Targeted Non Viral Gene Therapy for Duchenne Muscular Dystrophy

• Applicant: Dr. Sören Turan
• Subject Area: Human Genetics
• Term: from 2013 to 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 239221667

Duchenne Muscular Dystrophy (DMD) is a genetic disease that leads to widespread muscle degeneration, including failure of the heart and diaphragm. The genetic defect of DMD is caused by mutations of the dystrophin gene, leading to functional failure of the DMD protein. Viral based gene therapy of DMD is limited due to the extended size of the dystrophin gene. In this context non viral gene therapy is the appropriate method of choice. Our aim is to further optimize plasmid-based non viral gene therapy in terms of efficiency and safety. First, we want to generate murine and human DMD -/- disease/patient specific induced pluripotent stem cells (iPSC) with stable integrating reprogramming cassettes in defined safe harbor regions. These safe harbor regions have the beneficial properties to allow active transcription of transgenes. However, they do not interact with the surrounding genomic landscape, which is the essential requirement to circumvent side effects arising from insertional mutagenesis. We will follow three strategies for the generation of safe harbor iPSCs being lentiviral safe harbor iPSC, PHIC31-pseudo attP safe harbor iPSC and AAVS1 safe harbor iPSC. In order to correct DMD we will stably integrate therapeutic donor plasmids into these safe harbor loci using recombinase-mediated cassette exchange (RMCE). Subsequently, we will differentiate successfully targeted and characterized iPSC clones into muscle progenitors and select for these via FACS sorting. Finally we will analyze the differentiated cells for functional rescue of DMD in vitro. In summary, we present a novel concept for targeted non viral gene therapy of Duchenne Muscular Dystrophy by integration of RMCE and safe harbor platforms to guarantee stable sustained long term expression of DMD without any site effects of insertional mutagenesis and silencing.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Michele Calos, Ph.D.