2nd Year Research Reviews – 2008

2008 Second year reviews

In 2008 the National MPS Society awarded two MPS II grants, one MPS II grant and five general MPS research grants.

Dr. Nicola Brunetti-Pierri
Telethon Institute of Genetics and Medicine
Naples, Italy
HDAd gene therapy for lysosomal storage disorders

Correction of the neurological manifestations of lysosomal storage diseases has been elusive so far. The goal of our project was to develop a safe and effective strategy for correction of the central nervous system manifestations of MPS II which could be potentially applicable to other lysosomal storage diseases as well.

Helper-dependent adenoviral (HDAd) vectors which are devoid of all viral genes can infect post-mitotic cells, including cells of the central nervous system (CNS) and are particularly attractive for brain-directed gene therapy. We have showed that intrathecal injections of HDAd resulted in extensive transduction of ependymal cells and sustained expression of the transgene up to one year post-administration. We have also demonstrated, for the first time, the ability of HDAd injected by this route of delivery to transducer neuronal cells. The transduced neuroepithelial cells can be potentially used to secrete therapeutic proteins into the cerebrospinal fluid (CSF) and provide them via cross-correction to non-transduced cells. Targeting of neuronal cells and long-term transgene expression make this approach attractive for the treatment of several neurologic diseases, including lysosomal storage disorders. An HDAd vector encoding the iduronate sulfatase has been injected by intrathecal injection in a mouse model of Mucopolysaccharidosis II and we are currently processing brain samples to investigate whether correction of central nervous system manifestations has occurred.

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

With the support from the National MPS Society, we have made significant progress toward developing a new approach designed to treat both the neurological and non?neurological symptoms of MPS. This approach is based on drugs which modify GAGs so that the deficient enzyme is not required to degrade them. Our progress has moved through three important stages of drug development: i) development of methods to quantify disease in human MPS cell samples, ii) testing of candidate drugs in this model system, iii) testing of candidate drugs in mouse models of MPS. The following is a brief description of our progress:

Development of methods to quantify disease in cultured human MPS cells.

The first goal of our proposed research was to develop a laboratory system based on cells from patients with MPS that would allow us to test potential treatments in the lab. A testing system like this is essential to discovering new drugs which can modify GAGs so that the deficient enzyme is not required to degrade them. We were very successful in this aim with the development of the Sensi?Pro Assay. This assay can sensitively and specifically measure the amount of GAGs that have accumulated in the lysosomes of MPS patient samples or cells. While we originally developed the Sensi?Pro Assay to guide our own drug development efforts, we found (through a series of collaborations with MPS experts) that the assay was also very useful in other ways. We have found that it can also detect MPS disease in urine, tissue, blood, and cerebrospinal fluid in addition to human cells. The assay has also been found to be very useful in measuring treatment response, thus it can help in the development of other new treatments and can help clinicians monitor MPS patients and optimize treatment decisions. We are also conducting experiments to show how the assay could be used as a newborn screen for MPS.

Testing of candidate treatments in the Sensi?Pro Assay: With this testing system developed, we then tested over 100,000 drug candidates and found several promising drug candidates which reduce the lysosomal accumulation of GAGs in MPS I, II, and III (A, B, and C). This exciting milestone demonstrated the principle of modifying GAGs so that the deficient enzymes are not required to degrade them. In order to move these drug candidates toward clinical testing, we first need to improve their characteristics. To accomplish this, we began synthesizing and testing a large number of derivatives of the drug candidates. Through this ongoing effort, we have improved the characteristics of the drug candidates, and the most effective candidates are currently being evaluated for properties that are required for a safe and effective therapy.

Testing of drug candidates in mouse models of MPS: We obtained the mouse models of MPS I, II, and IIIA. We originally proposed to test our compound in the MPS II mouse, but our MPS II mouse colony is not large enough yet for testing drug candidates. Because our treatment is also effective in MPS IIIA, we tested the most promising drug candidate in mice with MPS IIIA. While this initial study is small, we were very excited to find that the lysosomal accumulation of GAGs was reduced in the brains of the MPS IIIA mice. Also, the mice displayed no adverse effects from treatment over the 20 day study. Larger efficacy studies in MPS I, II, and III are planned for the near future. We are also happy to report that we have recently been awarded an NIH Small Business Innovative Research grant to further our efforts to develop a new approach to treating MPS. We are thankful for the ongoing support of the MPS Society which is helping fund the development of this new approach to treating MPS.

Dr. Andrea Ballabio
TIGEM (Telethon Institute of Genetics & Medicine)
Naples, Italy
Modulation of autophagy as a potential therapeutic approach for MPS

Recent studies highlight the prominent role of inefficient autophagic-dependent mitochondria dysfunction in the pathogenesis of neurodegenerative autophagic diseases as Huntington. Mitochondria are organelles recognized as central players in cell death. Proper recycling of dysfunctional mitochondria relies on PINK1/Parkin -dependent selective autophagy (a process known as mitophagy) that avoids the release of pro-apoptotic factors.
These studies prompted us to investigate the role of mitochondria in the pathogenesis of MPSs. Our recent findings demonstrated that dysfunctional mitochondria accumulate, together with other toxic substrates such as p62-aggregates and poly-ubiquitinated proteins, as a consequence of autophagic stress in two mouse models of mucopolysaccharidoses, MSD and MPS-IIIA.

During the second year of MPS funding we decided to investigate more in depth the role of the lysosomal-mitochondrial axis dysfunction in the cellular pathology of both MSD and MPS-IIIA mice. Moreover, as planned in our proposal, we evaluated the recovery of normal mitochondrial function in the brain and liver of MDS and MPS-IIIA mice treated with rapamycin.

We observed that dysfunctional mitochondria accumulate, in a different fashion, in liver and brain from the MPS mouse models analyzed. These organelles showed reduced membrane integrity, low ATP content and significant morphological changes (being fragmented in brain and giant in liver). Such morphological/functional changes lead to cytochrome c release and apoptotic cell death in the liver of the affect mice. We also analyzed the mitochondrial function in the MSD and MPS-IIIA mice treated with rapamycin. After 5 weeks of treatment we observed a significant reduction of dysfunctional mitochondria together with a partial rescue of the pathological phenotype in treated mice. We have obtained the following results:

1) The mitochondria recovered a normal morphology in affected mice treated with rapamycin.
2) We did not detect any release of cytochrom c from liver tissue of affected mice treated with rapamycin, thus meaning that rapamycin successfully enhances the clearance of damaged mitochondria.
3) We have also detected a reduction in the levels of pro-inflammatory cytokines in treated mice. This could be due to either the removal of toxic substrates (aggregate proteins and dysfunctional mitochondria), or to rapamycin immunosuppressive activity, or both.

Together our data provide new evidence that the mitochondrial-lysosomal axis differentially contributes to neurodegeneration and to the systemic features in severe models of mucopolysaccharidoses and that induction of autophagy could be a feasible therapeutic approach for this type of inherited disorders. Importantly, our data need to be extended to evaluate whether the rapamycin treatment was indeed effective in rescuing neurodegenerative processes and behavior phenotype in the affected mice.

Dr. Brian Bigger
Royal Manchester Children’s Hospital
Manchester, UK
The effect of heparan sulphate on stem cell homing and engraftment in MPS I

When stem cells are transplanted into patients, they must find their way from the bloodstream into the bone marrow by following signals called chemokines, which are produced by the bone marrow. In recent years, it has become clear that chemokines interact with glycosaminoglycans, heparan sulphate in particular, in order to function normally. Heparan sulphate (HS) is one of the stored molecules in MPS I, as well as MPS II, MPS III and VII. Here, we examine how HS accumulation in MPS I could be affecting homing of stem cells to the bone marrow during bone marrow transplantation, and thus contribute to graft failure.

Year 1: MPS I Bone Marrow Cells Accumulate Excess Highly Sulphated Heparan Sulphate

We demonstrated that MPS I bone marrow cells do not only store HS inside the cells, butoutside cells in the extracellular matrix (ECM). The ECM is a sticky material made up of proteins and sugars which anchors and displays chemokines for interaction with homing cells. In the photographs (above left), heparan sulphate is stained fluorescent green, and there is clearly more associated with MPS I cells (bottom image). In the graph (above right), each bar represents the percentage of disaccharide building blocks that have sulphate groups. MPS I bone marrow has a dramatic increase in sulphated disaccharides in HS. This shows that MPS I HS is much more highly sulphated than normal. The amount of sulphation is important in chemokine-HS interactions.

Year 2: High Levels of Highly Sulphated Heparan Sulphate are Inhibitory to Stem Cell Homing

Experiments in vivo showed decreased homing in MPS I recipients of bone marrow transplants (above left). Work in vitro showed that heparan sulphate decreased the ability of bone marrow stem cells to home to the chemokine SDF-1, which is the most important chemokine for bone marrow transplant. This work showed that at low concentrations, the inhibition of homing was not dependent on the sulphation state of HS, however at high concentrations, sulphation state became important. A binding experiment (above right) showed that this was because SDF-1 will only bind to highly sulphated forms of HS at high concentrations it does not bind to desulphated forms at all.

We concluded that the decreased homing seen in transplants to MPS I recipients was caused by the unique combination of HS that is both highly sulphated and in massive excess in MPS I bone marrow cells, which causes SDF-1 to become trapped in super-sticky ECM, preventing it from signalling to transplanted cells. This is a disease-specific defect in homing that could be contributing to graft failure in the clinic.

This work was presented at the Harden Conference on Heparan Sulphate in Cambridge in 2009 , where it was awarded the Mituzani Foundation Poster Prize. It was also selected for oral presentation at the Clinical and Laboratory Sciences Showcase, University of Manchester 2009.

Dr. Adriana M Montano
Saint Louis University School of Medicine
St Louis, MO
Identification of genes for keratin sulfate biosynthesis: toward development of RNAi mediated therapy

Study has extended to 2011

Dr. Mark S. Sands
Washington University School of Medicine
St. Louis, MO


1) determine the effects of reduced lysosomal recycling on the energy imbalance in affected cells, and 2) determine the effects of dietary intervention on the progression of disease.

We previously showed that there was a significant energy imbalance in the form of depleted adipose stores in mouse models with lysosomal storage diseases (three of which were MPS disorders). We hypothesized that the energy imbalance was due to interrupted lysosomal recycling of macromolecules and the subsequent increase in energy expenditure required for the synthesis of new macromolecules. In order to further test this hypothesis we measured the levels of over 1,500 metabolites in liver homogenates from MPS I mice. Molecules directly involved in energy utilization such as simple sugars, nucleic acids and lipids were depleted. This is consistent with the animals being in a state of nutrient depravation. Interestingly, the levels of amino acids, amino acid derivatives and dipeptides were increased suggesting that protein catabolism, perhaps due to increased autophagy, is at least partially fulfilling intermediary metabolism. We showed that autophagy is increased in the livers of both MPS I and VII mice. The initial observation of energy imbalance also led us to hypothesize that a diet rich in energy (simple sugars and lipids) would decrease the severity of the disease. Therefore, we put both MPS I and VII mice on a high fat, simple sugar diet. Although there was no significant increase in life span or retinal function, most of the abnormalities identified on the metabolomic screen approached normal levels. In addition, the animals were now able to increase their adipose stores and autophagy was reduced to near normal levels. These data support our hypotheses that interrupted lysosomal recycling leads to a significant energy imbalance and that nutritional intervention can ameliorate some of the biochemical abnormalities associated with these diseases. This work was recently published [Woloszynek JC, et al., (2009) J. Biol. Chem. 284:29684-29691].

This grant also partially supported a study in which we combined adeno-associated virus (AAV)-mediated, CNS-directed gene therapy with bone marrow transplantation (BMT) in the murine model of MPS IIIB. We hypothesized that systemic therapy (BMT) would synergize with CNS-directed gene therapy to increase efficacy in MPS IIIB which has been relatively refractory to most therapies. CNS-directed, AAV-mediated gene therapy alone provided the greatest clinical benefit. The addition of BMT added little in the way of efficacy and actually decreased the life span slightly. This study was recently published [Heldermon C, et al. (2010) Mol. Ther. 18:873-880]. We have initiated another study in the MPS IIIB mouse where we combined CNS-directed AAV-mediated gene therapy with systemic lentiviral-mediated gene therapy. The rationale for this study is that the systemic lentiviral-mediated gene therapy would result in persistent high levels of circulating NaGlu activity that will enhance the effects of the CNS-directed therapy. It has been shown in another model of MPS that persistent high levels of circulating enzyme can result in some CNS correction. The preliminary data (14-16 months of age) suggest that the combination of AAV-mediated CNS-directed gene therapy combined with systemic gene therapy provides the greatest clinical benefit.


Woloszynek JC, Kovacs A, Ohlemiller KK, Roberts M, Sands MS: Metabolic adaptations to interrupted glycosaminoglycan recycling. J Biol Chem, 284:29684, 2009.

Heldermon C, Ohlemiller KK, Herzog E, Vogler C, Qin E, Wozniak DF, Tan Y, Orrock J, Sands MS: Therapeutic efficacy of bone marrow transplant, intracranial AAV-mediated gene therapy or both in the mouse model of MPS IIIB. Mol. Ther., 18:873, 2010.

Dr. Marta Serafini
Dulbecco Telethon Institute at M.Tettamanti Research Center Clinica Pediatrica Univ.
Monza, Italy
Marrow mesenchymal stem cell therapy for MPS I

The overall aim of ongoing experiments on this project is to establish a new stem cell-based therapy to improve the outcome of hematopoietic cell transplantation with a particular emphasis on the skeletal defects affecting MPS-I patients. For this purpose, we have used two complementary cell systems: on one side human mesenchymal stem cells (hMSCs) and on the other murine mesenchymal stem cells (mMSCs).

We have successfully isolated hMSCs from healthy donors and three MPS-I patients. We have evaluated their intrinsic osteogenic differentiative potential in vitro and, so far, our data indicate that genetically affected cells do not differ from healthy donors-derived cells, suggesting that microenvironment and/or signalling from neighbouring cells might be responsible for the observed phenotype at skeletal level.

In order to investigate the best source of stem cells, hMSCs have been isolated from healthy donors also from umbilical-cord blood, amniotic fluid and umbilical cord with an increasing efficiency in our system (around 70% for bone marrow and amniotic fluid, around 80% for digested umbilical cord and around 25% for umbilical cord blood). These cells differentiate in vitro in adipocytes, chondrocytes and osteoblasts and we are now evaluating their in vivo potential.

In parallel, we sought to isolate and characterise mMSCs. So far two protocols have been used and tested as it is widely accepted that mMSCs isolation protocols are less established. With our methods by the first passages we obtain a mixed population with an average of 50% of MSCs assessed by detection of markers as CD73, CD29, CD44, SCA1, CD36 and negativity for B220, CD11b and CD45 (by antibody staining), and good potential to differentiate in adipocytes, osteoblasts and chondrocytes. We are now in the process of isolating and culturing mMSCs using an inactivated feeder layer of murine fibroblasts, as a mean to increase the homogeneity and efficiency of mMSCs isolation.

In order to test the rescue capability of HCT with or without mesenchymal stem cells (MSCs), we planned to use the mouse model as our experimental system. To date, different murine models have been reported with mutations in the IDUA gene. Originally we established to use an immunodeficient mouse, the NOD/SCID/MPS-I (Garcia-Rivera et al., 2007) infused with human-derived MSCs.

Before starting the transplant experiments, we analysed the skeletal phenotype of this and other mutant models and we found that the range of bone-defects varied in penetrance and severity across the mutants. This situation could be compared to the differences observed in phenotype in MPS-I patients, representing a valuable tool to study the feasibility of MSCs transplant also in less severe forms of MPS-I.

In the last few months we have isolated GFP+ mMSCs and transplanted them into the different MPS-I mouse models. At defined time points after the infusion, we plan to evaluate the skeletal phenotype improvement, with or without hematopoietic stem cells. We have also tested several injection sites as MSCs are technically challenging to infuse for their intrinsic tendency to adhere. As demonstrated by our and other groups, intravenous injections are often lethal to the recipient animal, and cells tend to colonise the lungs and the heart but cells are rarely detectable in other inner organs and/or bones. We are now evaluating in site injections, intra-cardiac injections and possibly the use of cells containing-scaffold materials.

Dr. Richard Steet
University of Georgia Research Foundation
Athens, GA
Investigation of the cartilage pathogenesis of ML II and MPS

Defining the pathogenic mechanisms of MPS and MPS-related disorders is important since it will potentially aid in the development of new therapies. Our proposed research was designed to take advantage of the power and speed of the zebrafish system to identify factors that contribute to the cartilage pathogenesis in mucolipidosis II or ML-II, with the goal of directly investigating the contribution of these factors towards the disease process. With ongoing support from the MPS Society, we have now identified and confirmed that several enzymes – including cathepsins K, L and S and matrix metalloproteinase 13 – are upregulated in ML-II zebrafish embryos as well as feline ML-II tissues (Petrey A., Flanagan-Steet H., Nairn A., Moremen K., Haskins, M. and Steet R., manuscript in preparation). The activity of the cathepsins is elevated in ML-II zebrafish at time periods when only marginal activity can be detected in wild type embryos, suggesting that sustained or unregulated expression of these hydrolases occurs in response to impaired mannose phosphorylation. This abnormal expression may also compound the hypersecretion of these enzymes caused by defective mannose 6-phosphate dependent lysosomal targeting. Our current studies are focused on 1) localizing the increased cathepsin expression within specific tissues of our zebrafish model and 2) testing whether a reduction in this activity by genetic or pharmacological means will have therapeutic potential. In the coming years, we plan to translate any positive outcomes from our zebrafish experiments to studies in the feline ML-II model in collaboration with Dr. Haskins at the University of Pennsylvania.

Another facet of our MPS Society-funded project was to generate models for other MPS disorders using the zebrafish system, with the hope of using these models to explore whether common pathogenic mechanisms exist between ML-II and MPS disorders. Although we were able to reduce the activity of beta-glucuronidase (GUSB; the cause of MPS VII), in zebrafish embryos to less than 5% of wild type levels, no obvious phenotypes were observed. These data suggest that the relatively short duration of morpholino-induced gene suppression may limit our ability to model the more progressive MPS disorders in this organism. This finding led us to seek new ways to establish stable genetic zebrafish mutants with sustained loss of enzyme activity. In a fortunate respond to our request, GUSB was selected by an NIH-funded initiative that is tasked with generating zinc-finger nuclease constructs suitable for the stable suppression of genes in zebrafish. The development of a zebrafish model for MPS VII will be ongoing in the second half of this year in our lab and will expand the experimental systems available to investigate MPS pathogenesis.

As a new investigator, I am truly grateful to the MPS Society and the families for their support of this research project. Funding from the Society has proven vital in our ability to pursue higher risk experiments and we remain hopeful that the avenues of research that stem from this work can be translated into therapies for MPS and MPS-related disorders.