Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes.
BACKGROUND
The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 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 alpha2-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.
Allamand
Valérie
V
Inserm, U582, Paris, France. v.allamand@institut-myologie.org
Bidou
Laure
L
Arakawa
Masayuki
M
Floquet
Célia
C
Shiozuka
Masataka
M
Paturneau-Jouas
Marion
M
Gartioux
Corine
C
Butler-Browne
Gillian S
GS
Mouly
Vincent
V
Rousset
Jean-Pierre
JP
Matsuda
Ryoichi
R
Ikeda
Daishiro
D
Guicheney
Pascale
P
D000599:Amino Acids, Diamino / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D018389:Codon, Nonsense / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D005839:Gentamicins / Q000494:pharmacology*; D006801:Humans; D007797:Laminin / Q000235:genetics*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009136:Muscular Dystrophies / Q000235:genetics*; D009218:Myosins / Q000378:metabolism; D041681:NIH 3T3 Cells; D020871:RNA Stability / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D000599:Amino Acids, Diamino; D018389:Codon, Nonsense; D005839:Gentamicins; D007797:Laminin; D012333:RNA, Messenger; C100200:negamycin; D009218:Myosins