October 18, 2021

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The tortuous road and complicated development history of mRNA vaccines

The tortuous road and complicated development history of mRNA vaccines.

The tortuous road and complicated development history of mRNA vaccines

The tortuous road and complicated development history of mRNA vaccines.

At the end of 1987, Robert Malone conducted a landmark experiment in which he mixed multiple strands of messenger RNA with fat droplets, and found that the human cells added to it had absorbed mRNA and produced protein.

Realizing that this discovery may have far-reaching potential in medicine, Malone, a graduate student at the Salk Institute for Biological Research in La Jolla, California, later took down some notes, signed and dated them.

He wrote on January 11, 1988 that if cells can produce protein from the mRNA delivered to them, it is possible to “treat RNA as a drug”. Later, Malone’s experiments showed that frog embryos can absorb this mRNA. This is the first time that fat droplets have been used to simplify the process of mRNA entry into organisms.

But the road to success is tortuous. Malone’s experiment itself borrowed from the work of other researchers. After many years, people believed that mRNA was too unstable and expensive to be used as a medicine or vaccine. Dozens of academic laboratories and companies are dedicated to researching this idea, trying to find the correct formula of fat and nucleic acid-the cornerstone of mRNA vaccine.

Today’s mRNA vaccine innovations were invented after Malone worked in the laboratory for several years, including chemically modified RNAs and different types of fat vesicles that inject them into cells. Despite this, Malone, who calls himself the “mRNA vaccine inventor”, believes that his work has not received enough recognition, “I have been eliminated by history,” he told the “Nature” magazine.

The tortuous road and complicated development history of mRNA vaccines

With the beginning of the Nobel Prize, the debate about who is the pioneer of this technology is heating up, and with the announcement of the Nobel Prize next month, the speculation is becoming more and more intense. But formal awards limited to a few scientists will not recognize the many contributors to the development of mRNA medicine. In fact, the road to mRNA vaccines draws on the work of hundreds of researchers for more than 30 years.

Origin of mRNA

Malone’s experiment did not come out of thin air. As early as 1978, scientists used fatty membrane structures called liposomes to transport mRNA into mouse and human cells to induce protein expression.

Liposomes package and protect mRNA, and then fuse with the cell membrane to deliver genetic material to the cell. These experiments themselves are based on many years of work on liposomes and mRNA.

Both were discovered in the 1960s (see “The History of mRNA Vaccines”, figure below).

The tortuous road and complicated development history of mRNA vaccines

However, few researchers at the time considered mRNA as a drug product—especially because there was no way to make genetic material in the laboratory. Instead, they hope to use it to study basic molecular processes. Most scientists reuse rabbit blood, cultured mouse cells, or other animal-derived mRNA.

In 1984, the situation changed when Krieg and Douglas Melton, a developmental biologist at Harvard University in Cambridge, Massachusetts, and other members of the team led by molecular biologists Tom Maniatis and Michael Green used RNA synthetase (taken from a virus) and others Tools to biologically produce active mRNA in the laboratory-a core method that is still in use today. Krieg then injected the lab-made mRNA into frog eggs and showed that it really works.

Both Melton and Krieg said that they mainly use synthetic mRNA as a research tool to study gene function and activity. In 1987, after Melton discovered that mRNA can be used to activate and prevent protein production, he helped form a company called Oligogen (later renamed Gilead Sciences in Foster, California) to explore the use of synthetic RNA to block target gene expression Method in order to treat the disease. But no one in his laboratory or their collaborators thought of a vaccine.

The tortuous road and complicated development history of mRNA vaccines
Paul Krieg (left) and Douglas Melton (right), they are studying the method of synthesizing mRNA in the laboratory

“In general, RNA is known for its incredible instability,” Krieg said. “Everything around RNA is carefully obscured.” This may explain why the Harvard University Technology Development Office chose not to be the organization’s Patent application for RNA synthesis method. Instead, the Harvard researchers simply handed over their research results to Promega, a laboratory supplies company based in Madison, Wisconsin, that provides researchers with RNA synthesis tools.

Patent dispute

A few years later, Malone followed the strategy of the Harvard team to synthesize mRNA, but he added a new liposome-a positively charged liposome, which strengthened the material and the negatively charged mRNA backbone. The ability to combine. These liposomes were developed by Philip Felgner.

The tortuous road and complicated development history of mRNA vaccines
Philip Felgner (left) and Robert Malone

Although Malone successfully used liposomes to deliver mRNA into human cells and frog embryos, he did not obtain a Ph.D. He and his supervisor Salk gene therapy researcher Inder Verma fell out and ended his postgraduate study in 1989 and went to Vical, a recently founded startup in San Diego, California, works for Felgner. There, their work with collaborator Madison at the University of Wisconsin showed that lipid-mRNA complexes can stimulate protein production in mice.

The tortuous road and complicated development history of mRNA vaccines
An excerpt from the notebook of Malone’s lab, describing the process of injecting synthetic mRNA into mice in 1989

Then things became a mess. Vical (in cooperation with the University of Wisconsin) and Salk began applying for patents in March 1989. But Salk quickly abandoned his patent claims, and in 1990, Verma joined Vical’s advisory board.

Malone argued that Verma and Vical had reached a behind-the-scenes deal, and the related intellectual property rights belonged to Vical. Malone was listed as one of many inventors, but he no longer profited from subsequent licensing transactions as he did in any patents issued by Salk. Malone concluded: “They make a fortune from the fruits of my thoughts.”

Verma and Felgner categorically denied Malone’s allegations. “This is total nonsense,” Verma told Nature, saying that the decision to abandon the patent application depends on Salk’s technology transfer office.

Malone left Vical in August 1989 on the grounds of disagreement with Felgner on “scientific judgment” and “the credit of my intellectual contribution.” He completed medical school and received a year of clinical training, and then worked in academia, where he tried to continue research on mRNA vaccines, but it was difficult to obtain funding. (For example, in 1996, he applied to a research institute in California for funding to develop an mRNA vaccine to combat seasonal coronavirus infections, but was unsuccessful.) In the end, Malone focused on DNA vaccines and delivery technology.

In 2001, he began to engage in commercial work and consulting work. In the past few months, he has begun to publicly criticize the safety of mRNA vaccines his research has helped to achieve. For example, Malone said that the protein produced by vaccines can damage human cells, and the risks of vaccination outweigh the benefits to children and young people-other scientists and health officials have repeatedly refuted this claim.

Product manufacturing challenges

In 1991, Vical reached a multi-million-dollar research cooperation and licensing agreement with Merck, one of the world’s largest vaccine developers. Merck scientists evaluated mRNA technology in mice with the goal of creating a flu vaccine, but then abandoned this method. “The cost and feasibility of manufacturing stopped us,” said Jeffrey Ulmer, a former Merck scientist.

Researchers at a small biotechnology company called Transgène in Strasbourg, France, feel the same way. In 1993, a team led by Pierre Meulien collaborated with industry and academia to prove for the first time that mRNA in liposomes can trigger a specific antiviral immune response in mice. (Another exciting development occurred in 1992, when scientists at the Scripps Research Institute used mRNA to replace a defective protein in mice to treat metabolic disorders. However, independent laboratories report similar success requires nearly two Ten years time.)

Transgène researchers applied for a patent for their invention and continued to study mRNA vaccines. But Meulien is now the head of the Innovative Medicines Initiative, a public and private company headquartered in Brussels. He estimates that he needs at least 100 million euros ($119 million) to optimize the platform-he does not intend to Such a “tricky, high-risk” company asked the boss for so much money, and the patent lapsed after Transgène’s parent company decided to stop paying the expenses needed to maintain its operations.

Pierre Meulien

Like Merck’s team, Meulien’s team turned to focus on DNA vaccines and other vector-based delivery systems. The DNA platform eventually produced some licensed vaccines for veterinary applications—for example, to help prevent infections in fish farms. Just last month, Indian regulators urgently approved the world’s first DNA vaccine for human use to help fight COVID-19. However, for reasons that are not fully understood, DNA vaccines have been slow to succeed in humans.

Ulmer believes that, despite this, the collaborative advancement of the industry around DNA technology has also brought benefits to RNA vaccines. From manufacturing considerations and regulatory experience to sequence design and molecular insights, “many of the things we learn from DNA can be directly applied to RNA,” he said, “it provides the basis for the success of RNA.”

Continuous struggle

In the 1990s and most of the 21st century, almost all vaccine companies considering mRNA research chose to invest their resources in other areas. The traditional view is that mRNA is too easy to degrade and its production cost is too high. “This is an ongoing struggle,” said Peter Liljeström, a virologist at the Karolinska Institute in Stockholm, who first created a “self-amplifying” RNA vaccine 30 years ago.
In 1989, Matt Winkler founded Ambion, the first laboratory supplies company focused on RNA in Austin, Texas. He said: “RNA is too difficult to handle”, “If you (at the time) ask me if you can inject RNA into If someone has a vaccine, I will laugh at you face to face.”

The idea of ​​an mRNA vaccine is more popular in the oncology community, even though it is used as a therapeutic agent rather than a disease prevention. Starting with the work of gene therapist David Curiel, several academic scientists and startups have explored whether mRNA can be used to fight cancer. If cancer cells express the protein encoded by mRNA, then injecting it into the body may train the immune system to attack these cells.

Curiel, now at Washington University School of Medicine in St. Louis, Missouri, has had some successful experience with mice. But he said that when he approached Ambion about commercialization opportunities, the company told him: “We don’t think this technology has any economic potential.”

Another cancer immunologist achieved greater success and established the first mRNA therapy company in 1997. Eli Gilboa recommends extracting immune cells from the blood and enticing them to extract synthetic mRNA that encodes tumor proteins. These cells are then injected back into the body, where they can mobilize the immune system to attack the latent tumor.

Gilboa and his colleagues at Duke University Medical Center in Durham, North Carolina proved this in mice. By the end of the 1990s, academic collaborators had begun human trials, and Gilboa’s commercial subsidiary Merix Bioscience (later renamed Argos Therapeutics, now known as CoImmune) soon began its own clinical research. It wasn’t until a few years ago that a late candidate vaccine failed in a large trial that this approach looked promising.

But Gilboa’s work had an important impact. It inspired the founders of German companies CureVac and BioNTech (the two largest mRNA companies today) to start researching mRNA. CureVac’s Ingmar Hoerr and BioNTech’s Uğur Şahin both told Nature that after learning about what Gilboa did, they wanted to do the same thing, but inject mRNA directly into the body.

Ingmar Hoerr (left) founded CureVac, and cancer immunologist Eli Gilboa (right) founded the first mRNA therapy company

Start accelerator

Hoerr was the first to succeed. In 2000, he reported in Tübingen, Germany, that direct injection can cause an immune response in mice. That year, he founded CureVac (also based in Tübingen). But it seems that few scientists or investors are interested in it. At a conference where Hoerr showed early mouse data, he said, “There was a Nobel Prize winner standing in the first row and saying,’What you are telling us here is all shit – all shit.'” Hoerr declined to disclose the name of the Nobel Prize winner).

In the end, funds slowly flowed in. Within a few years, human trials began. Steve Pascolo, the company’s chief scientific officer at the time, was the first research subject: He injected himself with mRNA, but there was still a white scar on his leg that resembled the head, and a dermatologist performed a needle biopsy there for analysis. Soon after, a more formal trial began, which involved tumor-specific mRNA for skin cancer patients.

Şahin and his immunologist wife Özlem Türeci also began studying mRNA in the late 1990s, but it took longer than Hoerr to start the company. They worked at the Johannes Gutenberg University in Mainz, Germany for many years, obtained patents, papers and research grants, and then launched a business plan to billionaire investors in 2007. Şahin said: “If it succeeds, it will be a breakthrough.” He received a seed fund of 150 million euros.

Özlem Türeci (left) and Uğur Şahin(Right) Co-founded BioNTech, an mRNA vaccine company

In the same year, an emerging mRNA start-up company called RNARx received a grant from the US government: a small business grant of $97,396. The company’s founder, biochemist Katalin Karik and immunologist Drew Weissman, both made some key findings at the University of Pennsylvania (UPENN) in Philadelphia at the time: changing part of the mRNA encoding helps to synthesize mRNA through cellular innate immune defense .
Some basic insights

Karikó has been working on transforming mRNA into a drug platform throughout the 1990s, although funding agencies have consistently rejected her funding application. In 1995, after repeated rejections, she could have chosen to leave UPenn or accept a demotion and salary reduction, but she chose to stay and continue her unremitting pursuit, improve Malone’s plan, and try to induce a kind of interaction between the cells. Treatment-related large and complex proteins.

Katalin Karikó helped prove that chemical modification of RNA can protect the molecule from the body’s immune defenses

(The tortuous road and complicated development history of mRNA vaccines.)

In 1997, she started collaborating with Weissman, who had just established a laboratory at the University of Pennsylvania. They plan to jointly develop an HIV/AIDS vaccine based on mRNA. But when Karikó’s mRNA is injected into mice, they trigger a large-scale inflammatory response.

She and Weissman quickly found the reason: the synthesized mRNA stimulated a series of immunosensors called Toll-like receptors, which are the first reactors of pathogen danger signals. In 2005, the two reported that rearranging the chemical bond on a nucleotide uridine in mRNA to produce an analog called pseudouridine, which seemed to prevent the body from recognizing it as an enemy.

Few scientists at the time realized the therapeutic value of these modified nucleotides. But the scientific community quickly realized their potential. In September 2010, a team led by Derrick Rossi, a stem cell biologist who was working at Boston Children’s Hospital in Massachusetts at the time, described how to use modified mRNAs to transform skin cells into embryonic stem cells and then into contracted muscle tissue. One discovery caused a sensation. Rossi was named one of 2010’s “Important People” in Time magazine, and he co-founded a company Moderna in Cambridge.

Moderna tried to authorize UPenn to apply for a modified mRNA patent for the invention of Karikó and Weissman in 2006, but it was too late. After failing to reach a license agreement with RNARx, the University of Pennsylvania chose to pay quickly. In February 2010, the company (now called Cellscript) granted an exclusive patent to a small laboratory reagent supplier in Madison. It will continue to receive hundreds of millions of dollars in sublicensing fees from Moderna and BioNTech, the two companies that were the founders of the first batch of new coronavirus mRNA vaccines, and their products contain modified mRNA.

At the same time, RNARx ran out of $800,000 in small business grant funds before and after Karikó joined BioNTech in 2013 (retaining UPenn’s subsidiary appointment) and ceased operations.

Drew Weissman collaborated with Karikó to discover the advantages of modifying mRNA

(The tortuous road and complicated development history of mRNA vaccines.)

Debate about pseudouridine

Researchers are still debating whether Karikó and Weissman’s findings are critical to a successful mRNA vaccine. Moderna has always used modified mRNA-its name is a combination of these two words, but some other people in the industry have not.

Researchers at the Human Gene Therapy Department of Shire, a pharmaceutical company in Lexington, Massachusetts, concluded that if the correct “cap” structure is added and all impurities are removed, unmodified mRNA can produce an equally effective product. “It depends on the quality of the RNA,” said Michael Heartlein, who led Shire’s research and continued to advance the technology at TranslateBio in Cambridge, where Shire later sold its mRNA portfolio to the company. (Shire is now part of Takeda Corporation in Japan)

Although Translate has some human data showing that its mRNA does not cause a worrying immune response, it still needs to be clinically proven: its COVID-19 vaccine candidate is still in early human trials. French pharmaceutical giant Sanofi is convinced of the prospects of this technology: In August 2021, it announced plans to acquire Translate for US$3.2 billion. (Heartlein left last year and established another company in Waltham, Massachusetts called Maritime Therapeutics)

At the same time, CureVac has its own immune mitigation strategy, including changing the gene sequence of mRNA to minimize the content of uridine in its vaccine. 20 years of research seems to have yielded results, and early trials of the company’s experimental vaccines against rabies and COVID-19 have proven successful. But in June, data from later trials showed that CureVac’s candidate coronavirus vaccine is far less protective than Moderna or BioNTech’s vaccines.

Based on these results, some mRNA experts now believe that pseudouridine is an important part of the technology. Therefore, they believe that Carrick and Weissman’s findings are one of the key enabling contributions to gain recognition and rewards. “The real winner is modified RNA,” said Jake Becraft, co-founder and CEO of Strand Therapeutics, a Cambridge-based synthetic biology company dedicated to mRNA-based therapies.

Not everyone is so sure. InBev, CEO of Suzhou Abogen Biosciences, said: “There are many factors that may affect the safety and effectiveness of mRNA vaccines, and chemical modification of mRNA is just one of them.” The company is a Chinese company that has an mRNA vaccine against COVID-19 and is currently in the late stage of clinical trials. (This product is called ARCoV and uses unmodified mRNA)

Fat breakthrough

As for the key technology, many experts emphasized another innovation that is vital to mRNA vaccines-an innovation that has nothing to do with mRNA. It is the tiny fat bubbles called lipid nanoparticles or LNPs that protect the mRNA and transport it to the cell.

This technology comes from the laboratories of Pieter Cullis, a biochemist at the University of British Columbia in Vancouver, Canada, and several companies he founded or led. Since the late 1990s, they took the lead in using LNP to deliver nucleic acid strands that silence gene activity. One such treatment, patisiran, is now approved for a rare genetic disease.

Pieter Cullis

(The tortuous road and complicated development history of mRNA vaccines.)

After gene silencing therapy began to show promise in clinical trials, in 2012, the two companies of Cullis turned to explore the opportunities of LNP delivery systems in mRNA-based drugs. For example, Vancouver Acuitas Therapeutics, led by CEO Thomas Madden, has partnered with Weissman’s team at the University of Pennsylvania and several mRNA companies to test different mRNA-LNP preparations. One of the COVID-19 vaccines from BioNTech and CureVac can now be found. Moderna’s LNP blend is not much different.

Nanoparticles have a mixture of four kinds of fat molecules: three are helpful for structure and stability; the fourth is called ionizable lipid, which is the key to the success of LNP. This substance is positively charged under laboratory conditions and has similar advantages to the liposomes developed by Felgner and tested by Malone in the late 1980s. However, the ionizable lipids proposed by Cullis and his business partners are converted to neutral charges under physiological conditions (such as those in the blood), which limits the toxic effects on the body.

In addition, Ian MacLachlan, a former executive of several companies associated with Cullis, said that this four-fat cocktail can keep the product on the pharmacy shelf for a longer time and maintain its stability in the body. He said: “It is this whole set of medicine tools and materials that led to the pharmacology we now have.”

Ian MacLachlan (left) and Thomas Madden (right)

(The tortuous road and complicated development history of mRNA vaccines.)

By the mid-2000s, a new method of mixing and manufacturing these nanoparticles had been devised. It involves using a “T-connector” device to combine fat (dissolved in alcohol) with nucleic acid (dissolved in an acid buffer). When the two solution streams are combined, the components spontaneously form dense LNPs.It turns out that it is a more reliable technology than other methods of manufacturing mRNA-based drugs.

Andrew Geall, the current chief development officer of Replicate Bioscience in San Diego, said that once all the parts are put together, “just like a fantasy, we finally have a scalable process.”

Geall led the first team to combine LNP and RNA vaccines at Novartis’s American Center in Cambridge in 2012. Now every mRNA company uses some variants of this LNP delivery platform and manufacturing system-although who owns the relevant patents is still the subject of legal disputes.

For example, Moderna and a subsidiary of Cullis-Vancouver’s Arbutus Biopharma-fought over who owns the rights to the LNP technology found in Moderna’s COVID-19 vaccine.

The birth of an industry

By the late 2000s, several large pharmaceutical companies began to enter the mRNA field. For example, in 2008, Novartis and Shire both established mRNA research departments-the former (led by Geall) focused on vaccines, and the latter (led by Heartlein) focused on treatments.

In 2012, the Defense Advanced Research Projects Agency decided to start funding industry researchers to research RNA vaccines and drugs. This decision promoted the establishment of BioNTech, and other start-ups soon joined the competition.

Moderna is one of the companies built on this work. By 2015, it had raised more than $1 billion and promised to use mRNA to induce cells in the body to make its own drugs to repair diseases caused by protein deficiency or defects. When the plan failed, Moderna, led by CEO Stéphane Bancel, chose to prioritize vaccine production.

Derrick Rossi (left) and Stéphane Bancel (right) of Moderna

(The tortuous road and complicated development history of mRNA vaccines.)

This initially disappointed many investors and bystanders, because vaccine platforms seemed less transformative and less profitable. By the beginning of 2020, Moderna had sent nine candidate mRNA vaccines for infectious diseases into humans for testing, but none of them had a successful dunk, and only one entered a larger trial stage.

But when the new coronavirus broke out, Moderna quickly stood out, producing a prototype vaccine within a few days of the viral genome sequence being online. The company subsequently collaborated with the National Institute of Allergy and Infectious Diseases (NIAID) to conduct mouse research and human trials in less than ten weeks.

BioNTech has also taken a fully armed approach. In March 2020, the company partnered with New York-based Pfizer Pharmaceuticals (Pfizer), and subsequent clinical trials were conducted at a record speed, from the first human trial to emergency approval in less than 8 months.

Both licensed vaccines use modified mRNA formulated in LNP. Both also contain a sequence encoding a form of the SARS-CoV-2 spike protein that takes a shape more suitable for inducing protective immunity. Many experts say that the protein tuning designed by NIAID vaccinologist Barney Graham and the University of Texas at Austin structural biologist Jason McLellan and Scripps’ Andrew Ward is also a valuable contribution, even though it is a coronary Viral vaccines are unique, not as general platform mRNA vaccines.

When discussing the credit for mRNA discovery, some people’s anger is related to who owns a profitable patent. But most of the basic intellectual property rights can be traced back to claims made by Felgner, Malone and their colleagues at Vical in 1989 (and Liljeström in 1990). These documents are only valid for 17 years from the date of publication, so they are now public.

Even the Karikó-Weissman patent, which was licensed and filed by Cellscript in 2006, will expire in the next five years. According to industry insiders, this means that it will soon be difficult to obtain patents on broad claims regarding the delivery of mRNA in lipid nanoparticles, although the company can reasonably assign specific mRNA sequences (such as a form of spike protein) or proprietary Some lipid preparations have applied for patents.

Companies are working hard. Moderna is a major player in the field of mRNA vaccines. The company has conducted experimental injections in clinical tests for influenza, cytomegalovirus and a series of other infectious diseases. Last year it obtained two patents involving the extensive use of mRNA to produce secreted proteins. But many people in the industry told Nature that they think these may be challenging.

Eric Marcusson, chief scientific officer of Providence Therapeutics, an mRNA vaccine company in Calgary, Canada, said: “We don’t think there are a lot of patents that can be applied for, of course, they can’t be enforced.”

The Nobel debates

As for who should win the Nobel Prize, the names that appear most often in conversations are Karikó and Weissman. The two have won multiple awards, including one of the breakthrough awards ($3 million, the most profitable award in science) and the prestigious Princess of Asturias Technology and Scientific Research Award in Spain.

Also receiving the Asturias Prize are Felgner, Şahin, Türeci and Rossi, as well as Sarah Gilbert and AstraZeneca, the vaccinologist behind the COVID-19 vaccine developed by the University of Oxford in the United Kingdom, which uses viral vectors instead of mRNA. .

Cullis’s only recent honor is the US$5,000 Founder’s Award from the Controlled Release Association, an organization of professional scientists researching sustained-release drugs)

Others believe that Carrick should be widely recognized, which is her contribution to the laboratory’s discoveries. “Not only is she an amazing scientist, she is also a force in the field,” said Anna Blakney, an RNA bioengineer at the University of British Columbia.

Blakney praised Karikó for giving her the opportunity to speak at an important conference two years ago, when she was still in a junior postdoctoral position (before Blakney co-founded the Cambridge vaccine company Vax Equity, the company focused on self-amplified RNA technology). Karikó “is actively working to improve others when she has been underestimated throughout her career.”

Although some people involved in mRNA development, including Malone, think they deserve more recognition, others are more willing to share the spotlight. When it comes to lipid delivery systems, Cullis said, “We are talking about hundreds of people who have been working together to make these LNP systems so that they are really ready for prime time.”

“Everyone just added something gradually—including me,” Karikó said.

In retrospect, many people said that they are happy that mRNA vaccines are transforming humans, and they may have made valuable contributions in the process. “I’m very excited to see this,” Felgner said. “Everything we thought was going to happen – it’s happening now.”

The tortuous road and complicated development history of mRNA vaccines.

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