Validated high throughput microneutralization assay for anti-AAV2 neutralizing antibodies (NAbs) in clinical trials in support of gene therapy 2247247

Abstract Introduction Recombinant adeno-associated (AAV) viral vectors are being widely used in gene therapy. One of the challenges for AAV-based therapy is the presence of pre-existing neutralizing antibodies (NAbs) that humans develop against AAV throughout life. These NAbs inhibit the binding of viral vector to the cells resulting in failure of therapeutic gene delivery. Evaluating pre-existing anti-AAV antibodies is required for many AAV-based clinical trials and approved gene therapies. CTL developed a novel anti-AAV2 NAb assay based on enumeration of viable cells infected with AAV2 carrying a fluorescent reporter. Methods Green Fluorescent Protein (GFP)-carrying AAV2 vector was used to transduce target cells. The vector was incubated with positive/negative controls or with test sample and then added to the target cells and GFP-fluorescent cells were counted thereafter. The percentage of neutralization was calculated based on the ratio between the number of GFP cells after incubation with and without the sample. A sample was considered positive for NAbs with a reduction of vector transduction greater or equal to 50%. Results CTL screened 78 human samples of three matrix types (CSF, Serum, Plasma) as well as samples from non-human primates, cats and pigs. Positive and negative samples were identified by anti-AAV2 NAb assay. Dilution starting points were defined to resolve the matrix effect observed during testing serum samples. Specificity of viable cell-based anti-AAV2 NAb assay was proven and the assay was validated. This assay showed sensitivity comparable to ELISA luciferase assay and can be used for testing NAb in different species. Conclusion CTL developed and validated a highly reliable and sensitive NAb assay with direct acquisition and enumeration of fluorescent AAV2-GFP transduced cells which allows high throughput evaluation in preclinical as well as clinical trials. The potential advantage of this assay is the ability to quantify the individual virus-infected cells without additional steps. Funding Source private (funded by Cellular Technology Limited) Topic Categories Technological Innovations in Immunology (TECH)