Gene targeting and delivery of therapeutic oligonucleotides

Abstract: Research in gene therapeutic strategies involving oligonucleotides (ONs) constitutes a growing field with several clinical products already approved and many more under intense investigation. The recent advances have mainly involved the Antisense ON platform, with the aim of modulating gene expression on the RNA level. In contrast, targeting DNA using antigene ONs is a much less explored therapeutic option, but has been shown to be able to provide a potent alternative for the modulation of gene expression. Many genetic diseases originate in mutations in the genome, and by directly targeting these using anti-gene ONs could potentially have advantages over RNA based options, including lower dosage and reduced toxicity. In this thesis, several anti-gene therapeutic approaches involving ONs are presented, optimized and evaluated both in vitro and in cells. In Paper I, a novel approach for the generation of Zorro-LNA using click chemistry is developed, in order to join the two antigene ONs involved in a 3´-5´- 5´-3´orientation. This strategy replaces the use of reverse LNA phosphoroamidites and provides a screening platform suitable for optimizing new Zorro-LNA constructs. In paper II, single stranded ONs targeting the CAG-repeat region in the Huntingtin gene is used to down-regulate the mutant protein responsible for Huntington´s disease. The ONs are active in patient-derived fibroblasts and can efficiently reduce both mRNA and protein levels up to seven days following transfection and naked uptake delivery strategies. We also demonstrate, in different assays, that the mode of action is through ON binding to DNA, and not through RNA interactions. In paper III, we optimize different bisLNA anti-gene ON constructs for efficient DNA strand invasion using novel chemistries and intercalators. We demonstrate the selective binding through both Hoogsteen and Watson-Crick hydrogen bonding to supercoiled DNA in a physiological environment. In addition, the DNA binding is assessed in bacterial cells and detected using rolling circle amplification. Paper IV evaluates the specific delivery to cancer cells using aptamer-mediated uptake of LNA-containing ONs. The effect on the aptamer plasticity is investigated using chemical modifications, both in the cargo ON and in the aptamer itself, as well as the influence of the construct properties on cellular uptake in two different cell lines. Taken together, the results presented in this thesis aims at advancing the anti-gene based ON therapeutic strategies in terms of efficacy and specific delivery.