title: Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds.
abstract: Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells.
authors.affiliation: Department of Molecular and Cellular Genetics, UMR Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France; Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: Ilya
authors.initials: I
authors.lastname: Dovydenko
authors.affiliation: Department of Molecular and Cellular Genetics, UMR Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Tarassov
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: Alya
authors.initials: A
authors.lastname: Venyaminova
authors.affiliation: Department of Molecular and Cellular Genetics, UMR Génétique Moléculaire, Génomique, Microbiologie (GMGM), Strasbourg University - CNRS, Strasbourg 67084, France. Electronic address: n.entelis@unistra.fr.
authors.forename: Nina
authors.initials: N
authors.lastname: Entelis
mesh_terms: D004337:Drug Carriers; D006801:Humans; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D012313:RNA / Q000008:administration & dosage*
chemical_list: D004337:Drug Carriers; D012313:RNA
keywords: Antireplicative RNA; Cell delivery; Mitochondrial diseases; Mitochondrial drug delivery; RNA conjugates; RNA therapeutics