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.

E Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal—Tailored Acceleration of AAV Evolution

“…103,104 Another robust strategy acting on the level of specificity is de-targeting of AAV gene expression from unwanted off-target cells through the incorporation of binding sites for miRNAs (small, endogenously expressed, regulatory RNAs that inhibit gene expression) that are absent in on-target cells but abundant in off-target cells. 105,106 Altogether, we thus believe it is fair and warranted to consider AAV as the most flexible, most exciting, and most auspicious DNA and protein toolbox for generation of optimized vectors for human gene therapy that we have at our disposal to date.…”

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

“…82 Such a tissue-specific microRNA-based detargeting strategy can be used to silence transgene expression in the skeletal muscle in addition to the liver, to achieve relatively pure cardiac specificity. 83 Different types of inducible transcription systems, in which transgene expression is modulated by small molecules, have been engineered into the AAV backbone, essentially by adapting the systems broadly used in other vector platforms. 84 Currently, the 2 most considered methods to achieve pharmacologically inducible transcription are the tetracycline-inducible (TetOn) or tetracycline-repressible (TetOff) systems 85 and the method based on rapamycin-induced dimerization of FKBP12 (FK506 binding protein 12)-and mammalian target of rapamycin-derived proteins.…”

Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity

“…The use of miRNA-binding sites for cell type-specific detargeting to attenuate transgene immunogenicity was first described using lentiviral vectors to suppress transgene expression in different hematopoietic lineages (21,22). miRNA-mediated detargeting can also be used to assist tissue-specific expression by posttranscriptional control to restrict spuriously transcribed transgenes that result from the wide transduction profiles of rAAVs (23)(24)(25)(26). Many miRNAs have been reported to be involved in the regulation and development of macrophages, DCs, and B cells (27)(28)(29).…”