THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2008; 10: 217–224. Published online 12 December 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.1140 Drug-induced readthrough of premature stop codons leads to the stabilization of laminin α 2 chain mRNA in CMD myotubes Val´ erie Allamand 1,2 * ,† Laure Bidou 3,4† Masayuki Arakawa 5 C´ elia Floquet 3,4 Masataka Shiozuka 5 Marion Paturneau-Jouas 1,2 Corine Gartioux 1,2 Gillian S. Butler-Browne 6 Vincent Mouly 6 Jean-Pierre Rousset 3,4 Ryoichi Matsuda 5 Daishiro Ikeda 7 Pascale Guicheney 1,2 1 Inserm, U582, Paris, France 2 Universit´ e Pierre et Marie Curie-Paris 6, UMR-S582, Institut de Myologie, IFR14, Paris, France 3 IGM, Universit´ e Paris-Sud, UMR 8621, Orsay, F-91405, France 4 CNRS, Orsay, F-91405, France 5 Department of Life Sciences, The University of Tokyo at Komaba., 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan 6 Inserm UMR-S787, Institut de Myologie, Facult´ e de M´ edecine Piti´ e-Salpˆ etri` ere, Paris, F-75013, France 7 Microbial Chemistry Research Center, Numazu Bio-Medical Research Institute, 18–24 Miyamoto, Numazu City, Shizuoka 410-0301, Japan *Correspondence to: Val´ erie Allamand, Inserm U582, Institut de Myologie, Groupe Hospitalier Piti´ e-Salpˆ etri` ere, 47 Bd de l’Hˆ opital, F-75651 Paris Cedex 13, France. E-mail: v.allamand@institut-myologie.org † These authors contributed equally to this work. Received: 6 July 2007 Revised: 17 September 2007 Accepted: 19 October 2007 Abstract Background The most common form of congenital muscular dystrophy is caused by a deficiency in the α2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. Methods Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. Results We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin α2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. Conclusions Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein. Copyright  2007 John Wiley & Sons, Ltd. Keywords congenital muscular dystrophy; laminin α2 chain; premature termination codon; antibiotic-mediated readthrough; nonsense-mediated mRNA decay Introduction Congenital muscular dystrophy (CMD) is a clinically and genetically het- erogeneous group of neuromuscular disorders with autosomal recessive inheritance. The ‘classic’ (or occidental) form of CMD is characterized by clinical manifestations mainly affecting skeletal muscle [1–4]. A spe- cific deficiency of the α2 chain of laminin-211 is responsible for about 30 to 40% of these cases (MDC1A, MIM#607855). Numerous mutations have now been identified in the LAMA2 gene encoding the α2 chain of laminin, leading to either complete or partial protein deficiency [5–9]. Notably, 20 to 30% of the mutations identified in the LAMA2 gene are nonsense mutations leading to a premature termination codon (PTC). Copyright  2007 John Wiley & Sons, Ltd.