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Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement

Abstract

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 (P79–98) 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 P79–98 –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.

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Fig. 1: Scheme of pDNA equipment with peptide and of plasmids used.
Fig. 2: Linkage of P78-98 to TFO.
Fig. 3: Triple helix formation between FAM-TFO and pDNA.
Fig. 4: Linkage of TFO-P79-98 to pGeneGrip and pDYS.
Fig. 5: In vivo transfection efficacy.

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Acknowledgements

We warmly thank “Association Française contre les Myopathies” (AFM, Evry, France), ‘Vaincre la mucoviscidose’ (VLM, France) and “Association Gaetan Saleun” (Brest, France).

Funding

This study was co-funded by “Association Française contre les Myopathies” (Strategic project ##20609) (AFM, Evry, France) and ‘Vaincre la mucoviscidose’ (project RF2016050182) (VLM, France). DM was co-founded by AFM and VLM. CG received a PhD fellowship from University of Orléans and Région Centre Val de Loire.

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CG, DM, and CG conducted the in vitro experiments and acquired data. YTG and KP conducted the in vivo experiments and acquired data. PM and TM analyzed data and wrote the manuscript. TM and PM obtained funding acquisition. All authors critically reviewed and approved the final manuscript.

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Correspondence to Tristan Montier or Patrick Midoux.

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Girardin, C., Maze, D., Gonçalves, C. et al. Selective attachment of a microtubule interacting peptide to plasmid DNA via a triplex forming oligonucleotide for transfection improvement. Gene Ther (2022). https://doi.org/10.1038/s41434-022-00354-1

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  • DOI: https://doi.org/10.1038/s41434-022-00354-1

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