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Molecular Stem Cell Biology of Lysosomal Storage Diseases

Our aim is to understand the molecular mechanisms of Lysosomal Storage Diseases and to develop novel treatment options based on regenerative medicine and gene therapy.

Lysosomal Storage Diseases
Lysosomes are small organelles involved in the degradation and recycling of macromolecules. Uptake of macromolecules by lysosomes occurs by endocytosis or autophagy, and is regulated by the nutritional state of the cell. The lysosome signals to the nucleus and back to ensure a highly regulated system for control of metabolism and energy status. Various types of macromolecules can be degraded in the lysosome by one of 60 known lysosomal hydrolases recognizing nucleic acids, proteins and their modifications, and lipids.  In the case of a mutation in a lysosomal enzyme, specific macromolecules cannot be degraded and accumulate in the lysosome. This causes lysosomal dysfunction and a cascade of problems, eventually resulting in pathology of tissues and organs, such as muscle, cartilage, bone, visceral organs, and the central nervous system. The nature of pathology depends on the type of enzyme that is defective. As a group, lysosomal storage diseases occur at a frequency of 1:5000.

Pompe disease
In Pompe disease, the lysosomal enzyme acid alpha glucosidase (GAA), responsible for the degradation of glycogen into glucose, is defective. Many pathogenic mutations in the GAA gene have been characterized by us and others (www.pompecenter.nl). In Pompe disease, glycogen taken up by lysosomes cannot effectively be degraded anymore. This leads to lysosomal glycogen accumulation. Initially, lysosomes increase in size and number. At a certain point, they become to large and rupture, releasing their content in the cell and causing cell death. In Pompe disease, mainly skeletal muscle cells are affected, while only in the most severe form of the disease, cardiac cells are also affected. This can result in respiratory insufficiency due to a weak diaphragm, mobility problems, fatigue, problems with speech, swallowing, and hypertrophic cardiomyopathy. The disease is very heterogeneous and can become evident anywhere during life ranging from onset at birth to onset at 60-70 years of life. This depends on the type of mutation and the level of residual enzyme activity. Babies with Pompe disease, if left untreated, die within 1.5 year of life. Children, juvenile or adult patients can become ventilator and wheelchair dependent.

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Enzyme Replacement Therapy
Fortunately, a treatment is available for Pompe patients. This Enzyme Replacement Therapy (ERT) was originally developed in our research center by Dr. Arnold Reuser and Prof. Ans van der Ploeg, and consists of recombinant GAA that is produced in vitro (using cultured cells) and applied to patients through infusion.The success of ERT is based on a naturally occurring mechanism in which (a small part of) GAA is normally secreted by cells and taken up again through the mannose 6-phosphate receptor. The infused enzyme is taken up from the blood through the same route, using mannose 6-phosphate receptors. A similar principle is now used for the treatment of other lysosomal storage diseases. These include the mucopolysaccharidoses, of which several forms (I, II, and VI) are treated with ERT and also researched in our center.

Limitations of ERT
ERT is effective, but it also has a number of limitations, including the need for life long biweekly lengthy infusions. The clinical response to ERT is heterogeneous. This may be caused by the difficulty for recombinant GAA applied via the blood to reach muscle cells. Another drawback is the generation of an antibody response that can interfere with enzyme activity. In addition, ERT is extremely expensive preventing its reimbursement by insurance companies in a number of countries. Also in the Netherlands this topic is currently subject to public discussion. For these reasons, we are searching for alternative ways to treat this disease. Our approaches are generic and potentially relevant for lysosomal storage diseases and muscle disorders in general.

Mouse model
In our center we have generated a mouse model for classic infantile Pompe disease. This provides a very important tool to analyse the molecular mechanisms of this disease. It also enables the preclinical testing of potential novel therapies.

Human patient biopsies
Our center has collected many biopsies from patients with Pompe disease. These form an invaluable resource for basic research. The clinical status at the time of biopsy has been recorded, which enables us to relate our findings to the clinical status of the patients. We study both the natural course of the disease and the response to ERT.



Mutation analysis
Many mutations in the GAA gene that can cause Pompe disease have been characterized by our Center and others. These are listed in http://cluster15.erasmusmc.nl/klgn/pompe/mutations.html. More information on clinical aspects including clinical research can be found on http://www.erasmusmc.nl/klinische_genetica/research/lijnen/pompe_center/?lang=en.
Information on the effect of a mutation on the GAA mRNA or protein is important to devise strategies for novel therapies. In addition, it is important to know which mutation is pathogenic and to which extent for diagnostic purposes such as newborn screening programs. Novel mutations are still found to date, and we are continuing our research in this area.

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Regenerative medicine
Recent advances in stem cell research and molecular biology has opened new options for translational research based on regenerative medicine and gene therapy. A key and Nobel price-awarding finding from the Yamanaka lab in 2006 was that somatic cells, originally thought to be the endstate of differentiation, are plastic and can be reprogrammed to a pluripotent state by expression of only 3-4 transcription factors. The cells generated resemble embryonic stem cells and are known as induced pluripotent stem (iPS) cells. Another key finding in the field was the discovery that many adult organs and tissues harbour so-called adult stem cells capable of tissue renewal and repair after injury. These two cell types form the basis of our current research.


Gene therapy
Major progress has also been made recently in ways to alter the eukaryotic genome. This has important implications not only for basic science but also for the development of safe options for gene therapy by avoiding retro- or lentiviral vectors. Whereas retro- or lentiviral vectors integrate into the genome in an uncontrolled manner, methods based on homologous recombination enable precise genome engineering at any location of interest. One of the most promising methods that we are pursuing is by using TAL effector nucleases (Nature Methods: Method of the year 2011). These proteins introduce a double stranded break that is repaired by the cell in an imprecise way (to create a knock out) or, when a donor sequence is provided, in a precise way to create insertions or corrections. These options to modify the human genome in a controlled manner offer a novel strategy for correction of pathogenic mutations and for preclinical testing of a gene therapy approach.