CRISPR-Cas9

Stealthy stem cells to treat disease

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Gene-editing strategies that allow stem cells to evade the immune system offer hope for universal cell-replacement therapies

At the centre of the image, a molecular structure (orange) of CRISPR-Cas9 protein, with DNA (blue) passing through.
Gene-editing systems, such as CRISPR-Cas9, can be used to give stem cells immune-evasive properties

After decades of development, the dream of regenerative medicine has become a clinical reality — in part. Researchers can now cultivate stem cells in a laboratory, transform them into specialized cell types and then transplant them into people to alleviate disease.

In theory, this strategy promises an endless supply of replacement parts for ailing and ageing bodies: neurons to combat Parkinson’s disease, insulin-producing pancreatic cells to reverse type 1 diabetes, heart muscle cells to enhance cardiac function, and more.

But there’s a catch: therapies derived from stem cells must be customized to the patient — a process that is both slow and expensive. Or they can be made using donor cells. But, because the immune system tends to reject foreign cells, these ‘allogeneic’ off-the-shelf treatments require the concurrent administration of immune-dampening medicines — a strategy that raises the risk of complications such as infection and cancer.

Now, researchers are exploring a third approach — one that could fully realize the vision of mass-produced cell therapies for everyone, without the need for immune suppression.

By harnessing the power of gene-editing techniques, particularly CRISPR–Cas systems, to endow stem cells with immune-evasive properties, researchers can fashion stem cells that circumvent the immune system’s recognition mechanisms. They can also incorporate fail-safe features to ensure that the cells can be eliminated in the event of unforeseen complications. Such ‘stealth’ cells could, in principle, underpin a wide range of cell-replacement therapies, and billions of dollars have been invested in this work over the past five years.

The idea still requires validation. Only a small number of people have so far received any form of cell-replacement therapy derived from immune-edited stem cells, and no clinical results have yet been publicly disclosed. But with more products of this kind slated to enter human testing later this year, researchers are optimistic.

“We know in theory that it will work,” says Torsten Meissner, an immunologist at Beth Israel Deaconess Medical Center in Boston, Massachusetts, who points to the natural precedent of immune evasion to underscore his conviction: “Tumours have figured it out. Viruses have figured it out. Pregnancy is the other example.” Now, he says, biotechnology companies just need to work out how to emulate the same tactics for therapeutic gain.

Incognito mode

Strategies differ, but there are some gene edits that all researchers agree must underpin any universal stem-cell-derived therapy. There is also widespread consensus that the optimal product should incorporate as few edits as possible, both to minimize the potential for unintended genetic consequences and to streamline manufacturing and regulatory approval.

Beyond that, the scientific community is divided. The complexities of the immune system have fuelled spirited debates over the exact genetic manipulations necessary to create a cell therapy that is both capable of bypassing immune defences and delivering meaningful health benefits.

“The immune system is pervasive and persistent,” says Charles Murry, a cardiovascular pathologist at the University of Washington in Seattle and chief executive of StemCardia in Seattle, one of a growing number of biotechnology companies developing gene-editing strategies to overcome immune barriers in regenerative cell treatments.

It might take the immune system a while to find donor cells, Murry notes, “but find them it does. It’s ancient, smart and has lots of tricks up its sleeves.” Researchers must, therefore, be equally crafty when designing cells to evade it.

In most cases, the process starts by disrupting at least one part of the cell’s major histocompatibility complex (MHC), a cluster of proteins that functions like a molecular identity card, displaying unique pieces of cellular information that tell the immune system’s foot soldiers — a group of cells known as T lymphocytes — whether the cell is friend or foe.

“That’s the ‘universal’ element of the universal donor cell,” Murry explains. This edit strips the transplanted cell of its enemy identity, allowing it to seamlessly blend into its new environment and evade T-cell detection.

But the lack of MHC expression also presents a problem. Without the usual distinguishing markers of either ally or adversary, the edited cell becomes susceptible to attack by a different set of immune actors — natural killer (NK) cells, which have evolved to target and eliminate abnormal cells, including those without the proper MHC signatures.

To counteract this vulnerability, some researchers reintroduce genes that encode specific MHC antigens — ones that allow the cell to temper NK cells without inciting T-cell responses. Others are putting in genes that express ‘checkpoint’ proteins, molecules designed to directly curb the activity of NK cells.

Sana Biotechnology in Seattle, which favours the latter approach, reported last year that just three edits — two to eliminate MHC expression and one to boost expression of a checkpoint protein called CD47 — were sufficient to shield cells of rhesus monkeys (Macaca mulatta) from the animals’ immune systems1. It also showed that human cells, modified in the same manner, could ameliorate diabetes when transplanted into a mouse model2.

In November, Sana announced that it had the go-ahead to begin testing, in people, of donated human pancreatic cells that had been edited in this way. Trials of a stem-cell-derived product are likely to follow.

But not everyone has managed to replicate the findings around CD47. And with conflicting reports about how best to restrain NK-cell activity, stem-cell biologist Audrey Parent at the University of California, San Francisco (UCSF), sees that piece of the immune-evasion puzzle as the primary bottleneck in the field. “The NK cell part is not resolved yet,” she says.

Covert agents

Disagreement around NK-cell inhibition arises, at least in part, from the various methods laboratories use to assess the modified cells’ ability to evade immune detection. Although most research groups evaluate their edited stem cells in engineered mice with human-like immune systems, these ‘humanized’ models cannot faithfully replicate the complete immune response that cell products will face in people’s bodies.

Round orange cells, some covered with blue secretions, are seen in an islet of Langerhans from the pancreas
Pancreatic cells could potentially be edited to treat diabetes

Conversely, others generate gene-edited stem cells from monkeys and transplant them into other monkeys, mirroring the clinical scenario with humans. But this strategy is constrained by ethical concerns and the expense of experimentation with primates. Moreover, monkeys, although genetically similar to people, have distinct immune systems that might not faithfully reflect human responses.

Deepta Bhattacharya, an immunologist at the University of Arizona in Tucson, favours a different approach. When it comes to pushing the boundaries of immune evasion, he advocates evaluating universal gene-edited products that are intended for human use in mice with fully intact, natural immune systems. If cell therapies can pass this cross-species test, he reasons, they should be well-suited for transplantation into any human recipient.

Early this year, Bhattacharya and his colleagues reported that human stem cells containing a battery of 12 gene edits could survive in mice for months, with no signs of immune recognition or rejection3.

“A few of [the edits] we don’t think we actually need,” Bhattacharya says. But some edits that he considers crucial for thwarting rejection target a branch of the body’s natural defence mechanism known as the complement system. This system acts as a first line of defence against potential invaders by preparing antibodies to mark and eliminate foreign cells.

“Antibodies are tricky,” says Chad Cowan, co-founder and chief executive of Clade Therapeutics, a Boston-based biotech firm that is developing stem-cell-derived therapies for cancer and autoimmune conditions. (Bhattacharya is also a scientific co-founder.) “I think we’ve solved the cellular side of the equation,” Cowan says. “But antibodies actually turn out to be a bigger barrier than we thought.”

Clade’s solution, currently unpublished, involves engineering cells to secrete an enzyme that degrades and incapacitates nearby antibodies, thereby neutralizing the complement system. Another approach comes from Sonja Schrepfer, head of the hypoimmune platform at Sana who, together with UCSF heart surgeon Tobias Deuse and their colleagues, reported last year that overexpression of a protein that binds and disables antibodies can achieve the same result4.

Neither approach has been vetted in people — and, as molecular endocrinologist Timothy Kieffer at the University of British Columbia in Vancouver points out: “Strategies to thwart the highly evolved immune system are numerous, but are only hypothetical until proven otherwise.”

“The true test can only come in clinical trials,” he says.

Kieffer is also chief scientific officer of Fractyl Health, a metabolic therapeutics company in Lexington, Massachusetts. But two years ago, while serving as chief scientific officer for ViaCyte in San Diego, California, Kieffer played a pivotal part in launching the first clinical study of a stem-cell-derived product that incorporated immune-cloaking edits.

This pioneering product, developed in collaboration with biotech firm CRISPR Therapeutics in Boston, was named VCTX210. Designed to help people with type 1 diabetes to produce their own insulin, the product incorporated a suite of four gene edits collectively intended to enhance immune evasion and bolster cell survival. A subsequent version of this therapy, termed VCTX211, included an additional two edits, each aimed at further enhancing the robustness and functionality of the cells.

Invisibility shield

How effective these therapies were at sidestepping immune detection and improving the control of type 1 diabetes remains unclear. As Nature went to press, no results had been publicly disclosed. And both Vertex (which acquired ViaCyte in 2022, but is now working on separate stem-cell-derived therapies, using gene-editing technologies from CRISPR Therapeutics) and CRISPR Therapeutics (which now wholly owns the VCTX210 and VCTX211 assets) declined to comment on their immune-evasive cell-therapy programmes.

Also unclear is whether any safety concerns emerged in these trials. This matter is of utmost importance to researchers such as Kieffer because, as he explains, “concerns arise with manipulating the genome of cells for therapy, particularly when the goal is to endow them with an invisibility cloak that could be problematic should the cells become dangerous to the recipient”.

In the ViaCyte-CRISPR-Therapeutics trials, the companies took the precautionary step of encapsulating their immune-evasive cells in small, sticking-plaster-sized pouches, which are then implanted beneath the person’s skin. These devices contain pores that allow blood vessels to enter, providing oxygen and nutrients to the metabolically active cells inside, but prevent any therapeutic cells from escaping. If any unanticipated issues arise, they can be swiftly retrieved before rogue cells cause widespread damage.

Another safety measure involves the integration of genetic fail-safe features into the edited cells themselves. These features include drug-inducible suicide genes that can be activated by administration of a relatively benign medication. Researchers are also adorning modified cells with surface proteins that can be targeted with clinically approved antibody drugs, thereby achieving the same goal of cell destruction should any transplants turn cancerous or problematic in other ways.

In the end, the optimal safety strategy — not to mention the ideal amount of gene editing necessary to tamp down immune responses — can vary with the disease. A ready-to-use cell therapy for cancer does not necessarily need to incorporate the same design features as one tailored for diabetes, for instance, given the differences in the immune system that these cell products will confront and the distinct risk–benefit consideration in each disease. “There is no one catch-all solution,” Meissner says.

Certain parts of the body, including the eye and the brain, also enjoy an ‘immune privileged’ status, meaning that only a limited set of immune cells can enter them. This has led companies such as BlueRock Therapeutics in Cambridge, Massachusetts, which is developing off-the-shelf stem-cell-derived therapies for Parkinson’s disease, to tailor their immune-editing strategies accordingly. “There are some unique opportunities when you’re in the brain,” says BlueRock’s head of immunology, Greg Motz.

Those opportunities won’t be the last word on universal cell therapies, of course. Rather, Murry expects to see incremental advancement in the field, with short-term wins and losses informing long-term editing strategies.

“I would love it to be perfect out of the gates, but that’s not realistic,” Murry says. “This is going to be like peeling an onion.”

Source: Nature

CRISPRed Pigs: Precision Porcine Gene Editing Combats PPRS Virus Threat.

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Scientists are seeking FDA approval to bring the edited pigs to market.

Using CRISPR-Cas9 technology, British animal genetics firm Genus has generated a population of pigs that are resistant to the deadly PRRSV infection. [Genus]

CRISPR-based genome editing has already been successfully applied to sickle cell disease and additional companies are working on developing their CRISPR-based therapeutics. It seems only natural that the technology would make its way to animals. Now a team of scientists at Genus, a British animal genetics company with research facilities in Wisconsin and Tennessee, have developed a new generation of CRISPR-edited pigs that are resistant to porcine reproductive and respiratory syndrome (PRRS) virus, a disease that has had a widespread impact on porcine populations around the world for decades. 

Details of exactly how the pigs were edited are published in a new report published in The CRISPR Journal titled, “Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.” 

The Genus team describes a scaled gene editing program that introduced “a single modified CD163 allele into four genetically diverse, elite porcine lines.” The work produced healthy pigs that “resisted PRRS virus infections as determined by macrophage and animal challenges.” Edited pigs showed “no signs of infection or viral replication in lung and lymph node tissue when challenged with PRRSV.” The Genus team believes this is potentially the first integration of CRISPR gene editing into a livestock breeding program and could completely eliminate a major infectious disease in swine. 

“This is a milestone study illustrating the potential of CRISPR-based technologies for commercial livestock breeding,” said Rodolphe Barrangou, PhD, professor of food, bioprocessing, and nutrition sciences at North Carolina State University and editor-in-chief of The CRISPR Journal. “A commercially-relevant proof of concept that genome editing can be used to generate healthy PRRS-resistant pigs that are otherwise indistinguishable from the parent population sets the stage for deploying that approach for other diseases and traits of commercial interest.” 

“They were able to generate in a couple of generations a founder population of breeding boars (10–15 per line) and gilts to serve as a gene edited nucleus herd for ultimate commercial pork production and sale using classical breeding,” said Alison Van Eenennaam, PhD, an extension specialist in animal genomics and biotechnology in the department of animal science at the University of California, Davis. “There are more sophisticated approaches to guarantee the edited allele is in a homozygous state and absent off-target indels in all animals produced, e.g., using  edited porcine embryonic stem cells, but at the end of the day the approach they used did the job.”

Genus works on improving the genetics of livestock to ensure healthy, robust pigs and cattle for agricultural production, Elena Rice, PhD, Genus’ CSO and head of research and development and a co-author on the paper told GEN. “Livestock have a lot of diseases, and there are several that [are] just really devastating for farmers,” Rice said. Until the recent emergence of African swine flu, PRRS infections topped that list. 

Murky beginnings 

Also known as blue-ear pig disease, the first PRRS cases in the United States were reported in 1987. While the exact origins of the virus are murky, its impact is all too clear. The disease is now found in pig production facilities around the world, accounting for the death of as much as 20% of livestock produced annually. Its impact is environmental, psychological, and personal—not to mention economically devastating for the swine industry. By some estimates, annual losses in the United States exceed $600 million. 

Multiple vaccines have been developed to stop the spread of PRRS, but with limited effectiveness, according to Rice. As explained in the paper, the PRRS virus has “a high rate of mutation due to an error-prone viral RNA-dependent RNA polymerase and a significant rate of genetic recombination.”  

Vaccines are typically administered when the pigs are already showing symptoms, but by then it is often too late to save them. Even if the pigs survive the infection, their immune systems are usually too weak to combat secondary infections like pneumonia that can develop. Just a few infected pigs are enough to decimate an entire herd. Attempts to breed PRRS-resistant pig populations have also not worked out well to date.

Alongside breeding efforts, scientists in academia have also studied the genetic basis for PRRS infections. It turns out that several genes are involved in viral infection, including CD163, which encodes the entry receptor for the virus. In pigs, this particular protein is expressed on the surface of macrophages and monocytes and mediates inflammation among other functions. 

Genus’ work builds on the research into the role of CD163. “We had some evidence from university studies that we could edit a single host gene to confer resistance,” Brian Burger, PhD, senior research manager at Genus and first author on The CRISPR Journal paper, told GEN. “The challenge was: how do you go from that proof of concept work to a commercial breeding program?” It seemed like the ideal opportunity to bring CRISPR technology to bear on animal disease.

CRISPR’s efficiency also made it the right fit for editing animals. Finding the right edit for a gene without introducing dangerous off-target effects requires a lot of trial and error. “Ethically and morally, it’s very important [that] we designed the whole process so that we eliminate unnecessary production of animals,” Rice told GEN. 

Farm to table?

To edit the genomes, the Genus scientists injected CRISPR-Cas9 editing reagents into the genomes of pig zygotes. Their goal was to make a precise deletion in CD163 that removed a single exon encoding the domain that directly interacts with the virus. Importantly, the edit did not impact CD163’s function in the new population. They also genotyped the edited animals to ensure that the edit was consistent across the animals, there were no unforeseen off-target effects, and there was enough genetic diversity within the potential breeding population. Edited pigs that passed muster were then moved through the breeding process to establish a population of pigs that are PRRS-resistant. The paper also covers details of how the team optimized their editing reagents to ensure that their work would translate to a commercial breeding program, Burger added. 

The repercussions of this work could be major. Genus is not the only company working on PRRS by targeting CD163. But Genus certainly hopes to be the first to market with its edited pigs. It has its sights set on getting the edited pigs through the FDA’s regulatory process. 

Rice told GEN that the company is also working with regulatory agencies in other countries to seek broader approval as well. “Everything is going well, but it’s just a long process,” she said. ”The FDA is making a lot of effort to create a much better environment for gene editing [in livestock]. We’re learning together as we go through the process.”

Barrangou noted that “regulatory approval will be critical” and that “there are already established Ag-relevant frameworks for crops, notably in the U.S. and very recently in the EU that set the stage and precedence.” Furthermore, “the enthusiasm related to last week’s EU vote on the use of new genomic techniques and plant breeding technologies in Ag” makes the publication of this study “very timely and encouraging,” he added. 

Van Eenennaam has some reservations about the approval process in the United States. “The United States FDA is alone in the world in regulating ‘intentional’ genetic alterations including single base pair deletions as an animal drug, and requiring a ‘new animal drug’ approval for commercialization. This expensive regulatory path is basically a non-starter for smaller companies,” she told GEN in an email. 

Furthermore, “Until the pigs are approved, they are all considered unsalable new animal drugs and therefore cannot enter commerce or the food supply,” she added. “That means all of the 435 edited pigs produced in the paper in the development and testing process need to be incinerated, composted, or buried. Such a multiyear endeavor requires very deep pockets.” And the road to approval will likely be quite long. She pointed to the multi-year approval timeframe that was required for the genetically engineered AquAdvantage salmon as an example of just how long the process can be.

But regulatory approval is just the first hurdle. Genus scientists hope their pigs will be widely disseminated in the livestock industry to stop losses due to PRRS. Building PRRS-resistant piglets requires two gene-edited parents. Genus plans to sell aliquots of semen from gene-edited males to breeders who could use it to begin breeding programs that ultimately produce PRRS-resistant pigs after a few generations. 

If all goes well, the public could be faced with the choice of eating pork from gene-edited animals. Historically, the conversation around the production and consumption of genetically modified foods has been very polarizing. But the public response in this case may not follow historical trends given the central reason for the gene editing in the first place. Early consumer research conducted by Genus indicates that consumers are more open to CRISPR-edited foods when there is a good reason for the genetic modification. Given the impact PRRS has had on the livestock industry, there is a clear benefit here, Rice said.