Anti-gene oligonucleotides : DNA binding and therapeutic application

Sammanfattning: The field of nucleic acid therapeutics has in the last decade experienced substantial growth. This is evident from a multitude of new publications, an increasing number of clinical trials and several approved therapeutics on the market. Therapeutic oligonucleotides (ONs) are designed to treat mostly genetic disorders, caused by the expression of non-functional or toxic ribonucleic acids (RNA) or proteins. They can be divided into protein-, RNA- and DNA targeting ONs. While the first two have already had approved therapies, the last is still in its infancy. This thesis focuses on the improvement and assessment of DNA targeting ONs, also termed anti-gene ONs. Paper I evaluates a strand-invading anti-gene ON (AGO) for the treatment of Huntington’s disease (HD) and its mRNA down-regulating effect during in vitro differentiation from induced pluripotent stem cells to neurons. It shows that the locked nucleic acid (LNA)/DNA mixmer ON with a phosphorothioate (PS) backbone directed against the repeat region of the huntingtin (HTT) gene downregulates HTT mRNA and protein without affecting the process of differentiation. In Paper II, anti-gene LNA/DNA mixmer clamp-type ONs, which hybridize to the target by forming both Watson-Crick (WC) and Hoogsteen hydrogen bonds, are optimized to achieve improved invasion into double stranded DNA. By positioning an intercalating moiety between or on the WC and Triplex Forming ON (TFO) arms, the AGOs achieve efficient invasion at nanomolar concentrationsin vitro. Furthermore, the corresponding PS modified ONs are tested in HD cell model for their effect on mRNA expression, where they cause significant downregulation of HTT mRNA. In Paper III, a non-B-DNA structure formed by a sequence in the MYC gene promoter is analyzed in vitro. Furthermore, the effect of this structure, more specifically an H-DNA, on the strand-invasion efficiency of LNA/DNA mixmer tail-clamp AGOs was evaluated. The results show that the invasion of the ON can be positively influenced by an H-DNA if the ON does not have an intercalating moiety.