Gene Therapy is one of the most recent and fast-growing fields of medicine, which promises to change the way we approach and treat different diseases, including many which were considered incurable even a decade ago. Recombinant adeno-associated virus (rAAV) is the most commonly used vector for therapeutic gene/RNA delivery because of its safety profile, limited immunogenicity and broad tissue tropism. Despite being the least immunogenic therapeutic viral vector AAV can evoke anti-drug antibodies (ADAs), which may be either pre-existing or developed after the onset of treatment. The absence of ADAs, especially neutralizing antibodies (NAbs), is often a prerequisite for enrolment in gene therapy trials, therefore, developing sensitive and robust assays for these antibodies is crucial.
We discuss the process of developing and validating tests for detecting ADAs and NAbs. Given that the host immune response may be triggered by the AAV vector capsid, the therapeutic gene/ RNA, and the translated protein, a multi-tiered approach is required. There are numerous challenges in developing ADA tests. These include: selection of the optimal methodology, choice of cell line for NAb assays, determining the appropriate cut-off point, and utilizing appropriate positive controls. Here we share our experience in overcoming these challenges and set out best-practice criteria.