Adeno-associated virus (AAV)-derived vectors are now among the most effective tools in gene therapy, to the point that several treatments have obtained regulatory approval, including onasemnogene abeparvovec, administered to thousands of patients with spinal muscular atrophy. Despite a relatively modest inflammatory profile, these vectors can nonetheless activate the immune system, not only against the viral capsid but also against the transgene product itself. Such a response compromises therapeutic efficacy, through neutralization of the circulating protein or rejection of transduced cells. Attention to this phenomenon has increased following the occurrence of serious adverse events — cytotoxic myositis and myocarditis — in young patients with Duchenne muscular dystrophy treated with vectors encoding a microdystrophin. Yet the early events that trigger CD8+ T-cell activation remained poorly defined.
To characterize these kinetics, the authors investigated antigen presentation following intramuscular administration of AAV in mice, using a model system expressing the SIINFEKL epitope together with adoptive transfer of transgenic (OT-I) CD8+ T cells. Viral genomes were detected in the draining lymph nodes as early as one hour after injection. Transgene-derived peptides began to be presented to CD8+ T cells from the first day onward, leading to progressive activation and the first cell divisions by day four. Ablation of the injection site demonstrated that AAV particles reaching the draining lymph nodes during the first hour were sufficient to induce transgene-specific cytotoxic CD8+ T cells, highlighting the decisive role of this very early phase.
The team then compared several vectors carrying muscle-specific promoters and regulatory sequences (ACTA1, desmin, MHCK7, SPc5-12). Although described as muscle-specific, all displayed variable transgene expression in dendritic cells in vitro, correlating with the intensity of lymphocyte activation in vivo: promoters associated with high early antigen presentation induced a robust T-cell response, whereas reduced presentation was accompanied by the absence of CD8+ T cells. This work thus shows that promoter specificity modulates leaky expression in antigen-presenting cells and shapes the cytotoxic response.
The study was conducted in female mice, with the main findings confirmed in males. The authors specify that it focused on local administration, the reference system for studying immunogenicity, while noting that they observed comparable early presentation upon systemic administration with clinically relevant serotypes. They suggest that incorporating assays that quantify these initial antigen presentation events in antigen-presenting cells into the assessment of AAV vector immunogenicity would be a useful contribution to gene therapy design.