A high-sensitivity assay for predicting human innate immune responses to rAAV vectors

Stanford researchers have developed a high-sensitivity cell-based assay for predicting the innate immune response to recombinant adeno-associated virus.

Recombinant adeno-associated virus (rAAV) has enabled the development of transformative gene therapies for disorders ranging from spinal muscular atrophy to hemophilia B. However, the use of rAAV is limited by its immunogenicity, which can result in reduced therapeutic efficacy as well as dangerous and even deadly reactions in patients. While rAAV engineering promises less immunogenic vectors, these efforts are hindered by a lack of tools for predicting immunogenicity pre-clinically. Animal models have very different immune systems from humans, while cell-based models are non-physiological and display poor immune responses to rAAV. Assays employing human peripheral blood mononuclear cells (PBMCs) are similarly limited by wide variability between donors and low sensitivity.

To address this challenge, Stanford researchers developed a new tool for predicting innate immune responses to rAAV. rAAV capsids are first opsonized (tagged for recognition by the immune system) with an anti-AAV monoclonal antibody and then incubated with human PBMCs. PBMC immune activation can be measured by cytokine production or the expression of T-cell activation markers. Researchers showed that opsonization enabled immune responses from otherwise unresponsive PBMCs and discrimination between closely related but differentially immunogenic rAAVs.

Stage of Development
In vitro: assay elicits an rAAV-induced immune response in otherwise unresponsive PBMCs that increases with exposure to unmethylated CpGs