NEW YORK – Massachusetts-based biotech startup Korro Bio has developed an RNA editing technology platform that it believes will help it create therapeutics for a variety of diseases, without some of the complications involved in CRISPR-based editing.
The company, which recently came out of stealth mode, is attempting to use the transient editing properties of nucleotide deamination, an endogenous RNA-modifying process that happens naturally in cells.
In the body, deamination takes place in the liver and the kidney. Spontaneous deamination is the hydrolysis reaction of cytosine into uracil — in DNA, this process is corrected for by the removal of uracil by uracil-DNA glycosylase, generating an abasic site, which is then recognized by endonucleases that repair the resulting lesion by replacing it with another cytosine.
The most common single nucleotide mutation is spontaneous deamination of 5-methylcytosine into thymine and ammonia. In DNA, this reaction can be corrected by the enzyme thymine-DNA glycosylase, which removes the thymine base in a G-T mismatch. Deamination of guanine results in a post-replicative transition mutation where the original G-C base pair transforms into an A-T base pair. And deamination of adenine results in a post-replicative transition mutation where the original A-T base pair transforms into a G-C base pair.
Korro Bio's technology, which is based on the RNA editing research of company Cofounder Josh Rosenthal, relies on the action of RNA-editing enzymes encoded by the ADAR (adenosine deaminase acting on RNA) gene. ADAR enzymes bind to double-stranded RNA (dsRNA) and convert adenosine to inosine by deamination. The inosine is then read as guanosine.
Importantly for Korro Bio, ADAR is an RNA-binding protein and edits RNA through post-transcriptional modification of messenger RNA (mRNA) transcripts by changing the nucleotide content of the RNA. That means the company's technology doesn't induce any double-stranded breaks in DNA as CRISPR-based editing techniques can do, and that the edits it makes aren't permanent changes to the genome. Its aim is to selectively edit mRNA to make changes to specific codons.
"We avoid issues around gene editing which are permanent modification. By modifying the RNA, we're actually doing a transient edit," explained Korro Bio Cofounder and Senior Vice President of Technology Andrew Fraley. "Those RNAs are temporary molecules in the cells because they're being retranscribed from the DNA, but we're never actually changing the original information."
The company also believes this is a better approach to gene editing-based therapeutics because this process happens naturally in cells.
"If I take a cell out of your body and crack it open, this editing system was actually already in there. And there's also this element that's transient, so it's not like CRISPR-Cas9, or a zinc finger, or a TALEN, where you've got to put it in [the cell]," added company Cofounder and Chairman Nessan Bermingham. "The reality is that we're co-opting an endogenous system in your cell to do exactly what it's there to do."
Bermingham — who served as founding CEO of CRISPR-based biopharmaceuticals developer Intellia Therapeutics — noted that an important part of creating gene editing-based therapeutics is the verification and validation of targets in the body. Avoiding permanent changes to the genome allows for the developers of such therapeutics to continue modifying and improving their treatments as new research emerges on the effects of gene mutations on disease and on the possible side effects of editing.
"People are now looking at CRISPR-Cas9 targeting of the TTR gene, but what got them comfortable with doing that was all the work that had been done on antisense oligonucleotides, an siRNA against that target where it was a transient knockdown, and tracking those patients for an extended period of time to see the impact that it would have on them," Bermingham said. "So, as we think about the information that's coming through, how do we think about a transient intervention that's clinically relevant but does not potentially expose you to that risk of a permanent, non-reversible modification from an editing standpoint?"
Korro Bio's concept of editing RNA, where the changes are transient, may be one way to achieve those goals, he added.
"Unlike the other technologies that are out there, we're talking about a surgical modification of a single base versus cutting a piece of DNA with indel insertion or indel formation, or a gene therapy putting a whole new vector into a cell and expressing as much of a protein as you possibly can," Bermingham explained. "Actually reverting a base or changing a single base while maintaining a regulatory sequence made a lot more sense to us. I think we should be striving for a natural level of protein in the cell."
Indeed, Fraley said, recruiting endogenous proteins by providing the cell with a synthetic oligonucleotide or synthetic piece of modified RNA is "very much a drug substance or drug product." The goal is to recruit the endogenous protein in a way that drives the editing at a specific base, he added.
By eschewing the use of CRISPR nucleases altogether, the company also believes its therapeutics will be easier to deliver into cells than other gene editing systems — "We're not talking about a large structural construct that we're trying to fit into a cell. We're talking about relatively small oligonucleotides," Bermingham said — and that the potential for permanent off-target edits to the genome is greatly reduced.
"If you have a knock-out approach or a base editing approach, that's a permanent change to a cell, and any off-target activity you have is permanent," Fraley said. "Any off-target [activity] you might pick up from RNA editing is a transient off-target problem — it goes away if you stop dosing your compound. You change your chemistry then you can change your off-target profile to make it more specific. So, you have the ability to tune the chemistry."
The company is now focusing on building up an internal R&D group to begin drug development. Korro Bio received $4 million in seed financing from Atlas Venture, as well additional investment worth an undisclosed amount from Atlas and New Enterprise Associates. Bermingham said the funding has provided enough capital for the company to move forward with its plans.
The firm has filed for patent protection of its technology. But it doesn't yet have any ongoing clinical trials and is also keeping the specific indications it intends to develop therapeutics for under wraps for the time being.
Bermingham noted that some of the areas of the body Korro Bio is most focused on include the liver, eye, ear, muscle, and gut. He's optimistic that the firm could have a product on the market in about three to five years, taking into account the pace of development and the regulatory process.
"We will look [to put a product on the market] around the next three years. Given the data we have in hand today, given the fact that we've already moved into in vivo studies, around that three-year timeframe is reasonable," he added. "The caveat is that one can never predict these things exactly. It could go faster, or it could go slower. This is a new therapeutic approach, so we just have to be cognizant of that."
Importantly, Fraley noted, it's precisely the fact that the company is pursuing a new therapeutic approach that makes it stand out from other editing-based therapeutics companies. "From my perspective, the science is fascinating," he said. "RNA editing presents a new opportunity to go after more [indications] and have a new modality that's going to be hitting diseases and treating patients that current technologies just can't do."