GENE THERAPY SOLUTIONS

TESSA: SCALABLE AAV
PRODUCTION

Our proprietary self-silencing adenoviral vectors deliver high titre, high quality AAV to gene therapy pioneers. We engineer TESSA™ vectors to encode your gene or capsid of interest for high quality manufacture of AAV seed stocks. before process development and rAAV manufacture at any grade or scale.

"Overall, TESSA™ enables us to test new constructs much faster and cheaper now which is why we like to keep using it."

Dr Harald Hartweger, The Rockefeller University

Tetracycline enabled self silencing adenovirus (TESSA™) technology

TESSA™ vectors deliver a step-change in AAV manufacture; an easily scalable, contaminant-, chemical- and transfection free technology.

TESSA™ vectors take advantage of the adenoviral lifecycle, which contains two temporal phases; early and late. The early phase provides all the adenoviral help needed for high fidelity, high titre AAV manufacture. The late phase leads to adenovirus contamination. We’ve engineered supremely tight regulation of the adenoviral late region, which is wholly responsible for producing adenovirus structural proteins. Using TESSA™ technology, full early region expression, which provides all the help needed for AAV production, is still enabled, but the late region gene expression is turned off, meaning there is no adenoviral structural protein production. This represses adenovirus production by 99.99999 -100% during an AAV manufacturing run, while increasing rAAV yield, packaging efficiency and infectivity compared to both Ad5 helper plasmid or wild-type adenovirus.

Stable integration of AAV Rep and AAV Cap into the TESSA™ vector allows efficient and simultaneous delivery of all the necessary components for AAV replication in a single agent.

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All of this [TESSA + plasmid vs TESSA + AAV] was way better than helper free triple transfection in the same experiment, which gave us 26-180 times less yield depending on what you compare.

How it works

There are two stages to AAV manufacture with TESSA™. The first is to produce initial AAV seed stock. This can be done by plasmid transfection, for example with our transient AAV system, with a second TESSA™ vector encoding the AAV genome and gene of interest (a process we call TESSA 2.0 and which gives more efficient delivery than transfection), or any alternative existing approach.

Overall, producing AAV2 using two TESSA™ vectors gives approximately a 40-fold increase
in AAV particle yields compared to the helper free system. We also observe a >2400 fold cumulative increase in AAV2 infectious yield (1 in 6 genome-containing AAV2 particles is infectious from TESSA, compared to 1 in 1200 from plasmid based transfection; see data below), and an increased percentage of full capsids from 2-5% up to 70%.

In the second stage, called TESSA Pro, this AAV seed stock and another TESSA-AAV-Rep-Cap vector can be used to co-infect HEK293 cells. This leads to further AAV replication, so increasing the final AAV yield. Interestingly, whether you use AAV seed stock generated using two TESSA vectors or three plasmid transfection, the final AAV yield is comparable (see data below).

Advantages

TESSA™ technology is fully scalable and significantly reduces the number of input materials required for AAV manufacture. It’s ideally suited for rapid and continuous production of GMP quality AAV, reducing the potential for batch to batch variation. Ultimately, TESSA™ technology will result in improved cost of goods, and improved safety of gene therapies.

Adenoviral structural genes are regulated by negative feedback loop

A Tet repressor (TetR) binding site is inserted into the Major late promoter, while the TetR gene itself is encoded within the late region. This creates a negative feedback loop, where adenoviral structural proteins can only be produced in the presence of doxycycline.

Using two TESSA™ vectors improves rAAV yield and infectivity

Left: using two TESSA vectors (blue) to produce rAAV results in a 42-fold improvement in rAAV2 yield compared to the helper free system (grey). Right: rAAV2 produced using two TESSA vectors (blue) is also significantly more infectious than rAAV2 produced using the helper free system (grey) by TICD50. In fact, 1 in 6 rAAV2 particles containing a genome made from TESSA vectors is infectious compared to 1 in 1200 from plasmids (grey).

Adding TESSA™ to rAAV seed stock continues rAAV propagation

Two viruses in, one virus out: The initial AAV seed stock can be produced from either a normal 3-plasmid transient system, a TESSA™ vector plus gene of interest (GOI) plasmid, or two TESSA™ vectors (right). Once the initial AAV seed stock has been produced, this can be harvested and used alongside more TESSA™ vector to co-infect cells and produce more of the AAV. This step could be repeated as part of a continuous manufacturing process.

Source of rAAV seed stock does not affect final yield

Left: Despite giving a higher initial yield (column 3, blue) than the helper free system (column 5, grey), when that rAAV seed stock is used to co-infect HEK293 cells with a second TESSA vector, the resulting rAAV2 yields are comparable (columns 1 and 2, blue). Right: However, while still higher than with helper free production (grey), infectious AAV2 yields are slightly lower after a second round of rAAV2 production (columns 1 and 2, vs column 3).

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WuXi Advanced Therapies is a global Contract Testing, Development
and Manufacturing Organization (CTDMO) dedicated to accelerating
progress and time to market for cell and gene therapies.

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