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.

U1 snRNA as an Effective Vector for Stable Expression of Antisense Molecules and for the Inhibition of the Splicing Reaction / Martone, Julie; Fernanda Gabriella, Angelis; DE ANGELIS, FERNANDA GABRIELLA; Bozzoni, Irene. - STAMPA. - 867(2012), pp. 239-257. - METHODS IN MOLECULAR BIOLOGY. [10.1007/978-1-61779-767-5_16].

U1 snRNA as an Effective Vector for Stable Expression of Antisense Molecules and for the Inhibition of the Splicing Reaction

MARTONE, Julie;DE ANGELIS, FERNANDA GABRIELLA;BOZZONI, Irene
2012

Abstract

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.
2012
EXON SKIPPING, Methods in Molecular Biology
9781617797668
9781617797675
lentivirus; u1 snrna; luciferase constructs for exon skipping; fibroblast reprogramming; duchenne muscular dystrophy
02 Pubblicazione su volume::02a Capitolo o Articolo
U1 snRNA as an Effective Vector for Stable Expression of Antisense Molecules and for the Inhibition of the Splicing Reaction / Martone, Julie; Fernanda Gabriella, Angelis; DE ANGELIS, FERNANDA GABRIELLA; Bozzoni, Irene. - STAMPA. - 867(2012), pp. 239-257. - METHODS IN MOLECULAR BIOLOGY. [10.1007/978-1-61779-767-5_16].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/460576
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