


Elizabeth F. Neufeld, Ph.D.
David Geffen School of Medicine at UCLA, Los Angeles, CA
Making a minigene suitable for gene therapy for MPS IIIB


Calogera M. Simonaro, PhD
Mount Sinai School of Medicine, New York, NY
A Novel Approach for the Growth & Expansion of Bone Marrow-Derived Mesenchymal Stem Cells in Mucopolysaccharidosis Type IV and Other Mucopolysaccharidoses

The overall goal of this research project was to evaluate the use of a recombinant enzyme (acid ceramidase, rAC) produced and studied in our laboratory to improve the outcome of cell-based therapies for the MPS diseases.  An important and debilitating feature of MPS is progressive cartilage destruction leading to the development of severe arthritic joint disease.  At the present time there are no suitable methods to prevent these abnormalities in MPS, and current enzyme replacement (ERT) and bone marrow transplantation (BMT) therapies have limited effects.  We have previously found that glycosaminoglycan (GAG) storage in MPS activates numerous inflammatory and other signaling pathways, leading to cartilage cell (i.e., chondrocyte) death and cartilage destruction.   Among the many changes in MPS, there is an elevation of the pro-death fat or lipid called ceramide.

Based on this we proposed that rAC could be used to improve the survival and integrity of MPS chondrocytes, both in the laboratory (cell culture) and in animal models of MPS.  rAC is the enzyme that degrades the pro-death lipid ceramide, providing a basis for this hypothesis.  During the course of this project we determined that the addition of rAC to normal animal (human, rat, horse etc) chondrocytes maintained in the laboratory improved their growth and quality, as determined by several established methods to assess cartilage integrity (e.g., expression of cartilage-specific collagen etc).   Moreover, we found that these effects were even more pronounced using chondrocytes obtained from several animal models of MPS.  Due to the GAG accumulation in MPS cells and subsequent downstream changes, these cells grow very poorly and lose their cartilage-like properties, even more than normal cells.  In the presence of rAC, these features were significantly improved.

We also tested the effects of rAC on the growth and properties of stem cells obtained from the bone marrow of MPS animals (i.e., bone marrow mesenchymal stem cells, MSC).  We found that addition of rAC to normal and MPS bone marrow cells increased the production of MSC about 2-fold, and also significantly improved their ability to become chondrocytes.  We tested this using bone marrow from several MPS animal models, and found similar results.

We also obtained bone marrow from mice expressing a protein called green fluorescence protein (GFP), and also found similar results.  Based on these observations we have begun to evaluate whether cells (MSC or chondrocytes exposed to rAC) grow better after they are transplanted into MPS animals, and whether they have an enhanced effect on repairing or preserving the cartilage disease.  These studies have been initiated and are ongoing.

Thus, our ongoing goal continues to be to develop improved methods to repair the defective cartilage in MPS patients.   Funds from this grant have provided essential information that has moved us closer to this goal.


Katrin Kollmann, PhD  (Partnership grant with Insieme per Gabriel)
University Medical Center Hamburg-Eppendorf , Hamburg, Germany
Skeletal abnormalities in mucolipidosis II alpha/beta – Pathomechanisms and therapeutic strategies

The lysosomal storage disease mucolipidosis type II (MLII) is caused by defects in the GlcNAc-1-phosphotransferase. The phosphotransferase is an enzyme complex composed of six subunits (a2b2g2) that catalyzes the first step in the formation of the mannose 6-phosphate (M6P) recognition markers on lysosomal hydrolases. The M6P recognition marker is important for the efficient transport of newly synthesized lysosomal proteins/hydrolases to lysosomes. In MLII with mutations in the gene encoding the alpha/beta subunits of the complex (MLII alpha/beta), lysosomal hydrolases are not modified with M6P residues, and therefore many lysosomal hydrolases are missorted and do not reach lysosomes. The deficiency of hydrolases in the lysosomes leads to lysosomal dysfunction and the accumulation of undegraded material in different cell types of the body. Severe skeletal abnormalities accompanied by a decline in mobility and chronic joint pain are features of MLII alpha/beta.

We generated a mouse model for MLII alpha/beta by the insertion of a mutation into the murine Gnptab gene (c.3082insC) that is homologous to the mutation GNPTAB c.3145insC detected in MLII patients. The MLII mice show all characteristic biochemical alterations and clinical features found in the human MLII disease and allow the analysis of underlying pathogenetic cellular mechanisms. MLII mice show an increased lethality, reduced mean body weight and body length, and display skeletal alterations like abnormal spine curvature and osteoporosis1. We investigated the bone pathology in detail by biochemical, histomorphometric, histochemical and immunological methods to characterize alterations and identify pathomechanisms affecting the bone metabolism.

Electron microscopic analyses demonstrated the formation of storage lysosomes in osteocytes and osteblasts but not in osteoclasts. To analyze the targeting defect in the bone we cultured primary cells like osteoblasts and osteoclasts of wildtype and the MLII mice and determined the steady state expression level, sorting, proteolytic processing and the half-live of several enzymes such as tartrate resistant acid phosphatase (TRAP), cathepsin D, Z and K. Pulse-chase and real-time PCR experiments on cultured fibroblasts, osteoblasts and osteoclasts indicated that the rate of synthesis is similar in MLII cells compared to wildtype cells whereas the sorting efficiency to lysosomes was affected resulting in their hypersecretion into the medium. The extent of missorting, however, depends on the lysosomal enzyme examined. Thus, β-hexosaminidase and TRAP, were found to be highly reduced in MLII fibroblasts, osteoclasts and osteoblasts whereas the steady state expression level of proteins like cathepsin D were unchanged.

Our data indicate that subpopulations of lysosomal hydrolases appear to be more affected by the loss of M6P residues than others transported to lysosomes via M6P-independent pathways. Whereas in osteoclasts the missorting of lysosomal proteins lead to an increased bone resorption capacity in vitro the consequences of their mistargeting in osteoblasts are unclear. Microarray analyses carried out in cultured osteoblasts and osteoclasts from wildtype and MLII mice revealed changes in the expression of several genes which have to be confirmed by independent methods. Current studies are focussed on i) the isolation and identification of osteoblast-specific M6P-containing proteins that are directly involved in the regulation of bone remodelling, and ii) the pharmacological intervention of altered bone metabolism in MLII.

1 Kollmann K, Damme M, … (2012) Lysosomal Dysfunction Cause Neurodegeneration in Mucolipidosis II “Knock-in” Mice. Brain, in press

This work was presented on the ESGLD (European Study Group on Lysosomal Diseases) workshop in Helsinki 2011, where it was selected for oral presentation. At the annual conference of the APS 2012 (Working group for paediatric metabolic disorders in the german society for children medicine) it was awarded the poster prize.

General Grants

Dr. Andrea Ballabio  (Partnership grant with Caterina Marcus Foundation)
TIGEM, Naples, Italy
Modulating lysosomal function to treat MPS


Aim 1: Development of tools for in vivo TFEB activation

Aim 2: Evaluation of the therapeutic effects of in vivo TFEB overexpression in MPSIIIA mice.

To study the effects of TFEB overexpression in vivo in both wild-type mice and in the mouse models of MPSIIIA, we generated a conditional gain-of-function (TFEB-COND-GAIN) mouse line.  Time-and/or tissue-specific expression of the transgene can be obtained by crossing the transgenic mouse line with a strain carrying the CRE recombinase.  As a first test of the system we generated two founder lines specifically overexpressing Tcfeb in the liver using the Albumin-CRE strain that expresses CRE in the hepatocytes. These lines show different levels of TFEB overexpression. One overexpresses TFEB approximately 3-fold normal levels, while the other approximately 20-fold. We observed that TFEB overexpression in liver resulted in the activation of TFEB target genes.  To generate brain specific expression of TFEB, the TFEB-COND-GAIN mice were crossed with a NESTIN-CRE strain, that expresses CRE in the brain and central nervous system.  Unfortunately, we observed that the first line tested showed embryonic lethality.   We believe this could be due a wider expression pattern of NESTIN-CRE in the developing embryo, and by the high levels of TFEB overexpression. We are repeating the experiments with the founder line that shows lower expression levels of TFEB and by using a different brain specific CRE line, GFAP-CRE that has a more restricted expression pattern.

In the meantime, we had obtained very encouraging results on the function of TFEB overexpression in cellular models of lysosomal storage disorders (LSDs), both MPSIIIA and multiple sulfatase deficiency (MSD).  We had evidence that TFEB overexpression increased lysosomal exocytosis in cultured HeLa cells, we tested whether we observed the same effect in mouse embryonic fibroblasts derived from the murine models of MSD and MPSIIIA.  TFEB overexpression in these cells types resulted in a significant increase of LAMP1 on the plasma membrane and of lysosomal enzymes into the culture medium, hallmarks of lysosomal exocytosis.  This indicates that LSD cells efficiently respond to TFEB-mediated induction of lysosomal exocytosis.  Therefore, we evaluated the effect of TFEB overexpression on the clearance of GAGs in glia differentiated neuronal stem cells (NSCs) isolated from mouse models of MSD and MPSIIIA. TFEB overexpression resulted in a striking reduction of alcian blue-stained GAGs in both MSD and MPSIIIA NSC-derived glial cells (Figure 1A). The latter result was further confirmed by pulse-and-chase experiments using H3 glucosamine to label GAGs, showing a significant reduction of the levels of labeled GAGs after 48 hr of chase in both MSD and MPSIIIA NSC-derived glial cells overexpressing TFEB (Figure 1B). Finally, EM analysis revealed that TFEB-mediated clearance of GAGs in TFEB-overexpressing MSD and MPS-IIIA cells was associated with both significant reduction of cellular vacuolization and recovery of normal cellular morphology (Figure 1C).  These results indicate that TFEB overexpression results in an increased lysosomal exocytosis that leads to increased cellular clearance.

We have completed the generation of an adeno-associated type 2/9 virus (AAV2/9) that carries TFEB-3xflag under the control of a strong TBG promoter.  As a first pilot study we injected this vector systemically into adult multiple sulfatase deficiency (MSD) mice. This mouse model allowed us to treat the mice with a systemic injection, that is technically less challenging than direct intra-cerebral injections.   To this end, we injected systemically AAV2/9-TFEB-3xflag into adult MSD mice. One month after injection, several tissues were collected to monitor transduction efficiency and GAG storage. AAV-mediated TFEB delivery resulted in efficient TFEB transduction and significant reduction of GAG staining in liver and skeletal muscles, as detected by alcian blue staining and GAG quantification (Figure 2A,B). Subsequently, we investigated whether TFEB-mediated clearance of GAGs resulted in the reduction of the pathologic hallmarks of MSD, such as macrophage infiltration and apoptosis. We found a striking reduction of CD68-positive cells in AAV-TFEB injected MSD mice compared with untreated mice (Figure 2C). Most importantly, we also observed a significant reduction of TUNEL-positive cells (Figure 2D) (Medina el at Development Cell Volume 21, Issue 3, 421-430). These results indicate that TFEB activation of lysosomal exocytosis reduced both primary accumulation of GAGs and secondary pathological processes associated with LSDs such as inflammation and cell death. Our next challenge is to treat MPSIIIA mice with this vector in the brain directly.



















Dr. Alisdair B. Boraston
Department of Biochemistry and Microbiology, University of VictoriamVictoria, Canada
Discovery and assessment of inhibitor-based chemical chaperones as potential agents for the treatment of mucopolysaccharidosis IIIB.

HYPOTHESIS – Mutant forms of human NAGLU, which cause the MPS IIIB phenotype, are destabilized by the mutations, but not rendered non-functional, and can be chaperoned to the lysosome by specific inhibitors of a-N-acetylglucosaminidase (NAGLU) inhibitors (chemical chaperones) to result in elevated levels of lysosomal NAGLU activity.

GENERAL APPROACH – 1) Generation of potent and selective inhibitors of NAGLU. We are combining synthetic chemistry and X-ray crystallographic analysis of a model protein in complex with synthesized inhibitors to generate compounds that are selective for NAGLU. 2) Candidate inhibitors are being assessed in a Chinese Hamster Ovary (CHO) cell model of MPSIIIB. The readout in this model assay is increased NAGLU concentration and activity in lysosomes upon treatment with our compounds.

BACKGROUND RESULTS – Prior to receiving funding from NMPSS we established using biochemical, structural, and cellular assays that inhibitory compounds based on piperidine and indolizine scaffolds could be made and would function as reasonably effective and selective chemical chaperones in our cellular assay, providing the basis for this work. Our endeavours were to focus on expanding the number of compounds that display the required properties for chaperones namely affinity, solubility, bioavailability and selectivity as well as expanding the model system to include other naglu mutations.


PROGRESS Towards investigating inhibitor scaffolds using synthetic methodologies that potentially could result in compounds that are potent inhibitors of NAGLU, we have prepared a large number of compounds and are at different stages of their evaluation as inhibitors of NAGLU. Efforts to date have centered on the use of scaffolds demonstrated to be important in inhibiting glycosidases in general. Due to the synthetic difficulties that have resulted in some of the scaffolds syntheses, during the study we decided to first focus on hydroximolactone and piperidine scaffolds (see above figure) as these could be prepared in a more robust fashion.


We have completed the synthesis of a library of compounds for these scaffolds and are at different stages of evaluating them as inhibitors of NAGLU. Initial results have shown whilst they are modestly potent against NAGLU, these studies and the evaluation of roughly 20 crystal structures of bacterial NAGLU in complex with these inhibitors reveal them to lack selectivity for the enzyme over a functionally related human enzyme. This information, although disappointing, will guide us in the future preparation of selective compounds for NAGLU.

Despite the compounds above not being selective for NAGLU, we have been assessing the chemical chaperone potential of them in a CHO cell model of MPSIIIB that incorporates a single known mutation of naglu. We have assayed the most potent compounds found to date as chemical chaperones in the model against the six mutants that we had prior to receiving funding from NMPSS. Nine new mutants have been prepared in this study, bringing the total to fifteen mutants in our library. Again it was disappointing to find that of the compounds that have been assayed, even though they were not toxic at high concentrations, they were not able to increase the levels of mutant NAGLU activity above control levels with any of our mutants. The nine new mutants were also not active against our initial piperidine and indolizine compounds that we had prior to receiving funding from NMPSS. We are now assessing the observed weaker binding compounds of NAGLU, as well we will assess the compounds that are yet to be evaluated as inhibitors of NAGLU, as chemical chaperones in future work. These results have also guided us in the future development of compounds that display the required properties for chaperones namely affinity, solubility and bioavailability.