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Splice-correcting phosphorothioate RNA antisense oligonucleotides with 2′-O-methyl modifications (ASO) are promising therapeutic agents for several disorders caused by aberrant splicing. However, their usefulness is hindered by the lack of efficient delivery. Unmodified 25 kDa polyethylenimine (PEI) has shown potential for plasmid delivery but seems to be less efficient for short nucleic acid sequences. Herein, we have evaluated several amino acid modified PEI molecules as carriers for ASO. By characterization of their properties, such as size, stability and transfection into mammalian cells, we have identified tyrosine-modified PEI (PEIY) as an efficient ASO delivery system. HeLa705 cells containing an aberrant luciferase gene, interrupted by a mutated β-globin intron, were used to assess the splice correction effectiveness mediated by the various modified PEI/ASO polyplexes. PEIY has a self-assembly nature, as opposed to the highly cationic parent polymer, which is relevant for the stability of the PEIY/ASO complexes. As a result, at an optimal ratio of 20:1 (+/−), the complexes that formed significantly corrected the splicing on both the mRNA and the protein levels. ASO formulated with PEIY enhanced luciferase activity up to 450-fold. This increase was three times higher than that produced by the commercially available transfection agent Lipofectamine. PEIY/ASO polyplexes resulted in at least 80% correct splicing of the transcript. Moreover, extremely low doses of ASO (0.025 μM) showed significant splice correction represented by 150-fold increase of luciferase activity and 47% mRNA correction. Our findings suggest key parameters for formulating active complexes and reveal a new platform that can be further developed for ASO in vivotargeting.

Importance of the field:Gene therapy is regarded as one of the most promising therapeutic approaches, as it has the potential to treat disorders by correcting malformations at the nucleic acids.Areas covered in this review:Some of the most recent developments in the process of plasmid DNA vector design and formulation are reviewed with a special focus on: different formulations of nanovectors and a summary of successful cases reported; requirements for systemic administration; and functionalization of the nanocarriers by use of targeting entities.What the reader will gain:An understanding of the different physiological barriers and a comprehensive review of the recent strategies used to overcome these obstacles. Particular attention is given to formulations for intravenous administration, colloidal stability properties and different targeting entities used.Take home message:Overall, vector formulation must take into account the administration route and inherent physiological barriers. Critical parameters for the success of pDNA nanovectors are: particles size, colloidal stability of the formulation and interaction between the carrier and plasmid DNA. Highly relevant is the fact that this interaction should be balanced to offer protection to degradation as well as allow dissociation of the therapeutic nucleic acid for obtaining maximal activity.