Category Archives: Genetic Engineering

One of the most powerful tools available to biologists these days is CRISPR-Cas9, a combination of specialized RNA and protein that acts like a molecular scalpel, allowing researchers to precisely slice and dice pieces of an organism's genetic code.

But even though CRISPR-Cas9 technology has offered an unprecedented level of control for those studying genetics and genetic engineering, there has been room for improvement. Now, a new technique developed at Caltech by biology graduate student Shashank "Sha" Gandhi in the lab of Marianne Bronner, Distinguished Professor of Biology and director of the Beckman Institute, is taking CRISPR-Cas9 accessibility to the next level.

In a paper appearing in the journal Development, Gandhi and members of Bronner's lab describe the new technique, which has been designed specifically to disable or remove genes from a genome. This is known as "knocking out" a gene.

Gene knockout is an important method for studying what genes do because researchers can compare the behavior of a cell that has a working gene to the behavior of a cell in which that gene has been disabled. CRISPR-Cas9 has already been used for this, usually alongside genetic material that encodes a fluorescent protein, which makes it easy to identify cells from which a gene has been removed; cells with a knocked-out gene will glow.

One drawback of the technique, however, is that each part of the payload that makes it workthe Cas9 protein, the guide RNA, and the code for the fluorescent proteinhave to be delivered separately using a technique called electroporation, which opens the membranes of cells by zapping them with electricity. This can result in some cells receiving only some of the pieces. Thus, a cell could receive the code for making fluorescent protein, but not end up with a knocked-out gene. Or a cell could end up with its targeted gene knocked out, but not have the code to make fluorescent protein. Either case makes it more challenging for researchers to study how the cells are behaving.

"The advent of CRISPR has allowed people like me and Sha to work on just about any organism," Bronner says. "The caveat is that not every cell gets the same cocktail."

Another, bigger problem, has been that these CRISPR-Cas9 combinations often did not work across species lines because of a component known as a U6 promotera DNA sequence that tells a cell's machinery when and where to start making the guide RNA from a plasmid, a short loop of DNA that can be easily introduced into cells. A promoter that works in a fruit fly's genome will not necessarily work in that of a mouse, for example.

"The problem with the U6 promoter is that every species has its own version, so if you're working on a new system, you might not have a U6 to use," says Gandhi.

The new tool developed by Gandhi and other researchers in Bronner's lab eliminates both problems. Whereas the old CRISPR-Cas9 delivered each part of their payload separately, the new tool packages them together on a single plasmid. Because the plasmid contains all three required parts, the problem of a cell receiving only one or two of them is eliminated. The team's design also bypasses the need for using a U6 promoter, thereby enabling CRISPR-Cas9-based editing across multiple species.

"If you use plasmids for your CRISPR-Cas9 knockout experiments in an organism, this is the best way so far," Gandhi says.

Gandhi says that there is a growing interest in the tool among other research teams.

"We've actually been getting requests from around the world from people who are interested in using these tools in their research," he says. "I think that it will take a little bit of time for the tool to really pick up, but we've already received a lot of requests."

Bronner adds that her lab is working with Niles Pierce, professor of applied and computational mathematics and bioengineering, to develop more elaborate implementations of the tool.

"We're working on making this tool conditional, so you could say 'I want to lose gene X when gene Y turns on,' and the results are looking really promising," she says.

The paper describing their work, titled, "A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos," appears in the April 1 issue of the journal Development. Co-authors are biology graduate student Weiyi Tang; senior postdoctoral scholar in biology and biological engineering; Michael L. Piacentino, senior postdoctoral scholar research associate in biology and biological engineering; former postdoctoral scholars Yuwei Li and Felipe M. Vieceli; graduate student Hugo A. Urrutia; and Jens B. Christensen from the University of Copenhagen.

Funding for the research was provided by the National Institutes of Health and the American Heart Association.

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Researchers Improve Efficiency and Accessibility of CRISPR - Caltech

REDWOOD CITY, Calif., June 10, 2021 (GLOBE NEWSWIRE) -- Codexis, Inc., a leading enzyme engineering company enabling the promise of synthetic biology, today announced the expansion of its strategic collaboration and license agreement with Takeda Pharmaceutical Company Limited (Takeda) for the research and development of an additional gene therapy for a lysosomal storage disorder bringing the total number of programs under the agreement to four.

Under the terms of the original March 2020 agreement, Codexis leveraged its CodeEvolver protein engineering platform to generate novel gene sequences encoding enzyme variants that are tailored to enhance efficacy by increasing activity, stability, and cellular uptake. Takeda is combining these improved transgenes with its gene therapy capabilities to develop novel candidates for the treatment of rare genetic disorders.

We are thrilled to expand our collaboration with Takeda to advance novel gene therapies for the treatment of rare diseases. Over the past year, our CodeEvolver technology has generated novel genetic sequences that encode more efficacious enzymes for the potential treatment of Fabry and Pompe Diseases, as well as an undisclosed blood factor deficiency. Codexis and Takeda are excited about the prospect for each of these improved sequences to enable differentiated gene therapies for patients with rare genetic diseases, stated John Nicols, Codexis President and CEO.

Gjalt Huisman, Codexis Senior Vice-President, Biotherapeutics added, Within a year of embarking on our collaboration, the Codexis and Takeda research teams have made tremendous progress in generating and evaluating engineered gene sequences for the three separate therapeutic indications. We are proud that based on the results to date Takeda has exercised its option to initiate a fourth program.

Terms of AgreementUnder the terms of the original agreement, the parties began collaborative work on three initial programs. Takeda had the contractual option to expand the collaboration into a fourth program. Codexis is responsible for the creation of novel enzyme sequences for advancement as gene therapies into pre-clinical development. Takeda is responsible for the pre-clinical and clinical development and commercialization of gene therapy products resulting from the collaboration programs. Subject to the terms of the agreement, Codexis is eligible to receive an upfront payment, reimbursement for research and development fees, development and commercial milestone payments, and low- to mid-single digit percentage royalties on sales of any commercial product developed through programs initiated under the agreement.

About Codexis, Inc.Codexis is a leading enzyme engineering company leveraging its proprietary CodeEvolver platform to discover and develop novel, high performance enzymes and novel biotherapeutics. Codexis enzymes have applications in the sustainable manufacturing of pharmaceuticals, food, and industrial products; in the creation of the next generation of life science tools; and as gene therapy and biologic therapeutics. The Companys unique performance enzymes drive improvements such as: reduced energy usage, waste generation and capital requirements; higher yields; higher fidelity diagnostics; and more efficacious therapeutics. Codexis enzymes enable the promise of synthetic biology to improve the health of people and the planet. For more information, visit http://www.codexis.com.

Forward-Looking StatementsTo the extent that statements contained in this press release are not descriptions of historical facts regarding Codexis, they are forward-looking statements reflecting the current beliefs and expectations of management made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995, including Codexis expectations regarding the prospects for the development and future commercialization by Takeda of novel gene therapies for specified target indications. You should not place undue reliance on these forward-looking statements because they involve known and unknown risks, uncertainties and other factors that are, in some cases, beyond Codexis control and that could materially affect actual results. Factors that could materially affect actual results include, among others: Codexis dependence on its licensees and collaborators; the regulatory approval processes of the FDA and comparable foreign authorities are lengthy, time consuming and inherently unpredictable; results of preclinical studies and early clinical trials of product candidates may not be predictive of results of later studies or trials; even if we or our collaborators obtain regulatory approval for any products that are developed during a collaboration, such products will remain subject to ongoing regulatory requirements, which may result in significant additional expense; and there may be potential adverse effects to Codexis business if our collaborators products are not received well in the markets. Additional information about factors that could materially affect actual results can be found in Codexis Annual Report on Form 10-K filed with the Securities and Exchange Commission (SEC) on March 1, 2021 and in Codexis Quarterly Report on Form 10-Q filed with the SEC on May 7, 2021, including under the caption Risk Factors and in Codexis other periodic reports filed with the SEC. Codexis expressly disclaims any intent or obligation to update these forward-looking statements, except as required by law.

Investor Contact:Argot PartnersStephanie Marks/Carrie McKimCodexis@argotpartners.com(212) 600-1902

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Codexis and Takeda Expand Strategic Collaboration and License Agreement to Discover Additional Gene Therapy for a Fourth Rare Genetic Disorder -...

Opinion

Recently, I did something I had not done in a long time. I ate in a restaurant with my family. Actually, we ate on the outdoor patio, since my kids are too young to be vaccinated and we are somewhat more squeamish than average about COVID, but it was nevertheless a refreshing return to normality and a welcome rest from battling traffic on the way to the Delaware seashore.

I ordered a salad with blackened salmon. If we make the trip again, I will make a different choice.

Thats because last week, biotech company AquaBounty Technologies Inc. announced that it is harvesting several tons of genetically modified salmon, which will soon be sold at restaurants and other away-from-home dining retailers around the country. So far just one distributor Philadelphia-based Samuels and Son Seafoodhas reportedly said that it will be selling the novel salmon. But AquaBounty has announced plans to sell its salmon via food service channels across the Midwest and East Coast.

By selling to restaurants and cafeterias, rather than retailers, AquaBounty can avoid the federal GMO labeling law. And this sets a troubling precedent. Consumers who do not want to eat GMO fish will have to avoid salmon altogether when dining out.

There are many reasons why someone might not wish to consume meat from genetically engineered animals. They may not trust the U.S. Food and Drug Administrations (FDAs) safety assessment of the food. FDA conducted a lengthy review process of AquaBountys salmon, and concluded that it was no different in its nutrition profile and levels of hormones than conventional farm-raised salmon. But the salmon is a novel food, and some consumers may justifiably want to take a wait and see approach.

Other consumers may have concerns about the environmental risks associated with bioengineering animals, including the risk of transgenic contamination, whereby escaped GMO species crossbreed with native fish. Last year, a federal court ruled in favor of the advocacy group Center for Food Safety, ordering FDA to conduct an environmental assessment of its AquaBounty approval that takes the risk of fish escaping and reproducing in the environment into account. However, the judge allowed FDAs approval to stand pending completion of that assessment, because he deemed the near-term risk of such environmental harm to be low.

A consumer may worry that genetically engineering animals could harm animal welfare, just as conventional breeding has in some cases, or even that genetic engineering is fundamentally incompatible with the increasing recognition that livestock animals (and all sentient animals) deserve some moral standing, independent of their value as a commodity.

A consumer may see genetically engineered animals as synonymous with a corporate takeover of the food system, or as an affront to indigenous communities who have traditionally depended on wild salmon. This concern features prominently in Aramarks statement explaining its decision not to serve GMO salmon.

Aramark is not alone. Compass Group, Sodexo, Costco, Kroger, Walmart and Whole Foods have all pledged not to sell GMO salmon, at the behest of groups like the Center for Food Safety. But plenty of other outlets have made no such commitment, including (as far as I can tell) the restaurant where my family ate the other day.

AquaBounty and its supporters have a lot of good responses to concerns about GMO salmon, and ultimately, their arguments may win out in the court of public opinion. Personally, while I might aspire to one day eat an exclusively vegan, locavore diet that makes the world a better place with every bite, I might try the GMO salmon myself at some point.

But not like this. Consumers deserve to know whether the salmon on the menu comes from the first ever genetically engineered animal approved for human consumption. Food safety aside, consumers deserve the opportunity to consider the ethical and political issues enmeshed in genetically engineered animals before chowing down. At the very least, they should have the chance to notice whether this novel food tastes any different than its conventional counterpart. Pretending like this information is not important is an insult to the public, and creates the risk of a backlash that could erode confidence in both genetic engineering technology and the food system as a whole.

From what I know about FDAs food safety assessment, AquaBountys salmon seems safe to eat. But food safety concerns will nevertheless lead me to abstain from salmon of questionable origin for the foreseeable future. I want what I eat to contribute to a safer food system. And all else equal, a safer food system is a more transparent food system. How much transparency do we need? Reasonable people will disagree. But for me, secretly serving unsuspecting restaurant patrons genetically engineered salmonor pork or whatever else gets approved by FDAdeserves protest.

Thats a shame because a lot of difficult problemsfrom climate change to antibiotic resistance to invasive speciesmight conceivably get easier with the help of genetic engineering. But without public support, or trust, the technology is more likely to just serve the bottom line of a few unscrupulous companies.

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Not ready to eat GMO animals? Then you might not want to order the salmon - Food Safety News

Marianne Bronner and Shashank Gandhi (Credit: Caltech)

One of the most powerful tools available to biologists these days is CRISPR-Cas9, a combination of specialized RNA and protein that acts like a molecular scalpel, allowing researchers to precisely slice and dice pieces of an organisms genetic code.

But even though CRISPR-Cas9 technology has offered an unprecedented level of control for those studying genetics and genetic engineering, there has been room for improvement. Now, a new technique developed at Caltech by biology graduate student Shashank Sha Gandhi in the lab ofMarianne Bronner, Distinguished Professor of Biology and director of the Beckman Institute, is taking CRISPR-Cas9 accessibility to the next level.

In a paper appearing in the journalDevelopment, Gandhi and members of Bronners lab describe the new technique, which has been designed specifically to disable or remove genes from a genome. This is known as knocking out a gene.

Gene knockout is an important method for studying what genes do because researchers can compare the behavior of a cell that has a working gene to the behavior of a cell in which that gene has been disabled. CRISPR-Cas9 has already been used for this, usually alongside genetic material that encodes a fluorescent protein, which makes it easy to identify cells from which a gene has been removed; cells with a knocked-out gene will glow.

One drawback of the technique, however, is that each part of the payload that makes it workthe Cas9 protein, the guide RNA, and the code for the fluorescent proteinhave to be delivered separately using a technique called electroporation, which opens the membranes of cells by zapping them with electricity. This can result in some cells receiving only some of the pieces. Thus, a cell could receive the code for making fluorescent protein, but not end up with a knocked-out gene. Or a cell could end up with its targeted gene knocked out, but not have the code to make fluorescent protein. Either case makes it more challenging for researchers to study how the cells are behaving.

The advent of CRISPR has allowed people like me and Sha to work on just about any organism, Bronner says. The caveat is that not every cell gets the same cocktail.

Another, bigger problem, has been that these CRISPR-Cas9 combinations often did not work across species lines because of a component known as a U6 promotera DNA sequence that tells a cells machinery when and where to start making the guide RNA from a plasmid, a short loop of DNA that can be easily introduced into cells. A promoter that works in a fruit flys genome will not necessarily work in that of a mouse, for example.

The problem with the U6 promoter is that every species has its own version, so if youre working on a new system, you might not have a U6 to use, says Gandhi.

The new tool developed by Gandhi and other researchers in Bronners lab eliminates both problems. Whereas the old CRISPR-Cas9 delivered each part of their payload separately, the new tool packages them together on a single plasmid. Because the plasmid contains all three required parts, the problem of a cell receiving only one or two of them is eliminated. The teams design also bypasses the need for using a U6 promoter, thereby enabling CRISPR-Cas9-based editing across multiple species.

If you use plasmids for your CRISPR-Cas9 knockout experiments in an organism, this is the best way so far, Gandhi says.

Gandhi says that there is a growing interest in the tool among other research teams.

Weve actually been getting requests from around the world from people who are interested in using these tools in their research, he says. I think that it will take a little bit of time for the tool to really pick up, but weve already received a lot of requests.

Bronner adds that her lab is working with Niles Pierce, professor of applied and computational mathematics and bioengineering, to develop more elaborate implementations of the tool.

Were working on making this tool conditional, so you could say I want to lose gene X when gene Y turns on, and the results are looking really promising, she says.

The paper describing their work, titled, A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos, appears in the April 1 issue of the journalDevelopment. Co-authors are biology graduate student Weiyi Tang; senior postdoctoral scholar in biology and biological engineering; Michael L. Piacentino, senior postdoctoral scholar research associate in biology and biological engineering; former postdoctoral scholars Yuwei Li and Felipe M. Vieceli; graduate student Hugo A. Urrutia; and Jens B. Christensen from the University of Copenhagen.

Funding for the research was provided by the National Institutes of Health and the American Heart Association.

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Caltech Researchers Improve Usefulness of Powerful Tool Used to Edit Genes Pasadena Now - Pasadena Now

This image shows specialized lung cells (resembling a beaded necklace) that may mount a cytokine storm in response to some viral infections. Credit: UC San Diego Health Sciences

Gene expression patterns associated with pandemic viral infections provide a map to help define patients immune responses, measure disease severity, predict outcomes and test therapies for current and future pandemics.

Researchers at University of California San Diego School of Medicine used an artificial intelligence (AI) algorithm to sift through terabytes of gene expression data which genes are on or off during infection to look for shared patterns in patients with past pandemic viral infections, including SARS, MERS and swine flu.

Two telltale signatures emerged from the study, published today (June 11, 2021) in eBiomedicine. One, a set of 166 genes, reveals how the human immune system responds to viral infections. A second set of 20 signature genes predicts the severity of a patients disease. For example, the need to hospitalize or use a mechanical ventilator. The algorithms utility was validated using lung tissues collected at autopsies from deceased patients with COVID-19 and animal models of the infection.

These viral pandemic-associated signatures tell us how a persons immune system responds to a viral infection and how severe it might get, and that gives us a map for this and future pandemics, said Pradipta Ghosh, MD, professor of cellular and molecular medicine at UC San Diego School of Medicine and Moores Cancer Center.

From a simple blood draw, gene expression patterns associated with pandemic viral infections could provide clinicians with a map to help define patients immune responses, measure disease severity, predict outcomes and test therapies. Credit: UC San Diego Health Sciences

Ghosh co-led the study with Debashis Sahoo, PhD, assistant professor of pediatrics at UC San Diego School of Medicine and of computer science and engineering at Jacobs School of Engineering, and Soumita Das, PhD, associate professor of pathology at UC San Diego School of Medicine.

During a viral infection, the immune system releases small proteins called cytokines into the blood. These proteins guide immune cells to the site of infection to help get rid of the infection. Sometimes, though, the body releases too many cytokines, creating a runaway immune system that attacks its own healthy tissue. This mishap, known as a cytokine storm, is believed to be one of the reasons some virally infected patients, including some with the common flu, succumb to the infection while others do not.

But the nature, extent and source of fatal cytokine storms, who is at greatest risk and how it might best be treated have long been unclear.

When the COVID-19 pandemic began, I wanted to use my computer science background to find something that all viral pandemics have in common some universal truth we could use as a guide as we try to make sense of a novel virus, Sahoo said. This coronavirus may be new to us, but there are only so many ways our bodies can respond to an infection.

The data used to test and train the algorithm came from publicly available sources of patient gene expression data all the RNA transcribed from patients genes and detected in tissue or blood samples. Each time a new set of data from patients with COVID-19 became available, the team tested it in their model. They saw the same signature gene expression patterns every time.

In other words, this was what we call a prospective study, in which participants were enrolled into the study as they developed the disease and we used the gene signatures we found to navigate the uncharted territory of a completely new disease, Sahoo said.

By examining the source and function of those genes in the first signature gene set, the study also revealed the source of cytokine storms: the cells lining lung airways and white blood cells known as macrophages and T cells. In addition, the results illuminated the consequences of the storm: damage to those same lung airway cells and natural killer cells, a specialized immune cell that kills virus-infected cells.

We could see and show the world that the alveolar cells in our lungs that are normally designed to allow gas exchange and oxygenation of our blood, are one of the major sources of the cytokine storm, and hence, serve as the eye of the cytokine storm, Das said. Next, our HUMANOID Center team is modeling human lungs in the context of COVID-19 infection in order to examine both acute and post-COVID-19 effects.

The researchers think the information might also help guide treatment approaches for patients experiencing a cytokine storm by providing cellular targets and benchmarks to measure improvement.

To test their theory, the team pre-treated rodents with either a precursor version of Molnupiravir, a drug currently being tested in clinical trials for the treatment of COVID-19 patients, or SARS-CoV-2-neutralizing antibodies. After exposure to SARS-CoV-2, the lung cells of control-treated rodents showed the pandemic-associated 166- and 20-gene expression signatures. The treated rodents did not, suggesting that the treatments were effective in blunting cytokine storm.

It is not a matter of if, but when the next pandemic will emerge, said Ghosh, who is also director of the Institute for Network Medicine and executive director of the HUMANOID Center of Research Excellence at UC San Diego School of Medicine. We are building tools that are relevant not just for todays pandemic, but for the next one around the corner.

Reference: 11 June 2021, eBiomedicine.DOI: 10.1016/j.ebiom.2021.103390

Co-authors of the study include: Gajanan D. Katkar, Soni Khandelwal, Mahdi Behroozikhah, Amanraj Claire, Vanessa Castillo, Courtney Tindle, MacKenzie Fuller, Sahar Taheri, Stephen A. Rawlings, Victor Pretorius, David M. Smith, Jason Duran, UC San Diego; Thomas F. Rogers, Scripps Research and UC San Diego; Nathan Beutler, Dennis R. Burton, Scripps Research; Sydney I. Ramirez, La Jolla Institute for Immunology; Laura E. Crotty Alexander, VA San Diego Healthcare System and UC San Diego; Shane Crotty, Jennifer M. Dan, La Jolla Institute for Immunology and UC San Diego.

Funding: National Institutes for Health, UC San Diego Sanford Stem Cell Clinical Center, La Jolla Institute for Immunology Institutional Funds, and VA San Diego Healthcare System

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AI Trained With Genetic Data Predicts How Patients With Viral Infections Including COVID-19 Will Fare - SciTechDaily

Can we reprogram existing life at will?

To synthetic biologists, the answer is yes. The central code for biology is simple. DNA letters, in groups of three, are translated into amino acidsLego blocks that make proteins. Proteins build our bodies, regulate our metabolism, and allow us to function as living beings. Designing custom proteins often means you can redesign small aspects of lifefor example, getting a bacteria to pump out life-saving drugs like insulin.

All life on Earth follows this rule: a combination of 64 DNA triplet codes, or codons, are translated into 20 amino acids.

But wait. The math doesnt add up. Why wouldnt 64 dedicated codons make 64 amino acids? The reason is redundancy. Life evolved so that multiple codons often make the same amino acid.

So what if we tap into those redundant extra codons of all living beings, and instead insert our own code?

A team at the University of Cambridge recently did just that. In a technological tour de force, they used CRISPR to replace over 18,000 codons with synthetic amino acids that dont exist anywhere in the natural world. The result is a bacteria thats virtually resistant to all viral infectionsbecause it lacks the normal protein door handles that viruses need to infect the cell.

But thats just the beginning of engineering lifes superpowers. Until now, scientists have only been able to slip one designer amino acid into a living organism. The new work opens the door to hacking multiple existing codons at once, copyediting at least three synthetic amino acids at the same time. And when its 3 out of 20, thats enough to fundamentally rewrite life as it exists on Earth.

Weve long thought that liberating a subset ofcodons for reassignment could improve the robustness and versatility of genetic-code expansion technology, wrote Drs. Delilah Jewel and Abhishek Chatterjee at Boston College, who were not involved in the study. This work elegantly transforms that dream into a reality.

Our genetic code underlies life, inheritance, and evolution. But it only works with the help of proteins.

The program for translating genes, written in DNAs four letters, into the actual building blocks of life relies on a full cellular decryption factory.

Think of DNAs lettersA, T, C, and Gas a secret code, written on a long slip of crinkled paper wrapped around a spool. Groups of three letters, or codons, are the cruxthey encode which amino acid a cell makes. A messenger molecule (mRNA), a spy of sorts, stealthily copies the DNA message and sneaks back into the cellular world, shuttling the message to the cells protein factorya sort of central intelligence organization.

There, the factory recruits multiple translators to decipher the genetic code into amino acids, aptly named tRNAs. The letters are grouped in threes, and each translator tRNA physically drags its associated amino acid to the protein factory, one by one, so that the factory eventually makes a chain that wraps into a 3D protein.

But like any robust code, nature has programmed redundancy into its DNA-to-protein translation process. For example, the DNA codes TCG, TCA, AGC, and AGT all encode for a single amino acid, serine. While it works in biology, the authors wondered: what if we tap into that code, hijack it, and redirect some of lifes directions using synthetic amino acids?

The new study sees natures redundancy as a way to introduce new capabilities into cells.

For us, one question was could you reduce the number of codons that are used to encode a particular amino acid, and thereby create codons that are free to create other monomers [amino acids]? asked lead author Dr. Jason Chin.

For example, if TCG is for serine, why not free up the othersTCA, AGC, and AGT for something else?

Its a great idea in theory, but a truly daunting task in practice. It means that the team has to go into a cell and replace every single codon they want to reprogram. A few years back, the same group showed that its possible in E. Coli, the lab and pharmaceuticals favorite bug. At that time, the team made an astronomical leap in synthetic biology by synthesizing the entire E. Coli genome from scratch. During the process, they also played around with the natural genome, simplifying it by replacing some amino acid codons with their synonymssay, removing TCGs and replacing them with AGCs. Even with the modifications, the bacteria were able to thrive and reproduce easily.

Its like taking a very long book and figuring out which words to replace with synonyms without changing the meaning of sentencesso that the edits dont physically hurt the bacterias survival. One trick, for example, was to delete a protein dubbed release factor 1, which makes it easier to reprogram the UAG codon with a brand new amino acid. Previous work showed that this can assign new building blocks to natural codons that are truly blankthat is, they dont encode anything naturally anyways.

Chins team took this much further.

The team cooked up a method called REXER (replicon excision for enhanced genome engineering through programmed recombination)yeah, scientists are all about the backcronymswhich includes the wunderkind gene editing tool, CRISPR-Cas9. With CRISPR, they precisely snipped out large parts of theE. coli bacterial genome, made entirely from scratch inside a test tube, and then replaced more than 18,000 occurrences of extra codons that encode for serine with synonym codons.

Because the trick only targeted redundant protein code, the cells were able to go about their normal businessincluding making serinebut now with multiple natural codons free. Its like replacing hi with oy, making hi now free to be assigned a completely different meaning.

The team next did some house cleaning. They removed the cells natural translatorsthe tRNAsthat normally read the now-defunct codons without harming the cells. They introduced new synthetic versions of tRNAs to read the new codons. The engineered bacteria were then naturally evolved inside a test tube to grow more rapidly.

The results were spectacular. The superpowered strain, Syn61.3(ev5), is basically a bacterial X-Men that grows rapidly and is resistant to a cocktail of different viruses that normally infect bacteria.

Because all of biology uses the same genetic code, the same 64 codons and the same 20 amino acids, that means viruses also use the same codethey use the cells machinery to build the viral proteins to reproduce the virus, explained Chin. Now that the bacteria cell can no longer read natures standard genetic code, the virus can no longer tap into the bacterial machinery to reproducemeaning the engineered cells are now resistant to being hijacked by almost any viral invader.

These bacteria may be turned into renewable and programmable factories that produce a wide range of new molecules with novel properties, which could have benefits for biotechnology and medicine, including making new drugs, such as new antibiotics, said Chin.

Viral infection aside, the study rewrites whats possible for synthetic biology.

This will enable countless applications, said Jewel and Chatterjee, such as completely artificial biopolymers, that is, materials compatible with biology that could change entire disciplines such as medicine or brain-machine interfaces. Here, the team was able to string up a chain of artificial amino acid building blocks to make a type of molecule that forms the basis of some drugs, such as those for cancer or antibiotics.

But perhaps the most exciting prospect is the ability to dramatically rewrite existing life. Similar to bacteria, weand all life in the biosphereoperate on the same biological code. The study now shows its possible to get past the hurdle of only 20 amino acids making up the building blocks of life by tapping into our natural biological processes.

Next up, the team is looking to potentially further reprogram our natural biological code to encode even more synthetic protein building blocks into bacterial cells. Theyll also move towards other cellsmammalian, for example, to see if its possible to compress our genetic code.

Image Credit: nadya_il from Pixabay

Originally posted here:
Scientists Used CRISPR to Engineer a New 'Superbug' That's Invincible to All Viruses - Singularity Hub

June 10, 2021

Investments Totaling $250M Have Catalyzed Research and Clinical Trials Across Many Disciplines

By Ariel Bleicher and Cyril Manning

UC San Francisco is launching a new initiative to propel the development of living therapeutics a category of treatments broadly defined as human and microbial living cells that are selected, modified, or engineered to treat or cure disease and bring them quickly to patients.

The Living Therapeutics Initiative (LTI) will bring together UCSFs vast scientific and clinical expertise to accelerate research and quickly advance promising therapies to clinical trials for patients who have few, if any, good treatment options. As a federation of established UCSF initiatives, the LTI will allow disparate research and clinical programs to share information, toolsand platforms. Early this fall, the initiative will launch a $50 million grants program, made possible by philanthropy, to fund UCSF faculty living-therapeutics projects.

The Living Therapeutics Initiative creates a seamless continuum from the earliest stages of discovery all the way through to patient treatment in our hospitals, said UCSF Chancellor Sam Hawgood, MBBS. This process will span discovery, translational development, manufacturing therapeutic products, executing clinical trials, and securing regulatory approval for novel therapeutics. It will transform how we approach some of the most difficult diseases.

The Living Therapeutics Initiative creates a seamless continuum from the earliest stages of discovery all the way through to patient treatment in our hospitals.

Chancellor Sam Hawgood, MBBS

Over the past few years, UCSF has raised philanthropic gifts and made institutional commitments totaling more than $250 million to support living therapeutics-related efforts across the University.

Living therapeutics have been called a new third pillar of medicine, following small-molecule drugs (relatively simple compounds that can be chemically manufactured) and biologics (proteins and other molecules synthesized within microorganisms or cells).

CAR-T-cell therapies, which were among the first living therapeutics, have already proven lifesaving for patients with certain blood cancers. These advances can be replicated in other disciplines, and modification of cells to deliver these therapies is going to become a major new modality for many, many diseases, said Alan Ashworth, PhD, FRS, president of the UCSF Helen Diller Family Comprehensive Cancer Center and chair of the LTI steering committee.

Researchers across UCSF are already building the next generation of cellular therapies to treat diseases including solid tumors, autoimmunity, neurodegeneration, diabetesand infectious diseases. These therapies will be smarter, safer, and more effective than CAR-T, thanks to recent breakthroughs in cell engineering and gene editing.

The whole idea of living therapeutics is to take advantage of normal cellular processes and make them more efficient, said Michelle Hermiston, MD, PhD, clinical director of the UCSF Pediatric Immunotherapy Program. But there often is a big gap between what happens in the lab and what gets to the patient. With the LTI, were leveraging the scientific community at UCSF to bring new and novel therapies to kids and adults that they cant get anywhere else. Were at a point where these therapies are going to revolutionize how we treat disease.

Michelle Hermiston, MD, PhD: With the LTI, were leveraging the scientific community at UCSF to bring new and novel therapies to kids and adults that they cant get anywhere else. Were at a point where these therapies are going to revolutionize how we treat disease. Photo byMarco Sanchez

A number of these therapies could be realized in clinical trials with astonishing speed. One example is CAR-T therapies for solid tumors that have so far proven difficult to treat. Researchers and clinicians at UCSF are working together to develop CAR-T therapies for brain tumors. The science behind these efforts is at an advanced stage, and the cellular product is primed for production in UCSFs new cell-manufacturing facility as soon as it is up and running, said Ashworth.

Heritable disorders caused by single-gene mutations are also a prime target for living therapeutics. Clinical trials of CRISPR therapies for sickle cell disease, for example, are already underway at UCSF Benioff Childrens Hospital Oakland. Researchers are also using CRISPR to cure severe combined immunodeficiency syndrome and repair a genetic mutation in T cells that causes immune deficiency.

Such cell therapies could even be used in utero to treat diseases before birth. Pediatric and fetal surgeon Tippi MacKenzie, MD, is running the worlds first clinical trial using blood stem cells transplanted before birth. In this trial, the cells are donated by the mother and transfused into her fetus to treat alpha thalassemia major, a fatal blood disorder. Future therapies might genetically edit a fetuss own cells to repair the mutation that causes the disease.

Tippi MacKenzie, MD: Right now, UCSF has amazing expertise in the basic science of stem cell biology, and we have world-class clinical capabilities at UCSF Health. The Living Therapeutics Initiative is bringing these pieces together. Photo by Noah Berger

This is a transformational time in medicine, said MacKenzie, co-director of the UCSF Center for Maternal-Fetal Precision Medicine. Right now, UCSF has amazing expertise in the basic science of stem cell biology, and we have world-class clinical capabilities at UCSF Health. The Living Therapeutics Initiative is bringing these pieces together.

Further down the line, cell therapies may be used to restore or regenerate tissues that have been damaged by aging or disease. Such applications might include restoring cardiac function, regenerating neurons, and even engineering immune cells to treat HIV and other infectious diseases, particularly for immunocompromised patients.

Were just at the infancy of cell engineering hacking the genome of cells and getting them to behave in controllable ways, Ashworth said. Its like a computers operating system. The early ones were clunky and didnt work so well, but now theyre incredibly sophisticated. Thats the trajectory were on.

Not all living therapeutics are derived from human cells, however. We now understand that we also have trillions of microbial genomes within us that affect our bodies function, said Susan Lynch, PhD, director of the UCSF Benioff Center for Microbiome Medicine. We can manipulate the composition and activities of these microbial genomes in ways that will lead to better health overall.

Susan Lynch, PhD: We now understand that we also have trillions of microbial genomes within us that affect our bodies function. We can manipulate the composition and activities of these microbial genomes in ways that will lead to better health overall. Photo by Barbara Ries

Faculty members engaged in microbiome research across campus have demonstrated the tremendous potential of microorganisms isolated from the human gut and elsewhere in our bodies as therapies for a wide range of diseases. For example, microbial dysfunction in the infant gut characterized by the enrichment of particular microbial genes and their products drive immune dysfunction and can be used to predict the development of allergy and asthma in childhood.

Perturbed microbial ecosystems across the human body have been linked to autoimmune disease, metabolic syndromes such as obesity and diabetes, skin diseases, and even multiple sclerosis. Gut microbes can also produce and contribute to drug metabolism influencing pharmacologic function and bioavailability, opening up the possibility of microbial pharmacology as a key component of precision patient treatment.

The LTI will connect tools and expertise from across the ecosystem of UCSF initiatives and partner institutions working to advance cell-based therapeutics.

These initiatives and institutions include clinical services at UCSF Medical Center and UCSF Benioff Childrens Hospitals; the Chan Zuckerberg Biohub; the Gladstone-UCSF Institute of Genomic Immunology; the Innovative Genomics Institute; the Parker Institute for Cancer Immunotherapy; and UCSFs Bakar Computational Health Sciences Institute, Bakar ImmunoX Initiative, Benioff Center for Microbiome Medicine, Cell Design Institute, and Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research. Most recently, UCSF announced a partnership with Thermo Fisher Scientific for the co-development of a specialized facility for making cell-based immunotherapies and other cell-therapy products.

In addition to Alan Ashworth as chair, steering committee members include:

Michelle Hermiston, MD, PhD, clinical director of the UCSF Pediatric Immunotherapy Program

Wendell Lim, PhD, chair and Byers Distinguished Professor of cellular and molecular pharmacology;director of the Cell Design Institute

Tippi MacKenzie, MD, co-director of the UCSF Center for Maternal-Fetal Precision Medicine; pediatric fetal surgeon

Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology

Qizhi Tang, PhD, immunologist and Director of the Department of Surgerys Transplantation Research Lab

Jeffrey Wolf, MD, director of the UCSF Helen Diller Family Comprehensive Cancer CentersMyeloma Program

In addition to administering the $50 million in funding through an internal grant process, the LTI steering committee will help with coordination and strategy, such as thinking through regulatory issues, submitting applications to the U.S. Food and Drug Administration, and designing and evaluating clinical trials. Their evaluation of funding proposals will prioritize high-need, high-impact projects designed to lead to clinical trials.

Well be funding new ways of engineering cells for therapeutic benefit as well as the mechanics of getting those cells into the clinic, Ashworth said. Well learn from the clinical trials and then go back into the lab to design better versions of these therapies, iterating rapidly between lab and clinic. Thats at the heart of the Living Therapeutics Initiative.

Original post:
Living Therapeutics Initiative Will Accelerate Development and Delivery of Revolutionary Treatments - UCSF News Services

NEW YORK, June 07, 2021 (GLOBE NEWSWIRE) -- IN8bio, Inc. (IN8bio or the Company), a clinical-stage biopharmaceutical company focused on the discovery and development of innovative gamma delta T-cell therapies utilizing its DeltEx platform, today announced an update from the ongoing Phase 1 clinical trial of INB-200, its DeltEx drug resistant immunotherapy (DRI), MGMT-gene modified gamma delta T-cells in patients with newly diagnosed GBM. INB-200 was co-administered to patients undergoing the standard-of-care therapy for GBM during the temozolomide (TMZ) maintenance treatment.

The Phase 1 clinical trial of INB-200 (NCT04165941) is the first-in-human trial of a genetically modified gamma delta T-cell therapy. The therapy was well-tolerated with no observed infusion reactions, cytokine release syndrome (CRS), neurotoxicity or dose limiting toxicities (DLTs). The clinical program also cleared a data safety monitoring board (DSMB) review earlier in 2021 and enrollment for cohort 2 has been initiated. The trial is being conducted by Dr. Burt Nabors at the ONeal Comprehensive Cancer Center at the University of Alabama at Birmingham (UAB). The clinical trial poster was presented at the 2021 ASCO Annual Meeting from June 4-8.

Our DeltEx DRI platform combines the advantages of gamma delta T-cells with proprietary genetic engineering and next-generation cell therapy manufacturing that addresses the challenges of treating solid tumor cancers, said William Ho, Chief Executive Officer, and co-founder. Given the potential safety concerns of cellular therapies for solid tumor cancers, we are encouraged by this clinical update from our INB-200 trial in GBM patients. We believe that our Phase 1 program provides early evidence that gamma delta T-cells modified to be chemotherapy resistant are well-tolerated, with indications of clinical activity. Based on the initial safety profile, IN8bio has initiated Cohort 2 of this study, in which patients will receive three repeat doses of our DeltEx DRI product, INB-200.

The study is an open-label Phase 1 clinical trial evaluating DeltEx DRI, gamma delta T-cells genetically modified to express proteins that confer resistance to alkylating chemotherapies, in newly diagnosed GBM patients. Gamma delta T-cells are collected from the patient, expanded, activated and genetically modified with a proprietary process developed at IN8bio. Following surgery to remove the tumor and treatment with TMZ and radiation, patients in cohort 1 received a single intracranial dose of INB-200, during their TMZ maintenance phase. The primary endpoints of this Phase I trial are based on safety and tolerability, with secondary endpoints based on biologic response, progression free and overall survival.

The results of the study to date suggest that our INB-200 treatment is well-tolerated in lymphodepleted patients. Immunologic monitoring data presented at ASCO demonstrates that peripheral circulating T, Natural Killer (NK), and gamma delta T-cells decline and are suppressed during radiation + TMZ and remain as such through maintenance TMZ therapy. An advantage of this clinical approach in newly diagnosed GBM is that TMZ, the standard-of-care therapy, serves as the lymphodepleting agent for the cellular therapy. To date, two of three patients remain alive at 10 and nine months respectively. One treated patient was infused with INB-200 in May 2020 and, despite multiple poor prognostic factors including male sex, older age, MGMT-unmethylated and IDH wild-type GBM survived for 15.6 months post-diagnosis with stable disease before expiring from causes not related to GBM progression or INB-200 infusion. Based on the results to date, the Phase 1 study has initiated enrollment of patients in the second cohort, in which each patient will receive 3 repeat doses of INB-200, a DeltEx DRI product.

About IN8bioIN8bio is a clinical-stage biopharmaceutical company focused on the discovery, development and commercialization of gamma-delta T-cell product candidates for solid and liquid tumors. Gamma-delta T-cells are a specialized population of T-cells that possess unique properties, including the ability to differentiate between healthy and diseased tissue. These cells embody properties of both the innate and adaptive immune systems and can intrinsically differentiate between healthy and diseased tissue. IN8bios DeltEx platform employs allogeneic, autologous and genetically modified approaches to develop cell therapies, designed to effectively identify and eradicate tumor cells. IN8bio is currently conducting two investigator-initiated Phase 1 clinical trials for its lead gamma-delta T-cell product candidates: INB-200 for the treatment of newly diagnosed glioblastoma and INB-100 for the treatment of patients with leukemia undergoing hematopoietic stem cell transplantation. IN8bio also has a broad portfolio of preclinical programs focused on addressing other solid tumor types. For more information about IN8bio and its programs, please visit http://www.IN8bio.com.

About the DeltEx platformThe DeltEx platform is designed to overcome many of the challenges associated with the expansion, genetic engineering, and scalable manufacturing of gamma-delta T-cells. This approach enables the expansion of the cells, ex vivo, for administration of potentially therapeutic doses to patients, harnessing the unique properties of gamma-delta T-cells, including their ability to broadly recognize cellular stress signals on tumor cells. The DeltEx platform is the basis of a deep pipeline of innovative product candidates designed to effectively target and potentially eradicate disease and improve patient outcomes.

Forward Looking StatementsCertain statements herein concerning the Companys future expectations, plans and prospects, including without limitation, the Companys current expectations regarding the advancement of its product candidates through preclinical studies and clinical trials and the prospects for such candidates and underlying technology, constitute forward-looking statements. The use of words such as may, might, will, should, expect, plan, anticipate, believe, estimate, project, intend, future, potential, or continue, the negative of these and other similar expressions are intended to identify such forward looking statements. Such statements, based as they are on the current expectations of management, inherently involve numerous risks and uncertainties, known and unknown, many of which are beyond the Companys control. Consequently, actual future results may differ materially from the anticipated results expressed in such statements. Specific risks which could cause actual results to differ materially from the Companys current expectations include: scientific, regulatory and technical developments; failure to demonstrate safety, tolerability and efficacy; final and quality controlled verification of data and the related analyses; expense and uncertainty of obtaining regulatory approval, including from the U.S. Food and Drug Administration; and the Companys reliance on third parties, including licensors and clinical research organizations. Do not place undue reliance on any forward-looking statements included herein, which speak only as of the date hereof and which the Company is under no obligation to update or revise as a result of any event, circumstances or otherwise, unless required by applicable law.

Company Contact:IN8bio, Inc.Kate Rochlin, Ph.D.+1 646.600.6GDT (6438)info@IN8bio.com

Investors:Solebury TroutJulia Balanova+ 1 646.378.2936jbalanova@soleburytrout.com

Media:Burns McClellan, Inc.Ryo Imai / Robert Flamm, Ph.D.+1 212.213.0006 - ext. 315 / 364rimai@burnsmc.com / rflamm@burnsmc.com

Read more:
IN8bio Completes Treatment of First Cohort in Phase 1 Clinical Trial with Gamma Delta T-Cell Therapy in Patients with Newly Diagnosed Glioblastoma...

The European Commission recently published a report on new genomic techniques, including CRISPR gene editing, which was expected to havemajor implications for their regulation in the European Union (EU). As of today, the EU is blocking the introduction of next generation crops, regulating them as GMOs, which means theyve been all but banned under the continents precautionary principle-infused regulatory system.

Developers and supporters of gene editing technologies thought the report would accelerate the introduction of these products in the European market. However, far from introducing a strategy to end the European deadlock on these new biotechnologies, this report only announced further discussions. Further, EU political inaction may well comfort the leaders of China and the United States on these biotechnologies, two countries that are rushing to exploit these cutting edge tools.

New gene technologies hold promise in agriculture, industry and medicine, and the European Commission report recognizes this. In fact, the pioneering scientists involved with the most popular gene editing techniques (termed CRISPR-Cas), Emmanuelle Charpentier and Jennifer Doudna, were awarded with the 2020 Nobel Prize in Chemistry.

It cannot have escaped the attention of the Europe Commission that the continent is trailing far behind the US and China in all applied areas of these technologies. It is also obvious that the EU regulation of GMOs (a legal concept, often denounced by scientists as having no scientific or technical basis) has contributed to the backlash on these GMOs, which mainly aretransgenic plants. There is at least one consensus in this dossier: if these new genomic techniques are regulated as GMOs, it will not be possible to develop them for commercial purposes in Europe, and costly obstacles will have to be overcome before import is authorized.

A previous European official report (in 2011) already stated that The legislative framework as it operates today is not meeting needs or expectations, or its own objectives. But nothing has been done to solve the problem at the EU political level. What happened was actually quite the opposite: the regulatory burden increased further, while leaving uncertainties about the future of new biotechnologies. Inevitably, when politicians are inactive, the power of judges increases, and this happened in the EU. In 2018, the Court of Justice of the European Union (CJEU) concluded that a broad category of biotechnologies known as mutagenesis are GMOs and are, in principle, subject to the obligations laid down by the GMO Directive.

This means that these new genomic techniques, which often are mutagenesis techniques (they surgically modify genetic traits), fall within the scope of the EU GMO legislation. The current report by the European Commission was expected toprovide answers on how to overcome this major difficulty. It has not.

The pro-biotech side may be satisfied in the short term, because this report explicitly recognizes that products of new genomic techniques

have the potential to contribute to the objectives of the EUs Green Deal and in particular to the farm to fork and biodiversity strategies and the United Nations sustainable development goals.

The EUs proposedGreen Deal has the ambitious aim to make Europe the first climate neutral continent. Reactions from supporters of organic and regenerative agriculture, who are unequivocally opposed to biotechnology, were negative. According to IFOAM Organics Europe:

A weakening of the rules on the use of genetic engineering in agriculture and food is worrying news and could leave organic food systems unprotected including their ability to trace GMOs throughout the food chain to avoid contaminations that lead to economic losses and to live up to organic quality standards and consumer expectations. Organic producers urge the Commission and the Member States to maintain the existing regulatory framework and seriously consider the impact of the proposed regulatory scenario on organic food & farming, consumer choice and access to agrobiodiversity.

The rest is here:
Viewpoint: While most of Europe remains in thrall of crop biotechnology rejectionism, sustainability promises of CRISPR gene editing may soon lead to...

The COVID-19 pandemic has disrupted lives the world over for more than a year. Its death toll will soon reach three million people. Yet the origin of pandemic remains uncertain: The political agendas of governments and scientists have generated thick clouds of obfuscation, which the mainstream press seems helpless to dispel.

In what follows I will sort through the available scientific facts, which hold many clues as to what happened, and provide readers with the evidence to make their own judgments. I will then try to assess the complex issue of blame, which starts with, but extends far beyond, the government of China.

By the end of this article, you may have learned a lot about the molecular biology of viruses. I will try to keep this process as painless as possible. But the science cannot be avoided because for now, and probably for a long time hence, it offers the only sure thread through the maze.

The virus that caused the pandemic is known officially as SARS-CoV-2, but can be called SARS2 for short. As many people know, there are two main theories about its origin. One is that it jumped naturally from wildlife to people. The other is that the virus was under study in a lab, from which it escaped. It matters a great deal which is the case if we hope to prevent a second such occurrence.

Ill describe the two theories, explain why each is plausible, and then ask which provides the better explanation of the available facts. Its important to note that so far there isno direct evidencefor either theory. Each depends on a set of reasonable conjectures but so far lacks proof. So I have only clues, not conclusions, to offer. But those clues point in a specific direction. And having inferred that direction, Im going to delineate some of the strands in this tangled skein of disaster.

After the pandemic first broke out in December 2019, Chinese authorities reported that many cases had occurred in the wet marketa place selling wild animals for meatin Wuhan. This reminded experts of the SARS1 epidemic of 2002, in which a bat virus had spread first to civets, an animal sold in wet markets, and from civets to people. A similar bat virus caused a second epidemic, known as MERS, in 2012. This time the intermediary host animal was camels.

The decoding of the viruss genome showed it belonged a viral family known as beta-coronaviruses, to which the SARS1 and MERS viruses also belong. The relationship supported the idea that, like them, it was a natural virus that had managed to jump from bats, via another animal host, to people. The wet market connection, the major point of similarity with the SARS1 and MERS epidemics, was soon broken: Chinese researchers found earlier cases in Wuhan with no link to the wet market. But that seemed not to matter when so much further evidence in support of natural emergence was expected shortly.

Wuhan, however, is home of the Wuhan Institute of Virology, a leading world center for research on coronaviruses. So the possibility that the SARS2 virus had escaped from the lab could not be ruled out. Two reasonable scenarios of origin were on the table.

From early on, public and media perceptions were shaped in favor of the natural emergence scenario by strong statements from two scientific groups. These statements were not at first examined as critically as they should have been.

We stand together to strongly condemn conspiracy theories suggesting that COVID-19 does not have a natural origin, a group of virologists and others wrote in theLanceton February 19, 2020, when it was really far too soon for anyone to be sure what had happened. Scientists overwhelmingly conclude that this coronavirus originated in wildlife, they said, with a stirring rallying call for readers to stand with Chinese colleagues on the frontline of fighting the disease.

Contrary to the letter writers assertion, the idea that the virus might have escaped from a lab invoked accident, not conspiracy. It surely needed to be explored, not rejected out of hand. A defining mark of good scientists is that they go to great pains to distinguish between what they know and what they dont know. By this criterion, the signatories of the Lancet letter were behaving as poor scientists: They were assuring the public of facts they could not know for sure were true.

It later turned out that the Lancet letter had beenorganized and draftedby Peter Daszak, president of the EcoHealth Alliance of New York. Daszaks organization funded coronavirus research at the Wuhan Institute of Virology. If the SARS2 virus had indeed escaped from research he funded, Daszak would be potentially culpable. This acute conflict of interest was not declared to the Lancets readers. To the contrary, the letter concluded, We declare no competing interests.

Virologists like Daszak had much at stake in the assigning of blame for the pandemic. For 20 years, mostly beneath the publics attention, they had been playing a dangerous game. In their laboratories they routinely created viruses more dangerous than those that exist in nature. They argued that they could do so safely, and that by getting ahead of nature they could predict and prevent natural spillovers, the cross-over of viruses from an animal host to people. If SARS2 had indeed escaped from such a laboratory experiment, a savage blowback could be expected, and the storm of public indignation would affect virologists everywhere, not just in China. It would shatter the scientific edifice top to bottom, anMIT Technology Revieweditor, Antonio Regalado,saidin March 2020.

A second statement that had enormous influence in shaping public attitudes was aletter(in other words an opinion piece, not a scientific article) published on 17 March 2020 in the journalNature Medicine. Its authors were a group of virologists led by Kristian G. Andersen of the Scripps Research Institute. Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus, the five virologists declared in the second paragraph of their letter.

Unfortunately, this was another case of poor science, in the sense defined above. True, some older methods of cutting and pasting viral genomes retain tell-tale signs of manipulation. But newer methods, called no-see-um or seamless approaches, leave no defining marks. Nor do other methods for manipulating viruses such as serial passage, the repeated transfer of viruses from one culture of cells to another. If a virus has been manipulated, whether with a seamless method or by serial passage, there is no way of knowing that this is the case. Andersen and his colleagues were assuring their readers of something they could not know.

The discussion part of their letter begins, It is improbable that SARS-CoV-2 emerged through laboratory manipulation of a related SARS-CoV-like coronavirus. But wait, didnt the lead say the virus hadclearlynot been manipulated? The authors degree of certainty seemed to slip several notches when it came to laying out their reasoning.

The reason for the slippage is clear once the technical language has been penetrated. The two reasons the authors give for supposing manipulation to be improbable are decidedly inconclusive.

First, they say that the spike protein of SARS2 binds very well to its target, the human ACE2 receptor, but does so in a different way from that which physical calculations suggest would be the best fit. Therefore the virus must have arisen by natural selection, not manipulation.

If this argument seems hard to grasp, its because its so strained. The authors basic assumption, not spelt out, is that anyone trying to make a bat virus bind to human cells could do so in only one way. First they would calculate the strongest possible fit between the human ACE2 receptor and the spike protein with which the virus latches onto it. They would then design the spike protein accordingly (by selecting the right string of amino acid units that compose it). Since the SARS2 spike protein is not of this calculated best design, the Andersen paper says, therefore it cant have been manipulated.

But this ignores the way that virologists do in fact get spike proteins to bind to chosen targets, which is not by calculation but by splicing in spike protein genes from other viruses or by serial passage. With serial passage, each time the viruss progeny are transferred to new cell cultures or animals, the more successful are selected until one emerges that makes a really tight bind to human cells. Natural selection has done all the heavy lifting. The Andersen papers speculation about designing a viral spike protein through calculation has no bearing on whether or not the virus was manipulated by one of the other two methods.

The authors second argument against manipulation is even more contrived. Although most living things use DNA as their hereditary material, a number of viruses use RNA, DNAs close chemical cousin. But RNA is difficult to manipulate, so researchers working on coronaviruses, which are RNA-based, will first convert the RNA genome to DNA. They manipulate the DNA version, whether by adding or altering genes, and then arrange for the manipulated DNA genome to be converted back into infectious RNA.

Only a certain number of these DNA backbones have been described in the scientific literature. Anyone manipulating the SARS2 virus would probably have used one of these known backbones, the Andersen group writes, and since SARS2 is not derived from any of them, therefore it was not manipulated. But the argument is conspicuously inconclusive. DNA backbones are quite easy to make, so its obviously possible that SARS2 was manipulated using an unpublished DNA backbone.

And thats it. These are the two arguments made by the Andersen group in support of their declaration that the SARS2 virus was clearly not manipulated. And this conclusion, grounded in nothing but two inconclusive speculations, convinced the worlds press that SARS2 could not have escaped from a lab. A technical critique of the Andersen letter takes it down inharsher words.

Science is supposedly a self-correcting community of experts who constantly check each others work. So why didnt other virologists point out that the Andersen groups argument was full of absurdly large holes? Perhaps because in todays universities speech can be very costly. Careers can be destroyed for stepping out of line. Any virologist who challenges the communitys declared view risks having his next grant application turned down by the panel of fellow virologists that advises the government grant distribution agency.

The Daszak and Andersen letters were really political, not scientific, statements, yet were amazingly effective. Articles in the mainstream press repeatedly stated that a consensus of experts had ruled lab escape out of the question or extremely unlikely. Their authors relied for the most part on the Daszak and Andersen letters, failing to understand the yawning gaps in their arguments. Mainstream newspapers all have science journalists on their staff, as do the major networks, and these specialist reporters are supposed to be able to question scientists and check their assertions. But the Daszak and Andersen assertions went largely unchallenged.

Doubts about natural emergence.Natural emergence was the medias preferred theory until around February 2021 and the visit by a World Health Organization (WHO) commission to China. The commissions composition and access were heavily controlled by the Chinese authorities. Its members, who included the ubiquitous Daszak, kept asserting before, during, and after their visit that lab escape was extremely unlikely. But this was not quite the propaganda victory the Chinese authorities may have been hoping for. What became clear was that the Chinese had no evidence to offer the commission in support of the natural emergence theory.

This was surprising because both the SARS1 and MERS viruses had left copious traces in the environment. The intermediary host species of SARS1 was identifiedwithin four monthsof the epidemics outbreak, and the host of MERS within nine months. Yet some 15 months after the SARS2 pandemic began, and after a presumably intensive search, Chinese researchers had failed to find either the original bat population, or the intermediate species to which SARS2 might have jumped, or any serological evidence that any Chinese population, including that of Wuhan, had ever been exposed to the virus prior to December 2019. Natural emergence remained a conjecture which, however plausible to begin with, had gained not a shred of supporting evidence in over a year.

And as long as that remains the case, its logical to pay serious attention to the alternative conjecture, that SARS2 escaped from a lab.

Why would anyone want to create a novel virus capable of causing a pandemic? Ever since virologists gained the tools for manipulating a viruss genes, they have argued they could get ahead of a potential pandemic by exploring how close a given animal virus might be to making the jump to humans. And that justified lab experiments in enhancing the ability of dangerous animal viruses to infect people, virologists asserted.

With this rationale, they have recreated the 1918 flu virus, shown how the almost extinct polio virus can be synthesized from its published DNA sequence, and introduced a smallpox gene into a related virus.

These enhancements of viral capabilities are known blandly as gain-of-function experiments. With coronaviruses, there was particular interest in the spike proteins, which jut out all around the spherical surface of the virus and pretty much determine which species of animal it will target. In 2000 Dutch researchers, for instance, earned the gratitude of rodents everywhere bygenetically engineeringthe spike protein of a mouse coronavirus so that it would attack only cats.

Virologists started studying bat coronaviruses in earnest after these turned out to be the source of both the SARS1 and MERS epidemics. In particular, researchers wanted to understand what changes needed to occur in a bat viruss spike proteins before it could infect people.

Researchers at the Wuhan Institute of Virology, led by Chinas leading expert on bat viruses, Shi Zheng-li or Bat Lady, mounted frequent expeditions to the bat-infested caves of Yunnan in southern China and collected around a hundred different bat coronaviruses.

Shi then teamed up with Ralph S. Baric, an eminent coronavirus researcher at the University of North Carolina.Their workfocused on enhancing the ability of bat viruses to attack humans so as to examine the emergence potential (that is, the potential to infect humans) of circulating bat CoVs [coronaviruses]. In pursuit of this aim, in November 2015 they created a novel virus by taking the backbone of the SARS1 virus and replacing its spike protein with one from a bat virus (known as SHC014-CoV). This manufactured virus was able to infect the cells of the human airway, at least when tested against a lab culture of such cells.

The SHC014-CoV/SARS1 virus is known as a chimera because its genome contains genetic material from two strains of virus. If the SARS2 virus were to have been cooked up in Shis lab, then its direct prototype would have been the SHC014-CoV/SARS1 chimera, the potential danger of which concerned many observers and prompted intense discussion.

If the virus escaped, nobody could predict the trajectory,saidSimon Wain-Hobson, a virologist at the Pasteur Institute in Paris.

Baric and Shi referred to the obvious risks in their paper but argued they should be weighed against the benefit of foreshadowing future spillovers. Scientific review panels, they wrote, may deem similar studies building chimeric viruses based on circulating strains too risky to pursue. Given various restrictions being placed on gain-of function (GOF) research, matters had arrived in their view at a crossroads of GOF research concerns; the potential to prepare for and mitigate future outbreaks must be weighed against the risk of creating more dangerous pathogens. In developing policies moving forward, it is important to consider the value of the data generated by these studies and whether these types of chimeric virus studies warrant further investigation versus the inherent risks involved.

That statement was made in 2015. From the hindsight of 2021, one can say that the value of gain-of-function studies in preventing the SARS2 epidemic was zero. The risk was catastrophic, if indeed the SARS2 virus was generated in a gain-of-function experiment.

Inside the Wuhan Institute of Virology.Baric had developed, and taught Shi, a general method for engineering bat coronaviruses to attack other species. The specific targets were human cells grown in cultures and humanized mice. These laboratory mice, a cheap and ethical stand-in for human subjects, are genetically engineered to carry the human version of a protein called ACE2 that studs the surface of cells that line the airways.

Shi returned to her lab at the Wuhan Institute of Virology and resumed the work she had started on genetically engineering coronaviruses to attack human cells. How can we be so sure?

Because, by a strange twist in the story, her work was funded by the National Institute of Allergy and Infectious Diseases (NIAID), a part of the US National Institutes of Health (NIH). And grant proposals that funded her work, which are a matter of public record, specify exactly what she planned to do with the money.

The grants were assigned to the prime contractor, Daszak of the EcoHealth Alliance, who subcontracted them to Shi. Here are extracts from the grants for fiscal years 2018 and 2019. (CoV stands for coronavirus and S protein refers to the viruss spike protein.)

Test predictions of CoV inter-species transmission. Predictive models of host range (i.e. emergence potential) will be tested experimentally using reverse genetics, pseudovirus and receptor binding assays, and virus infection experiments across a range of cell cultures from different species andhumanized mice.

We will use S protein sequence data,infectious clone technology, in vitro and in vivo infection experiments and analysis of receptor binding to test the hypothesis that % divergence thresholds in S protein sequences predict spillover potential.

What this means, in non-technical language, is that Shi set out to create novel coronaviruses with the highest possible infectivity for human cells. Her plan was to take genes that coded for spike proteins possessing a variety of measured affinities for human cells, ranging from high to low. She would insert these spike genes one by one into the backbone of a number of viral genomes (reverse genetics and infectious clone technology), creating a series of chimeric viruses. These chimeric viruses would then be tested for their ability to attack human cell cultures (in vitro) and humanized mice (in vivo). And this information would help predict the likelihood of spillover, the jump of a coronavirus from bats to people.

The methodical approach was designed to find the best combination of coronavirus backbone and spike protein for infecting human cells. The approach could have generated SARS2-like viruses, and indeed may have created the SARS2 virus itself with the right combination of virus backbone and spike protein.

It cannot yet be stated that Shi did or did not generate SARS2 in her lab because her records have been sealed, but it seems she was certainly on the right track to have done so. It is clear that the Wuhan Institute of Virology was systematically constructing novel chimeric coronaviruses and was assessing their ability to infect human cells and human-ACE2-expressing mice, says Richard H. Ebright, a molecular biologist at Rutgers University and leading expert on biosafety.

It is also clear, Ebright said, that, depending on the constant genomic contexts chosen for analysis, this work could have produced SARS-CoV-2 or a proximal progenitor of SARS-CoV-2. Genomic context refers to the particular viral backbone used as the testbed for the spike protein.

The lab escape scenario for the origin of the SARS2 virus, as should by now be evident, is not mere hand-waving in the direction of the Wuhan Institute of Virology. It is a detailed proposal, based on the specific project being funded there by the NIAID.

Even if the grant required the work plan described above, how can we be sure that the plan was in fact carried out? For that we can rely on the word of Daszak, who has been much protesting for the last 15 months that lab escape was a ludicrousconspiracy theoryinvented by China-bashers.

On December 9, 2019, before the outbreak of the pandemic became generally known, Daszak gave aninterviewin which he talked in glowing terms of how researchers at the Wuhan Institute of Virology had been reprogramming the spike protein and generating chimeric coronaviruses capable of infecting humanized mice.

And we have now found, you know, after 6 or 7 years of doing this, over 100 new SARS-related coronaviruses, very close to SARS, Daszak says around minute 28 of the interview. Some of them get into human cells in the lab, some of them can cause SARS disease in humanized mice models and are untreatable with therapeutic monoclonals and you cant vaccinate against them with a vaccine. So, these are a clear and present danger:

Interviewer: You say these are diverse coronaviruses and you cant vaccinate against them, and no anti-viralsso what do we do?

Daszak: Well I thinkcoronavirusesyou can manipulate them in the lab pretty easily. Spike protein drives a lot of what happen with coronavirus, in zoonotic risk. So you can get the sequence, you can build the protein, and we work a lot with Ralph Baric at UNC to do this. Insert into the backbone of another virus and do some work in the lab. So you can get more predictive when you find a sequence. Youve got this diversity. Now the logical progression for vaccines is, if you are going to develop a vaccine for SARS, people are going to use pandemic SARS, but lets insert some of these other things and get a better vaccine.

The insertions he referred to perhaps included an element called the furin cleavage site, discussed below, which greatly increases viral infectivity for human cells.

In disjointed style, Daszak is referring to the fact that once you have generated a novel coronavirus that can attack human cells, you can take the spike protein and make it the basis for a vaccine.

One can only imagine Daszaks reaction when he heard of the outbreak of the epidemic in Wuhan a few days later. He would have known better than anyone the Wuhan Institutes goal of making bat coronaviruses infectious to humans, as well as the weaknesses in the institutes defense against their own researchers becoming infected.

But instead of providing public health authorities with the plentiful information at his disposal, he immediately launched a public relations campaign to persuade the world that the epidemic couldnt possibly have been caused by one of the institutes souped-up viruses. The idea that this virus escaped from a lab is just pure baloney. Its simply not true, he declared in an April 2020interview.

The safety arrangements at the Wuhan Institute of Virology.Daszak was possibly unaware of, or perhaps he knew all too well, thelong historyof viruses escaping from even the best run laboratories. The smallpox virus escaped three times from labs in England in the 1960s and 1970s, causing 80 cases and 3 deaths. Dangerous viruses have leaked out of labs almost every year since. Coming to more recent times, the SARS1 virus has proved a true escape artist, leaking from laboratories in Singapore, Taiwan, and no less than four times from the Chinese National Institute of Virology in Beijing.

One reason for SARS1 being so hard to handle is that there were no vaccines available to protect laboratory workers. As Daszak mentioned in the December 19 interview quoted above, the Wuhan researchers too had been unable to develop vaccines against the coronaviruses they had designed to infect human cells. They would have been as defenseless against the SARS2 virus, if it were generated in their lab, as their Beijing colleagues were against SARS1.

A second reason for the severe danger of novel coronaviruses has to do with the required levels of lab safety. There are four degrees of safety, designated BSL1 to BSL4, with BSL4 being the most restrictive and designed for deadly pathogens like the Ebola virus.

The Wuhan Institute of Virology had a new BSL4 lab, but its state of readiness considerably alarmed the State Department inspectors who visited it from the Beijing embassy in 2018. The new lab has a serious shortage of appropriately trained technicians and investigators needed to safely operate this high-containment laboratory, the inspectors wrote in acableof January 19, 2018.

The real problem, however, was not the unsafe state of the Wuhan BSL4 lab but the fact that virologists worldwide dont like working in BSL4 conditions. You have to wear a space suit, do operations in closed cabinets, and accept that everything will take twice as long. So the rules assigning each kind of virus to a given safety level were laxer than some might think was prudent.

Before 2020, the rules followed by virologists in China and elsewhere required that experiments with the SARS1 and MERS viruses be conducted in BSL3 conditions. But all other bat coronaviruses could be studied in BSL2, the next level down. BSL2 requires taking fairly minimal safety precautions, such as wearing lab coats and gloves, not sucking up liquids in a pipette, and putting up biohazard warning signs. Yet a gain-of-function experiment conducted in BSL2 might produce an agent more infectious than either SARS1 or MERS. And if it did, then lab workers would stand a high chance of infection, especially if unvaccinated.

Much of Shis work on gain-of-function in coronaviruses was performed at the BSL2 safety level, as is stated in her publications and other documents. She has said in an interviewwithSciencemagazine that [t]he coronavirus research in our laboratory is conducted in BSL-2 or BSL-3 laboratories.

It is clear that some or all of this work was being performed using a biosafety standardbiosafety level 2, the biosafety level of a standard US dentists officethat would pose an unacceptably high risk of infection of laboratory staff upon contact with a virus having the transmission properties of SARS-CoV-2, Ebright says.

It also is clear, he adds, that this work never should have been funded and never should have been performed.

This is a view he holds regardless of whether or not the SARS2 virus ever saw the inside of a lab.

Concern about safety conditions at the Wuhan lab was not, it seems, misplaced. According to afact sheetissued by the State Department on January 15, 2021, The U.S. government has reason to believe that several researchers inside the WIV became sick in autumn 2019, before the first identified case of the outbreak, with symptoms consistent with both COVID-19 and common seasonal illnesses.

David Asher, a fellow of the Hudson Institute and former consultant to the State Department, provided more detail about the incident at aseminar. Knowledge of the incident came from a mix of public information and some high end information collected by our intelligence community, he said. Three people working at a BSL3 lab at the institute fell sick within a week of each other with severe symptoms that required hospitalization. This was the first known cluster that were aware of, of victims of what we believe to be COVID-19. Influenza could not completely be ruled out but seemed unlikely in the circumstances, he said.

Comparing the rival scenarios of SARS2 origin.The evidence above adds up to a serious case that the SARS2 virus could have been created in a lab, from which it then escaped. But the case, however substantial, falls short of proof. Proof would consist of evidence from the Wuhan Institute of Virology, or related labs in Wuhan, that SARS2 or a predecessor virus was under development there. For lack of access to such records, another approach is to take certain salient facts about the SARS2 virus and ask how well each is explained by the two rival scenarios of origin, those of natural emergence and lab escape. Here are four tests of the two hypotheses. A couple have some technical detail, but these are among the most persuasive for those who may care to follow the argument.

Start with geography. The two closest known relatives of the SARS2 virus were collected from bats living in caves in Yunnan, a province of southern China. If the SARS2 virus had first infected people living around the Yunnan caves, that would strongly support the idea that the virus had spilled over to people naturally. But this isnt what happened. The pandemic broke out 1,500 kilometers away, in Wuhan.

Beta-coronaviruses, the family of bat viruses to which SARS2 belongs, infect the horseshoe batRhinolophus affinis, which ranges across southern China. The bats range is 50 kilometers, so its unlikely that any made it to Wuhan. In any case, the first cases of the COVID-19 pandemic probably occurred in September, whentemperatures in Hubei provinceare already cold enough to send bats into hibernation.

What if the bat viruses infected some intermediate host first? You would need a longstanding population of bats in frequent proximity with an intermediate host, which in turn must often cross paths with people. All these exchanges of virus must take place somewhere outside Wuhan, a busy metropolis which so far as is known is not a natural habitat ofRhinolophusbat colonies. The infected person (or animal) carrying this highly transmissible virus must have traveled to Wuhan without infecting anyone else. No one in his or her family got sick. If the person jumped on a train to Wuhan, no fellow passengers fell ill.

Its a stretch, in other words, to get the pandemic to break out naturally outside Wuhan and then, without leaving any trace, to make its first appearance there.

For the lab escape scenario, a Wuhan origin for the virus is a no-brainer. Wuhan is home to Chinas leading center of coronavirus research where, as noted above, researchers were genetically engineering bat coronaviruses to attack human cells. They were doing so under the minimal safety conditions of a BSL2 lab. If a virus with the unexpected infectiousness of SARS2 had been generated there, its escape would be no surprise.

The initial location of the pandemic is a small part of a larger problem, that of its natural history. Viruses dont just make one time jumps from one species to another. The coronavirus spike protein, adapted to attack bat cells, needs repeated jumps to another species, most of which fail, before it gains a lucky mutation. Mutationa change in one of its RNA unitscauses a different amino acid unit to be incorporated into its spike protein and makes the spike protein better able to attack the cells of some other species.

Through several more such mutation-driven adjustments, the virus adapts to its new host, say some animal with which bats are in frequent contact. The whole process then resumes as the virus moves from this intermediate host to people.

In the case of SARS1, researchers have documented the successive changes in its spike protein as the virus evolved step by step into a dangerous pathogen. After it had gotten from bats into civets, there were six further changes in its spike protein before it became a mild pathogen in people. After a further 14 changes, the virus was much better adapted to humans, and with a further four, theepidemic took off.

But when you look for the fingerprints of a similar transition in SARS2, a strange surprise awaits. The virus has changed hardly at all, at least until recently. From its very first appearance, it was well adapted to human cells. Researchers led by Alina Chan of the Broad Institute compared SARS2 with late stage SARS1, which by then was well adapted to human cells, and found that the two viruses were similarly well adapted. By the time SARS-CoV-2 was first detected in late 2019, it was already pre-adapted to human transmission to an extent similar to late epidemic SARS-CoV, theywrote.

Even those who think lab origin unlikely agree that SARS2 genomes are remarkably uniform. Baric writes that early strains identified in Wuhan, China, showed limited genetic diversity, which suggests that the virus may have been introduced from a single source.

A single source would of course be compatible with lab escape, less so with the massive variation and selection which is evolutions hallmark way of doing business.

The uniform structure of SARS2 genomes gives no hint of any passage through an intermediate animal host, and no such host has been identified in nature.

Proponents of natural emergence suggest that SARS2 incubated in a yet-to-be found human population before gaining its special properties. Or that it jumped to a host animal outside China.

All these conjectures are possible, but strained. Proponents of a lab leak have a simpler explanation. SARS2 was adapted to human cells from the start because it was grown in humanized mice or in lab cultures of human cells, just as described in Daszaks grant proposal. Its genome shows little diversity because the hallmark of lab cultures is uniformity.

Proponents of laboratory escape joke that of course the SARS2 virus infected an intermediary host species before spreading to people, and that they have identified ita humanized mouse from the Wuhan Institute of Virology.

The furin cleavage site is a minute part of the viruss anatomy but one that exerts great influence on its infectivity. It sits in the middle of the SARS2 spike protein. It also lies at the heart of the puzzle of where the virus came from.

The spike protein has two sub-units with different roles. The first, called S1, recognizes the viruss target, a protein called angiotensin converting enzyme-2 (or ACE2) which studs the surface of cells lining the human airways. The second, S2, helps the virus, once anchored to the cell, to fuse with the cells membrane. After the viruss outer membrane has coalesced with that of the stricken cell, the viral genome is injected into the cell, hijacks its protein-making machinery and forces it to generate new viruses.

But this invasion cannot begin until the S1 and S2 subunits have been cut apart. And there, right at the S1/S2 junction, is the furin cleavage site that ensures the spike protein will be cleaved in exactly the right place.

The virus, a model of economic design, does not carry its own cleaver. It relies on the cell to do the cleaving for it. Human cells have a protein cutting tool on their surface known as furin. Furin will cut any protein chain that carries its signature target cutting site. This is the sequence of amino acid units proline-arginine-arginine-alanine, or PRRA in the code that refers to each amino acid by a letter of the alphabet. PRRA is the amino acid sequence at the core of SARS2s furin cleavage site.

Viruses have all kinds of clever tricks, so why does the furin cleavage site stand out? Because of all known SARS-related beta-coronaviruses, only SARS2 possesses a furin cleavage site. All the other viruses have their S2 unit cleaved at a different site and by a different mechanism.

How then did SARS2 acquire its furin cleavage site? Either the site evolved naturally, or it was inserted by researchers at the S1/S2 junction in a gain-of-function experiment.

Consider natural origin first. Two ways viruses evolve are by mutation and by recombination. Mutation is the process of random change in DNA (or RNA for coronaviruses) that usually results in one amino acid in a protein chain being switched for another. Many of these changes harm the virus but natural selection retains the few that do something useful. Mutation is the process by which the SARS1 spike protein gradually switched its preferred target cells from those of bats to civets, and then to humans.

Mutation seems a less likely way for SARS2s furin cleavage site to be generated, even though it cant completely be ruled out. The sites four amino acid units are all together, and all at just the right place in the S1/S2 junction. Mutation is a random process triggered by copying errors (when new viral genomes are being generated) or by chemical decay of genomic units. So it typically affects single amino acids at different spots in a protein chain. A string of amino acids like that of the furin cleavage site is much more likely to be acquired all together through a quite different process known as recombination.

Recombination is an inadvertent swapping of genomic material that occurs when two viruses happen to invade the same cell, and their progeny are assembled with bits and pieces of RNA belonging to the other. Beta-coronaviruses will only combine with other beta-coronaviruses but can acquire, by recombination, almost any genetic element present in the collective genomic pool. What they cannot acquire is an element the pool does not possess. And no known SARS-related beta-coronavirus, the class to which SARS2 belongs, possesses a furin cleavage site.

Proponents of natural emergence say SARS2 could have picked up the site from some as yet unknown beta-coronavirus. But bat SARS-related beta-coronaviruses evidently dont need a furin cleavage site to infect bat cells, so theres no great likelihood that any in fact possesses one, and indeed none has been found so far.

The proponents next argument is that SARS2 acquired its furin cleavage site from people. A predecessor of SARS2 could have been circulating in the human population for months or years until at some point it acquired a furin cleavage site from human cells. It would then have been ready to break out as a pandemic.

If this is what happened, there should be traces in hospital surveillance records of the people infected by the slowly evolving virus. But none has so far come to light. According to the WHOreport on the origins of the virus, the sentinel hospitals in Hubei province, home of Wuhan, routinely monitor influenza-like illnesses and no evidence to suggest substantial SARSCoV-2 transmission in the months preceding the outbreak in December was observed.

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Viewpoint: Why the Wuhan lab escape theory explaining the origin of the global pandemic isn't going away anytime soon - Genetic Literacy Project