P6: Development of therapies

Subproject Speakers
Berlin:               Simone Spuler, MD, Prof.
Paris/Versailles:  Luis Garcia, PhD

Dysferlin Muscular Dystrophy (DysfMD) results from mutations in the gene encoding dysferlin (DYSF) and has typically an adult-onset of clinical symptoms and leads to loss of ambulation within 10-15 years after onset. Dysferlin is involved in sarcolemmal repair; however, its function is only partially understood. Its proper function is linked to the expression of caveolin-3 at the plasma membrane. Dysferlin binds also to annexin. Caveolin-3 and annexin are components of "lipid rafts". Lipid rafts are glycosphingolipid (GSL)- and cholesterol-enriched membrane domains that float in the phospholipid-rich portion of the membrane and regulate cell signaling. Lipid rafts are heterogeneous and their exact definition is still a matter of debate. However, GSLs and glycosylphosphatidylinositol (GPi)-anchored proteins are considered as a common denominator of lipid rafts. The group of Simone Spuler has recently shown that the Gpi-anchored complement-inhibitory factor CD55 (decay-accelerating factor, DAF) is down-regulated in muscle cells from DysfMD patients. This renders dysferlin-deficient muscle fibers more susceptible to complement attack and accelerates the course of the disease. Therefore, three proteins associated with lipid rafts, namely caveolin, annexin, and Gpi-anchored proteins, are associated with dysferlin. We hypothesize that dysferlin-deficiency leads to lipid-raft disassembly and that the disturbances in lipid rafts are the major cause of CD55 downregulation.

The group of Dr Luis Garcia developed a gene therapy strategy in animal models for muscular dystrophies, the dystrophin deficient mdx mouse and GRMD dog. They achieved persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. Using this AAV U7 mediated exon-skipping strategy, sustained production of functional dystrophin at physiological levels was achieved for entire groups of muscles resulting in a functional and morphological correction of the muscular dystrophy. However, exon skipping is not suitable if skipped exons diminish or abolish protein function. This problematic situation constitutes a limit for most proteins involved in muscle diseases, including dysferlin, which appears to include not only modular parts, but also indispensable parts. To address this eventuality, a therapy was developed for muscular dystrophy, so-called trans-splicing, whereby novel AAV U7 vectors encode for a non-mutated exon that replaces the mutated exon during the splicing process.
In this project we will apply highly advanced visualization techniques to study the sarcolemma and the role of dysferlin in lipid rafts. We will investigate the consequences of dysferlin-deficiency on the organization of these membrane components. These techniques will provide a test system to study the efficiency of novel gene therapies on cultures of DysfMD myoblasts. We further will develop a tailored gene therapy approach, which permit to repair rare dysferlin-mutations by spliceosome-mediated RNA trans-splicing, and we will investigate the therapeutic effect of trans-splicing on membrane morphology and function.



Maximilian Berger (MD student)

Charité Universitätsmedizin Berlin

Working title
Alterations in the proteomic profile of sporadic Inclusion Body Myositis - a mass spectrometry based investigation

 


 

Alejandro Léon Betancourt (MD student)
Charité Universitätsmedizin Berlin

Working title
Missensemutations in dysferlinopathy targeting skeletal muscle by pharmacologically active small peptides coupled to magnetic nanoparticles


 
Helena Escobar Fernandez
, MyoGrad doctoral student 2011-2015


Completed doctoral project:

A cell-based gene therapy approach for dysferlinopathy using Sleeping Beauty transposon

Date and place of thesis defense:
November 18, 2015 in Berlin
Doctoral degree from FU Berlin



Petra Gimpel,  MyoGrad doctoral student 2013-2017

Completed doctoral project:
Mechanisms of non-centrosomal MTOC formationat the nucleus in muscle cells

Date and place of thesis defense:
September 4, 2017 in Berlin
Doctoral degree from FU Berlin
Joint doctoral degree from UPMC, Paris VI and from FU Berlin



Leonie Heidt (MD student)
Charité Universitätsmedizin Berlin

Working title
Dysferlinopathy caused by protein misfolding: The murine animal model Dysf-MMex38



Séverine Kunz, MyoGrad doctoral student 2010-2014

Completed doctoral project:

Characterization of the sarcolemma in limb-girdle muscular dystrophy

Date and place of thesis defense:
October 22, 2014 in Paris
Joint doctoral degree from UPMC, Paris VI and from FU Berlin



Henning Langer,  MyoGrad doctoral student

Master, Biology of Human Performance and Health
Maastricht University, The Netherlands

Working title
The biochemistry of skeletal muscle plasticity



Jakub Malcher, MyoGrad doctoral student
Master, Biotechnology
Adam Mickiewicz University, Poznan

Working title
Trans-splicing and exon skipping as strategies for gene therapy in dysferlinopathy in a new knockin mouse model
 

Vinko Palada, MyoGrad doctoral student 2013-2017

Completed doctoral project:
Molecular mechanisms of muscle pain associated with myotonic dystrophy
type II

Date and place of thesis defense:
March 13, 2017 in Berlin
Doctoral degree from FU Berlin



Susanne Philippi
, MyoGrad doctoral student 2010-2014


Completed doctoral project:

RNA-based therapies for dysferlinopathies


Date and place of thesis defense:
September 25, 2014 in Paris
Joint doctoral degree from UPMC, Paris 6 and from FU Berlin



William Roman, MyoGrad doctoral student 2012-2016

Completed doctoral project:
Positioning nuclei at the periphery of skeletal muscle cells

Date and place of thesis defense:
September 27, 2016 in Paris
Joint doctoral degree from UPMC, Paris 6 and from FU Berlin