NEW YORK (GenomeWeb) – A collaboration between a veterinary oncologist, synthetic DNA manufacturer Gen9, and design software firm Autodesk claims to have created the largest synthetic viral genome to date, which will be used in clinical trials to treat cancer in dogs using oncolytic viruses.
The synthetic canine adenovirus Type 2 (CAV-2) genome measures between 32 and 33 kilobases, a record, based on a recent literature search, Gen9 VP of Research and Development Devin Leake told GenomeWeb. He noted that several synthetized bacterial genomes have been larger.
"This is not a standard product or a length we typically provide," he said. "This is a proof of concept that we are interested in publishing so that we can show people what can be done."
It's a tumor-destroying virus that could blaze the trail for cancer treatment in dogs — and perhaps one day, humans. Auburn University veterinarian Bruce Smith has been working with oncolytic viruses for years. "We're specifically talking about a virus engineered to replicate, but only in a tumor cell," he said. Recent research suggests that the virus can not only cause the cell to explode, but also recruit the innate immune system.
For him, a synthetic viral genome represents a major leap in technology. "It brings virus creation down from 12 months to two," he said. He's already begun clinical trials using a recombinant virus, showing that the one-year survival rate with amputation and chemotherapy is only about 20 percent.
But the biggest gains could still be ahead, David Curiel, a professor at Washington University and a past collaborator with Smith, told GenomeWeb. "The specific value is it provides the technical means to potentially make highly designed viruses that match a patient's pathobiology — a so-called personalized virus."
With human virotherapy advancing in parallel with research in dogs, synthetic viruses have the potential to one day treat human tumors.
The collaboration got rolling in January 2015, when Smith received a cold call from Andrew Hessel, an employee of the industrial design software firm Autodesk. Synthetic viral genomes are one of Hessel's pet interests, going back to his time working for Amgen, he told GenomeWeb.
"Autodesk's Bio/Nano group was founded with the understanding there were few design tools for nano-scale materials," Hessel said. "We made many design tools but none that were reaching into these scales or domains. We'd like software tools that really enable creators at these types of materials," including viral genomes.
He's already used his company's tools to design bacteriophages and came across Smith's work coincidentally, but he immediately saw an application for his ideas on the potential for synthetic genomes to change biology.
"[Smith] was one of the first people I had come across using a recombinant virus for a clinical trial," Hessel said. He sent an email immediately. "I said, 'How would you like to upgrade your technology?'"
Hessel had also been tracking the DNA synthesis field as a whole and Gen9 in particular, which has developed a high-throughput platform for making DNA with the ability to print longer pieces using scaffolding technology. Hessel had previously collaborated with Gen9 on his project to synthesize the Phi-X bacteriophage. He suggested to Smith that Gen9 could simply print the CAV-2 genome for him.
"I thought, wow, that's a revolution," Smith said. Soon, the three were collaborating on taking the classically constructed CAV-2 virus, first developed in the lab of Washington University researcher David Curiel, and making a synthetic copy.
"When we got together to discuss if it would be possible to synthesize a 30,000 base pair genome, we knew that it would not be a trivial task, but that the project was definitely in Gen9's sweet spot," Leake said. "Our error correction tech and ability to do long-length assembly made us an ideal candidate for this project."
At the time, Smith was already using the virus in clinical trials to treat osteosarcoma in dogs, a cancer of the bone. To make sure the virus only replicates in tumor cells, replication is linked to a promoter for the osteocalcin gene. While the virus infects lots of cells, it will only replicate in cells with up-regulated osteocalcin.
Smith already had access to the sequenced genome for this version of the virus, so it wasn't difficult to turn that into a synthetic version.
"This type of application is perfect for DNA synthesis because of the exquisite control that you gain," Leake said.
That's not to say there weren't any challenges, but Gen9 was able to synthesize the virus without any major hiccups. "Since we had never synthesized a viral genome that long, my original estimation was that we'd be able to do this in two months," Leake said. "I was off by a couple of weeks." It took about 10 weeks, he said, but having completed it once, he sees room for improvement. "I think that we can do better than two months," he said.
Because the synthetic virus was an exact copy of the original, Hessel designed a DNA "watermark" to help distinguish it from the recombinant virus in the lab. This did raise several design challenges that Hessel had to deal with by trial and error.
"You want to make sure the inserted genetic segment doesn't break the genome," he said. Other considerations: Does it produce a stem-loop structure? Is it unique? Will it amplify properly? "Those are just some of the concerns," he said. "There are enough tools that allow you to do this work, but it wasn't a drag and drop process, which it should be."
At the moment, the 102 base-pair stretch simply encodes the date of design and the identities of the collaborators. But Hessel sees the potential for it to encode more. "You could point to a much more complete description file online," he said.
Smith has already taken the synthetic virus and validated in vitro that it replicates like the original. "We've seen that," he said. The next step is to actually treat dogs with it, to show that it behaves the same way in vivo.
So far, the recombinant virus has shown some efficacy, but nothing far and away beyond the current standard of care, which includes either amputation or intensive chemotherapy, or both. But Smith has ideas on how to increase efficacy by tweaking the delivery and design processes.
"It took us about 10 weeks to synthesize the virus and it's taken an equivalent amount of time to put it into cells and become ready to put into the patients," he said. "That's almost half a year." Using recombinant virus can take two, even three times as long, but some dogs have only months to live. If Gen9 can optimize synthesis and Smith can optimize treatment preparation, "that's getting to the point where it would be very clinically relevant," he said.
Moreover, there are design considerations that could improve the virus' ability to kill tumor cells. "Right now there's no specificity for infection," Smith said. CAV-2 only kills specific tumor cells, but infects lots of cell types. "That's a huge sink for the virus, going to a place where it does no good at all."
He's got an idea to design the virus to bind receptors up-regulated in tumor cells. "You can use fewer viruses or have more virus go to the tumor cells," he said. And by changing the promoter, Smith could get the virus to attack different types of tumor cells. This is especially important for further developing the field.
"Working with dogs in the oncolytic space would be a major gateway to development in humans," Hessel said. It was the potential for this kind of treatment in humans that got Hessel excited about Smith's research in the first place. It's just one of the beneficial uses he sees for synthetic DNA, a technology for which he's somewhat of an evangelist.
"Dogs have tumors very similar to what we see in humans," Smith said, often featuring the same driver mutations and developing in the same way. "Having data in dogs would greatly speed translation into humans," he said.
Curiel agrees. "If the synthetic virus acts like the genetically constructed virus, then you've provided the rationale to advance this approach to humans," he said. Virotherapy design is perhaps even more advanced in human adenoviruses than it is for CAV-2. "We know more about [human adenoviruses], and our ability to rationally engineer them is more advanced than for other viruses," he said.