Biologists based in the U.K. have engineered adeno-associated viruses (AAVs) to better fill their capsids and with the capability to improve manufacturing yields.
The team, from University College London (UCL), has tinkered with proteins that help package viral DNA into empty capsids, as well as proteins that help viruses leave the cells used to produce them.
According to Darren Nesbeth, PhD, a professor of engineering biology at UCL, “You can go pretty crazy in an engineering faculty as you’re not a clinician. My research opens doors and stimulates thoughts, rather than us having to generate immediate efficacy data.”
The team attached a flexible linker to VP2, a key structural protein on the outside of viral capsids. They used the linker to tether VP2 to Rep40, a native AAV protein that acts to ratchet viral genomes into the viral capsid.
“The concept is simple, in a way,” explains Nesbeth. “Normally, VP2 and Rep40 collide in the nucleus, but by permanently tethering them, we hoped to fill more capsids per unit time.”
Batches of a gene therapy with many capsids empty of viral genomes are less effective, and it’s more likely that a patient’s immune system will generate a harmful immune response.
The team also used the flexible linker to attach a transcription activator-like effector (TALE) protein they found in the academic literature to VP2. According to Nesbeth, TALEs were used for genetic engineering before the widespread adoption of CRISPRs.
The intention of the VP2-linker-TALE was to insert up to three partner DNA sequences into the AAV payload. “Again, the concept is that the capsid will actively grab genomes out of the surrounding environment,” Nesbeth says.
The team found that by including both a TALE and TALE target payloads, the AAV had more encapsulated DNA. They are now doing ongoing work to prove that the mechanisms are those they hypothesized.
In addition, the team experimented with changing the membrane-associated accessory protein (MAAP) that helps AAV leave its host cell. The team found that by swapping MAAP5 to MAAP8, a poor secretor could be changed to a good secretor.
Likewise, the team found that secretion was improved by swapping MAAP5 to a MAAP found to be the ancestor of many good secretors, which they called HeatMAAP. All their cells remained healthy during this process.
“People have suspected that this lever [MAAPs] exists for controlling AAV secretion, and we’ve shown that it might do,” explains Nesbeth. “It also seems that ancestral MAAPs might help us gain fine control over AAV secretion for manufacturing purposes.”
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