Novel Combinatorial MicroRNA-Binding Sites in AAV Vectors Synergistically Diminish Antigen Presentation and Transgene Immunity for Efficient and Stable Transduction.

Ishani Dasgupta,Manish Muhuri,Jennifer Chen,Phillip W L Tai,Jia Li,Yukiko Maeda,Ran He,Guangping Gao,Motahareh Arjomandnejad,Katelyn Sylvia,Anoushka Lotun,Wei Zhan,Sangeetha Manokaran,Qin Su,Thomas Nixon,Allison M Keeler

doi:10.3389/fimmu.2021.674242

Abstract

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.

Highlights

Adeno associated virus (AAV) vector-mediated gene therapies have emerged as the platforms of choice for the treatment of monogenic diseases
Two copies of binding sites for these miRNAs were individually cloned into the Recombinant AAVs (rAAVs) expression cassettes to generate a library of 26 vectors (Table 1)
Host immune responses against rAAV1-encoded transgene products after intramuscular delivery have been reported to cause the clearance of transduced fibers and loss of transgene expression [88,89,90,91]

Summary

Introduction

Adeno associated virus (AAV) vector-mediated gene therapies have emerged as the platforms of choice for the treatment of monogenic diseases. Recombinant AAVs (rAAVs) have been proven to confer long-lasting and safe transgene expression in a variety of human tissues [3,4,5,6]. AAV vectors are known to possess a weak immunological footprint, in part, because of their relative inability to transduce antigen presenting cells (APCs). Human clinical trials have demonstrated how B and T cell immune responses directed against the AAV capsid, likely arising after natural infection with wild-type AAV, might potentially impact gene transfer safety and efficacy in patients [3]. The quality and intensity of humoral and cellular immune responses can vary depending on the transgene DNA composition, vector tropism to APCs, route of administration, and the tissue target

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