Nucleic acid based therapeutic approaches

Abstract: Nucleic acid therapy comprises several approaches based on nucleic acid as the active therapeutic component to treat human disease. Antisense and short interfering RNA (siRNA) are two gene-silencing techniques in this category. Based on nucleic acid sequence recognition both techniques inhibit gene expression. This is highly attractive whenever there is deleterious expression of genetic material, as for example in viral diseases or cancer. in many of these diseases effective treatments are limited and/or toxic; therefore there is a need for new alternative therapeutics. We aimed to improve these genesilencing techniques to facilitate future therapeutic application. We have made efforts in facilitating the use of viral vectors for nucleic acid delivery, but in particular, we have used the synthetic nucleotide analogue locked nucleic acid (LNA) in both antisense oligonucleotides and siRNA. As therapeutic targets we inhibited the human pathogens human immunodeficiency virus type 1 (H IV-1) and severe acute respiratory syndromeassociated corona virus (SARS-CoV) in cell culture. The results demonstrate that LNA is advantageous in both antisense and siRNA techniques. Starting with delivery, as one major obstacle for nucleic acid based therapeutics, we aimed to facilitate the use of recombinant adeno-accosiated virus (AAV). In certain applications viruses can serve as suitable delivery vehicles for gene-silencing techniques. AAV has favourable properties for use as delivery vehicle, however there has been limited use of AAV due to production difficulties. We developed an alternative method for production that utilized baculoviruses for transfer of needed helper genes. We showed expression of the helper genes from the baculovirus and production of infectious recombinant AAV. The protocol may lead to improved AAV production and facilitate increased use of recombinant AAV. Continuing with the development of synthetic antisense oligonucleotides, we targeted the conserved HIV-1 dimerization initiation site with LNA modified antisense oligonucleotides, LNA/DNA mix-mers. We showed improved inhibition of dimerization by using LNA/DNA mix-mers and activation of RNase H by LNA/DNA mix-mers containing at least six consecutive DNA bases. We subsequently demonstrated inhibition of HIV-1 replication. LNA improves the antisense oligonuleotides and can function on a therapeutic target. Using the more recently discovered genesilencing technique siRNA, we targeted the SARS-CoV RNA dependent RNA polymerase. The siRNA inhibited SARSCoV replication both when delivered pre and post infection. This study shows an example of how genetic information on an emerging pathogen can rapidly be converted to an antiviral tool by the means of siRNA. Finally, we combined LIMA with siRNA to a compound we term siLNA. We showed siLNA compatibility with the siRNA machinery in association with higher nuclease resistance and enhanced strand bias. siLNA provides a possibility to reduce undesired, off-target effects of siRNA. We also applied siLNA to inhibit SARS-CoV and showed improved efficiency over unmodified siRNA on certain target sites. LNA brings many favourable features to siRNA beneficial to future therapeutic use. Taken together, these studies improve certain technical aspects of gene-silencing techniques and show potential applications of nucleic acid based therapeutics.