BioCensors: Parsing Out the Genetic Payload of Viruses by Differential Biorecognition of Capsid Features

Gene therapies are poised to revolutionize medicine by addressing diseases previously deemed incurable. Viral vectors that deliver therapeutic genes to target organs are central to these therapies. However, current production systems to generate functional vectors also generate by-products such as empty or incorrectly filled viruses. Detecting these defective particles is critical for ensuring the efficacy and safety of gene therapies. However, available analytical tools are labor-intensive and costly. This project leverages the discovery that the surface structure of a virus depends on the genetic material it carries. The project will use molecular fingers called "ligands" that are engineered to bind to the surface of functional viruses differently than they bind to defective ones. This binding will be analyzed using label-free biosensing technologies with high sensitivity and accuracy. This research has three aims: first, to study ligand interactions with full and empty viruses; second, to integrate these ligands with Extended-Gate Field Effect Transistor (EGFET) biosensors, creating an "electrochemical dictionary" for interpreting the ligand-to-virus binding events; and third, to validate this technology with therapeutic viruses. This approach could be extended to detecting disease-causing viruses and engineering sensors for environmental or industrial applications. Biotech and biopharma communities will be engaged through professional presentations, academic meetings, and student involvement, fostering the adoption of this biosensing technology in research and industry. Quantifying viral titer and gene loading is critical for assessing viral infectivity and ensuring the safety and efficacy of gene therapies utilizing viral vectors to deliver therapeutic transgenes. Recent findings on the relationships between viral capsid biomolecular features, genetic payloads, and virion transduction activity have significant implications for viral vector manufacturing and diagnostic analysis. Adeno-Associated Viruses (AAVs) exemplify this potential, as their capsid surface signals the presence of therapeutic genes. However, AAV production often yields heterogeneous mixtures, including empty and misloaded capsids that can induce genotoxicity and immunogenicity. Current analytical assays for quantifying the titers of capsid, encapsulated transgenes, and infectious units are laborious and inaccurate, causing delays and uncertainty in treating patients. To address these challenges, this project will develop "Bio-Censors" that use affinity ligands recognizing capsid features specific to gene-loaded versus empty AAVs to simultaneously quantify capsid and transgene titers. The research approach integrates peptide ligands with pH-controlled affinity for AAVs, microfluidic devices for pH tuning, and multiplexed EGFET biosensors. In Aim 1, peptide ligands' differential recognition of AAV capsids using surface plasmon resonance-electrochemical impedance spectroscopy will be investigated. Aim 2 focuses on engineering EGFET Bio-Censors to enhance detection and discrimination. In Aim 3, the Bio-Censors’ performance will be validated with therapeutic AAVs in complex fluids. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.