Viable Viruses

Over the past 20 years, the use of viruses as gene therapy vehicles has exploded; though the rare diseases field has seen particularly promising breakthroughs, gene therapies are now being explored for a wider range of therapeutic areas. The success of any gene therapy is dependent on the accuracy of the genetic material underpinning the viral vectors; therefore, if the development and use of safe and efficacious viral gene therapies is going to be scaled to even more areas, researchers must be able to ensure vector quality during the discovery, design, and manufacturing. And that demands a complete and precise view of the viral genome.  

Perhaps the most actively investigated gene therapy research vehicle is the recombinant adeno-associated virus (rAAV), which can be engineered to deliver DNA to target cells. The rAAV genome is a single stranded DNA molecule of around 4.7 kb. rAAVs are a good option for gene therapies because they are non-pathogenic, meaning most individuals will not build an immune response that rejects the therapy by eliminating the virus or cells that have been successfully modified. Other advantages of rAAVs include their low toxicity, reduced host genome integration, and strong vector persistence. 

The challenge in rAAV gene therapy research stems from the difficulty in achieving the right level of vector quality. Researchers need to see the entire 4.7 kb length of the viral genome with a very high degree of accuracy to ensure they have confidence in any genetic changes they propose, while allowing them to discover truncated products and chimaeras. However, short-read and quantitative polymerase chain reaction (qPCR)-based methods of genomic sequencing most commonly used today typically miss these important changes because the results they produce are limited to only 50-300 bases – significantly less than the 4,700 bases that make up an rAAV strand.

The positive news for researchers is that recent landmark developments in long-read genomic sequencing have delivered next generation technologies capable of delivering much deeper insights into rAAV genomes. For example, third generation single molecule, real-time sequencing allows researchers to comprehensively sequence the entire 4.7 kb rAAV genome in a single long-read – crucially, without breaking the genome into smaller pieces. And because molecular integrity is maintained, difficult to detect variations are more likely to be revealed. In contrast, qPCR methods can only confirm the size of the AAV molecule or provide short-reads that must be stitched together.

We’re already seeing long-read sequencing redefining the quality control standards of viral vector preparations; for example, in a study published in Cell, researchers from the University of Massachusetts Medical School used long-read sequencing to reveal impurities that were missed by short-read methods. The long-read technology identified sequences originating from the plasmid backbone and helper plasmids, showing that vector populations contained between 1.3 and 2.3 percent of unexpected material that affected the quality of the plasmids. The discovery highlights the potential degree of foreign products in rAAV-based therapeutic vectors that can be exposed with long-read sequencing. 

Third-generation sequencing is also being used to identify host integration events, which have been associated with tumorigenesis in mice. In one example, researchers exploring liver cells employed long-reads to uncover chromosomal integrations at a surprisingly high frequency of 1–3 percent both in vitro and in vivo. I believe such depth of knowledge is key to understanding all potential safety risks in new gene therapy research.

Advancements in genomic technologies mean rAAV research is being applied faster and further than ever to a myriad of conditions. The scaling of gene therapy research has the potential to reshape the therapeutic landscape, but there are still technical challenges that must be overcome to ensure safe and effective products. In my view, third generation long-read sequencing will be at the center of the new era of rAAV research, affording scientists complete and accurate insights into viral genomes to power their studies. And as long-read sequencing continues to become even faster and more accessible, I expect the field to produce more effective – and safer – gene therapies for an increasingly broad range of diseases.