Anti-gene oligonucleotide clamps invade dsDNA and downregulate huntingtin expression

Anti-gene oligonucleotides belong to a group of therapeutic compounds, which, in contrast to antisense oligonucleotides, bind to DNA. Clamp anti-gene oligonucleotides bind through a double-stranded invasion mechanism. With two arms connected by a linker, they hybridize to one of the DNA strands forming Watson-Crick and Hoogsteen hydrogen bonds. Here, we investigated the design of 30 locked nucleic acid-DNA mixmers with or without a strong intercalating moiety (M3) to target polypurine⋅polypyrimidine sequences from the <i>MYC</i> or the <i>Huntingtin</i> gene. Introducing M3 as a linker proved to be essential for strand invasion. Additional M3 at the end of the Watson-Crick- or the Hoogsteen-binding strand could be beneficial depending on the clamp orientation. Invasion was superior when the linker was located adjacent to sequences with high GC content. For <i>in vivo</i> application, we assessed strand-invasion of phosphorothioate-modified clamp anti-gene oligonucleotides. While the binding kinetics was slower than for the corresponding phosphodiester clamps, equal invasion was eventually reached. Lastly, we demonstrated, that the clamp anti-gene oligonucleotide targeting a <i>single</i> site in the template strand of the <i>Huntingtin</i> gene induces significant mRNA down-regulation in patient-derived fibroblasts, boding well for the anti-gene therapeutic concept.