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A systematic and multidisciplinary approach towards understanding and therapy of the inborn lysosomal storage disease alpha-mannosidosis

   
Project

QLK3-2001-02458

Cell factory area

3.1.3

EU Contribution

1 557 640 Euro

Duration

36 months

Type

Research project

Starting date

01-10-2001

Keywords
alpha-mannosidosis
lysosomal
oligomannose species
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ABSTRACT
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Alpha-mannosidosis is a rare, degenerative and chronic disease with multiple factors contributing to the degrees of progression. Since the patients are born healthy and exhibit a slow progression of clinical symptoms, an effective therapy initiated at early age could contribute to a normal evolution. This project is undertaken to reach such a therapy. Due to the complexity of the disease, the project requires an international effort with a plethoria of different techniques and disciplines involved. The aim is to reach a critical mass of understanding on both the molecular and pathophysiological level, and to use this knowledge to develop new strategies for therapy, using a mouse model of the disease.

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OBJECTIVES
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The main object of this project is to obtain a therapy for the lysosomal storage disease alpha-mannosidosis. To reach this goal the project will focus on the following objectives:

  • Characterising the three-dimensional structure and active site of lysosomal alpha-mannosidase.
  • Reaching molecular and pathophysiological understanding of alpha-mannosidosis.
  • Using the increased understanding to develop new strategies for enzyme therapy of alpha-mannosidosis.
  • Determining the effects of these therapeutic strategies on a mouse model of alpha-mannosidosis.
  • Characterising the spectrum of disease-causing mutations in the European population.

The other aims of the project are:

  • Understanding the cellular oligomannose metabolism and its importance in pathology and
  • Determining the structure and function of the glycosylhydrolase family # 38, based on molecular modeling.

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DESCRIPTION OF THE WORK
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The project involves the understanding of the lysosomal storage disease alpha-mannosidosis, and the search for therapy using a mouse model of the disease.

The project fits under the generic activities QOL-7.1 and 8.1 due to the increased understanding of alpha-mannosidosis, and the cell factory activity QOL-3.1.3 due to the design of new therapeutic strategies.

The understanding of alpha-mannosidosis comprises different levels:

  • Gene
  • Protein
  • Pathophysiology.

On the gene level the disease causing mutations in all European countries will be determined and genetic counseling of human alpha-mannosidosis established.

On the protein level the three-dimensional structure, the active site and intracellular transport of lysosomal alpha-mannosidase will be characterised.

On the pathophysiology level the abnormal oligomannose metabolism in fibroblast cells and serum from patients will be investigated, and the clinical consequences of these changes evaluated.

The search for a therapy includes:

  • Enzyme production
  • Therapy of a mouse model.

On enzyme production high producer cells of human and mouse recombinant enzymes will be generated and these enzymes will subsequently be produced and purified in small-scale cell factories. To improve the therapeutic values the recombinant enzymes will be modified by site directed mutagenesis or chemical modification. The uptake and therapeutic effects of these different forms of the enzyme will be studied on cell cultures.

Using the mouse model the most promising enzyme isoforms will be investigated for short term and long-term effects using different histochemical and biochemical analysis of the animal tissues. Gene therapy as an alternative approach to enzyme therapy, will be evaluated continuously.

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DELIVERABLES
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The Project will result in the following deliverables:

  • Determining the three-dimensional structure of lysosomal alpha-mannosidase and the class 2 alpha-mannosidase family.
  • Characterising the structure of the active site of lysosomal alpha-mannosidase and the class 2 alpha-mannosidase family.
  • Finding the post translational modifications of lysosomal alpha-mannosidase and determining their functions on the intracellular transport.
  • Studying the proteins from endoplasmic reticulum and Golgi that interact with lysosomal alpha-mannosidase.
  • Large-scale production of mouse and human lysosomal alpha-mannosiase.
  • Determining the safety and efficiency of enzyme replacement therapy on a mouse model.
  • Characterising the levels and nature of oligomannose species in the endoplasmic reticulum, the cytosol and secretions of fibroblasts from patients.
  • Reporting on the structure and function of cytosolic and serum alpha-mannosidase.
  • The effect of serum oligomannosides from patients on components of the immune system.
  • The correlation between the genotype and phenotype of alpha-mannosidosis.
  • 1Reporting the European spectrum of disease-causing mutations.

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CONSORTIUM
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COORDINATOR
  Ole K. Tollersrud
Medical Biochemistry
Univ.Tromsö
9037 Tromsö, Norway
Tel: +47 77626313
Fax: +47 77626506
olekt@fagmed.uit.no

PARTNERS
  E. Hough
Physical Chemistry
Univ.Tromsö
9037 Tromsö, Norway
Tel: +47 77644073
Fax: +47 77644737
edward.hough@chem.uit.no

O. Nilssen
Medical Genetics
Univ.Tromsö
9037 Tromsö, Norway
Tel: +47 77645410
Fax: +47 77645430

oivindn@fagmed.uit.no

D. Malm
Medicine
Univ.Tromsö
9037 Tromsö, Norway
Tel: +47 77626000
Fax: +47 77626863
dag.malm@rito.no

K. von Figura
Biochemie 2
Univ. Göttingen
37073 Göttingen, Germany
Tel: +49 551395948
Fax: +49 551395979
kfigura@gwdg.de

P. Saftig
Biochemie 2
Univ. Göttingen
37073 Göttingen, Germany
Tel: +49 551395932
Fax: +49 551395979
psaftig@gwdg.de

A. Orlacchio
Scienze Biochimiche e
Biotecnologie Moleculare
Univ. Perugia
06100 Perugia, Italy
Tel: +39 0755857443
Fax: +39 0755857434
orly@unipg.it

T. Beccari
Scienze Biochimiche e
Biotecnologie Moleculare
Univ.Perugia
06100 Perugia, Italy
Tel: +39 0755857439
Fax: +39 0755857443
tbeccari@unipg.it

S. McSweeney
Macromol. Cryst.
ESRF
38043 Grenoble, France
Tel: +33 476882362
Fax: +33 476882160
seanmc@esrf.fr

J.C. Michalski
Lab. Chimie Biol.
Univ. Lille 1
59655 Villeneuve d'Ascq, France
Tel: +33 320434146
Fax: +33 320436555
Jean-Claude.Michalski@univ-lille1.fr

J.P. Zanetta.
Lab. Chimie Biol.
Univ. Lille 1
59655 Villeneuve d'Ascq, France
Tel: +33 320434010
Fax: +33 320436555
jean-pierre.zanetta@univ-lille.fr

R. Cacan
Lab. Chimie Biol.
Univ. Lille 1
59655 Villeneuve d'Ascq, France
Tel: +33 320434430
Fax: +33 320436555
rene.cacan@univ-lille1.fr

J. Fogh
Roskildevej 12C
HemeBiotech A/S
Hilleroed, Denmark
Tel: +45 48250054
Fax: +45 48251054
jf@hemebiotech.com
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