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

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.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders

“…For example, incorporating binding sites for tissueor cell-type-specific endogenous micro-RNAs (miRNAs) in the transgene can de-target expression from a tissue and therefore reduce off-target transgene expression. [45][46][47][48][49] Finally, complementary DNA (cDNA) can be modified for higher transgene expression, such as codon optimization. However, codon optimization is most effective when accompanied by a reduction of CpG con- The PAGES epidemiological sample of individuals with autistic disorder was analyzed for rare structural variation (aneuploidies and damaging CNV) and for point mutations (damaging SNV and indels).…”

AAV Vector-Mediated Antibody Delivery (A-MAD) in the Central Nervous System

“…VHH expression can be driven within the cell-type of interest using specific promoters, overcoming issues such as (potential) toxicity resulting from off-target, multi-organ transgene overexpression, or deleterious immune responses resulting from the (undesired) transduction of antigen presenting cells (APCs) ( 166 , 167 ).…”

FSHD Therapeutic Strategies: What Will It Take to Get to Clinic?

“…These include: (1) removing immunogenic Cas9 epitopes (immunodominant epitopes have been removed from SpCas9 while preserving its cutting efficacy) [ 86 ], (2) interfering with processing and presentation of Cas9 antigens, (3) using orthologs from nonpathogenic bacteria, (4) inducing immune tolerance, and (5) transient immunosuppression [ 84 ]. miRNA-based detargeting has also emerged as a way to specifically repress expression of immunogenic transgenes in antigen-presenting cells by incorporating binding sites for macrophage-specific miRNAs into rAAV vectors [ 87 , 88 ].…”