1st Year Research Reviews – 2011


Prof Vito Ferro
School of Chemistry & Molecular Biosciences, the University of Queensland
“Small molecule chaperones for EET for MPS II”

The aim of this project is the development of small molecule drugs for MPS II. Conventional small molecule drugs can be taken orally as a pill and have the potential to reach the brain in order to treat the more severe forms of MPS II, unlike enzyme replacement therapy (Elaprase) which can’t cross the “blood-brain barrier”. Our approach is to develop compounds for so-called Enzyme Enhancement Therapy (or EET; aka “Pharmalogical Chaperone Therapy”). This is an approach to treatment that has shown great promise in other lysosomal storage disorders, e.g., Gaucher’s and Fabry disease, but has yet to be tried for the mucopolysaccharidoses such as MPS II. EET works by having a small molecule drug (a “chaperone”) attach itself to the defective enzyme, in this case iduronate sulfatase, and stabilizing it so it can do its intended job: to degrade the mucopolysaccharides in the cell. In order to prepare compounds for EET that are suitable for testing we need to synthesize small molecules that resemble the sugar iduronic acid, the component of the mucopolysaccharides that is degraded by iduronate sulfatase. The first step of this process is to prepare iduronic acid itself and then to make some chemical modifications to it. Iduronic acid is quite a complex sugar and is not commercially available, so in the first year of this project we have focused on developing methods of preparing iduronic acid from cheap and readily available glucose. We have investigated three different methods and one of them has so far shown the most promise. We have been able to prepare an important derivative of iduronic acid with modifications in the desired parts of the molecule. Our next aim is to transform this key intermediate into a range of compounds for testing as chaperones for iduronate sulfatase.


Drs. Patricia Dickson and Stephen Kaler
UCLA Harbor, Los Angeles, CA
Choroid plexus-directed gene therapy as a source of intrathecal NAGLU-IGF2 for Sanfilippo B syndrome

The goal of this project is to study whether the choroid plexus could be made to produce NAGLU-IGF2 into the ventricles of the brain, and whether this will improve brain lysosomal storage in Sanfilippo B syndrome mice. The choroid plexus is responsible for production of cerebrospinal fluid, and if it could be made to produce a therapeutic enzyme, it would serve as a potentially permanent source of that enzyme for the brain. We produced a construct containing the gene encoding NAGLU (the enzyme that is deficient in Sanfilippo B syndrome) fused to IGF2 (insulin-like growth factor 2). Manufactured forms of NAGLU lack mannose 6-phosphorylation, limiting their uptake into cells. To circumvent this problem, we attached IGF2 to NAGLU. IGF2 binds the mannose 6-phosphate receptor so that it can get NAGLU into cells without mannose 6-phosphate. Our studies in cells showed that IGF2 greatly improves intracellular uptake of NAGLU. This project began on July 1, 2011. Year 1 milestones achieved include: 1) re-establishment of the Sanfilippo B mouse colony in our laboratory, 2) production of adeno-associated viral vectors containing NAGLU-IGF2, 3) verification that the vectors produce intact, active NAGLU-IGF2 enzyme and 4) injection of adeno-associated vectors into the brain of normal rats. In year 2, we plan to complete the study the distribution of NAGLU-IGF2 in the brain of normal rats, select an effective dose, and perform a study to evaluate its distribution and effectiveness in Sanfilippo B mice. These experiments will provide proof-of-concept for choroid-plexus directed gene therapy for Sanfilippo B syndrome using NAGLU-IGF2.

General Grants

Alberto Auricchio   (review will be available September, 2012)
Fondazione Telethon, Naples, Italy
Gene therapy of MPS VI

Adriana M. Montaño
Saint Louis University
Role of inflammation in pathogenesis of MPS IVA

Morquio A disease (Mucopolysaccharidosis IVA, MPS IVA) is an autosomal recessive disorder, caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Patients with Morquio A disease have accumulation of the glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate, mainly in bone and cartilage, causing systemic skeletal dysplasia. The broad goals of this research are to characterize the immune profile of Morquio A mouse model and to elucidate the role of cartilage and bone inflammatory reactions in the pathogenesis of Morquio A disease through investigation of secreted inflammatory factors involved in cartilage destruction and bone remodeling.

1. Characterization of the immune profile of the Morquio A mouse model.

Immune response to enzyme replacement therapy (ERT) is the principal limitation in the effectiveness of the treatment. The first step in the characterization of the immune profile of the knock-out Morquio A mouse model is to investigate the immune response after ERT.

We have found that Morquio A mice undergoing ERT have: i) the highest immune humoral response towards the recombinant human GALNS enzyme at 14 weeks of treatment, and ii) the highest cellular response at 16 weeks of treatment. This is consistent with previous observations where age of the mice and length of treatment play a role in the levels of immune response.











Figure 1. Humoral response against human GALNS used for ERT in knock-out Morquio A mice. Four mice were treated with 18 i.v. infusions with human GALNS (filled bars) or PBS (open bars).










Figure 2. Cellular response against human GALNS used for ERT in knock-out Morquio A mice. Mice were treated by 16, 22 or 24 i.v. weekly infusions with human GALNS. Splenocytes were stimulated in-vitro with human GALNS (150 g/ml).

2. Characterization of expression profiles of genes associated to the pathogenesis of Morquio A disease.

We compared differences in inflammation profile of cartilage between Morquio A and wild type mice. We have found that there is up-regulation of several genes which play important roles in autophagy and apoptosis. We are in the process of quantifying and comparing these results at various ages in cartilage and bone cells of Morquio A and wild type mice.

Richard Steet, Ph.D.
University of Georgia, Athens, GA
Blockade of cathepsin activity and TGF-beta signaling as a therapeutic approach for LSDs

Investigating the molecular pathogenesis of lysosomal diseases such as the MPS and MPS-related disorders is a promising avenue towards the development of new therapies and can aid our understanding of the mechanisms that contribute to the onset and progression of disease symptoms.  Over the past several years, we have taken advantage of a zebrafish ML-II model to investigate the pathogenic mechanisms that underlie the cartilage defects associated with this disease.  We have identified and confirmed several target proteins (including cathepsin proteases and matrix metalloproteinases or MMPs) that exhibit increased and sustained activity in ML-II zebrafish embryos and hypothesize that inhibition of this excessive protease activity would result in therapeutic correction of ML-II associated phenotypes.  In support of this hypothesis, we have demonstrated that inhibition of cathepsin K by genetic and pharmacological means leads to substantial correction of the cartilage defects in ML-II embryos as well as a surprising reduction in the activity of other proteases.

Over the past year, we have focused our efforts on determining whether other proteases such as cathepsin L and MMPs contribute to the disease process.  Our results demonstrate that cathepsin L is subject to the same sustained activation in ML-II zebrafish embryos that we previously observed for cathepsin K (Petrey et al, Disease Mechanisms and Models, 5(2) 177-90 (2012)).  These results are significant since they point to a common mechanism whereby this class of proteases is abnormally activated from immature forms.  We believe this activation arises from the hypersecretion of these enzymes into the extracellular space upon loss of mannose 6-phosphate dependent lysosomal targeting.  This hypothesis is supported by 1) the observation that levels of the mannose 6-phosphate recognition marker are greatly reduced or absent on the highly active, mature forms of cathepsins K and L in ML-II embryos and, 2) the direct visualization of cathepsin K exclusively within the extracellular space of developing cartilage of ML-II (but not control) zebrafish.  Our near-term goals include an assessment of whether cathepsin L inhibition is capable of producing the same therapeutic benefit in cartilage as we noted with cathepsin K suppression.  We have also obtained a specific inhibitor to MMP-13, an enzyme whose activity is increased in ML-II zebrafish.  We intend to treat our ML-II model with this inhibitor and determine whether any of the phenotypes can be rescued or improved when this protease activity is decreased.  Initial results indicate that this inhibitor can act on zebrafish MMP.  Lastly, we have begun to manipulate the TGF-beta signaling pathway in ML-II zebrafish embryos to determine how altered regulation of this pathway relates to the disease phenotypes.

Since increased cathepsin and MMP activity, and dysregulation of the TGF-beta signaling pathway are common features of many MPS disorders, we believe our results on ML-II will inform our general understanding of the pathogenesis of other LSDs.  Furthermore, our findings indicate that abnormal protease activation (in addition to increased expression) is an important factor that should be considered in assessing the pathogenesis of these diseases.  Finally, the demonstration that a reduction in cathepsin activity can provide some therapeutic benefit suggests that further investigation into small molecule protease inhibitors for the treatment of MPS and MPS-related disorders is warranted.  We thank the MPS Society for their continued support of this research.