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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.

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Acknowledgements

We thank Luke Roode (Division of Molecular Pharmacy, UNC Eshelman School of Pharmacy) for his critical reading of this manuscript. We appreciate James Wilson (Gene Therapy Center, University of Pennsylvania, USA) and Guangping Gao (Gene Therapy Center, University of Massachusetts Medical School) for their generous gift of AAV9 packaging plasmid. This manuscript was supported by grant AR 56394, AR45967 (to X Xiao).

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Qiao, C., Yuan, Z., Li, J. et al. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther 18, 403–410 (2011). https://doi.org/10.1038/gt.2010.157

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