Oligonucleotide therapies for RNA and DNA : modulation of natural splice-variants, DNA structure and characterization of new synthetic nucleotides and reporter cell lines

Oligonucleotide therapy is an evolving field having shown fast and important developments in the last years. From genetics to metabolic, inflammatory, immunodeficiency diseases, cancer and viral infections the medical applications for this type of therapy are becoming broader every day. However, the major challenge for these therapies is still the delivery; thus new chemistries, as well as improved delivery vectors are needed. Equally, the lack of relevant reporter systems hampers the characterization and progress of these emergent “drugs.”

This thesis work was aimed to address some of the gaps presented above. Thus, the introduction section starts with a brief overview of the gene therapy. It continues with an explanation of the oligonucleotide therapy strategies and technologies used for RNA and DNA targeting and ends with a clinical case for each approach. The methodology section explains the theoretical and practical aspects of the most relevant techniques in this study and the results section explain the rational and gives a brief presentation of the main results and conclusions for each paper.

Paper I presents a new splice-switching approach for treating diseases by regulating proteins at the splicing level. The new principle was to convert the normal splice form to a natural, but less abundant and inactive, splice variant to treat hypercholesterolemia. Paper II shows the development and characterization of a new collection of reporter cell lines for splice- switching. These reporter cell lines can serve as models for cell-type-specific screenings of different ON chemistries and delivery vectors. Paper III and IV demonstrate the development and characterization of a new nucleic acid chemical modification providing an oligomer with cell penetrating properties. Finally, in paper V a new mechanism for gene expression regulation at the genome level is presented.

List of scientific papers

I. RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form. Cristina S. J. Rocha, Oscar P. B. Wiklander, Lilian Larsson, Pedro M. D. Moreno, Paolo Parini, Karin E. Lundin, C.I. Evard Smith. Journal of Molecular and Cellular Cardiology. 2015: 82, 186-93
https://doi.org/10.1016/j.yjmcc.2015.03.009

II. Four novel splice-switch reporter cell lines: distinct impact of oligonucleotide chemistry and delivery vector on biological activity. Cristina S. J. Rocha, Karin E. Lundin, Mark A. Behlke, Rula Zain, Samir EL Andaloussi, C.I. Edvard Smith. Nucleic Acid Therapeutics. September 2016, ahead of print. [Accepted]
https://doi.org/10.1089/nat.2016.0631

III. Nuclease resistant oligonucleotides with cell penetrating properties. Stefan Milton, Dmytro Honcharenko, Cristina S. J. Rocha, Pedro M. D. Moreno, C. I. Edvard Smith and Roger Strömberg. Chemical Communications. 2015: 51(9), 4044-4047
https://doi.org/10.1039/c4cc08837a

IV. Fully and partially AECM-modified oligonucleotides. Synthesis and initial studies on cellular uptake and splice-switching activity in different reporter cell lines. Dmytro Honcharenko, Cristina S. J. Rocha, Jyotirmoy Maity, Ulf Tedebark, Stefan Milton, Rula Zain, C. I. Edvard Smith and Roger Strömberg. [Manuscript]

V. Disruption of higher order DNA structures in Friedreich’s ataxia (GAA)n repeats by PNA and LNA targeting. Helen Bergquist, Cristina S. J. Rocha, Rubén Álvarez-Asencio, Chi-Hung Ngyuen, Mark. W. Rutland, C. I. Edvard Smith, Liam Good, Peter E. Nielsen, Rula Zain. [Submitted]