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Nonviral transfection of mammalian cells can be performed with electrostatic complexes (polyplexes) between a plasmid DNA (pDNA) encoding a foreign gene and a cationic polymer. However, an excess of the cationic polymer is required for pDNA condensation and polyplexes formation, which generate in vivo toxicity. Here, we present a new concept of polyplexes preparation aiming to reduce the polymer quantity. pDNA was functionalized with 3,6,9-trioxaundecan-1- {4 - [(2-chloroethyl) ethylamino)] - benzylamino}, 11-azide, and polyethyleneimine (lPEI) with reducible dibenzocyclooctyl (SS-DBCO) groups allowing azide-alkyne cycloaddition between pDNA and lPEI after condensation. The size of polyplexes with DBCO-SS-lPEI was smaller than with lPEI due to a stronger DNA condensation thanks to linkages between polymer and pDNA preventing dissociation until disulfide bridges reduction. In vitro transfection showed that the amount of DBCO-SS-lPEI leading to the most efficient polyplexes was three times lower than lPEI. As expected, toxicity in mice was significantly reduced upon intravenous injection of DBCO-SS-lPEI polyplexes at doses where the lPEI polyplexes killed mice. This is probably due to the high stability of the DBCO-SS-lPEI polyplexes which prevented their aggregation in the pulmonary capillaries. Overall, this new concept of polyplexes with DBCO-SS-lPEI offering the possibility of administering higher doses of polyplexes than lPEI and their ability to pass the pulmonary barrier could be favorably exploited for transfection of distant organs or tissues, such as tumors., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

In nonviral gene therapy approaches, the linkage of signal molecules to plasmid DNA (pDNA) is of interest for guiding its delivery to the nucleus. Here, we report its linkage to a peptide (P 79-98 ) mediating migration on microtubules by using a triplex-forming oligonucleotide (TFO). pDNA of 5 kbp and 21 kbp containing 6 and 36 oligopurine • oligopyrimidine sites (TH), respectively, inserted outside the luciferase gene sequence were used. TFO with a dibenzocyclooctyl (DBCO) group in 3' end comprising some Bridged Nucleic Acid bases was conjugated by click chemistry with the peptide carrying an azide function in the C-terminal end. We found the formation of 6 and 18 triplex with pDNA of 5 kbp and 21 kbp, respectively. A twofold increase of the transfection efficiency was observed in the hind-limbs upon Hydrodynamic Limb Vein (HLV) injection in mice of naked P 79-98 -pDNA of 21 kbp. This work paves the way for the selective equipping of pDNA with intracellular targeting molecules while preserving the full expression of the encoded gene., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)