Gene-edited stem cells aim to reverse rheumatoid arthritis

At the root of anything good or bad in the human immune system is a question about recognition. When the immune system functions correctly, it clocks unrecognized bacteria and viruses. When it misbehaves, immune cells misidentify the body’s tissue as its own. Such autoimmune disorders attack the pancreas in type 1 diabetes, nerve fibers in multiple sclerosis, and joints in rheumatoid arthritis (RA). Recognition also stifles the drugs designed to treat immune symptoms: Our bodies can develop drug-specific antibodies that target them.

“Some people think RA is a well-controlled disease. But actually, if you look at the data, [the drugs are] all failing,” said Brian Freed, an immunologist at the University of Colorado Anschutz Medical Campus and the co-founder and Chief Science Officer at RheumaGen. Nearly 30 percent of patients fail their second- and third-line treatments within three years of diagnosis (1). “It's just a matter of time. Either you're a fast fail or slow fail.”

It's just a matter of time. Either you're a fast fail or slow fail.
- Brian Freed, RheumaGen

To Freed, this creates a compelling case for treating autoimmune conditions differently — to lean into the recognition problem.

In 2016, his lab zeroed in on a suspicious pocket in the structure of a protein linked to the immune attack in RA (2,3). The pocket — just a few links in a long, tangled chain of amino acids — was particularly attractive for collagen to bind to. Researchers hypothesized that this sensitivity can worsen RA symptoms in the joints (4). But what if that recognition of collagen disappeared? When Freed’s team then tweaked the molecular pocket in mice, immune cells stopped overreacting to collagen.

Based on later experiments in mice, Freed devised a treatment to replace this errant subset of a person’s immune cells with a near-identical, gene-edited version that avoids autoimmune confusion. And now, RheumaGen is performing IND-enabling studies to hopefully test the idea in humans in a clinical trial in 2026.

The type of protein the team targets is rarely linked so directly to treating a disease. “This is one of the first examples I've heard,” said Hojun Li of the University of California, San Diego, a hematologist and expert in gene editing who is not affiliated with Freed’s lab.

“It's a very tantalizing option,” Li said. “Rheumatologic disorders are pretty complicated.” Freed has a strong suspicion that the benefits could extend far beyond this one disease.

Match making and breaking

RA affects about one percent of the adult population, and it is up to three times more common in women than in men (5,6). While researchers can’t pinpoint a specific cause of RA or its onset, they have a decent understanding of how it progresses.

One of the mechanisms is by T cells making enemies out of collagen. Human leukocyte antigen (HLA) proteins on certain immune cells’ surfaces will stick to collagen and falsely label it as foreign.

Each person has about a dozen types of HLA molecules in their body. One HLA-B subtype, for example, is identical throughout a person’s body, but their HLA-B is very likely to be different from their neighbor’s. “There are literally tens of thousands of different HLA molecules,” Freed said. “The likelihood that you match somebody in your office is about one in a million.”

A 3D rendition of a dendritic cell is shown in pink and purple. — Dendritic cells can express HLA-DRB1 molecules on their surface. Researchers have linked certain variants of the HLA-DRB1 subtype to RA.
Credit: iStock.com/Design Cells

This is why when one person needs a kidney transplant, doctors ensure that the donor’s kidney has compatible molecular factors. If the donor organ is mismatched, its cells could fatally attack the recipient’s body.

Certain variants of the HLA-DRB1 subtype linked to RA are especially autoreactive to collagen, including a variant called *0401 (7,8). RheumaGen estimates based on this data that about half of people with treatment-resistant RA have this *0401 variant.

Freed and his team noticed that the *0401 variant changed what was normally a negatively charged structural pocket in the HLA protein to a positively charged one. This now positively charged *0401 pocket attracted negatively charged collagen to it, presenting it as foreign to the immune system.

Freed wondered if replacing one single amino acid in the pocket could erase the positive charge. The team focused on the 71^st amino acid in the HLA protein’s sequence, designing a CRISPR-Cas9 directed gene edit. The edit would swap the *0401 variant’s positively changed lysine (K) for a negatively charged glutamic acid (E). They called the therapy K71E.

If we alter this one amino acid, could we stop the disease?
- Brian Freed, RheumaGen

The swap worked like a charm in vitro (3). In one experiment with human stem cells, changing the pocket’s charge with K71E was enough to halt the autoimmune response. “They can't respond because we took away the trigger,” Freed said. He then wondered if this therapy might be a way to treat RA: “If we alter this one amino acid, could we stop the disease?”

Unresponsive and unrejected

The encouraging early evidence felt validating. But, to Freed, the K71E approach still seemed outlandish because of one obvious concern: The gene edit would result in a mismatched HLA on purpose. Wouldn’t the immune system reject it?

To answer this question, Freed and his team engineered mice to carry the gene for the different human HLA variants (9). One set of mice had the *0401 gene variant. Another had the *0401 gene with the K71E swapped amino acid (*0401^K71E). A third set had another HLA gene variant linked to RA, called *0101, which mismatches with *0401 in 10 places within the collagen binding pocket. They then transplanted small segments of skin between the mice, monitoring for signs of rejection.

As expected, transplants between mice with the same HLA genes took well. Also predictably, transplanting highly mismatched skin (*0101 to *0401, for example) was rejected across the board. But to Freed and his colleagues’ surprise, the one-position mismatch — *0401 and *0401^K71E mice — transplanted safely. None of the *0401 mice rejected *0401^K71E skin.

“It’s such a small change,” Freed said. “The immune system can’t tell the difference.”

Richard Freed, Brian’s nephew who co-founded RheumaGen in 2019, added, “There's a blind spot.”

The breakthrough allowed the Freeds to ponder a potential way to use this therapy to treat people with RA — a bone marrow transplant. HLA-equipped immune cells originate from stem cells in the bone marrow. Typical RA treatment quiets symptoms with immunosuppressants that don’t work indefinitely. But, replacing bone marrow stem cells with gene-edited stem cells containing the K71E edit could solve itself from the bottom up.

To see if this was the case, the researchers performed a bone marrow transplant on *0401 mice with stem cells from *0401^K71E mice. Not only did the *0401 mice accept the edited stem cells, but they were also no longer sensitive to collagen.

“Nobody would have thought that it would work, myself included,” Freed said. “The idea has always been that if you change an HLA molecule, it'll either get rejected, or it's going to present some protein and it's going to trigger an immune response and all hell will break loose.” But, he added, “it turns out that on a practical basis it doesn't necessarily happen.”

This breakthrough has now set the stage for a potential clinical trial of K71E in humans.

A delicate balance

Bone marrow transplants are generally aggressive procedures. For patients with leukemia, doctors will wipe out cancerous immune cells with chemotherapy or radiation therapy, then replenish the supply with stem cells that give rise to blood and immune cells. The combination of cancer, chemotherapy, and a vulnerable immune system can keep patients in a hospital for months.

RheumaGen envisions a rather different bone marrow transplant for their treatment. They would extract stem cells from a patient’s bone marrow, isolate just those expressing the relevant HLA-DRB1 gene, edit them with the K71E swap, and repopulate that particular cohort of T cells through an intravenous administration. A traditional bone marrow transplant would require chemotherapy to wipe out the patient’s old immune system to replace it with new stem cells, but because here the scientists are just replacing a small, edited subset of the individual’s own stem cells, they don’t need to take such an intense approach. “It really won't require hospitalization,” Freed said. “Wear a mask for a week. Come back in to check your blood counts and then basically just notice that the symptoms are going away.”

Recently, the team also discovered an entirely different HLA target that seems relevant for type 1 diabetes, multiple sclerosis, and RA. They hypothesize that swapping one amino acid at the 82^nd position in a different HLA protein could temper autoimmune damage in any of those three diseases. At the time of writing, RheumaGen is considering this new target for their potential RA clinical trial.

“It would be amazing if this would work,” Li said, cautioning that mouse studies are useful but limited predictors for autoimmune disease. A mouse’s immune system doesn’t share the same controls as a human’s. “You can't really recreate that until you do a clinical trial.”

The big open question is whether the therapy will replace an adequate percentage of the errant immune cells and whether those cells will survive once back in the body. “If you have like 50 percent of cells still remaining that don't have the edit, will you still correct anything?” Li wondered.

In mice, the team replaced about 70 percent of the *0401 cells in the bone marrow and spleen in their K71E experiments. They are aiming for at least 50 percent in a clinical trial with their proposed “less aggressive” bone marrow procedure, Freed said.

At the end of the day, it is still basically a bone marrow transplant. It's not an easy pill to swallow for patients.
- Nathan Yozwiak, Mass General Brigham

“It’s unlikely that we have to go all the way to 100 percent,” Richard Freed said. “We think that's enough to get the patients into remission [or] at least enough to get them to be under control with medication,” he said. If 50 percent replacement does not eliminate medication, patients in the study may receive a more aggressive initial dose to replace more original cells. The team will also examine other potential problems, like whether their therapy edits other genes away from the target, whether symptoms persist for other reasons, and, perhaps most importantly, patients’ feedback.

“Technically, it's been a revelation that [gene editing] works. But that's not all you need,” said Nathan Yozwiak, the Head of Research at Mass General Brigham’s Gene and Cell Therapy Institute. “At the end of the day, it is still basically a bone marrow transplant. It's not an easy pill to swallow for patients.”

If the trial does prove successful, Yozwiak wonders how accessible treatment will be. His team recently published an article advocating for research partnerships between academia and medical centers to improve patient acceptance, access, and commercial viability for cell therapies (10). Not every hospital can provide gene therapy; these types of treatments, like Casgevy for sickle cell anemia, are breakthroughs that come at tremendous cost.

“I'm really curious about how RheumaGen is going to build on the lessons from Casgevy,” Yozwiak said. “Are patients going to want this? Are they going to be able to afford this? And how serious does the disease have to be?”

RheumaGen speculates that the cost for their potential therapy should be lower, when compared to multimillion dollar gene therapies for rare diseases, because the patient pool is so large. “One percent of the global population has RA, for God's sake,” Richard Freed said. “They're spending 70, 80, 90,000 dollars a year just on the medication.”

Brian Freed remains optimistic about either approach because the HLA edit has been so surprisingly tolerable. “We jokingly call it ‘plastic surgery on the immune system.’ We just change a tiny, tiny amount,” he said. “Nobody ever did it because it would just be a little too risky. And now we're de-risking it.”

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