In situ capsid protein and DNA imaging platform for the characterization of therapeutic adeno-associated virus (AAV) titers - Project Summary Adeno-associated virus (AAV) is the leading gene delivery vector for treating different human diseases. Some AAV therapies have been approved for use in the clinic and constitute an essential step toward personalized gene therapy. However, despite its promise to treat many genetic diseases, AAV gene therapy still face many safety challenges that lead to several adverse events, including death. In most cases, these adverse events were related to the use of AAVs with high vector genome (vg) titers (>1E14 vg/kg). Despite following a multi-step purification process, these AAV preparations are often contaminated with non-therapeutic AAV particles, such as empty or partial AAV capsids (lacking or containing truncated vg) and cross-packaged DNA. These contaminants can contribute to adverse events at high AAV vg titers even when present in lower ratios. Accordingly, the success of AAV therapies relies on the use of high-quality AAV capsids (qualified to package a high majority of full therapeutic AAV vg and minimal levels of contaminants) and on the precise quantification of AAV titers to ensure optimal dosage, efficacy and safety. The current analytical methods used to ensure the production of high-quality AAV are not comprehensive and thus lack the precision needed for the accurate and reproducible quantification of AAV vg, capsid and contaminant titers. These limitations result in a misestimation of the empty/partial/full AAV capsid ratio, leading to the administration of either low AAV vg titers with sub-optimal therapeutic efficacy or high AAV vg titers with toxic levels of contaminants, potentially causing severe adverse events or death in treated patients. Therefore, there is an unmet need for developing analytical methods that provide an efficient, cost-effective, and easy implementation for the comprehensive, accurate, and reliable quantification of AAV capsid and genomic titers and AAV integrity. In this proposal, we are set to develop a unique and groundbreaking QC platform that we named “AAVBiochip.” It will be an essential in situ single particle analytical tool to determine capsid and vg titers and the ratio of empty/partial/full AAV capsids in a single assay. We will immobilize AAVs on the platform surface by electrostatic interactions for rapid imaging and analysis. In Aim 1, we will demonstrate the feasibility of our technology by using well-characterized AAV titers for which the capsid proteins, conformational epitope (3D structure) of assembled capsids, and ITR genomic regions will be targeted using specific antibodies and sensitive molecular beacons. We will also compare our measurements with standard methods for quantifying AAV titers, ensuring the accuracy and reliability of our results. In Aim 2, We will conduct a comprehensive benchmarking comparison using different AAV serotypes approved by the FDA for gene therapies. AAVs will be engineered to carry the green fluorescence protein (GFP) payload. For the analytical characterization of these AAV capsids, a combination of standard analytical methods and the AAVBiochip will be used. Overall, our work will enable a comprehensive, ultrasensitive and accurate AAV characterization technology that will offer significant advantages as a QC tool for any AAV-based gene therapy.
