New research focuses on methods that could change medicine-making worldwide. The scientists propose bursting cells open, removing their DNA and using the cellular gears inside to make therapies.
Rao and his team of collaborators, which spans multiple research institutions, believe they have a better approach that may change medicine-making worldwide. They suggest forgoing the concept of using living cells as medicine-producers. Instead, they propose breaking the cells and using the remaining cellular gears for assembling the therapeutic compounds. Instead of inserting the DNA into living cells, the team burst them open, and removed their DNA altogether. Yet, the residual molecular machinery of ribosomes, polymerases and other cogwheels still functioned the way it would in a cell. “Now if you drop your DNA drug-making instructions into that soup, this machinery starts making what you need,” Rao explains. “And because you're no longer worrying about living cells, it becomes much simpler and more efficient.” The collaborators detail their cell-free protein synthesis or CFPS method in their recent paper published in preprint BioAxiv.
While CFPS does not use living cells, it still needs the basic building blocks to assemble proteins from—such as amino acids, nucleotides and certain types of enzymes. These are regularly added into this “soup” to keep the molecular factory chugging. “We just mix everything in as a batch and we let it integrate,” says James Robert Swartz, professor of chemical engineering and bioengineering at Stanford University and co-author of the paper. “And we make sure that we provide enough oxygen.” Rao likens the process to making milk from milk powder.
For a variety of reasons—from the field’s general inertia to regulatory approval hurdles—the method hasn’t become mainstream. The pandemic rekindled interest in medicines that can be made quickly and easily, so it drew more attention to the technology.
The idea of a cell-free protein synthesis is older than one might think. Swartz first experimented with it around 1997, when he was a chemical engineer at Genentech. While working on engineering bacteria to make pharmaceuticals, he discovered that there was a limit to what E. coli cells, the workhorse darling of pharma, could do. For example, it couldn’t grow and properly fold some complex proteins. “We tried many genetic engineering approaches, many fermentation, development, and environmental control approaches,” Swartz recalls—to no avail.
“The organism had its own agenda,” he quips. “And because everything was happening within the organism, we just couldn't really change those conditions very easily. Some of them we couldn’t change at all—we didn’t have control.”
It was out of frustration with the defiant bacteria that a new idea took hold. Could the cells be opened instead, so that the protein-forming reactions could be influenced more easily? “Obviously, we’d lose the ability for them to reproduce,” Swartz says. But that also meant that they no longer needed to keep the cells alive and could focus on making the specific reactions happen. “We could take the catalysts, the enzymes, and the more complex catalysts and activate them, make them work together, much as they would in a living cell, but the way we wanted.”
In 1998, Swartz joined Stanford, and began perfecting the biochemistry of the cell-free method, identifying the reactions he wanted to foster and stopping those he didn’t want. He managed to make the idea work, but for a variety of reasons—from the field’s general inertia to regulatory approval hurdles—the method hasn’t become mainstream. The pandemic rekindled interest in medicines that can be made quickly and easily, so it drew more attention to the technology. For their BioArxiv paper, the team tested the method by growing a specific antiviral protein called griffithsin.
First identified by Barry O’Keefe at National Cancer Institute over a decade ago, griffithsin is an antiviral known to interfere with many viruses’ ability to enter cells—including HIV, SARS, SARS-CoV-2, MERS and others. Originally isolated from the red algae Griffithsia, it works differently from antibodies and antibody cocktails.
Most antiviral medicines tend to target the specific receptors that viruses use to gain entry to the cells they infect. For example, SARS-CoV-2 uses the infamous spike protein to latch onto the ACE2 receptor of mammalian cells. The antibodies or other antiviral molecules stick to the spike protein, shutting off its ability to cling onto the ACE2 receptors. Unfortunately, the spike proteins mutate very often, so the medicines lose their potency. On the contrary, griffithsin has the ability to cling to the different parts of viral shells called capsids—namely to the molecules of mannose, a type of sugar. That extra stuff, glued all around the capsid like dead weight, makes it impossible for the virus to squeeze into the cell.
“Every time we have a vaccine or an antibody against a specific SARS-CoV-2 strain, that strain then mutates and so you lose efficacy,” Rao explains. “But griffithsin molecules glom onto the viral capsid, so the capsid essentially becomes a sticky mess and can’t enter the cell.” Mannose molecules also don’t mutate as easily as viruses’ receptors, so griffithsin-based antivirals do not have to be constantly updated. And because mannose molecules are found on many viruses’ capsids, it makes griffithsin “a universal neutralizer,” Rao explains.
“When griffithsin was discovered, we recognized that it held a lot of promise as a potential antiviral agent,” O’Keefe says. In 2010, he published a paper about griffithsin efficacy in neutralizing viruses of the corona family—after the first SARS outbreak in the early 2000s, the scientific community was interested in such antivirals. Yet, griffithsin is still not available as an off-the-shelf product. So during the Covid pandemic, the team experimented with synthesizing griffithsin using the cell-free production method. They were able to generate potent griffithsin in less than 24 hours without having to grow living cells.
The antiviral protein isn't the only type of medicine that can be made cell-free. The proteins needed for vaccine production could also be made the same way. “Such portable, on-demand drug manufacturing platforms can produce antiviral proteins within hours, making them ideal for combating future pandemics,” Rao says. “We would be able to stop the pandemic before it spreads.”
Top: Describes the process used in the study. Bottom: Describes how the new medicines and vaccines could be made at the site of a future viral outbreak.
Image courtesy of Rao and team, sourced from An approach to rapid distributed manufacturing of broad spectrumanti-viral griffithsin using cell-free systems to mitigate pandemics.
Rao’s idea is to perfect the technology to the point that any hospital or pharmacy can load up the media containing molecular factories, mix up the required amino acids, nucleotides and enzymes, and harvest the meds within hours. That will allow making medicines onsite and on demand. “That would be a self-contained production unit, so that you could just ship the production wherever the pandemic is breaking out,” says Swartz.
These units and the meds they produce, will, of course, have to undergo rigorous testing. “The biggest hurdles will be validating these against conventional technology,” Rao says. The biotech industry is risk-averse and prefers the familiar methods. But if this approach works, it may go beyond emergency situations and revolutionize the medicine-making paradigm even outside hospitals and pharmacies. Rao hopes that someday the method might become so mainstream that people may be able to buy and operate such reactors at home. “You can imagine a diabetic patient making insulin that way, or some other drugs,” Rao says. It would work not unlike making baby formula from the mere white powder. Just add water—and some oxygen, too.
In a recent research trial, patients with Parkinson's disease reported that their symptoms had improved after stem cells were implanted into their brains. Martin Taylor, far right, was diagnosed at age 32.
For years, there's been little improvement in the standard treatment. Patients are typically given the drug levodopa, a chemical that's absorbed by the brain’s nerve cells, or neurons, and converted into dopamine. This drug addresses the symptoms but has no impact on the course of the disease as patients continue to lose dopamine producing neurons. Eventually, the treatment stops working effectively.
BlueRock Therapeutics, a cell therapy company based in Massachusetts, is taking a different approach by focusing on the use of stem cells, which can divide into and generate new specialized cells. The company makes the dopamine-producing cells that patients have lost and inserts these cells into patients' brains. “We have a disease with a high unmet need,” says Ahmed Enayetallah, the senior vice president and head of development at BlueRock. “We know [which] cells…are lost to the disease, and we can make them. So it really came together to use stem cells in Parkinson's.”
In a phase 1 research trial announced late last month, patients reported that their symptoms had improved after a year of treatment. Brain scans also showed an increased number of neurons generating dopamine in patients’ brains.
Increases in dopamine signals
The recent phase 1 trial focused on deploying BlueRock’s cell therapy, called bemdaneprocel, to treat 12 patients suffering from Parkinson’s. The team developed the new nerve cells and implanted them into specific locations on each side of the patient's brain through two small holes in the skull made by a neurosurgeon. “We implant cells into the places in the brain where we think they have the potential to reform the neural networks that are lost to Parkinson's disease,” Enayetallah says. The goal is to restore motor function to patients over the long-term.
Five patients were given a relatively low dose of cells while seven got higher doses. Specialized brain scans showed evidence that the transplanted cells had survived, increasing the overall number of dopamine producing cells. The team compared the baseline number of these cells before surgery to the levels one year later. “The scans tell us there is evidence of increased dopamine signals in the part of the brain affected by Parkinson's,” Enayetallah says. “Normally you’d expect the signal to go down in untreated Parkinson’s patients.”
"I think it has a real chance to reverse motor symptoms, essentially replacing a missing part," says Tilo Kunath, a professor of regenerative neurobiology at the University of Edinburgh.
The team also asked patients to use a specific type of home diary to log the times when symptoms were well controlled and when they prevented normal activity. After a year of treatment, patients taking the higher dose reported symptoms were under control for an average of 2.16 hours per day above their baselines. At the smaller dose, these improvements were significantly lower, 0.72 hours per day. The higher-dose patients reported a corresponding decrease in the amount of time when symptoms were uncontrolled, by an average of 1.91 hours, compared to 0.75 hours for the lower dose. The trial was safe, and patients tolerated the year of immunosuppression needed to make sure their bodies could handle the foreign cells.
Claire Bale, the associate director of research at Parkinson's U.K., sees the promise of BlueRock's approach, while noting the need for more research on a possible placebo effect. The trial participants knew they were getting the active treatment, and placebo effects are known to be a potential factor in Parkinson’s research. Even so, “The results indicate that this therapy produces improvements in symptoms for Parkinson's, which is very encouraging,” Bale says.
Tilo Kunath, a professor of regenerative neurobiology at the University of Edinburgh, also finds the results intriguing. “I think it's excellent,” he says. “I think it has a real chance to reverse motor symptoms, essentially replacing a missing part.” However, it could take time for this therapy to become widely available, Kunath says, and patients in the late stages of the disease may not benefit as much. “Data from cell transplantation with fetal tissue in the 1980s and 90s show that cells did not survive well and release dopamine in these [late-stage] patients.”
Searching for the right approach
There's a long history of using cell therapy as a treatment for Parkinson's. About four decades ago, scientists at the University of Lund in Sweden developed a method in which they transferred parts of fetal brain tissue to patients with Parkinson's so that their nerve cells would produce dopamine. Many benefited, and some were able to stop their medication. However, the use of fetal tissue was highly controversial at that time, and the tissues were difficult to obtain. Later trials in the U.S. showed that people benefited only if a significant amount of the tissue was used, and several patients experienced side effects. Eventually, the work lost momentum.
“Like many in the community, I'm aware of the long history of cell therapy,” says Taylor, the patient living with Parkinson's. “They've long had that cure over the horizon.”
In 2000, Lorenz Studer led a team at the Memorial Sloan Kettering Centre, in New York, to find the chemical signals needed to get stem cells to differentiate into cells that release dopamine. Back then, the team managed to make cells that produced some dopamine, but they led to only limited improvements in animals. About a decade later, in 2011, Studer and his team found the specific signals needed to guide embryonic cells to become the right kind of dopamine producing cells. Their experiments in mice, rats and monkeys showed that their implanted cells had a significant impact, restoring lost movement.
Studer then co-founded BlueRock Therapeutics in 2016. Forming the most effective stem cells has been one of the biggest challenges, says Enayetallah, the BlueRock VP. “It's taken a lot of effort and investment to manufacture and make the cells at the right scale under the right conditions.” The team is now using cells that were first isolated in 1998 at the University of Wisconsin, a major advantage because they’re available in a virtually unlimited supply.
Other efforts underway
In the past several years, University of Lund researchers have begun to collaborate with the University of Cambridge on a project to use embryonic stem cells, similar to BlueRock’s approach. They began clinical trials this year.
A company in Japan called Sumitomo is using a different strategy; instead of stem cells from embryos, they’re reprogramming adults' blood or skin cells into induced pluripotent stem cells - meaning they can turn into any cell type - and then directing them into dopamine producing neurons. Although Sumitomo started clinical trials earlier than BlueRock, they haven’t yet revealed any results.
“It's a rapidly evolving field,” says Emma Lane, a pharmacologist at the University of Cardiff who researches clinical interventions for Parkinson’s. “But BlueRock’s trial is the first full phase 1 trial to report such positive findings with stem cell based therapies.” The company’s upcoming phase 2 research will be critical to show how effectively the therapy can improve disease symptoms, she added.
The cure over the horizon
BlueRock will continue to look at data from patients in the phase 1 trial to monitor the treatment’s effects over a two-year period. Meanwhile, the team is planning the phase 2 trial with more participants, including a placebo group.
For patients with Parkinson’s like Martin Taylor, the therapy offers some hope, though Taylor recognizes that more research is needed.
BlueRock Therapeutics
“Like many in the community, I'm aware of the long history of cell therapy,” he says. “They've long had that cure over the horizon.” His expectations are somewhat guarded, he says, but, “it's certainly positive to see…movement in the field again.”
"If we can demonstrate what we’re seeing today in a more robust study, that would be great,” Enayetallah says. “At the end of the day, we want to address that unmet need in a field that's been waiting for a long time.”
Editor's note: The company featured in this piece, BlueRock Therapeutics, is a portfolio company of Leaps by Bayer, which is a sponsor of Leaps.org. BlueRock was acquired by Bayer Pharmaceuticals in 2019. Leaps by Bayer and other sponsors have never exerted influence over Leaps.org content or contributors.