We report the use of the U1 snRNA as a vector for the stable expression of 
antisense molecules against the splice junctions of specific dystrophin exons. 
The single-stranded 5' terminus of U1 can be replaced by unrelated sequences as 
long as 50 nucleotides without affecting both the stability and the ability to 
assemble into snRNP particles. Effective exon skipping has been obtained for 
different dystrophin exons by antisense sequences against 5' and 3' splice sites 
alone or in combination with ESE sequences. The efficacy of these molecules has 
been studied both in in vitro systems and in animals. In both cases the chimeric 
molecules, delivered as part of lentiviral or AAV vectors (De Angelis et al. 
Proc Natl Acad Sci USA 99:9456-9461, 2002; Denti et al. Proc Natl Acad Sci USA 
103: 3758-3763, 2006; Denti et al. Hum Gene Ther 17: 565-743, 2006; Denti et al. 
Hum Gene Ther 19: 601-608, 2008; Incitti et al. Mol Ther 18: 1675-1682, 2010), 
provided high skipping activity and efficient rescue of dystrophin synthesis. 
Moreover, the U1-antisense molecules, delivered to mice via systemic injection 
of recombinant AAV viruses, displayed body wide transduction, long-term 
expression, dystrophin rescue as well as morphological and functional benefit 
(Denti et al. Hum Gene Ther 19: 601-608, 2008). In this Chapter we report 
methods for producing U1-antisense expression cassettes in the backbone of 
lentiviral constructs and for testing their activity both in patients' derived 
myoblasts as well as in fibroblasts reprogrammed to muscle differentiation.