title: Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver.
abstract: Vectors based on adeno-associated virus (AAV) are effective in gene delivery in vivo. Tissue-specific gene expression is often needed to minimize ectopic expression in unintended cells and undesirable consequences. Here, we investigated whether incorporation of target sequences of tissue-specific microRNA (miRNA) into AAV vectors could inhibit ectopic expression in tissues such as the liver and hematopoietic cells. First we inserted liver-specific miR-122 target sequences (miR-122T) into the 3'-untranslated region (UTR) of a number of AAV vectors. After intravenous delivery in mice, we found that five copies of the 20mer miR-122T reduced liver expression of luciferase by 50-fold and β-galactosidase (LacZ) by 70-fold. Five copies of miR-122T also reduced mRNA levels of a secretable protein (myostatin propeptide) from the AAV vector plasmid by 23-fold in the liver. However, gene expression in other tissues, including the heart was not inhibited. Similarly, we inserted four copies of miR-142-3pT or miR-142-5pT, both hematopoietic lineage-specific, into the 3'-UTR of the AAV-luciferase vector. We wished to see whether they could prolong transgene expression by inhibiting expression in antigen-presenting cells. However, in vivo luciferase gene expression in major tissues declined with time, regardless of the miR-142 target sequences used. Quantitative analysis of the vector DNA in various tissues revealed that the decline of transgene expression in vivo was mainly because of promoter shut-off other than loss of AAV-transduced cells by immune destruction. Moreover, transgene expression was not detected in circulating mononuclear cells after delivering AAV9 vector with or without miR142T. These results demonstrate that liver-specific miR-122 target sequence in AAV vectors was highly efficient in reducing liver expression, whereas hematopoietic miR-142 target sequences were ineffective in preventing decline of AAV vector gene expression in nonhematopoietic tissues resulted from promoter shut-off.
authors.affiliation: Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. cqiao@email.unc.edu
authors.forename: C
authors.initials: C
authors.lastname: Qiao
authors.forename: Z
authors.initials: Z
authors.lastname: Yuan
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: B
authors.initials: B
authors.lastname: He
authors.forename: H
authors.initials: H
authors.lastname: Zheng
authors.forename: C
authors.initials: C
authors.lastname: Mayer
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: X
authors.initials: X
authors.lastname: Xiao
mesh_terms: D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008099:Liver / Q000378:metabolism*; D008156:Luciferases / Q000235:genetics; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D055435:Myostatin / Q000378:metabolism; D009928:Organ Specificity; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: C531907:MIRN122 microRNA, human; D035683:MicroRNAs; C529102:Mirn142 microRNA, mouse; C525912:Mstn protein, mouse; D055435:Myostatin; D008156:Luciferases; D001616:beta-Galactosidase