Synthetic oligonucleotides recruit ILF2/3 to RNA transcripts to modulate splicing

Abstract

We describe a new technology for recruiting specific proteins to RNA through selective recognition of heteroduplexes formed with chemically modified antisense oligonucleotides (ASOs). Typically, ASOs function by hybridizing to their RNA targets and blocking the binding of single-stranded RNA–binding proteins. Unexpectedly, we found that ASOs with 2′-deoxy-2′-fluoro (2′-F) nucleotides, but not with other 2′ chemical modifications, have an additional property: they form heteroduplexes with RNA that are specifically recognized by the interleukin enhancer-binding factor 2 and 3 complex (ILF2/3). 2′-F ASO–directed recruitment of ILF2/3 to RNA can be harnessed to control gene expression by modulating alternative splicing of target transcripts. ILF2/3 recruitment to precursor mRNA near an exon results in omission of the exon from the mature mRNA, both in cell culture and in mice. We discuss the possibility of using chemically engineered ASOs that recruit specific proteins to modulate gene expression for therapeutic intervention.

Pharmacology of a Central Nervous System Delivered 2′-O-Methoxyethyl–Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates

“…Hybridization of ISIS 396443 to ISS-N1 prevents the recruitment of the splicing repressor heterogeneous nuclear ribonucleoprotein A1 and A2, and results in almost complete SMN2 exon 7 inclusion in cell culture (Hua et al, 2008;Rigo et al, 2012). Robust splicing correction and SMN protein production are also observed in the central nervous system (CNS) of mice transgenic for SMN2 after central administration of ISIS 396443 (Hua et al, 2010).…”

“…Intracerebroventricular infusions and i.p. bolus injections (Rigo et al, 2012) were performed as previously described. For i.c.v.…”

“…Ten microliters of lysate was used to isolate RNA with an RNeasy 96 Kit (Qiagen) that included in-column DNA digestion with 50 U of DNase I (Invitrogen, Carlsbad, CA). Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed as previously described (Rigo et al, 2012). For Supplemental Fig.…”

2′β‐Fluoro‐Tricyclo Nucleic Acids (2′F‐tc‐ANA): Thermal Duplex Stability, Structural Studies, and RNase H Activation

“…[19] Indeed, a recent study has demonstrated that F-RNA antisense oligonucleotides can direct the recruitment of protein complex ILF2/3 to induce exon skipping, while corresponding 2'-O-(2-methoxyethyl) oligonucleotides are unable to do that. [20] Overall, introduction of fluorine substituent(s) constitutes an effective approach to improve the therapeutic potential of nucleic acid analogues.…”

Comparison of Duplex Stabilizing Properties of 2′-Fluorinated Nucleic Acid Analogues with Furanose and Non-Furanose Sugar Rings

“…FRNA-modified oligonucleotides (ONs) have found widespread use as nucleic acid aptamers, 1 ribozymes, 2 antisense agents, 3 allele-selective gene silencing, 4 splice-switching, 5 single-stranded siRNA, 6 and anti-microRNA 7 oligomers. FRNA-modified siRNAs possess unique gene silencing properties 8,9 and are currently being evaluated in human trials for the treatment of genetic disorders.…”