Abstract
The field of nucleic acid mediated gene silencing has been reinvigorated with the widespread adoption of RNA interference using siRNA. Since issues of delivery, stability, and duration of effect remain relevant to siRNA, and all other types of gene silencing nucleic acids, we are studying chemical modifications that may enhance the efficiency of molecules employed for this purpose. To this end, we synthesized 2′-deoxy-2′-fluoro-d-arabinonucleic acid (2′F-ANA) modifications of DNA as our prior work suggested that they would simultaneously raise the Tm of mRNA:DNA hybrids, increase resistance to nucleases, and very importantly, still permit RNaseH binding and catalysis of the target mRNA because the 2′F-ANA of the arabinose sugar ring projects, like DNA, into the major groove of the helix. Accordingly, we compared the gene silencing efficacy of 2′F-ANA modified oligonucleotides (ON) with traditional phosphorothioate (PS) antisense oligodeoxynucleotides (AS ODN) with respect to cellular delivery, intracellular stability, dose response, and duration of gene silencing in living myeloid leukemia cells. 2′F-ANA modification were made to form either “altimers” where triplets of nucleotides with F-ANA modified sugars alternated with triplets of nucleotides with deoxyribose sugars, or “gapmers” where 7, 2′fluorinated oligonucleotides flank 7 central unmodified nucleotides. The mRNA target for these comparative studies was a region within the c-myb mRNA that was previously shown by us to be accessible for hybridization in vivo (Nucleic Acids Res.32:5791, 2004). The nucleic acids were delivered by nucleofection into K562 cells. When analyzed at 24 hours, the PS ODN and 2′F-ANA ONs demonstrated an equivalent ability to silence c-myb mRNA and protein expression (>90% compared to untreated controls). Of significant interest however, the silencing effect of the 2′F-ANA ONs was still demonstrable 96 hours post nucleoporation whereas the PS ODN lost activity after 48 hours. Further, at doses where the traditional PS ODN had no silencing effect on c-myb mRNA or protein expression (1 μg/106 cells), the 2′F-ANA ON still gave >80% suppression of the target mRNA and protein. These effects were not dependent on delivery, which appeared to be equivalent for the two chemistries. Rather, when intracellular levels of delivered material were measured by semi-quantitative slot blotting, it was shown that intracellular levels of PS ODN declined rapidly after 24 hours, whereas ~90% of the 2′F-ANA introduced into the cells was still detectable 96 hours post nucleoporation. Whether this is due to relative inability to export the 2′F-ANA out of the cell, and or diminished intracellular degradation is being investigated. Although our primary results suggested that PS ODN are more rapidly degradated in cell lysate compared to 2′F-ANA ON. Therefore, our data suggest that 2′F-ANA AS ODN are efficient gene silencing molecules with advantages over PS ODN. These include significantly greater potency, and a duration of effect that is 3-4 times longer after a single dose. These findings suggest that appropriately targeted 2′F-ANA ON, in the form of single stranded antisense molecules, or siRNA, could well prove therapeutically useful for the treatment of cancer, and in other diseases where gene silencing is expected to beneficial.
Human and Canine Codon Optimization of Measles and Canine Distemper Viral Nucleic Acid Sequences Increases Sequence Identity and Protein Expression Compared to Wild Type and Human Codon Sub‐Optimized Constructs