title: Application of mutated miR-206 target sites enables skeletal muscle-specific silencing of transgene expression of cardiotropic AAV9 vectors.
abstract: Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206-mediated skeletal muscle-specific silencing of miR-206TS-bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1-mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3' portion of the miR-206, even enhanced the miR-206- mediated transgene repression. In vivo expression of m206TS-3G- and miR-122TS-containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs.
authors: Department of Applied Biochemistry, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors: Anja
authors: A
authors: Geisler
authors: Christian
authors: C
authors: Schön
authors: Tobias
authors: T
authors: Größl
authors: Sandra
authors: S
authors: Pinkert
authors: Elisabeth A
authors: EA
authors: Stein
authors: Jens
authors: J
authors: Kurreck
authors: Roland
authors: R
authors: Vetter
authors: Henry
authors: H
authors: Fechner
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000229:Dependovirus / Q000502:physiology*; D005786:Gene Expression Regulation; D023061:Gene Order; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics; D019076:Transgenes; D056189:Viral Tropism
chemical_list: C544547:MIRN1 microRNA, human; C546087:MIRN206 microRNA, human; D035683:MicroRNAs