Mechanism of action of GGA, a targeted oligonucleotide enhancer of splicing developed for the treatment of spinal muscular atrophy

Spinal muscular atrophy is the leading genetic cause of infant death, and much research has gone into the development of potential therapies for the disease. It is caused by a loss of the SMN1 gene. However, patients have the SMN2 gene, which contains a few silent mutations causing the skipping of exon 7 during splicing. One of the most promising therapeutic strategies involves the use of antisense oligonucleotides to rescue the splicing of SMN2 exon 7, allowing for production of full length SMN protein. One successful antisense oligonucleotide strategy involves the use of a bifunctional targeted oligonucleotide enhancer of splicing (TOES) (Skordis et al., 2003). This oligonucleotide, named GGA, consists of an annealing region that targets it to SMN2 exon 7 and a non-annealed enhancer tail domain, designed to recruit activator proteins and stimulate inclusion of exon 7. However, the precise mechanism by which GGA induces exon 7 inclusion was not fully understood at the time of design. This study has focused on investigation of the mechanism of action of GGA, in order to improve the therapeutic potential for GGA and future TOES. GGA was found to bind directly to SRSF1, an activator protein, via its enhancer tail domain. The tail domain forms a G-quadruplex structure in vitro (Smith et al., manuscript submitted). The presence of this structure in nuclear extracts was confirmed, and the enhancer domain was found to bind the G-quadruplex associated proteins CNBP and nucleolin. Stabilization of this structure using ligands reduced the efficacy of GGA, indicating that GGA does not form a G-quadruplex when it is actively stimulating SMN2 exon 7 inclusion. Single molecule methods revealed that the annealing domain of GGA, which anneals over an exonic splicing silencer shown to bind hnRNP A1 and/or Sam68 (Kashima et al., 2007; Pedrotti et al., 2010), reduces the number of Sam68 proteins bound per SMN2 RNA. These findings are consistent with the fact that the annealing region of GGA promotes U2AF65 binding and the enhancer tail domain promotes U2 snRNP binding to SMN2 transcripts (Smith et al., manuscript submitted; Smith, 2012).