NEW YORK – Akribion Genomics, currently a brand of German biotechnology company Brain Biotech, has developed new families of CRISPR nucleases that the group says will have applications in research, diagnostics, and therapeutics.
Dubbed G-dases, the novel CRISPR-associated nucleases include two classes: G-dase M and G-dase E. While G-dase M is functionally analogous to the classical CRISPR-Cas editing systems, G-dase E is an engineered nuclease that has shown novel RNA-guided collateral activities for targeted cell depletion, according to Oliver Grünvogel, a consultant who was brought on to support Akribion’s spinoff from Brain Biotech.
The genesis of G-dases, he said, was part of Brain Biotech’s attempt to circumvent the IP barriers posed by existing CRISPR editing tools. “Since the background of Brain Biotech is really strong in the discovery of enzymes, they started a search project for a new CRISPR-Cas system, with the intention to have something that they could use for themselves and with freedom to operate.”
Using a metagenomic screening approach, the company first identified more than 2,000 novel CRISPR nuclease sequences from numerous organisms that might have genome editing potential. “Since our intention was to stay away from the known IP spaces, we focused on novel systems,” Grünvogel said.
From those, the company further selected the 15 best-performing nucleases with low sequence homologies to establish CRISPR systems to build its G-dase M nuclease portfolio.
So far, G-dase M has demonstrated its CRISPR-Cas-like genome-editing capability in various organisms, including prokaryotes, yeasts, and human cell culture, Grünvogel said, adding that the company is still working on further characterizing these nucleases, especially their off-target effects.
G-dase M provides Brain Biotech with a way to perform commercial genome engineering with freedom to operate and is “on par” with conventional CRISPR-Cas-based systems, he said, but does not have big advantages over them.
“To be very honest, we see them very much as similar tools,” Grünvogel said. “Our key [objective] in the beginning was to allow Brain Biotech to have access to a tool that we can use, and, when possible, enable partners to also have that access.”
The company has already started to license its G-dase M nucleases to partners, with the first two deals inked and “a couple more in the pipeline,” he said.
The company also sought to further optimize other metagenome-derived nucleases to boost their performance by protein engineering. One of the best-performing candidates, which later was developed into G-dase E, showed what the company called a “surprising mode of action.”
“Some things were off; we saw huge toxic effects; somehow we had a very limited number of clones always, but the clones that we saw were with an unusually high percentage positive for the edits that we wanted,” Grünvogel said. “Then we figured out that it had a completely different mode of action [compared] to the classical CRISPR systems.”
G-dase E nuclease, activated by binding to its RNA target, showed a unique collateral activity of depleting RNA and DNA molecules indiscriminately, eventually leading to cell death. “It started to make sense that we always saw these lower clone numbers on the plates. Because if this was activated, it was killing all the cells that we had,” Grünvogel said.
So far, the company has tested G-dase E in a variety of sample types, including prokaryotes, yeast, and human cell culture. Overall, the nuclease has demonstrated “super strong” collateral cell depletion activity in those organisms as soon as it is activated by the target RNA sequence, Grünvogel noted.
In one mixed-culture experiment using cells tagged with green fluorescent protein (GFP) and red fluorescent protein (RFP), for instance, G-dase E engineered to target GFP was shown to kill about 98.5 percent of GFP-positive cells, while leaving the RFP-tagged cells alive.
With this new mode of action, G-dase E can potentially open the door to “fully novel applications” in research, diagnostics, and therapeutics, he said.
For one, researchers could leverage G-dase E to help achieve targeted cell depletion for different experiments. During genome editing, for instance, they can deploy conventional editing tools first followed by G-dase E, which can be programmed to target cells that have not been edited, potentially boosting the editing outcome.
Similarly, G-dase E could potentially be used for cell therapy, Grünvogel said, where it could help purify edited cells for ex vivo therapy before transferring them to the patient.
Beyond those applications, the company’s primary goal right now is to develop targeted cancer treatments using G-dase E. “This is now also the key focus for Akribion Genomics,” he said. “We really want to apply this first in oncology, because we now have a tool where we can look at the specific RNA biomarkers that are present within tumor cells and are able to deplete those cells.”
While G-dase E is a “very mature proof of concept,” he said, it still requires extensive study before it can be deemed safe to be used in patients. “What we are still missing at this point, and what we're also working on, is [to understand] how cell death is initiated,” he said.
Additionally, though company researchers have not observed any detrimental effects from G-dase E on healthy cells so far, these experiments were done in cell culture, Grünvogel pointed out, and further characterization in tissue systems is needed.
While Akribion considers cancer therapeutics its top priority, the company is also open to licensing the G-dase E technology for other commercial applications, as long as it is not interfering with the company’s primary business interests, Grünvogel said.
So far, the company has filed applications for at least eight major patent families for its G-dase platform. “Our first focus was IP,” Grünvogel noted, adding that now that the patent applications are out the door, the company also intends to put out publications to demonstrate the performance of G-dases to the broader scientific community.
Akribion is also gearing up to spin off from its parent company. “Brain Biotech has decided to spin [Akribion] off because the focus they have on industrial biotech just doesn't enable the pharma development side that we need to go into,” Grünvogel said.
As such, he said the current focus for the team is fundraising. “We are out now talking to potential investors and strategic partners, and really want to have sufficient funding to make it to the inflection points,” he added.