Reversible stabilization of DNA/PEI complexes by reducible click-linkage between DNA and polymer. A new polyplex concept for lowering polymer quantity.

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.

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

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.

CFTR and dystrophin encoding plasmids carrying both luciferase reporter gene, nuclear import specific sequences and triple helix sites.

Duchenne Muscular Dystrophy and Cystic Fibrosis are two major monogenetic diseases which could be treated by non-viral gene therapy. For this purpose, plasmid DNA (pDNA) coding for the functional genes requires its equipment with signal molecules favouring its intracellular trafficking and delivery in the nucleus of the target cells. Here, two novel constructions of large pDNAs encoding the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and full-length dystrophin (DYS) genes are reported. The expression of CFTR and DYS genes are driven respectively by the hCEF1 airway epithelial cells and spc5-12 muscle cells specific promoter. Those pDNAs encode also the luciferase reporter gene driven by the CMV promoter to evaluate gene delivery in animals by bioluminescence. In addition, oligopurine • oligopyrimidine sequences are inserted to enable equipment of pDNAs with peptides conjugated with a triple helix forming oligonucleotide (TFO). Furthermore, specific κB sequences are also inserted to promote their NFκB-mediated nuclear import. pDNA constructions are reported; transfection efficiency, tissue specific expression of CFTR and dystrophin in target cells, and triple helix formation are demonstrated. These plasmids are tools of interest to develop non-viral gene therapy of Cystic Fibrosis and Duchenne Muscular Dystrophy.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry.

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp. We show that the azido group of CEBA allows the linkage via click chemistry of CEBA-pDNA with a fluorophore or a peptide containing a dibenzocyclooctyne (DBCO) function. This new heterobifunctional reagent opens new ways to equip pDNA easily with signal molecules including peptides and nucleic acids without side products providing great interest for non-viral gene therapy.