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.
 

Theses in progress

Completed theses