Antisense Derivatives of U7 Small Nuclear RNA as Modulators of Pre‐mRNA Splicing

In vivo-expressed short splicing-modulating RNAs represent an interesting option for the gene therapy of various diseases. Moreover, they can be used to study the nature of splicing mutations, or for basic studies on alternative splicing (AS). Modified derivatives of the U7 small nuclear RNA (snRNA) involved in histone RNA 3′ end processing are particularly well suited to this type of approach. Two important features are the nuclear accumulation and high stability of the RNA as part of a small nuclear ribonucleoprotein particle. In particular, U7 derivatives containing two tandem antisense sequences directed against targets upstream and downstream of an exon can induce the efficient and specific skipping of that exon. U7 snRNA derivatives can also be equipped with an additional functional moiety; for example, an exonic splicing enhancer sequence capable of tethering a splicing activator protein of the SR family to the exon of interest, in order to promote the inclusion of this exon into the mRNA. In this chapter, the construction of such U7 derivatives, starting from the original U7 Sm OPT plasmid, will be described. U7 expression cassettes have been successfully introduced into many cell lines, primary cells or tissues, with the help of lentiviral and adeno-associated viral vectors; a procedure will also be described for subcloning into such viral delivery vectors. The U7 system is a good alternative to antisense oligonucleotides, because the splicing modulatory sequences can be expressed permanently and as part of a stable, nuclearly located, RNA.