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 MYC or the Huntingtin 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 in vivo 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 single site in the template strand of the Huntingtin gene induces significant mRNA down-regulation in patient-derived fibroblasts, boding well for the anti-gene therapeutic concept.