TESSA's helper-adenovirus system achieved a 10 to 30-fold increase in rAAV yield.Credit: PASIEKA/SPL/Getty Images
Gene therapies have been deployed in a growing list of therapeutic roles over the past decade, but producing the viruses that deliver them has not kept pace. “The challenge is how to manufacture large quantities of pure vectors at a reasonable cost,” says Leonard Seymour, professor of gene therapy at the University of Oxford.
To address this bottleneck, Seymour and colleagues developed a new way to manufacture rAAV using the ‘helper’ adenovirus technique. “We focused on helper-adenovirus production as it is relatively scalable,” says geneticist Ryan Cawood, chief scientific officer at OXGENE and WuXi Advanced Therapies, who led the discovery with Seymour. “They are also easy to manipulate, as demonstrated by the successful adenoviral COVID-19 vaccines.”
The drawback of conventional rAAV manufacturing is the risk of contamination if helper adenoviruses persist in the final product. The solution the team came up with is TESSA - tetracycline-enabled self-silencing adenovirus (Su, W. et al. Nature Comms 13, 1182 [2022]). TESSA represses itself after it has completed its helper function, effectively removing itself from the mix. The idea is a natural extension of the team’s work using viral ‘nanomachines’ for protein production. “For many years, we have been using viruses for therapeutic purposes,” says Seymour. “We realized that we could harness the power of one DNA virus to make another one. This is the principle we now call TESSA.”
The self-silencing adenovirus
In nature, adeno-associated viruses co-infect cells with an adenovirus. In the early phase of its life cycle, the adenovirus provides the machinery needed to generate new AAV particles; in its late phase, it produces structural proteins and assembles new copies of itself. The team hypothesized that if they could modify the adenovirus’s major late promoter (MLP) gene, they could truncate its life cycle while maintaining its helper functions.
They engineered an adenoviral vector to include a repressor protein (TetR) and its binding site (tetracycline operator, TetO) in the MLP. Adding these two elements means that once the MLP is active, it produces a repressing protein blocking the generation of new adenovirus particles. “By expressing TetR downstream of the tetracycline-sensitive element, the system turns itself completely off,” Seymour explains.
As well as scalability, the new approach has other advantages over alternative ‘helper-free’ rAAV manufacturing systems. “For all the serotypes tested, we demonstrated that the rAAV particles produced with the TESSA technology are better assembled,” says Cawood. The team achieved a 10 to 30-fold increase in rAAV yield, with particles 5- to 60-fold more infectious, and an almost complete absence of contaminating adenovirus.
The Oxford scientists went a step further, generating, for the first time, a stable vector containing the AAV rep and cap genes, known to be toxic to adenovirus. Combining this TESSA-RepCap with a second TESSA virus encoding the rAAV genome created TESSA2.0, which was able to increase the efficiency of manufacturing for difficult serotypes such as rAAV2.
By leveraging the advantages of a transfection-free helper system while sidestepping the downsides, rAAV manufacturing in the clinical and research fields can produce higher yields and superior quality. For Seymour, it’s a revolutionary combination: “TESSA technology will change everything.”