1st Year Research Reviews – 2008

Dr. Sara Cathey
Greenwood Genetics Center, North Charleston, SC
Natural history study for mucolipidosis
One year partnership grant with ISMRD.
Received 7-09

The Mucolipidosis Project of the Greenwood Genetic Center (GGC) began in 2005. The goals of the project are to establish databases of clinical and laboratory information about ML II and ML III alpha/beta, understand the disease course, provide natural history information to families and the medical community, and raise awareness of the mucolipidoses. These goals are being met and exceeded!

The largest group of individuals with ML II and ML III alpha/beta ever reported has been described in the medical literature. Families and doctors have a resource for reliable information. Scientists have access to samples for research. The ML Project is ongoing, allowing the natural course of the diseases to be more fully understood. Fifteen families attended the first ML Clinic at GGC in 2006. The majority of those families, plus others, will participate in GGCs Second ML Clinic, July 27-29, 2009. This is an opportunity to evaluate individuals over time. We can not only study disease progression, but also assess the impact of interventions. Understanding the natural course of disease builds the framework for successful treatments. What interventions work best, and when? Are there markers? of disease in the blood or urine that can be followed to guide therapies? Who are the best candidates for surgery? Which surgeries?

By supporting work like the ML Project, the National MPS Society continues to foster research, progress, and hope. Families remain the driving force of this incredible momentum. Your support and participation make great things possible.

Nicola Brunetti-Pierri, M.D.
Department of Molecular and Human Genetics
Baylor College of Medicine, Houston, TX
HDAd gene therapy for lysosomal storage disorders?
Received 7-09

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

Helper-dependent adenoviral (HDAd) vectors are devoid of all viral genes and result in long term transgene expression in the absence of chronic toxicity. Because of their ability to infect nondividing cells, including cells of the central nervous system, HDAd vectors are particularly attractive for brain-directed gene therapy. Using a vector expressing a reporter gene (the green fluorescent protein), we have showed that HDAd vectors administered via intratechal injection into the cerebrospinal fluid (CSF) resulted in minimal systemic toxicity and extensive transduction of neuroependymal cells as well as neuronal cells. Importantly, the expression has lasted for at least 3 months and we are planning to look also at longer time points (6 months- 1 year). Given the encouraging results obtained with the reporter gene, we are perfroming experiments in a mouse model of Mucopolysaccharidosis II injecting a vector encoding for the therapeutic gene. These experiments are crucial because they will clarify whether our approach will allow the correction of the central nervous system manifestation in the mice affected with Mucopolysaccharidosis II.

Brett E. Crawford, Ph.D.
Zacharon Pharmaceuticals Inc., La Jolla CA, 92037
“Glycosaminoglycan inhibitors as substrate reduction therapies for MPS II”
Received 7-09

Thanks to the support of the National MPS Society, we have made important progress during the first year of our research project. As proposed, our work has focused on two aspects of developing a substrate reduction therapy for MPS II: i) the development of methods to quantify GAG accumulation in cultured human MPS cells and ii) the testing of candidate GAG inhibitors in this model system.

In order to develop methods to quantify GAG accumulation in cultured MPS cells, we obtained cells from patients with MPS II from the National Institute of General Medical Sciences Human Genetic Cell Repository. With these cells we developed improved methods to quantify GAG accumulation in cultured MPS cells (the Sensi?Pro Substrate Assay). The Sensi?Pro assay was validated by successfully detecting reductions in GAG accumulation in MPS II cells treated with recombinant iduronate sulfatase. By developing and validating these methods, we now have an experimental model to test the effectiveness of candidate drugs by measuring their ability to impact GAG accumulation in MPS cells.

We have recently found that the assay could also be used clinically as it has the sensitivity to detect GAG accumulation in urine, serum, and cerebral spinal fluid of patients with MPS with several advantages over the alternative methods. Using the Sensi?Pro assay, we have screened our candidate GAG biosynthesis inhibitors to determine if any of these compounds reduce GAG accumulation in MPS II. Through these studies we have identified a series of compounds that reduce GAG accumulation in MPS II cells.

These exciting and positive results provide a foundation to accomplish the next stage of the research project: testing the most promising candidate drugs in the mouse model of MPS II. We have obtained the MPS II mouse model (strain courtesy of Joseph Muenzer, MD, PhD) and are well positioned to accomplish the next stage of the research project.

Dr. Andrea Ballabio
TIGEM (Telethon Institute of Genetics & Medicine), Naples, Italy
Modulation of autophagy as a potential therapeutic approach for MPS?”
Received 7-09

We recently generated new insights in the pathogenic mechanisms of mucoplysaccharidoses and more in general of lysosomal storage disorders. We demonstrated that a crucial lysosomal degradative pathway, autophagy, is severely impaired in two mouse models of mucopolysaccharidoses, Multiple Sulfatase Deficiency (MSD) and mucopolysaccharidosis type-IIIA (MPS-IIIA). As a consequence of a defect of autophagy we observed an abnormal accumulation of several autophagic substrates such as polyubiquitinated aggregates, p62 protein and dysfunctional mithocondria, that lead to cell death and tissue pathology in both models analyzed. During the first year supported by MPS funding, we began to evaluate the therapeutic effect of preventing/removing autophagic-dependent accumulation of toxic substrates in MSD and MPS-IIIA mouse models. We used both a drug delivery approach based on rapamycin administration and a genetic approach based on crossing affected mice with p62 KO mice.

Newborn (0-2 gg) and adult (3 weeks of age) MSD and MPS-IIIA mice received intraperitoneal injection of rapamycin (20mg/kg) three times a week during all treatment period. Mice were analyzed at two time points: 1) an early time point (5 weeks of age) for biochemical and histological analysis (both MPS-IIIA and MSD mice) and 2) a late time point for biochemical, histological and behavioral analysis. We chose 20 weeks of age as late time point for MPS-IIIA mice and 12 weeks of age as a for MSD mice because the most significant difference between normal and affected mice in terms of behavior were observed at this age.

The first group of treated mice (either newborn- or adult-treated mice) were analyzed at 5 weeks of age (early time point) and collected tissues (brain and liver) assessed for the accumulation of p62, alfa-synuclein and polyubiquitined proteins. In all affected mice (MSD and MPS-IIIA) the injection with rapamycin resulted in a significant decrease of toxic accumulation when compared to control PBS-injected affected mice. Importantly, the extent in the decrease of toxic accumulation was higher in mice treated at birth compared to mice treated at 3 weeks of age. We are now evaluating mitochondrial dysfunction and apoptotic cell death in the same tissues. These first data are very encouraging. The other experimental groups of mice (MSD and MPS-IIIA mice injected with rapamycin along with control normal and affected mice PBS-injected) will be analyzed at 20 weeks (MPS-IIIA) or at 12 weeks of age (MSD) to evaluate if the rapamycin treatment is able to recover a normal behavior in affected mice.

We plan to complete this task within 1 year.

The conditional p62 KO mice have been received from Dr Tanakas group (Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; Komatsu et al., 2007). These mice are now available in our animal facility and the colony has been established and expanded. These mice will be crossed with either MSD or MPS-IIIA mice. We plan to induce the ablation of p62 in specific tissues (brain and liver) to evaluate to which extent the abnormal accumulation of this substrate is involved in the tissue pathology and consequently if its ablation could prevent the pathology itself.
We plan to complete this task within 1 year.

Brian Bigger PhD
MPS Stem Cell Research Group
University of Manchester, Royal Manchester Children’s Hospital
The effect of heparan sulphate on stem cell homing and engraftment in MPS”
Received 7-09

Heparan Sulfate: A Helping Hand or A Sticking Point?

Heparan sulfate is stored in the cells of MPS I, II, III, and VII patients. In unaffected cells, heparan sulfate has a very important role in cell to cell signalling: allowing cells to talk to each other by interacting with molecules on the surface of nearby cells, or released into the space between cells. Like all the glycosaminoglycans or GAGs stored in the MPS diseases, it consists of a protein core with large chains of sugars attached to it. The sugar chains can have sulfate groups attached in a variety of patterns, which give them patches of negative charge. The patterns of sulfation differ between different kinds of cells and organs across the body. It is believed that these changing patterns of sulfation are important in deciding which signals get processed by the cell, and which signals get ignored.

This work focuses on MPS I Hurler, and bone marrow transplantation. When bone marrow is transplanted, the cells enter the blood stream, and need to find their way to the bone marrow by following signals coming from the bone marrow, like a homing beacon. It is known that heparan sulfate is involved in mediating the homing signal, and we are investigating whether this interaction is altered in MPS I Hurler, and whether this might be influencing the success of bone marrow transplantation for MPS I Hurler, and potentially other MPS diseases.

It is well known that large amounts of heparan sulphate and other glycosaminoglycans are stored inside the cells of MPS patients, but we wanted to prove that heparan sulphate is found on the outside of MPS I Hurler cells, where it could take part in signalling. The pictures below show an excess of heparan sulfate, (shown in green) on the surface of bone marrow stromal cells taken from mice with MPS I Hurler. Having shown that there is more heparan sulfate on the cell surface, specialised antibodies were used to show that this heparan sulfate had different sulfation patterns to heparan sulphate from unaffected cells. We found that MPS I Hurler heparan sulfate carries more of the type of sulfation that tends to lead to increased signalling. Indeed, we found that bone marrow cells could home across matrix (the material that fills the gaps between cells) containing MPS I Hurler heparan sulfate almost twice as well as across matrix from unaffected cells. In summary, so far we have found

MPS I Hurler bone marrow stromal cells accumulate heparan sulfate externally as well as internally,
Heparan sulfate from MPS I Hurler cells has a specific structure that favors homing to bone marrow and alters cell:cell signaling.

As this work continues, we hope to clarify the precise role of this altered heparan sulfate in the bone marrow homing and engraftment process, and find ways of manipulating this pathway to improve transplant success.

Adriana M Montano, PhD
Saint Louis University
School of Medicine – Dept of Pediatrics, St Louis, MO
Identification of genes for keratin sulfate biosynthesis: toward development of RNAi mediated therapy?”
Received 7-09

Mucopolysaccharidosis IVA (Morquio A disease) is caused by the deficiency of the enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The lack of this enzyme leads to the accumulation of undegraded glycosaminoglycans (GAGs): keratan sulfate (KS) and chondroitin-6- sulfate (CS) in all the cells of the body, especially in skeletal tissue.

Several treatment approaches have been focused on providing the deficient enzyme to Morquio A mouse models. Our approach is to partially block the synthesis of KS mainly in cartilage cells to stop the progression of the disease by attenuating one or more genes involved in the synthesis of KS (See the figure).

The complete set of genes involved in the synthesis of KS is unknown posing an obstacle for understanding the biochemical processes leading their accumulation. In order to overcome this problem, we have performed a detailed differential expression analysis and identified two candidate genes that may be involved in the synthesis of KS, and made progress towards their characterization. We have cloned the genes in an expression vector to study their effects in-vitro. In addition, we have cloned the genes in another vector that will facilitate the purification of the proteins resultant as the product of these genes. In-vitro analysis and the protein purification experiments are in progress. After the completion of these crucial experiments, we will focus, in the second year of our program, on (i) the suppression of these genes at the RNA level, and (ii) performing in-vitro tests in cartilage cells of Morquio A patients. The success of the second phase will provide a promising therapy technique for MPS IVA patients.

Mark S. Sands, Ph.D.
Washington University School of Medicine
Department of Internal Medicine, St. Louis, MO
Metabolic adaptations and phenotypic consequences of blocking lysosomal recycling?”
Received 7-09

Specific Aims:
1) Determine the effects of reduced lysosomal recycling on the energy imbalance in affected cells.
2) Determine the effects of dietary intervention on the progression of disease.


We generated preliminary data showing that there was a significant energy imbalance of unknown etiology in 5 murine models of different lysosomal storage disease (Woloszynek et al., 2007, J. Biol. Chem., 282:35765). The energy imbalance manifests as a significant decrease in adiposity. These data suggest that lysosomal storage disorders are diseases of deficiency as well as excess (lysosomal storage). We hypothesized that the energy imbalance was due to an increase in energy-intensive reactions required to maintain homeostasis in the context of a decreased pool of metabolites from lysosomal recycling. The goal of this research was to provide additional data supporting this hypothesis and determine the effects of an energy-rich diet on disease progression. We performed an unbiased metabolomics survey of the liver of MPS I animals. This analysis showed that simple sugars, nucleotides and lipids were decreased in the MPS I liver compared to normal. This supports the hypothesis that the animals are in a state of deficiency. The metabolomics analysis also showed an increase in amino acids, amino acid derivatives, dipeptides, and urea. These data suggest that increased protein catabolism is at least partially fulfilling intermediary metabolism. When MPS I animals were placed on a high fat, simple sugar diet (high fat diet) for 4 weeks, most of the abnormal metabolite levels approached normal. Consistent with the apparent increase in protein catabolism, we observed an increase in a marker of autophagy (LC3) in the livers of both MPSI and MPS VII animals. Autophagy was decreased in the livers of MPS I animals placed on the high fat diet, however, the decrease was not statistically significant (p=0.08). Interestingly, autophagy was significantly reduced and approached nearly normal levels in the livers of MPS VII animals maintained on a high fat, simple sugar diet from weaning. MPS VII animals maintained on the high fat diet from weaning had significantly increased body weight for the duration of the study (7 months). Unfortunately, there was no significant improvement in longevity, retinal function or cardiac morphology or function. These data strongly support the hypothesis that animals with LSDs are suffering from nutritional stress similar to starvation. Although a crude high fat, simple sugar diet resulted in only minimal clinical improvement, a more targeted nutrient approach may yet prove beneficial as an adjunct therapy as the mechanism of this energy imbalance is better understood. The data generated from this MPS Society-funded project is currently under review at the Journal of Biological Chemistry.

Year 2 funding:

We have made significant progress on this important area of research during the first year of funding. We are currently breeding the MPS I and MPS VII mutations onto the ATG5- and ATG6 (Beclin)-deficient animals. These gene knockouts interrupt normal autophagy. These double mutant animals will allow us to determine if the changes in autophagy in response to the energy imbalance are helpful or harmful.

Marta Serafini, PhD
STEMMPS Unit, Dulbecco Telethon Institute at M.Tettamanti Research Center Clinica Pediatrica Univ. Milano-Bicocca, Monza, Italy
Marrow mesenchymal stem cell therapy for MPS I?”
Received 7-09

The goal of our project is the identification of a novel cell therapy strategy capable of effectively alleviating disease manifestations, in particular at the skeletal level, still affecting MPS-I children after allogeneic hematopoietic stem cell transplantation.

To reach this goal, we are focusing on the following different aspects:

1. Optimize the culture of human mesenchymal stem cells (hMSC), a population of multipotent stem cells with interesting therapeutic potential and compare hMSC derived from healthy and MPS-I children of similar age.

We defined a new protocol to isolate hMSC clones and compared the properties of our cultures kept under hypoxic/normoxic conditions. We focused on the establishment and characterization of MSC lines from healthy young donors (age 1-10) and MPS-I patients (age 1-3). Lines have been maintained in long-term in vitro culture. We are particularly interested in the capacity of these cells to differentiate into the osteogenic lineage, as our approach aims to demonstrate the repair of MPS-I problems at the skeletal level. For this reason, we compared the in vitro osteogenic differentiation of the generated populations in a time course, monitoring the formation of mineralized matrix and the osteogenic transcription profile. The comparison of osteogenic properties between cells derived from healthy donors and MPS-I patients did not reveal significant differences. Ongoing experiments will evaluate within a few weeks if both the populations are able to establish the bone microenvironment in vivo.

2. Explore the potential of umbilical cord blood (UCB-MSC) and amniotic fluid (AF-MSC) as alternative sources of hMSC,.

Few reports have described an advantage of fetal hMSC in osteogenic differentiation potential over adult hMSC, which may represent optimal sources for bone repair and regeneration. We are currently working on isolation procedures to improve the success rate, which is 20% for UCM samples and 60% for AF samples. We fully characterized the populations obtained and compared their capacity to differentiate into mature osteoblasts and produce a mineralized matrix under permissive osteogenic conditions. The next step of our work will be the evaluation of our transplant experiments in mice to assess their osteogenic capacity.

3. Optimization of hMSC transduction with lentiviral vectors (LV).

To track hMSC transplanted in the NOD/SCID/MPS-I mice, we inserted the GFP reporter gene into hMSC. For this reason, GFP-transduction of hMSC was optimized, tuning vector dose and number of rounds of transduction. According to our generated protocol, we genetically modified the cells with an HIV-1-based lentiviral vector carrying the eGFP reporter gene (in collaboration with Dr. A.Biffi). Efficient GFP expression (range: 87-95% GFP positive cells) was observed three days after transduction and the percentage of GFP-expressing cells remained virtually unchanged in subsequent 10 passages, which correlated to 35 to 40 days post-transduction. We observed that LV-transduced hMSC have a lower proliferation capacity compared to untransduced cells.

To further evaluate whether lentiviral transduction altered the differentiation properties of hMSCs in vitro, we analysed the cells for the typical markers CD73, CD105, CD90 and CD146. We also induced the transduced cells to differentiate into cells of the mesenchymal lineages: adipocytes, osteocytes and chondrocytes. The data obtained are comparable with those from untransduced hMSC. In addition, chromosomal stability of the transduced cells was confirmed by karyotype analysis.

4. Establishment and optimization of the transplant protocol to evaluate the contribution of hMSC to MPS-I disease model.

We are comparing different transplantation protocols in the mouse model MPS-I in order to identify the one allowing for efficient MSC homing to the skeleton and phenotypic amelioration. In fact, for MSC trafficking experiments, the timing of delivery, number of cells delivered and site of MSC infusion may impact the engraftment efficiency and the destination of exogenously delivered cells. Completion of the work dedicated to the assessment of the feasibility and therapeutic efficacy of MSC transplantation in MPS-I mice is expected in the next few months. Through a direct collaboration with Dr. Gupta, we are scheduled to begin the transplant experiments in September 2009.

Richard Steet, Ph.D.
Assistant Professor of Biochemistry and Molecular Biology Complex Carbohydrate Research Center
University of Georgia Research Foundation, Athens, GA
Investigation of the cartilage pathogenesis of ML II and MPS?”
Received 7-09

The pathogenic mechanisms that underlie the bone and cartilage phenotypes in MPS and MPS-related disorders are only now beginning to emerge. Defining these mechanisms is important since it will potentially aid in the development of new therapies. Our proposal aims to take advantage of the power and speed of the zebrafish system to identify factors that contribute to the cartilage pathogenesis in mucolipidosis-II and directly test their contribution to the disease process. These studies will utilize our newly characterized zebrafish model for ML-II and will serve as a prelude for therapeutic development. We are also attempting to model additional MPS and MPS-related disorders in this model organism in order to better understand how loss of specific hydrolases results in the phenotypes noted in patients.

Over the past year, we have analyzed the gene expression profiles in wild type and ML-II whole zebrafish embryos and isolated zebrafish chondrocytes, demonstrating that several genes relevant to bone and cartilage homeostasis appear to be upregulated, including cathepsins and matrix metalloproteinases. These findings have been confirmed in chondrocyte and fibroblast-like synoviocyte samples from the feline ML-II model. Unlike MPS cartilage and synoviocyte cells, however, we have not seen increases in genes that express pro-inflammatory cytokines. These preliminary findings may suggest unique mechanisms of cartilage damage in ML-II that are independent of inflammation. Our goal in the coming year will be to reduce the expression of the upregulated proteases in the ML-II zebrafish and assess whether this reduction can suppress the cartilage phenotypes in our model. We are also actively investigating the mechanisms that lead to upregulation of these potentially damaging proteases.

Attempts have been made to generate models for other MPS disorders using the zebrafish system. As an entry point to this work, the activity of the GAG-degrading enzyme, beta-glucuronidase, was depleted in zebrafish embryos using antisense oligonucleotides called morpholinos. Although we were able to effectively suppress beta-glucuronidase activity to less than 5% of wild type levels, no obvious phenotypes were observed in these mutant embryos. This finding has prompted us to examine the lysosomal biology of zebrafish in greater detail. To do so, we determined the expression (by RT-PCR) and the activity (using fluorescent substrates) of several lysosomal hydrolases across a developmental timeline (0-5 days post-fertilization). Our results indicate that, unlike enzymes involved in the breakdown of protein-bound oligosaccharides, expression of GAG-degrading enzymes – such as alpha-iduronidase and beta-glucuronidase – does not significantly increase until later developmental stages (4-5 dpf). By this stage, many of the vital organ systems have already developed in the embryos. This fact, along with the possibility that GAG accumulation and turnover is relatively slow in developing zebrafish tissue, may limit our ability to model certain MPS disorders in this organism using our current antisense-based approach. Thus, we are now actively seeking new ways to genetically target these hydrolases in order to generate zebrafish mutants with sustained loss of enzyme activity.