UMMS Affiliation

Horae Gene Therapy Center; Department of Microbiology and Physiological Systems; VIDE Program; Department of Medicine; Department of Pediatrics; Li Weibo Institute for Rare Diseases Research

Publication Date


Document Type



Hemic and Immune Systems | Immunity | Immunology of Infectious Disease | Nucleic Acids, Nucleotides, and Nucleosides


Recombinant adeno-associated virus (rAAV) platforms hold promise for in vivo gene therapy but are undermined by the undesirable transduction of antigen presenting cells (APCs), which in turn can trigger host immunity towards rAAV-expressed transgene products. In light of recent adverse events in patients receiving high systemic AAV vector doses that were speculated to be related to host immune responses, development of strategies to mute innate and adaptive immunity is imperative. The use of miRNA binding sites (miR-BSs) to confer endogenous miRNA-mediated regulation to detarget transgene expression from APCs has shown promise for reducing transgene immunity. Studies have shown that designing miR-142BSs into rAAV1 vectors were able to repress costimulatory signals in dendritic cells (DCs), blunt the cytotoxic T cell response, and attenuate clearance of transduced muscle cells in mice to allow sustained transgene expression in myofibers with negligible anti-transgene IgG production. In this study, we screened individual and combinatorial miR-BS designs against 26 miRNAs that are abundantly expressed in APCs, but not in skeletal muscle. The highly immunogenic ovalbumin (OVA) transgene was used as a proxy for foreign antigens. In vitro screening in myoblasts, mouse DCs, and macrophages revealed that the combination of miR-142BS and miR-652-5pBS strongly mutes transgene expression in APCs but maintains high myoblast and myocyte expression. Importantly, rAAV1 vectors carrying this novel miR-142/652-5pBS cassette achieve higher transgene levels following intramuscular injections in mice than previous detargeting designs. The cassette strongly inhibits cytotoxic CTL activation and suppresses the Th17 response in vivo. Our approach, thus, advances the efficiency of miRNA-mediated detargeting to achieve synergistic reduction of transgene-specific immune responses and the development of safe and efficient delivery vehicles for gene therapy.


adeno-associated virus vectors, antigen presenting cells, gene therapy, miR-142, miR-223-3p, miR-652-5p, miR-BS, microRNA

Rights and Permissions

Copyright © 2021 Muhuri, Zhan, Maeda, Li, Lotun, Chen, Sylvia, Dasgupta, Arjomandnejad, Nixon, Keeler, Manokaran, He, Su, Tai and Gao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

DOI of Published Version



Muhuri M, Zhan W, Maeda Y, Li J, Lotun A, Chen J, Sylvia K, Dasgupta I, Arjomandnejad M, Nixon T, Keeler AM, Manokaran S, He R, Su Q, Tai PWL, Gao G. Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction. Front Immunol. 2021 Apr 28;12:674242. doi: 10.3389/fimmu.2021.674242. PMID: 33995418; PMCID: PMC8113644. Link to article on publisher's site

Journal/Book/Conference Title

Frontiers in immunology

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Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.