RESEARCH FOR A CURE

Updated September 2014

Below, you’ll find reports on research projects The Canadian MPS Society has funded since 2004, totaling $1,075,732.00.  

2014 Summer Studentship Research Grants Funded

The Society is pleased to announce it is funding two Summer Studentship Research Grants in 2014, each valued at $4,000:

Mucopolysaccharidoses Type IIIB (MPS IIIB, Sanfilippo syndrome B): Establishment of a disease model using cultured neuronal cells from skin fibroblast-derived induced pluripotent stem cells from patients

  • Recipient: Kelly Turner
  • Institution: University of Victoria
  • Supervisor: Dr. Francis Choy
  • Amount funded: $4,000
  • Click here to read Ms. Turner's Lay Language Research Report

LV-based gene correction for MPS IIIC

  • Recipient: Wided Akik
  • Institution: St. Justine University Hospital
  • Supervisor: Dr. Alexey Pshezhetsky
  • Amount funded: $4,000

2013-2 Research Grant:

The Canadian MPS Society, through its MPS II Research Fund, is pleased to announce it will be funding the following research grant for gene therapy research into MPS II (Hunter syndrome): 

Somatic and CNS gene delivery for the treatment of Hunter syndrome

  • Principal Investigator: Dr. Douglas McCarty
  • Institution: Center for Gene Therapy at Nationwide Children's Hospital in Columbus, Ohio.
  • Amount: $110,000 (two-year grant)

2013 Summer Studentship Research Grants Funded

The Society is pleased to announce it is funding two Summer Studentship Research Grants in 2013, each valued at $4,000:

Sleep Problems in Children with Morquio Syndrome (MPS-IV): A Prospective Cohort Study

  • Recipient: Jason Steinmetz
  • Institution: McGill
  • Supervisor: Dr. John Mitchell
  • Amount funded: $4,000

Rescue of mitochondrial dysfunction in Mucopolysaccharidosis IVA disease by treatment with an antioxidant combination

  • Recipient: Hsien Huey Tan
  • Institution: UVic
  • Supervisor: Dr. Patrick Walter
  • Amount funded: $4,000
  • Click here to read Ms. Tan's lay language report.

2013-4 Research Grant:

The Canadian MPS Society, through its MPS IV B  Research Fund, is pleased to announce it will be funding the following research grant for research into MPS IV B (Morquio B syndrome):

Morquio-Better Project - 2: Novel chaperones for GLB1-deficient diseases

  • Principal investigator: Dr. Eduard Paschke
  • Institution: Medical University of Graz (Austria)
  • Funds allocated: $60,000.00 (one year grant)

2012 Summer Studentship Research Grants Funded

The Society is pleased to announce it is funding two Summer Studentship Research Grants in 2012, each valued at $4,000:

Identification of potential pharmacological chaperones for treatment of MPS by high-throughput screening for inhibitors of human iduronidase

  • Recipient: Michael Jew
  • Institution: UBC
  • Supervisor: Prof. SG Withers
  • Amount funded: $4,000

Purification and uptake studies of alpha-N-acetylglucosaminidase

  • Recipient: Geoffrey Morris
  • Institution: UVic
  • Supervisor: Dr. Francis Choy
  • Amount funded: $4,000

This grant is being funded by a generous donation to the Society by the Sanfilippo Children’s Research Foundation.

The Canadian MPS Society's 2012-1 Research Grant:

Overcoming the engraftment defect in MPS I mice to allow non-myeloablative BMT

  • Principal Investigator:  Dr. Brian Bigger
  • Institution:  University of Manchester, UK
  • Funds allocated:  $50,000.00 (one year grant)

Summary of Research Project:

Mucopolysaccharidosis (MPS) type I is a lysosomal storage disorder caused by genetic defects in an enzyme necessary for the breakdown of glycosaminoglycans (GAGs).  This disorder varies in severity, but usually results in serious bone defects, neurological deterioration, poor function of organs and a shortened life span.

Severe MPS I (Hurler syndrome) can be treated with bone marrow transplantation (BMT), which provides a population of cells that produce the missing enzyme and supply it to cells in the rest of the body.  Until recently BMT in MPS I had poor survival rates in the clinic and the use of reduced intensity conditioning increases the chance of transplant rejection.  We have recently found that MPS I mice are more likely to reject transplant than normal mice, particularly when reduced intensity conditioning is used, which is representative of the difficulties observed in clinical transplantation.  Using high dose chemotherapy has improved BMT success in the clinic, but also increases the risks of organ toxicity, infection, infertility, and developing cancer later in life.

In this project we aim to characterise the reasons behind the engraftment defect in MPS I mice, and identify treatments that can overcome this to allow successful BMT.  We wish to make BMT safer for MPS I, by reducing the chemotherapy necessary for a successful transplant using the treatments that we identify in these initial experiments.  This would make BMT for children with Hurler syndrome less risky, as well as potentially making this therapy an option for attenuated forms of MPS I.

2012-4 Research Grant:

The Canadian MPS Society, through its MPS IV B  Research Fund, is pleased to announce it will be funding the following research grant for research into MPS IV B (Morquio B syndrome):

Morquio-Better Project

  • Principal investigator: Dr. Sylvia Stockler
  • Institution: BC Children's Hospital
  • Funds allocated: $50,000.00 (one year grant)

Summary of Research Project:
Morquio B Disease (MBD, or MPS IV B) is a very rare hereditary bone disease that causes painful bone deformity and loss of the ability to walk. MBD is caused by abnormalities (mutations) in the GLB1 gene, which is also responsible for another disease called GM1 gangliosidosis (GM1G). GM1G causes problems in the brain (neurodegeneration), including intellectual disability. Because both conditions are caused by defects in the same gene, it is not clear whether there is overlap. For example, when a child with bone disease is diagnosed with MBD, doctors are not able to predict whether this child will develop brain problems later in life. Ideally doctors would be able to identify which mutations on the GLB1 gene produce MBD with bone disease only. But, because there are so few individuals with MBD in the world, we do not know how many of them develop neurodegeneration later in life or what the abnormalities in their GLB1 gene are. New treatment strategies for some GLB1 mutations have been developed in laboratory models, but it is not clear which individuals with MBD they might benefit. Our goal is to lay critical groundwork for addressing this issue by establishing an international MBD patient registry.

  • One year of funding from the MPS Society will allow us to:
  • assemble a group of experts to oversee the registry
  • create a public MBD website that will provide educational materials and help us recruit registry participants
  • design and implement the registry
  • begin collecting patient data and populating the registry

The information collected in the registry will motivate more researchers and industry to work on this neglected disease, and it will provide an inventory of patients to re-contact for participation in future clinical trials. Thus, this project will directly contribute the knowledge, experience, and insights required to develop treatments for MBD.

2011 Summer Studentship Research Grants:

The Canadian MPS Society is pleased to have funded two Summer Studentship Research Grants in 2011:

Characterization of CNS Phenotype of MPS IIIC Mouse Model

  • Recipient: Katrin Resch
  • Institution: CHU St-Justine Research Centre, Montreal
  • Supervisor: Dr. Alexey Pshezhetsky
  • Amount funded: $4,000.00
  • Click here to read Ms. Resch's final research report

Development of Neuronal Cell Culture Models for MPS III (Sanfilippo syndrome)

  • Recipient: Michael James McLean
  • Institution: University of Victoria
  • Supervisor: Dr. Francis Choy
  • Amount funded: $4,000.00

2011-2 Research Grant:

The Canadian MPS Society, through its MPS II Research Fund, is pleased to announce it will be funding the following research grant for research into MPS II (Hunter syndrome): 

Utilising neuronal stem cell models to develop small molecule therapies for MPSII

  • Principal investigator: Dr. Emyr Lloyd-Evans
  • Institution: University of Cardiff, Wales
  • Funds allocated: $50,000.00 (one year grant)

Summary of research project:
We are aiming to develop a cellular model of Hunter Syndrome brain (MPSII). To do this we will
take stem cells from the MPSII mouse model and using careful conditions will change these cells
into neurons. This cellular model will help in studying the brain pathology of MPSII but can also
be used to test the effects of therapy on the MPSII brain. We will study both these aspects. We
are particularly interested in looking at the effects of a novel chemical compound we have
isolated from Malaysian plants. In some preliminary experiments, we found that one of these
compounds is capable of reducing storage in MPSII human skin cells but not in cells from other
lysosomal diseases. We aim to fully study the effects of this compound on MPSII neuronal stem
cells. Finally we are interested in using neuronal stem cells to study the order in which disease
events happen in MPSII. We have done this before in Niemann-Pick C1 disease5, where we
identified defects in lysosomal calcium homeostasis as an early disease event and a therapeutic
intervention point that enhances NPC1 mouse function. Such a study in MPSII will determine
the mechanisms leading to cellular pathogenesis and identify novel therapeutic intervention
points. This proposal is in line with the Canadian MPS Society’s remit and this RFA as the main
aims are to develop cellular tools for the MPS community, study novel therapies for MPSII and
determine the cellular pathogenic events that occur in the MPSII brain.

2010 CIHR SHOPP Program Partnership Grant:

The Canadian MPS Society is pleased to announce it is partnering with Canadian Institute of Health Research (CIHR) to fund a fellowship into Lysosomal Disease Research.  The fellowship is for 3 years and the Society and CIHR are both funding half of the $135,000.00 award (45,000.00 per year for 3 years).

The molecular mechanisms of microgliosis in GM2 ganglioside lysosomal storage diseases

  • Recipient: Elizabeth J. White
  • Institution: McMaster University
  • Supervisor: Suleiman A. Igdoura
  • Funds allocated: $135,000 (over 3 years, funded jointly by the Society and CIHR - $67,500.00 each total/$22,500.00 each per year for 3 years)

Summary of research project:
Lysosomal storage diseases cause progressive neurodegeneration and are characterized by an inherited genetic error in the breakdown and recycling of carbohydrate-linked lipids, which presents most prominently in cells of the central nervous system. Buildup of these substances in neurons leads to neuron dysfunction and death, as well as the over-activation of cells that act as the garbage collectors of the CNS known as microglia. Substances leaking from dying neurons interact with receptors on microglia to provide homing and 'eat me' signals that direct microglia to areas of damaged neurons. Unfortunately, the positive clearing activities of microglia are also associated with exacerbation of neuron cell death. This research focuses on the role of the microglia in the perpetuation of neuron death in lysosomal storage disease and how certain receptors on the cell surface of microglia participate in this process. We plan to knock out a receptor that is normally upregulated on activated microglia and determine how this affects the clinical progression of a mouse model of Sandhoff disease. This receptor could be a potential pharmaceutical target to inhibit the development of disease, and thus have an important impact on individuals living with lysosomal storage disease. 

To view the announcement on the CIHR website, go to http://www.cihr-irsc.gc.ca/e/193.html then follow the links to View Decisions / Funding Decisions Notifications / 2010.

2010 Summer Studentship Research Grants:

The Canadian MPS Society is pleased to be funding three Summer Studentship Research Grants in 2010: 

Mucopolysaccharidosis Type IIIB (Sanfilippo syndrome): Conjugation of Naglu to a synthetic protein transduction domain derived from the HIV-1 transactivator of transcription protein to allow Naglu to cross the BBB

  • Recipient: Sarah Truelson
  • Institution: University of Victoria
  • Supervisor: Dr. Francis Choy
  • Amount funded: $4,000.00
  • Click here to read Ms. Truelson's final research report 

Chaperone Therapy for MPS IIIC

  • Recipient: Vincent Boudreau
  • Institution: University of Montreal
  • Supervisor: Dr. Alexey Pshezhetsky
  • Amount funded: $4,000.00
  • Click here to read Mr. Boudreau's final research report.

“Charting the Experience of Mucopolysaccharide and Related Diseases at the Hospital for Sick Children”

  • Recipient: Maha Safey El-Din Saleh
  • Institution: The Hospital for Sick Children, Toronto
  • Supervisor: Dr. Julian Raiman
  • Amount funded: $4,000.00

The Canadian MPS Society's 2010-1 Research Grant:

Comprehensive study of pathogenic glycosaminoglycan storage in a murine model of MPS I

  • Principal Investigator:  Dr. Lorne Clarke
  • Institution:  University of British Columbia, Vancouver, BC
  • Funds allocated:  $100,000.00 (two year grant: $50,000.00 per year for two years)

Summary:
The mucopolysaccharidoses (MPSs) represent a group of complex progressive multi-system
diseases that are caused by metabolic defects in the degradation of glycosaminoglycans (GAGs)
Treatment in the form of enzyme replacement via either a direct route, recombinant enzyme
replacement (ERT) or via hematopeotic stem cell transplantation (HSCT) has been attempted for
most of these disorders. Long term ERT experience is now available for MPS I, II, and VI;
HSCT experience has been reported for most of the MPSs. It is now clear from the long-term
follow up studies of MPS patients that some effects of MPS disease appear to be alleviated by
treatment whereas others i.e. skeletal, CNS, cardiac and connective tissue are the most
recalcitrant. Although the primary metabolic defect leads to storage of glycosaminoglycans there
has been no comprehensive and exhaustive studies of “pathogenic glycosaminoglycan” storage
and accumulation in MPS animal models. The main reasons for this relates to the inability to
accurately and specifically measure the pathogenic GAG content of various organs, tissues and
bodily fluids. Previous studies have used either non-specific dye binding methods (DMB or
alcian blue) or the laborious carbazole method for measurement of GAGs. These techniques
reflect total non-specific GAG content of tissues rather than a measurement of the specific GAG
species that are predicted to be stored based on the lysosomal hydrolase which is deficient (ergo
“pathogenic glycosaminoglycans”). Since the amount of pathogenic GAG storage in MPS
tissues represents a small component of the total GAG content of tissues, these non-specific
measures of GAG content have not been particularly helpful in understanding the natural history
of the MPSs and the specific role that pathogenic GAGs may play. This proposal highlights a
comprehensive study that will shed light on the role that pathogenic GAG accumulation has in
the complex and progressive phenotype of severe MPS I. The studies put forth represent the
most thorough analysis of primary storage and its responsiveness in an MPS model to date. The
data will provide insight into the role of GAGs in and the utility of pathogenic GAG levels as
MPS disease biomarkers. In addition, this data will be critical for the future evaluation of small
molecule therapeutics in MPS.

One of the prime mandates of the Canadian MPS society is related to the support of research
that will ultimately lead to a cure for individuals with these devastating diseases. The
availability of animals models of the various MPS disorders has been one of the prime reasons
that this field has advanced so quickly. Now that enzyme replacement is in place it is clear that
further advancements in therapeutics requires us to more thoroughly understand the various
factors that are responsible for disease symptoms and their progression. This proposal outlines
the application of novel GAG analysis tools in order to more precisely understand the role that
primary GAG storage has in disease progression and recalcitrance after therapy in MPS I.

MPS II (Hunter Syndrome) Research Grant:

The Canadian MPS Society, through its MPS II Research Fund, funded the following research grant for research into MPS II: 

MPS II AAV Gene Therapy Research

  • Principal investigator: Dr. Joseph Muenzer
  • Institution: The University of North Carolina at Chapel Hill
  • Funds allocated: $50,000.00 (one year grant)
  • Click here to read Dr. Muenzer's Research Project Summary

The Canadian MPS Society's 2009 Research Grants:

The Canadian MPS Society funded the following three research grants in 2009:

Identification of pharmacological chaperones for treatment of MPS I

  • Principal investigator:  Dr. Allison Kermode
  • Institution:  Department of Biological Sciences, Simon Fraser University, Burnaby, BC
  • Funds allocated:  $50,000.00 (one year grant)

Summary:
Mucopolysaccharidosis I (MPS I) is a progressive lysosomal storage disease (LSD).  In severely affected children there are profound disturbances to the heart, brain and other organ systems.  Individuals that inherit this disease are deficient in the lysosomal enzyme, alpha-L-iduronidase.  Consequently, there is an inability of the lysosome to effect the stepwise degradation of certain glycosaminoglycans – namely dermatan sulfate and heparan sulfate – a process essential for normal growth and homeostasis of tissues.  Intravenous delivery of purified enzyme (enzyme replacement therapy or ERT) is an effective means to restore lysosomal enzyme deficiency.  However, the current methods used to commercially produce the enzymes for ERT are prohibitively costly.  In addition, in their present form, ERTs do not correct all aspects of disease pathology (e.g. skeletal symptoms and brain dysfunction), partly because of an inability of the recombinant enzyme to cross the blood-brain barrier.  Pharmacological chaperone therapy is an emerging approach to treat protein deficiencies – by transiently binding to a lysosomal enzyme, the chaperone (a small molecule) is able to increase residual enzyme activity, and thus potentially exhibit a significant therapeutic effect.  Capitalizing on our expertise in using plants as hosts to produce the normal (‘wild-type’) human alpha-L-iduronidase enzyme, the objectives of the present proposal are two-fold: (1) To develop a plant-based system to express mutant α-L-iduronnidase enzymes (i.e. those that underlie some forms of MPS I disease); (2) To use this plant-based expression system as a unique in vivo tool for the screening of small molecule libraries to identify potential therapeutics (‘pharmacological chaperones’) for treatment of MPS I disease.  The screening will take place at the McMaster University High Throughput Screening Facility.  The compounds identified will promote secretion of active enzyme from plant cells and the system will have significant advantages over current screening methods.  The efficacy and selectivity of any promising lead compounds will be subsequently verified.  This research will validate a novel, inexpensive and reliable method of drug discovery for human LSDs by in vivo screening methods based on transgenic plant cells.  This research fits in well with the objectives of the Canadian MPS Society to develop new ways of treating MPS I, a devastating childhood genetic disease.

A comparison of two biomarkers on treatment outcome in mucopolysacchride disease

  • Principal Investigator:  Dr. Brian Bigger
  • Institution:  University of Manchester, Giving for Living Research Centre, Manchester, UK
  • Funds allocated:  $50,000.00 (one year grant)
  • Click here to read Dr. Bigger's final research report

Summary:
Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders that lead to accumulation of glycosaminoglycans (GAGs) in cells.  Currently in the clinic MPS diseases are diagnosed by measuring urinary GAG levels and blood levels of the defective enzyme.  However urinary GAG levels change with age and blood enzyme levels can be masked by treatment, so these tools are not as useful in monitoring disease progression and response to treatment. 
Other biomarkers for MPS disease progression have been developed, including the ratio of urinary GAGs dermatan sulphate and chondroitin sulphate (DS/CS ratio) and levels of heparin cofactor II-thrombin complex (HCII-T).  These techniques both have limitations; therefore we aim to evaluate these biomarkers and compare their ability to measure outcomes of treatment.
Serum, plasma and dried blood spot samples are being collected from newly diagnosed MPS patients, MPS patients receiving treatment and mouse models of MPS.  We will use these samples to determine the ideal methods of sample collection, processing and storage, and to check the reliability of the test for HCII-T.  Response of HCII-T to treatment will be tested and compared to DS/CS ratio measurements.
Initial data confirms that addition of anti-coagulant to blood samples prevents detection of HCII-T, showing that blood cannot be drawn from central lines as  commonly occurs in clinics. HCII-T levels in MPS mice can also be successfully measured using dried blood spots.  We would like to develop HCII-T as a biomarker in human dried blood spots.  This less invasive sampling method could lead to development of newborn screening for MPS.
This work falls within the remit of the Canadian MPS Society, as development of a new tool to monitor MPS will expand options for diagnosis and newborn screening, as well as providing a better way to measure treatment outcomes.

Proteomic studies of skeletal disease in a murine model of MPS I

  • Principal Investigator:  Dr. Lorne Clarke
  • Institution:  University of British Columbia, Vancouver, BC
  • Funds allocated:  $100,000.00 (two year grant: $50,000.00 per year for two years)

Summary
The mucopolysaccharidoses (MPSs) are a group of complex progressive multi-system diseases that are caused by metabolic defects in the degradation of glycosaminoglycans (GAGs).  Although these disorders progressively affect almost every organ in the body the progressive bone and joint effects represents one of the most devastating features of these diseases.  Treatment in the form of enzyme replacement or hematopeotic stem cell transplantation (HSCT) has been attempted for most of these disorders.  Long term ERT experience is now available for MPS I, II, and VI; HSCT experience has been reported for most the MPSs,  It is now clear from the long-term follow up studies of MPS patients receiving either single or combinations of these treatments regimens that the effects of MPS disease on the skeleton and the brain are the most recalcitrant. Although the primary metabolic defect leads to storage of glycosaminoglycans it is now apparent that many of the disease manifestations and complications do not relate directly to GAG storage but to effects that this storage has on other cellular processes.  Understanding the various pathways involved as well as the mediators of such effects is critical for the development of therapeutics that will ultimately improve the skeletal manifestations of the MPSs.  This proposal highlights a proteomic based approach to study the pathophysiology and treatment responsiveness of skeletal disease in a murine model of MPS I.  This approach has the potential to uncover biomarkers that are reflective of skeletal disease as well as to identify altered pathways that would provide skeletal based therapeutic targets.  These studies will provide insights that may improve the long-term outcome of MPS patients and targets one of the most significant MPS disease manifestations.  In addition, the results from this pilot study will serve as a foundation for a future more comprehensive research application by our group on this topic.

2008 Society-Sponsored Research Project:

The lived experience of parents with children diagnosed with Mucopolysaccharidosis waiting for Enzyme Replacement Therapy

  • Researcher: Maria Maione, BScN
  • Research conducted at: Ryerson University
  • The Canadian MPS Society sponsored this thesis written by Maria Maione, which was submitted to Ryerson for her Master of Nursing degree in 2008.  Click here to read Ms. Maione's thesis.  Click here for Appendix D.  Click here for Appendix E.

2008 Summer Studentship Research Grants:

The Canadian MPS Society awarded one Summer Studentship Research Grant in 2008: 

Mucopolysaccharidosis Type IIIB (Sanfilippo syndrome):Affinity purification of human recombinant α-N-acetyl-glucosaminidase from cultured Sf9 cells for uptake studies and potential therapeutic treatment.

  • Recipient: Sarah Truelson
  • Research conducted at: Universtiy of Victoria, Victoria, BC
  • Supervisor: Dr. Francis Choy
  • Amount funded: $4,000.00
  • Click here to read Ms. Truelson's final research report. 

The Canadian MPS Society's 2008 Research Grants:


A monoclonal Antibody Based ELISA for Heparin Cofactor II- Thrombin Complex

  • Principal Investigator:  Lorne Clarke
  • Institution: University of British Columbia
  • Amount funded: $40,000.00 (including $20,000.00 from the MPS II Research Fund)

NAGLU – Apolipoprotein for treatment of Sanfilippo B syndrome

  • Principal Investigator: Patricia Dickson
  • Institution: Los Angeles Biomedical Research Institute
  • Amount funded: $40,000.00

Development of gene expression-targeted isoflavone therapy (GET IT) for Mucopolysaccharidosis type III

Professor Wegrzyn received funding for the first three years of this research project from the UK MPS Society.  He determined that long-term experiements are required in order to obtain conclusions on the efficacy and safety of the use of genistein in the MPS III mouse model. The Canadian MPS Society was pleased to be able to collaborate with MPS societies from several countries (UK, Japan, Switzerland, Germany, Spain, Ireland, USA, Sweden, Austria, and the Netherlands) to fund a fourth year of this research project.

  • Principal Investigator: Grzegorz Wegrzyn
  • Institution: University of Gdansk, Poland
  • Amount funded: $4,000.00 (~)

2007 Summer Studentship Research Grants:

Brain-targeted MPS II therapy delivered by microencapsulated cells

  • Recipient: Jason Mark Schwindt
  • Research conducted at: McMaster University Medical Centre
  • Supervisor: Dr. Murray Potter
  • Amount funded: $4,000.00

Final Report:

MPS II, or Hunter syndrome, is caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS).  In classical Hunter syndrome, IDS deficiency causes accumulation of glycosaminoglycans (GAGs) in lysosomes, bone abnormalities, organ enlargement, and childhood-onset of brain deterioration leading to early death.  Recently, enzyme replacement therapy (ERT) for MPS II has become available, with improvements in systemic symptoms reported in phase I/II[1] and II/III[2] clinical trials, but without any benefit for the central nervous system (CNS) involvement.
 Our laboratory has developed the use of microencapsulated cells for therapeutic enzyme delivery[3, 4].  In this technique, cells engineered to secrete the desired enzyme are enclosed in a protective microcapsule for transplantation.  The microcapsule allows long-term delivery of enzyme with one treatment by protecting the implanted cells from rejection by the host.  This technology has already been used to deliver therapeutic enzyme in MPS II fibroblasts[5] and mice[6], but not to the brain.
 One of the main hurdles to overcome in treating the brain is the blood-brain-barrier (BBB). The BBB is characterized by tight junctions and lack of fenestrations between the endothelial cells in the brain vessels [7-9] so that virtually nothing crosses the BBB  without the help of endogenous transport mechanisms [8, 10].   The Tat protein was isolated from the HIV virus and was shown to be important for cellular transduction of the virus[11].  Study of Tat revealed that the protein transduction domain for Tat was a short 11 amino acid sequence from the first exon[12].  This short sequence (designated TAT) was shown to be sufficient to deliver a variety of proteins into different organs, including the brain[13, 14], and specifically into the lysosome[15, 16].
 Our laboratory is seeking to develop peripherally administered CNS-targeted therapy for MPS II.  Specifically, IDS and TAT will be joined at the molecular level to form the fusion enzyme “TAT-IDS”.  This fusion enzyme will be secreted by microencapsulated cells, which will ultimately be implanted intraperitoneally into patients with MPS II.  An important consideration for cellular production of active IDS is the necessary post-translational activation of the enzyme by the formylglycine-generating enzyme (FGE).  This enzyme converts a highly conserved cysteine located at the N-terminal region into formylglycine (FGly) for activation [17]. Co-expression of FGE has been shown to increase the activation of recombinant sulfatases, including IDS[18, 19].
The specific aims of this summer project are:
   1. To construct vectors with TAT-fused and “unfused” IDS and the marker enzyme luciferase.
   2. To transfect these vectors into mouse cells to create high expressing cell lines for each of the constructs.  The expression, localization and activity of the constructs will be characterized in vitro.  IDS-expressing cells will be co-transfected with the SUMF1 gene to maximize IDS activity. A summer student funded by the Canadian MPS Society in 2006 and who continued to work in the lab as a 4th year undergraduate thesis student has already developed the SUMF1 vector.
We hope to obtain funding to continue this project as a graduate thesis.  The general outline of the remainder of the project is:
   3. To microencapsulate the cell lines for further characterization.   4. To study the in vitro effects of the fusion enzyme on the abnormal histology seen in IDS-deficient cell cultures.   5. To study the in vivo distribution of the marker “TAT-luciferase” in mice by implanting the microencapsulated cells.   6. To study the distribution and efficacy of TAT-IDS delivered by microencapsulated cells in a mouse model of MPS II.
 Our proposed therapy would allow a significant advance in the treatment of MPS II.  The fusion protein technology developed could be used with traditional ERT production and delivery methods, microencapsulation, or with other gene therapy methods.  Successful application of this technique in MPS II could lead to applications in most other LSDs and more common acquired brain diseases.


Evaluation and implementation of new fluorometric enzyme assays, for diagnosis and monitoring of patients with lysosomal disorders

  • Recipient: Patrick Wong
  • Research conducted at: University of British Columbia Children's and Women's Site
  • Supervisor: Dr Paula Waters
  • Amount funded: $4,000.00

Final Report:

Diagnosis of lysosomal storage disorders relies on the demonstration of deficient enzyme activities in patient samples such as leucocytes, serum and/or cultured fibroblasts. In the context of a clinical service laboratory, the choice of assay method and sample type is driven by the need to provide accurate diagnostic information in a timely manner. This becomes even more crucial as new therapies for such disorders become available.
 The Biochemical Genetics Laboratory at British Columbia’s Children’s Hospital is BC’s provincial reference laboratory for the diagnosis of inherited metabolic diseases. It is the only lab in BC performing diagnostic lysosomal enzymology, and also receives specimens for lysosomal testing from several other Canadian provinces. Our lab has a wide repertoire of diagnostic lysosomal enzyme assays. Many of these use artificial fluorometric substrates with simple, rapid methodologies which have been available for decades. However, several of our enzyme assays still rely on classical methods using
radiolabelled natural substrates, which are time-consuming, cumbersome, expensive and in some cases lack sensitivity. For many years there was no choice, as suitable alternative assays were not widely available for these enzymes, but as novel artificial substrates and methods are now becoming available we are systematically validating and implementing these for clinical use in our lab. For example, we have recently implemented a new assay method for galactocerebrosidase (Reference 1), which proved invaluable to us in the rapid diagnosis of a newborn affected with Krabbe disease and subsequent regular monitoring following cord blood transplant. However, it is known that the “normal” range for galactocerebrosidase (as measured with the natural substrate) is wide and that interpretation of “partially low” results can be challenging (Reference 2). We are
therefore continuing with more extensive collection and review of reference data using the fluorometric substrate. Patrick will participate in this work.
 Diagnosis of MPSII (Hunter disease) traditionally relies on a radiometric ion-exchange chromatographic assay of iduronate sulfatase, which requires a relatively large sample size and four days’ prior dialysis. This assay also suffers from increasing
“blank” values due to substrate deterioration, which adversely affects the signal/noise ratio and in practice can mean thatassay results on serum are not definitive and cultured fibroblasts are required. We are in the process of switching over to a
novel fluorometric assay (Reference 3) and have thus far found excellent discrimination between affected patients andnormal subjects in a rapid assay using only 10ìl serum. We plan to also apply this assay on white blood cells (something which was not feasible with the radioactive method, for sensitivity reasons) and expect this to allow rapid confirmation of diagnosis without recourse to fibroblasts. Patrick will participate in this work.
 Diagnosis of MPSIII (Sanfilippo disease) is often delayed for reasons including mild somatic features and false-negative urine MPS screening results (Reference 4). Conversely, MPSIII has been challenging and time-consuming to rule out, eg in older patients with behavioural abnormalities and equivocal “trace heparan sulfate” on TLC or electrophoresis. Now that fluorometric substrates have been developed for all four relevant enzymes (for types A-D), it would be valuable to have these simpler assays readily available for use. We currently have a fluorimetric method for type III B only. Patrick’s main summer project would focus on the introduction of newer assay methods for MPSIII A (heparan sulfamidase) and possibly, if time should permit, MPSIII C (acetyl-coA á-glucosaminideN-acetyl transferase).
 Summary of student involvement: Patrick will initially work alongside one of our medical lab technologists in the ongoing establishment of the Krabbe and Hunter assays, and will review the results and the background to those method workups with Dr Waters. Thus he will gain both hands-on familiarity with the basic techniques (eg cell isolation, lysate preparation, protein determination, fluorometric assays) and an overview of the necessary factors (including protein linearity, assay reproducibility, sample handling and stability to storage, quality control issues, development of appropriate reference ranges) to consider in implementing a clinical lab assay. He will then focus primarily on the heparan sulfamidase
(MPSIIIA) assay, designing and performing the necessary development and validation experiments under the guidance of
Dr Waters. Patrick will also have opportunity to observe other aspects of clinical laboratory diagnosis of Lysosomal disorders, including urine screening analysis of mucopolysaccharides and oligosaccharides, algorithms for appropriate testing in response to positive screen results or clinical suspicion, Tay-Sachs carrier screening, and our participation in external quality control schemes.  Dr Hilary Vallance (Biochemical Genetics Lab Director) will also be available as a cosupervisor to provide Patrick with broader exposure to these aspects. Overall, this summer experience will give Patrick a good introduction to the field of lysosomal disorders and should provide a further stimulus to his initial interest in this area.


Mucopolysaccharidosis Type III B (Sanfilippo syndrome): Expression and purification of human recombinant a-N-acetyl-glucosaminidase from cultured Sf9 cells for uptake studies and potential therapeutic treatment

  • Recipient: Tasha Brianne Kulai
  • Research conducted at: University of Victoria
  • Supervisor: Dr. F.Choy
  • Amount funded: $4,000.00

Final Report: 

Introduction- Mucopolysaccharidosis Type III (MPS III, Sanfilippo syndrome) is a group of inherited diseases resulting from deficient lysosomal enzymes that degrade heparan sulfate, an important ground substance in connective tissues. Sanfilippo syndrome is hallmarked by the accumulation of undegraded heparan sulfate in the brain and is lethal by the time the patients reach their late teens. Although it is feasible to treat the patients by injection of the missing enzyme, the administered enzyme fails to reach the brain because of the presence of the blood-brain barrier. There are 4 clinical forms of Sanfilippo syndrome (IIIA, B, C, and D) and Sanfilippo IIIB results from an inherited deficiency of lysosomal α-N-acetylglucosaminidase (Naglu). Progress report- Using the Sf9 cell expression system, we (Bandsmer et al, 2006) recently succeeded in expressing human recombinant Naglu that was fused to an eleven amino acid HIV-Tat protein transducing domain (PTD). This PTD has been demonstrated to confer its fusion partner the ability to permeate the cell membranes as well as the blood-brain barrier. We have ligated a cellulose binding domain (CBD) 5’ to PTD-Naglu for its purification to homogeneity using cellulose affinity column chromatography. PTD-Naglu was recovered from the column with a yield of ~15% by using Factor Xa that specifically cleaves the four amino acid residues (IQGR) inserted between CBD and PTD-Naglu.
Rationale and aims- Since both CBD and PTD-Naglu are macromolecules (Mr 20,000 and 83,000, respectively), we hypothesize that the enzyme purification yield using Factor Xa cleavage can be improved by introducing glycine and serine amino acid spacer arm linker (GGSGG) between the CBD and PTD-Naglu. This will open up the space between these huge macromolecules so that it is more accessible for proteolytic cleavage, thus improving the overall yield. We plan to obtain mg quantity of the purified enzyme for uptake study in vitro using cultured skin fibroblasts from controls and Sanfilippo patients, and in vivo using a Sanfilippo mouse model available from our collaborator Dr. Elizabeth Neufeld at UCLA.
Methods- Oligonucleotides that encode (GGSGG)n  residues (where GGSGG is the basic unit and n varies from 1 to 4) with flanking sequences that partially overlap at its 5’ with CBD and 3’ with TAT-Naglu will be custom synthesized, recombined into the expression construct, cloned and sequenced, and used to transform cultured Sf9 cells.  Recombinant enzyme secreted will be adsorbed onto a cellulose column and recovered after on-column Factor Xa digest. The yield will be compared from PTD-Naglu with spacer arm of various lengths.
Results expected and significance- We hypothesize that spacer arm (GGSGG)n introduced between the CBD and Naglu will improve enzyme purification yield, and spacer arm that permits maximum recovery will be selected for purification to obtain mg quantity of homogenous enzyme for uptake studies. If recombinant PTD-Naglu is found to cross the blood brain barrier and safe to use, our long term goal is to proceed to clinical trial in humans.


The Canadian MPS Society’s 2007 Research Grants:

Brain-targeted MPS II therapy delivered by microencapsulated cells

  • Research conducted by:  Dr. Murray Potter
  • Research conducted at:  McMaster University, Hamilton, ON
  • Funds allocated:   March 2007:   $20,000.00 (from MPS II Research Fund)
  • Funds allocated:   June 2007:  $20,000.00 (from MPS II Research Fund)
  • Click here to read Dr. Potter's final research report (in lay language)

Research Summary:
MPS II, or Hunter syndrome, is an inherited disorder affecting males caused by deficiency of the enzyme iduronate sulfatase.  This deficiency causes bone abnormalities, organ enlargement, brain deterioration and early death.  It is now possible to treat many features of MPS II by weekly injections of a purified form of the missing enzyme (enzyme replacement therapy, ERT). Unfortunately, the brain remains difficult to treat.  We are developing a new treatment that targets the brain by modifying the replacement enzyme so that it can better reach the brain.  The modification involves adding a targeting signal called “TAT” to the enzyme.  TAT has been successfully shown to deliver other proteins and enzymes to the brain in animal experiments.
Instead of directly injecting the modified enzyme, we will transplant cells that can produce the enzyme.  Transplantation removes one of the drawbacks of ERT, which is the need for weekly injections.  The cells will be placed inside a special microcapsule to protect them from rejection, allowing one transplantation procedure to last for months or years.   Microencapsulated cells have already successfully been used in animal experiments, including treatment of MPS II mice. 
We feel that this research will bring a treatment for the brain disease in MPS II and related disorders closer to reality.  This goal is directly related to one of the underlying purposes of the Canadian MPS Society, that of finding a cure for all MPS.

The role of serpins in the pathophysiology of MPS brain disease and evaluation of their role as potential biomarkers of disease

  • Researcher:  Dr. Lorne Clarke
  • Research conducted at:  University of British Columbia, Vancouver, BC
  • Funds allocated: March 2007:  $40,000.00 CAD

Research Summary:
The mucopolysaccharidoses (MPSs) represent a group of complex progressive multi-system diseases that are caused by metabolic defects in the degradation of glycosaminoglycans (GAGs).  The recent introduction of enzyme replacement (ERT) regimes for MPS I, MPS II and MPS VI has brought to light the importance of developing objective methods to evaluate patients and has sparked further research related to the pathophysiology of this complex group of disorders.  Many of the MPSs have significant and devastating involvement of the CNS.  Unfortunately there is a very poor understanding of the factors which underlie the brain manifestations of disease.  Understanding these factors will be key in the identification of therapeutic approaches which may alleviate these effects as well as possibly leading to the identification of objective measures that may reflect the degree of brain involvement.  We propose in this grant application, to expand on our initial observation of serpin complex formation in MPS I to determine the role that CNS serpins and their proteinases may play in MPS disease pathophysiology.

The Canadian MPS Society's 2006 Research Grant:

Evaluation of Heparin Cofactor II – Thrombin Complex as a Biomarker of MPS I

  • Researcher:  Dr. Lorne Clarke
  • Research conducted at:  University of British Columbia, Vancouver, BC
  • Funds allocated: Spring 2006:  $40,000.00 CAD
  • Click here to read Dr. Clarke's research report

Research summary:
The mucopolysaccharidoses (MPSs) represent a group of complex progressive multi-system diseases which are caused by metabolic defects in the degradation of glycosaminoglycans (GAGs).  The recent introduction of enzyme replacement  (ERT) regimes for MPS I and MPS II has brought to light the importance of developing objective methods to evaluate patients.  The clinical heterogeneity; manifest as variable age of onset, as well as variable rates of disease, clearly complicate the ability of physicians to accurately prognosticate clinical course for individual patients and leads to significant difficulty in objectively evaluating the effectiveness of treatment regimes.  The development of biomarkers that reflect disease severity, disease progression and responsiveness to treatment regimes will be an invaluable tool.  Dr. Clarke’s laboratory has identified a potential serum biomarker for MPS I and are critically evaluating the efficacy and usefulness of this biomarker in MPS I patients.

The Canadian MPS Society's Research Grants (2004-2005):

Proteomic Approaches to the Identification of Biomarkers of MPS Diseases

  • Researcher:  Dr. Lorne Clarke
  • Research conducted at:   University of British Columbia, Vancouver, BC.
  • Funds allocated: Spring 2005:  $30,000.00 CAD
  • Funds allocated: Spring 2004:  $35,000.00 CAD    

Research summary:
Mucopolysaccharidosis I (MPS I) is the most common of the generalized mucopolysaccharidoses and is considered the prototypical storage disease.  Accordingly, advances in the understanding of the pathogenesis of this disease and factors that play a role in the progression of MPS I, will likely be applicable to other MPS disorders. Clinical trials of direct enzyme replacement are currently underway and various MPS I animal models are available. While these developments hold great promise for those affected with MPS I, it is clear that many of the basic molecular/pathophysiological changes involved in the progression of MPS I and related disorders, remain to be elucidated.  Further understanding of these factors will be necessary in order to develop methods to accurately measure: the progression of disease, appropriate dosing and effectiveness of enzyme replacement and the identification of other possible therapeutic approaches that may need to be explored. We have shown that the targeted disruption of the murine a-L-iduronidase gene (IDUA) leads to a set of physiological responses in the mouse that closely resembles human MPS I and we propose to use this model to study the molecular pathophysiology of MPS I    (Russell et al. 1998).
Dr. Clarke and his team of researchers will use micro array analysis and isotope-coded affinity tag (ICAT) proteomics to investigate the pathogenesis of MPS I utilizing the murine MPS I model.
Ultimately their hope is to identify serum biomarkers of disease that may be useful in the evaluation of disease progression and response to therapy in children with MPS I. In addition, their more exploratory approach will lead to identification of other factors that may underlie the pathophysiology of MPS diseases. ICAT analysis of both immunocompetent and immunocompromised MPS I mice will represent one of the first applications of this type of proteomic analysis to a mouse model of disease.

Summer Studentship Research Grants (2004 - 2006):


Heparin Cofactor II – Thrombin Complex as a Biomarker of MPS I

  • Researcher: Jayant Shravah
  • Research conducted at:  UBC, Vancouver, BC
  • Amount allocated:  Spring/Summer 2006: $4,000.00
     

Co-transfect C2C12 mouse myoblasts with IDS and SUMFI, in an effort to achieve high expression and secretion of activated IDS (MPS II)

  • Researcher:  Alicia Donald
  • Research conducted at:  McMaster University, Hamilton, ON
  • Amount allocated:  Spring/Summer 2006: $4,000.00
     

Delivery of ARSA A to central nervous system using a fusion protein composed of ARSA A attached to sMTf

  • Researcher:  Soohun Chun
  • Research conducted at:  McMaster University, Hamilton, ON
  • Amount allocated:  Spring/Summer 2004: $4,000.00

Lysosomal Storage Disease Research Consortium (LSDRC) - Research funded in 2006:

Eain M. Cornford, PhD, Professor of Neurology, David Geffen School of Medicine at UCLA, Veterans Affairs Greater Los Angeles Healthcare System:

“Gene delivery across the blood-brain barrier in Lafora knockout mice”

Philip E. Dawson, PhD, Assoc. Professor, Cell Biology, Scripps Research Institute, LaJolla, CA:

“Potential of chemical chaperones and thioester reactive small molecules as potential therapeutic approaches in the treatment of infantile Batten disease”

Kostantin Dobrenis, PhD, Asst. Professor, Neuroscience, Co-Director, Center for Disorders of Lysosomal Metabolism, Albert Einstein College of Medicine:

"GM2 Gangliosidosis Therapy Using Neuronotropic Enzyme"

Angela Gritti, PhD, Institute for Stem Cell Research (SCRI), Milan, Italy:

“Neural Stem Cell Based Therapy for GM2 Gangliosidosis”

Synthia H. Mellon, PhD, Prof, Ob/Gyn. Reproductive Sciences, Univ. of California at San Francisco:

“Neurosteroid Therapy for Lysosomal Storage Disorders”

Thomas N Seyfried, PhD, Prof. of Biology, Boston College:

“Evaluate MJ-DGJ as a substrate reduction therapy, neural stem cells (NSCs), as a cross-correctional therapy, and caloric restriction (CR) as an anti-inflammatory therapy for ganglioside storage diseases”

Brian W. Soper, PhD, Research Staff Scientist, The Jackson Laboratory:

“MPS VII CNS Gene Therapy Using Neuronal Stem Cells” Canadian MPS Society funds allocated - Spring 2004:  $10,000.00 USD

 
Summary of LSDRC

The Canadian MPS Society was proud to be a member of the LSD Research Consortium (LSDRC), which partnered with the American National Institute of Neurological Disorders and Stroke (NINDS) for the purpose of a jointly sponsored program to provide funding towards preclinical or translational research specifically addressing the neurological aspects of LSDs.  On July 2, 2004, a Program Announcement (PA) was released by the NINDS soliciting applications for funding for research “focused on improving central nervous system (CNS) treatment outcomes, enhancing the effectiveness of delivery and targeting of cells, enzymes, drugs and genes into the brain.”*.  A total of approximately $1,050,000.00 US in grants was available from the NINDS and the Office of Rare Diseases (ORD).  Applications not funded by the NINDS were turned over to our group for consideration for funding within our funding capabilities. 
 
The Canadian MPS Society contributed $10,000.00 US toward this consortium:  a total of $310,000.00 US was contributed by the following American organizations: the National MPS Society, the National Tay Sachs and Allied Diseases Association, the Sanfilippo Syndrome Medical Research Foundation, Hunter’s Hope Foundation, and the National Neimann-Pick Disease Foundation.  The LSDRC was represented by a three member Executive Committee consisting of Barbara Wedehase (ED of National MPS Society), Jayne Gershkowitz (ED of National Tay Sachs and Allied Diseases), and Sissi Langford (Chair of Committee on Federal Legislation, National MPS Society).   We were excited to be a part of this collaboration. 
For more information on the LSDRC, please visit www.lsdresearch.org.  To read the complete Program Announcement, visit Http://grants.nih.gov/grants/guide/pa-files/PAS-04-120.html
*CENTRAL NERVOUS SYSTEM THERAPY DEVELOPMENT FOR LYSOSOMAL STORAGE DISORDERS (PAS—04-120) Lysosomal Storage Disease Research Consortium(LSDRC), National Institue of Neurological Disorders and Stroke (NINDS), Office of Rare Diseases (ORD)