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RNA-mediated pathogenesis is a recently developed disease model that proposes that certain types of mutant genes produce toxic transcripts that inhibit the activities of specific proteins. This pathogenesis model was proposed first for the neuromuscular disease myotonic dystrophy (DM), which is associated with the expansion of structurally related (CTG) n and (CCTG)n microsatellites in two unrelated genes. At the RNA level, these expansions form stable hairpins that alter the pre-mRNA splicing activities of two antagonistic factor families, the MBNL and CELF proteins. It is unclear which altered activity is primarily responsible for disease pathogenesis and whether other factors and biochemical pathways are involved. Here, we show that overexpression of Mbnl1 in vivo mediated by transduction of skeletal muscle with a recombinant adeno-associated viral vector rescues disease-associated muscle hyperexcitability, or myotonia, in the HSA LR poly(CUG) mouse model for DM. Myotonia reversal occurs concurrently with restoration of the normal adult-splicing patterns of four pre-mRNAs that are misspliced during postnatal development in DM muscle. Our results support the hypothesis that the loss of MBNL1 activity is a primary pathogenic event in the development of RNA missplicing and myotonia in DM and provide a rationale for therapeutic strategies designed either to overexpress MBNL1 or inhibit MBNL1 interactions with CUG and CCUG repeat expansions.microsatellite ͉ muscle ͉ pathogenesis ͉ splicing ͉ transgenic D uring mammalian embryonic and postnatal periods, tissues are initially formed and then extensively remodeled to meet the evolving demands of the developing organism. This postnatal remodeling process requires a dynamic series of biochemical events at both the transcriptional and posttranscriptional levels. For vertebrate skeletal muscle, myogenic precursor cells derived from somites migrate during embryogenesis and, ultimately, fuse to form primary and secondary myofibers (1). After birth, fetal muscle must be modified to perform adult functions, and this transition (e.g., a suckling versus a highly mobile adult mouse) requires the sequential replacement of fetal protein isoforms with their adult counterparts. For some muscle genes, this process involves skipping of fetal exons and͞or the inclusion of adult exons during pre-mRNA splicing. Remarkably, this normal splicing transition is altered specifically in the neuromuscular disease myotonic dystrophy (DM) (2-4).Adult-onset DM is a multisystemic degenerative disease (4). Characteristic features of this disorder include skeletal muscle myotonia (muscle hyperexcitability), weakness͞wasting, heart conduction defects, particulate subcapsular cataracts, and insulin insensitivity. The genetic basis of DM is unusual because it is associated with different unstable microsatellites in two unrelated genes. DM type 1 (DM1) is caused by the expansion of a (CTG) n repeat in the 3Ј UTR of the DMPK (dystrophia myotonica protein kinase) gene (5). Normal DMPK microsatellite lengt...

The clinical application of adeno-associated virus vectors (AAVs) is limited because of concerns about AAV integration-mediated tumorigenicity. We performed integration-site analysis after AAV1-LPL(S447X) intramuscular injection in five lipoprotein lipase-deficient subjects, revealing random nuclear integration and hotspots in mitochondria. We conclude that AAV integration is potentially safe and that vector breakage and integration may occur from each position of the vector genome. Future viral integration-site analyses should include the mitochondrial genome.