title: Design principles for bifunctional targeted oligonucleotide enhancers of splicing.
abstract: Controlling the patterns of splicing of specific genes is an important goal in the development of new therapies. We have shown that the splicing of a refractory exon, SMN2 exon 7, could be increased in fibroblasts derived from patients with spinal muscular atrophy by using bifunctional targeted oligonucleotide enhancers of splicing (TOES) oligonucleotides that anneal to the exon and contain a 'tail' of enhancer sequences that recruit activating proteins. We show here that there are striking agreements between the effects of oligonucleotides on splicing in vitro and on both splicing and SMN2 protein expression in patient-derived fibroblasts, indicating that the effects on splicing are the major determinant of success. Increased exon inclusion depends on the number, sequence and chemistry of the motifs that bind the activator protein SRSF1, but it is not improved by increasing the strength of annealing to the target site. The optimal oligonucleotide increases protein levels in transfected fibroblasts by a mean value of 2.6-fold (maximum 4.6-fold), and after two rounds of transfection the effect lasted for a month. Oligonucleotides targeted to the upstream exon (exon 6 in SMN) are also effective. We conclude that TOES oligonucleotides are highly effective reagents for restoring the splicing of refractory exons and can act across long introns.
authors: Department of Biochemistry, University of Leicester, Leicester LE1 9HN, Dubowitz Neuromuscular Centre, Institute of Child Health, UCL, London WC1N 1EH, UK.
authors: Nicholas
authors: N
authors: Owen
authors: Haiyan
authors: H
authors: Zhou
authors: Alexey A
authors: AA
authors: Malygin
authors: Jason
authors: J
authors: Sangha
authors: Lindsay D
authors: LD
authors: Smith
authors: Francesco
authors: F
authors: Muntoni
authors: Ian C
authors: IC
authors: Eperon
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