By deleting the gene for uridine monophosphate synthetase (UMPS), an enzyme in the pathway for uridine synthesis, researchers have made cells, including embryonic stem cells and T cells, dependent on dietary uridine.
The work, which was published in the July 13, 2020, online issue of Nature Biotechnology, adds a new potential way of controlling cell therapies.
Currently, for all their potential, cell therapies are a lot like the airplanes of the Wright Brothers era. As synthetic biologist Wendell Lim describes it, those planes could fly, “but it wasn’t very far, and it wasn’t very safe.”
To enable the medical equivalent of mass transportation via cell therapies, both academic and industry researchers are developing safety switches that could be used to trigger cell death in the event that a cell therapy goes awry.
The work now published in Nature Biotechnology takes something of the opposite approach to other attempts to engineer safety or suicide switches into cells.
Instead of engineering cells to express a protein that could function as a switch, “we CRISPRd these knockouts,” James Patterson told BioWorld, to make cells that lacked UMPS and, consequently, uridine.
By default, such cells will not grow, because cells that don’t have the enzyme cannot proliferate without added dietary uridine.
Patterson is a co-author on the paper, and the founder of Auxolytic Ltd., a company focused on the development of nutrient-based safety switches for cell therapies.
Auxolytic was founded in 2016, when Patterson, with the help of angel investment, “filed a piece of IP around the technology and put it into this company,” he said.
The company is “just still myself,” he added, and “the work I do is effectively just reaching out to people” to collaborate.
“That’s the business model; it’s built around partnerships,” he said, with no plans for an independent drug development arm.
“My PhD was in yeast biology,” Patterson explained, a field where “this idea of nutrient-based cell growth control, or auxotrophy, is really common. [And] the other thing that I’m really interested in is synthetic biology,” the engineering of new abilities into cells.
Putting the two together, he said, “we set about trying to demonstrate for the first time in human cells that auxotrophy is something we can engineer” in human cells.
Misbehave, and it’s no dinner for you!
For making the switch, the first order of business was to decide which proteins would make good candidates for deletion in human cells – “I spent a long time doing due diligence,” Patterson said.
An advantage of UMPS is that the enzyme turned out to have to useful functions in terms of a safety switch. It is necessary for the synthesis of uridine, a precursor of the RNA base uracil. Cells lacking uridine can’t make RNA, and also have trouble with DNA repair and other cellular functions, including lipid metabolism.
But UMPS also catalyzes 5-fluoroorotic acid into what are normally toxic metabolites, and so cells that lack the enzyme can be easily selected during the engineering process, because 5-fluoroorotic acid is not toxic to them.
In their paper, Patterson and colleagues at Stanford University and Rice University first showed that several different types of human cells were unable to survive without uridine in cell culture, and then moved on to mouse experiments with cells including T cells and embryonic stem cells.
Here, they showed that without supplementary dietary uridine, UMPS-edited embryonic stem cells were unable to form teratomas, and UMPS-edited primary human T cells were unable to mount an immune response, as measured by their ability to cause graft-vs.-host-disease.
There is, of course, no lack of uridine in the organism as a whole. But Patterson and his colleagues showed that circulating levels of uridine are not high enough to sustain growth of cells lacking the ability to make their own. Patterson acknowledged that cells could survive in niches where local concentrations of uridine are higher, but to date, the team has seen no evidence of such survival actually occurring.
Patterson noted that the dependence on dietary supplementation means that while “other switches are very all or nothing, we’re in a position to have more fine-grained control” over the proliferation rate of cells once they have been administered. Other advantages of the approach, he said, are that there are no issues with immunogenicity of an introduced protein, and no possibility of cells’ losing the expression of the off-switch protein.
Without any dietary uridine, it takes “around a week” for the levels of cells to decline, making it “less a cytokine storm switch, and more a long-term control,” Patterson said.
Whether the uridine approach could or should be combined with other switches “depends on the therapy itself and what safety issues you’re expecting,” he said. But there is “no technical reason why switches can’t be combined.”