Evaluating Neutralizing Antibodies Against AAVs Using a High-Sensitivity LacZ Reporter Assay

Background & Aim: Adeno-associated viruses (AAVs) have become a critical tool for delivering therapeutic genes in gene therapy applications. However, the presence of pre-existing AAV neutralizing antibodies (nAbs) in patients poses a significant risk by reducing the efficacy and safety of AAV-based therapies. Furthermore, the immune response following administration of AAV gene therapy products (DTP) can generate new neutralizing antibodies, complicating long-term treatment outcomes. Therefore, robust methods to measure the levels of nAbs in patients before and after therapy are essential for clinical success. Methodology: We have developed a novel LacZ reporter system tailored for multiple AAV serotypes, which enables sensitive and efficient detection of AAV neutralizing antibodies using a cell-based assay. Unlike traditional approaches requiring multi-step protocols and DNA extraction, this single-step method directly quantifies AAV-driven LacZ activity in cell lysates, offering a streamlined workflow with minimal handling and broad dynamic range. The signal remains stable for over one hour, ensuring consistent and reproducible readouts. Results: This innovative assay offers a powerful and reliable platform for assessing the immunogenicity of AAV-based gene therapies, providing a rapid and streamlined method for detecting neutralizing antibodies. Conclusion: Continued development of such advanced assay platforms is crucial for addressing key challenges in the field, supporting therapeutic research, and ensuring safer and more effective outcomes for patients undergoing AAV-based gene therapy.