DNA nuclear targeting sequences for enhanced non-viral gene transfer: An in vitro and in vivo study

Abstract An important bottleneck for non-viral gene transfer commonly relates to translocation of nucleic acids into the nuclear compartment of target cells. So-called 3NF are optimized short nucleotide sequences able to interact with the transcription factor NFκB, which can enhance the nuclear import of plasmid DNA (pDNA) carrying such motifs. In this work, we first designed a consistent set of six pDNA featuring a common backbone and only varying in their 3NF sequences. These constructions were then transfected under various experimental settings. In vitro, cationic polymer-assisted pDNA delivery in five human-derived cell lines showed the potential advantage of 3NF carrying pDNA in diverse cellular contexts. In vivo, naked pDNA were hydrodynamically delivered to muscle hind limbs in healthy mice; this direct accurate comparative (in absence of any gene carrier) revealed modest but consistent trends in favor of the pDNA equipped with 3NF. In summary, the results reported emphasize the implications of various parameters on NFκB-mediated pDNA nuclear import; under specific conditions, 3NF can provide modest to substantial advantages for pDNA gene transfer, in vitro as well as in vivo. This study thus further underscores the potential of optimized nuclear import for more efficient non-viral gene transfer applications.