Osaka, Japan – Gene therapy is at the cutting edge of medicine, with exciting new products being developed and used to treat a wide variety of genetic diseases. However, one major roadblock to delivering gene therapy quickly is the time-consuming purification process.

Now, researchers from The University of Osaka have reported a new technique that could help accelerate this process: a new way to detect and quantify components of the ‘delivery system’ for therapeutic genes. The work, recently published in Analytical Chemistry, represents an exciting advance for gene therapy.

Recombinant adeno-associated virus (rAAV) is a promising vector for delivering therapeutic genes to patients with genetic disorders. Cells are first engineered to produce viral particles that contain the therapeutic gene. However, only some of these particles are ‘full’, i.e., containing the therapeutic gene, while other particles are ‘empty’ and do not. Optimizing the ratio of full to empty particles is critical for maximizing clinical efficacy.

“An important quality control step in producing these rAAV-based vectors is determining the number of viruses that contain a complete genome and the number that are empty,” says lead author, Yuki Yamaguchi. “While several methods can accurately distinguish between full and empty particles once the virus has been purified, it is a time-consuming step that slows production.”

The problem is that host-cell proteins get in the way when researchers are trying to distinguish between full and empty particles. These proteins, found in the cell lysate and the conditioned medium, need to be removed, and this is what takes time.

To develop an efficient way of quantifying full versus empty particles, without the need for purification, a technique called mass photometry was used. This technique detects whether substances bind to a glass surface. The study tested the binding activity of full and empty viral particles within rAAVs, cell lysate, and the conditioned medium.

“The results were very promising,” explains Susumu Uchiyama, senior author. “We found that the viral particles, which deliver the therapy, were mostly unbound, while most of the host-cell proteins in the cell lysate and the conditioned medium were bound. In this way, the mass photometer was able to distinguish between the two types of molecules in a way that was quicker and just as accurate as conventional methods.”

Given that the full-to-empty particle ratio is a key measure of rAAV-based gene therapy’s quality and clinical effectiveness, this new technique is expected to improve rAAV production. The technique described here should speed up the manufacturing process, with positive effects for patients in need of genetic therapy.

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The article, “Direct Identification and Quantification of Recombinant Adeno-associated Virus in Crude Cell Lysate and Conditioned Medium by Mass Photometry,” is published in Analytical Chemistry. DOI: https://doi.org/10.1021/acs.analchem.5c00793 . About The University of Osaka
The University of Osaka was founded in 1931 as one of the seven imperial universities of Japan and is now one of Japan's leading comprehensive universities with a broad disciplinary spectrum. This strength is coupled with a singular drive for innovation that extends throughout the scientific process, from fundamental research to the creation of applied technology with positive economic impacts. Its commitment to innovation has been recognized in Japan and around the world, being named Japan's most innovative university in 2015 (Reuters 2015 Top 100) and one of the most innovative institutions in the world in 2017 (Innovative Universities and the Nature Index Innovation 2017). Now, Osaka University is leveraging its role as a Designated National University Corporation selected by the Ministry of Education, Culture, Sports, Science and Technology to contribute to innovation for human welfare, sustainable development of society, and social transformation.
Website: https://resou.osaka-u.ac.jp/en