Researchers have created a nanosensing platform capable of evaluating the quality of individual viral vector particles. Viral vectors have great potential in gene editing and therapies, but there is an urgent need for quality control methods to reduce potential side effects on patients. In response to this need, a team from Japan has developed a nanosensing technique that can distinguish between functional and defective viral vectors on a single-particle scale. This simple and cost-effective method could significantly enhance the progress of treatments for genetic disorders.
In recent decades, advancements in genetic manipulation technologies have progressed remarkably, bringing us closer to modifying genes in vivo. This development has the potential to unlock the possibilities of gene therapy and can herald a new chapter in medicine. Presently, the most promising gene therapy approaches utilize the existing molecular machinery found in viruses.
Adeno-associated virus (AAV) vectors, in particular, have captured considerable interest among scientists for their potential use as nucleic acid vaccines against diseases like COVID-19. However, during the manufacturing of AAV vectors, some particles may only have a partial genome while others might be completely empty. Such defective vectors can result in unintended side effects, highlighting the critical need for effective quality control measures during production.
To tackle this issue, a group of researchers from Japan has introduced an innovative nanosensing technique to analyze viral vector characteristics. Their recent study was published online on June 5, 2024, in ACS Nano. The research team includes Associate Professor Makusu Tsutsui and Professor Tomoji Kawai from the Institute of Scientific and Industrial Research at Osaka University; Lecturer Akihide Arima from the Institute of Nano-Life-Systems at Nagoya University; Specially Appointed Professor Yoshinobu Baba from the same institute, along with Project Researcher Mikako Wada and Assistant Professor Yuji Tsunekawa from the Institute of Medical Science at The University of Tokyo; and Professor Takashi Okada, who is also affiliated with the Institute of Medical Science at The University of Tokyo.
The proposed method involves measuring the ionic current passing through a nanopore when a voltage difference is applied to a solution containing AAVs. When the nanopore is clear, the ionic current remains stable. However, as a viral particle travels through the nanopore, it partially obstructs the flow of ions momentarily, creating a spike or pulse in the ionic current measurement.
Interestingly, full-genome AAV vectors are heavier and slightly bigger compared to empty or partially filled ones, making it possible to tell them apart when they pass through the nanopore. The faulty vectors create a distinct ‘signature’ in the measured ionic current that is easily distinguishable from that of full-genome vectors. The team confirmed this through various experiments, finite-element simulations, and theoretical analysis. “By designing an optimal sensor structure, we identified, for the first time, the sub-nanometer-scale differences in the size of viral vectors derived from genes,” Tsutsui explains.
This technique presents an easy and affordable way to ensure the quality of AAV vectors, which was previously reliant on more complicated methods such as mass photometry, transmission electron microscopy, and analytical ultracentrifugation. “This advancement could transform medicine by supplying a tool to fabricate AAV vectors of exceptionally high quality for safe and efficient gene therapy,” Tsunekawa emphasizes. “It could be essential for developing production and purification systems for AAV vectors,” he adds.
Furthermore, this technique not only applies to AAV vectors but also has potential for examining other types of viral vectors, potentially paving the way for effective gene therapy options and enhancing our understanding of viral biology. Ensuring the high quality of clinically used AAV vectors may also lead to lower dosages for patients, thus reducing side effects.
