Switching on transgene expression by correcting aberrant splicing using multi‐targeting steric‐blocking oligonucleotides

Abstract

Mutations leading to aberrant splicing are found as a cause of numerous pathologies. Splice-switching oligonucleotides (SSOs), which modify aberrant expression patterns of alternatively spliced mRNAs, are a novel means of potentially controlling such diseases. We used an experimental model in which a mutated beta-globin intron, carrying an aberrant splice site at nucleotide 705, interrupts the coding region of the luciferase reporter gene inserted in HeLa pLuc/705 cells. We have optimized delivery of splice correcting, steric-blocking 2'-O-methyl SSOs targeting the 705 mutated region (2'-O-Me SSO(705)) with DLS (DLS: delivery liposomal system) lipoplexes. Optimal luciferase activity for DLS/2'-O-Me SSO(705) was achieved at 100 nM and was detectable at concentrations as low as 10 nM in serum-containing culture medium, confirming the potential of DLS lipoplex-mediated nuclear SSO delivery as observed in cellular uptake studies. We confirmed by cytofluorometry and epifluorescence microscopy the high potential of the DLS lipoplex for cellular and nuclear oligonucleotide uptake. The DLS lipoplex was then used to directly compare the intracellular efficacy of various SSO chemistries and sequences in correction of aberrant splicing. 2'-O-Methoxyethyl-oligodeoxyribonucleoside phosphorothioates had a greater activity than 2'-O-methyl phosphodiester or 2'-O-methyl-phosphorothioate oligoribonucleotides. Targeting the splicing enhancer 623 region upstream was as efficient as targeting the 705 splice site, and, remarkably, simultaneous targeting of both sites was more efficient than treatment of the cells either with 2'-O-Me SSO(705) or 2'-O-Me SSO(623) alone. We demonstrated that SSOs can switch on luciferase activity in HeLa cells previously transfected with the pLuc/705 plasmid via the same DLS vector and provides a novel approach to modulate the expression of a transgene.