CRISPR diagnostics


Although clinical diagnostics take many forms, nucleic acid–based testing has become the gold standard for sensitive detection of many diseases, including pathogenic infections. Quantitative polymerase chain reaction (qPCR) has been widely adopted for its ability to detect only a few DNA or RNA molecules that can unambiguously specify a particular disease. However, the complexity of this technique restricts application to laboratory settings. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has underscored the need for the development and deployment of nucleic acid tests that are economical, easily scaled, and capable of being run in low-resource settings, without sacrifices in speed, sensitivity or specificity. CRISPR-based diagnostic (CRISPR-dx) tools offer a solution, and multiple CRISPR-dx products for detection of the SARS-CoV-2 RNA genome have been authorized by the US Food and Drug Administration (FDA). On page 941 of this issue, Jiao et al. (1) describe a new CRISPR-based tool to distinguish several SARS-CoV-2 variants in a single reaction.

There are multiple types of CRISPR systems comprising basic components of a single protein or protein complex, which cuts a specific DNA or RNA target programmed by a complementary guide sequence in a CRISPR-associated RNA (crRNA). The type V and VI systems and the CRISPR-associated endonucleases Cas12 (23) and Cas13 (45) bind and cut DNA or RNA, respectively. Furthermore, upon recognizing a target DNA or RNA sequence, Cas12 and Cas13 proteins exhibit “collateral activity” whereby any DNA or RNA, respectively, in the sample is cleaved regardless of its nucleic acid sequence (46). Thus, reporter DNAs or RNAs, which allow for visual or fluorescent detection upon cleavage, can be added to a sample to infer the presence or absence of specific DNA or RNA species (48).

Initial versions of CRISPR-dx utilizing Cas13 alone were sensitive to the low picomolar range, corresponding to a limit of detection of millions of molecules in a microliter sample. To improve sensitivity, preamplification methods, such as recombinase polymerase amplification (RPA), PCR, loop-mediated isothermal amplification (LAMP), or nucleic acid sequence–based amplification (NASBA), can be used with Cas12 or Cas13 to enable a limit of detection down to a single molecule (8). This preamplification approach, applicable to both Cas12 and Cas13 (67), enabled a suite of detection methods and multiplexing up to four orthogonal targets (7). Additional developments expanded CRISPR-dx readouts beyond fluorescence, including lateral flow (7), colorimetric (9), and electronic or material responsive readouts (10), allowing for instrument-free approaches. In addition, post–collateral-cleavage amplification methods, such as the use of the CRISPR-associated enzyme Csm6, have been combined with Cas13 to further increase the speed of CRISPR-dx tests (7). As an alternative to collateral-cleavage–based detection, type III CRISPR systems, which involve large multiprotein complexes capable of targeting both DNA and RNA, have been used for SARS-CoV-2 detection through production of colorimetric or fluorometric readouts (11).

FDA-authorized CRISPR-dx tests are currently only for use in centralized labs, because the most common CRISPR detection protocols require fluid handling steps and two different incubations, precluding their immediate use at the point of care. Single-step formulations have been developed to overcome this limitation, and these “one-pot” versions of CRISPR-dx are simple to run, operate at a single temperature, and run without complex equipment, producing either fluorescence or lateral flow readouts. The programmability of CRISPR makes new diagnostic tests easier to develop, and within months of the release of the SARS-CoV-2 genome, many COVID-19–specific CRISPR tests were reported and distributed around the world.

The broader capability for Cas enzyme–enhanced nucleic acid binding or cleavage has led to several other detection modalities. Cas9-based methods for cleaving nucleic acids in solution for diagnostic purposes have been combined with other detection platforms, such as destruction of undesired amplicons for preparation of next-generation sequencing libraries (12), or selective removal of alleles for nucleotide-specific detection (13). Alternatively, the programmable cleavage event from the Cas nuclease can be used to initiate an amplification reaction (14). Cas9-based DNA targeting has also been used for nucleotide detection in combination with solid-state electronics, promising an amplification-free platform for detection. In this platform, called CRISPR-Chip, the Cas9 protein binds nucleotide targets of interest (often in the context of the native genome) to graphene transistors, where the presence of these targets alters either current or voltage (15). By utilizing additional Cas9 orthologs and specific guide designs, CRISPR-Chip approaches have been tuned for single–base-pair sensitivity (15). Because they are integrated with electronic readers, CRISPR-Chip platforms may allow facile point-of-care detection with handheld devices.

Jiao et al. use a distinct characteristic of type II CRISPR systems, which involve Cas9, to develop a new type of noncollateral based CRISPR detection. Unlike Cas12s and Cas13, Cas9-crRNA complex formation requires an additional RNA known as the trans-activating CRISPR RNA (tracrRNA). By sequencing RNAs bound to Cas9 from Campylobacter jejuni in its natural host, the authors identified unexpected crRNAs, called noncanonical crRNA (ncrRNA), that corresponded to endogenous transcripts. Upon investigation of this surprising observation, it became clear that the tracrRNA was capable of hybridizing to semi-complementary sequences from a variety of RNA sources, leading to biogenesis of ncrRNAs of various sizes. Recognizing that they could program tracrRNAs to target a transcript of interest, the authors generated a reprogrammed tracrRNA (Rptr) that could bind and cleave a desired transcript, converting a piece of that transcript into a functional guide RNA. By then creating fluorescent DNA sensors that would be cleaved by the Rptr and ncrRNAs, the sensing of RNA by Cas9 could be linked to a detectable readout. This platform, called LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection), can be combined with gel-based readouts and enables multiplexed detection of several different sequences in a single reaction (see the figure).

Jiao et al. also combined LEOPARD with PCR in a multistep workflow to detect SARS-CoV-2 genomes from patients with COVID-19. Although more work is needed to integrate this Cas9-based detection modality into a single step with RPA or LAMP to create a portable and sensitive isothermal test, an advantage of this approach is the higher-order multiplexing that can be achieved, allowing multiple pathogens, diseases, or variants to be detected simultaneously. More work is also needed to combine this technology with extraction-free methods for better ease of use; alternative readouts to gel-based readouts, such as lateral flow and colorimetric readouts, would be beneficial for point-of-care detection.

In just 5 years, the CRISPR-dx field has rapidly expanded, growing from a set of peculiar molecular biology discoveries to multiple FDA-authorized COVID-19 tests and spanning four of the six major subtypes of CRISPR systems. Despite the tremendous promise of CRISPR-dx, substantial challenges remain to adapting these technologies for point-of-care and at-home settings. Simplification of the chemistries to operate as a single reaction in a matter of minutes would be revolutionary, especially if the reaction could be run at room temperature without any complex or expensive equipment. These improvements to CRISPR-dx assays can be achieved by identification or engineering of additional Cas enzymes with lower-temperature requirements, higher sensitivity, or faster kinetics, enabling rapid and simple amplification-free detection with single-molecule sensitivity.

Often overlooked is the necessity for a sample DNA or RNA preparation step that is simple enough to be added directly to the CRISPR reaction to maintain a simple workflow for point-of-care testing. In addition, higher-order multiplexing developments would allow for expansive testing menus and approach the possibility of testing for all known diseases. As these advancements are realized, innovative uses of CRISPR-dx will continue in areas such as surveillance, integration with biomaterials, and environmental monitoring. In future years, CRISPR-dx assays may become universal in the clinic and at home, reshaping how diseases are diagnosed.

Researchers Are Hatching a Low-Cost Coronavirus Vaccine


A new vaccine for Covid-19 that is entering clinical trials in Brazil, Mexico, Thailand and Vietnam could change how the world fights the pandemic. The vaccine, called NDV-HXP-S, is the first in clinical trials to use a new molecular design that is widely expected to create more potent antibodies than the current generation of vaccines. And the new vaccine could be far easier to make.

Existing vaccines from companies like Pfizer and Johnson & Johnson must be produced in specialized factories using hard-to-acquire ingredients. In contrast, the new vaccine can be mass-produced in chicken eggs — the same eggs that produce billions of influenza vaccines every year in factories around the world.

If NDV-HXP-S proves safe and effective, flu vaccine manufacturers could potentially produce well over a billion doses of it a year. Low- and middle-income countries currently struggling to obtain vaccines from wealthier countries may be able to make NDV-HXP-S for themselves or acquire it at low cost from neighbors.

“That’s staggering — it would be a game-changer,” said Andrea Taylor, assistant director of the Duke Global Health Innovation Center.

First, however, clinical trials must establish that NDV-HXP-S actually works in people. The first phase of clinical trials will conclude in July, and the final phase will take several months more. But experiments with vaccinated animals have raised hopes for the vaccine’s prospects.

“It’s a home run for protection,” said Dr. Bruce Innis of the PATH Center for Vaccine Innovation and Access, which has coordinated the development of NDV-HXP-S. “I think it’s a world-class vaccine.”

Vaccines work by acquainting the immune system with a virus well enough to prompt a defense against it. Some vaccines contain entire viruses that have been killed; others contain just a single protein from the virus. Still others contain genetic instructions that our cells can use to make the viral protein.

Once exposed to a virus, or part of it, the immune system can learn to make antibodies that attack it. Immune cells can also learn to recognize infected cells and destroy them.

In the case of the coronavirus, the best target for the immune system is the protein that covers its surface like a crown. The protein, known as spike, latches onto cells and then allows the virus to fuse to them.

But simply injecting coronavirus spike proteins into people is not the best way to vaccinate them. That’s because spike proteins sometimes assume the wrong shape, and prompt the immune system to make the wrong antibodies.

This insight emerged long before the Covid-19 pandemic. In 2015, another coronavirus appeared, causing a deadly form of pneumonia called MERS. Jason McLellan, a structural biologist then at the Geisel School of Medicine at Dartmouth, and his colleagues set out to make a vaccine against it.

They wanted to use the spike protein as a target. But they had to reckon with the fact that the spike protein is a shape-shifter. As the protein prepares to fuse to a cell, it contorts from a tulip-like shape into something more akin to a javelin.

Scientists call these two shapes the prefusion and postfusion forms of the spike. Antibodies against the prefusion shape work powerfully against the coronavirus, but postfusion antibodies don’t stop it.

Dr. McLellan and his colleagues used standard techniques to make a MERS vaccine but ended up with a lot of postfusion spikes, useless for their purposes. Then they discovered a way to keep the protein locked in a tulip-like prefusion shape. All they had to do was change two of more than 1,000 building blocks in the protein into a compound called proline.

The resulting spike — called 2P, for the two new proline molecules it contained — was far more likely to assume the desired tulip shape. The researchers injected the 2P spikes into mice and found that the animals could easily fight off infections of the MERS coronavirus.

The team filed a patent for its modified spike, but the world took little notice of the invention. MERS, although deadly, is not very contagious and proved to be a relatively minor threat; fewer than 1,000 people have died of MERS since it first emerged in humans.

But in late 2019 a new coronavirus, SARS-CoV-2, emerged and began ravaging the world. Dr. McLellan and his colleagues swung into action, designing a 2P spike unique to SARS-CoV-2. In a matter of days, Moderna used that information to design a vaccine for Covid-19; it contained a genetic molecule called RNA with the instructions for making the 2P spike.

Other companies soon followed suit, adopting 2P spikes for their own vaccine designs and starting clinical trials. All three of the vaccines that have been authorized so far in the United States — from Johnson & Johnson, Moderna and Pfizer-BioNTech — use the 2P spike.

Other vaccine makers are using it as well. Novavax has had strong results with the 2P spike in clinical trials and is expected to apply to the Food and Drug Administration for emergency use authorization in the next few weeks. Sanofi is also testing a 2P spike vaccine and expects to finish clinical trials later this year.

Dr. McLellan’s ability to find lifesaving clues in the structure of proteins has earned him deep admiration in the vaccine world. “This guy is a genius,” said Harry Kleanthous, a senior program officer at the Bill & Melinda Gates Foundation. “He should be proud of this huge thing he’s done for humanity.”The Coronavirus Outbreak ›

But once Dr. McLellan and his colleagues handed off the 2P spike to vaccine makers, he turned back to the protein for a closer look. If swapping just two prolines improved a vaccine, surely additional tweaks could improve it even more.

“It made sense to try to have a better vaccine,” said Dr. McLellan, who is now an associate professor at the University of Texas at Austin.

In March, he joined forces with two fellow University of Texas biologists, Ilya Finkelstein and Jennifer Maynard. Their three labs created 100 new spikes, each with an altered building block. With funding from the Gates Foundation, they tested each one and then combined the promising changes in new spikes. Eventually, they created a single protein that met their aspirations.

The winner contained the two prolines in the 2P spike, plus four additional prolines found elsewhere in the protein. Dr. McLellan called the new spike HexaPro, in honor of its total of six prolines.

The structure of HexaPro was even more stable than 2P, the team found. It was also resilient, better able to withstand heat and damaging chemicals. Dr. McLellan hoped that its rugged design would make it potent in a vaccine.

Dr. McLellan also hoped that HexaPro-based vaccines would reach more of the world — especially low- and middle-income countries, which so far have received only a fraction of the total distribution of first-wave vaccines.

“The share of the vaccines they’ve received so far is terrible,” Dr. McLellan said.

To that end, the University of Texas set up a licensing arrangement for HexaPro that allows companies and labs in 80 low- and middle-income countries to use the protein in their vaccines without paying royalties.

Meanwhile, Dr. Innis and his colleagues at PATH were looking for a way to increase the production of Covid-19 vaccines. They wanted a vaccine that less wealthy nations could make on their own.

The first wave of authorized Covid-19 vaccines require specialized, costly ingredients to make. Moderna’s RNA-based vaccine, for instance, needs genetic building blocks called nucleotides, as well as a custom-made fatty acid to build a bubble around them. Those ingredients must be assembled into vaccines in purpose-built factories.

The way influenza vaccines are made is a study in contrast. Many countries have huge factories for making cheap flu shots, with influenza viruses injected into chicken eggs. The eggs produce an abundance of new copies of the viruses. Factory workers then extract the viruses, weaken or kill them and then put them into vaccines.

The PATH team wondered if scientists could make a Covid-19 vaccine that could be grown cheaply in chicken eggs. That way, the same factories that make flu shots could make Covid-19 shots as well.

In New York, a team of scientists at the Icahn School of Medicine at Mount Sinai knew how to make just such a vaccine, using a bird virus called Newcastle disease virus that is harmless in humans.

For years, scientists had been experimenting with Newcastle disease virus to create vaccines for a range of diseases. To develop an Ebola vaccine, for example, researchers added an Ebola gene to the Newcastle disease virus’s own set of genes.

The scientists then inserted the engineered virus into chicken eggs. Because it is a bird virus, it multiplied quickly in the eggs. The researchers ended up with Newcastle disease viruses coated with Ebola proteins.

At Mount Sinai, the researchers set out to do the same thing, using coronavirus spike proteins instead of Ebola proteins. When they learned about Dr. McLellan’s new HexaPro version, they added that to the Newcastle disease viruses. The viruses bristled with spike proteins, many of which had the desired prefusion shape. In a nod to both the Newcastle disease virus and the HexaPro spike, they called it NDV-HXP-S.

PATH arranged for thousands of doses of NDV-HXP-S to be produced in a Vietnamese factory that normally makes influenza vaccines in chicken eggs. In October, the factory sent the vaccines to New York to be tested. The Mount Sinai researchers found that NDV-HXP-S conferred powerful protection in mice and hamsters.

“I can honestly say I can protect every hamster, every mouse in the world against SARS-CoV-2,” Dr. Peter Palese, the leader of the research, said. “But the jury’s still out about what it does in humans.”

The potency of the vaccine brought an extra benefit: The researchers needed fewer viruses for an effective dose. A single egg may yield five to 10 doses of NDV-HXP-S, compared to one or two doses of influenza vaccines.

“We are very excited about this, because we think it’s a way of making a cheap vaccine,” Dr. Palese said.

PATH then connected the Mount Sinai team with influenza vaccine makers. On March 15, Vietnam’s Institute of Vaccines and Medical Biologicals announced the start of a clinical trial of NDV-HXP-S. A week later, Thailand’s Government Pharmaceutical Organization followed suit. On March 26, Brazil’s Butantan Institute said it would ask for authorization to begin its own clinical trials of NDV-HXP-S.

Meanwhile, the Mount Sinai team has also licensed the vaccine to the Mexican vaccine maker Avi-Mex as an intranasal spray. The company will start clinical trials to see if the vaccine is even more potent in that form.

To the nations involved, the prospect of making the vaccines entirely on their own was appealing. “This vaccine production is produced by Thai people for Thai people,” Thailand’s health minister, Anutin Charnvirakul, said at the announcement in Bangkok.

In Brazil, the Butantan Institute trumpeted its version of NDV-HXP-S as “the Brazilian vaccine,” one that would be “produced entirely in Brazil, without depending on imports.”

Ms. Taylor, of the Duke Global Health Innovation Center, was sympathetic. “I could understand why that would really be such an attractive prospect,” she said. “They’ve been at the mercy of global supply chains.”

Madhavi Sunder, an expert on intellectual property at Georgetown University Law Center, cautioned that NDV-HXP-S would not immediately help countries like Brazil as they grappled with the current wave of Covid-19 infections. “We’re not talking 16 billion doses in 2020,” she said.

Instead, the strategy will be important for long-term vaccine production — not just for Covid-19 but for other pandemics that may come in the future. “It sounds super promising,” she said.

In the meantime, Dr. McLellan has returned to the molecular drawing board to try to make a third version of their spike that is even better than HexaPro.

“There’s really no end to this process,” he said. “The number of permutations is almost infinite. At some point, you’d have to say, ‘This is the next generation.’”

Covid-19 vaccines have alerted the world to the power of RNA therapies


Molecular biology is not a popularity contest. But if it were, it would be a partisan one. The evolutionary biologists would pledge their allegiance en masse to dna. The sequences contained in its regular coils knit together the stories of almost all life on the planet. Pharmacologists, being of a more practical bent, would instead vote for proteins. Proteins are not about sequence, but about shape; their complex, irregular outlines, and the ways that they can change, allow them to do almost all of the biological work that gets done in cells. And it is thanks to the way that particular drug molecules fit into those shapes that almost all drugs have their effects.Listen to this story

Enjoy more audio and podcasts on iOS or Android.

There would be only a small following for ribonucleic acid (rna), widely seen as a helpmeet molecule. It could be argued that the production of rna is dna’s main purpose; it is certainly true that the production of proteins would be nowhere without it. But it is a backstage operator, not a star; hewing wood and drawing water, hard working but hardly glamorous, appreciated only by devotees.

Or at least that was the case until vaccines made of rna started giving protection against covid-19 to millions of people around the world every day. Now Cinderella has gone to the ball. Not only are rna vaccines being considered for all sorts of other diseases, some of which have yielded to no other approach; other pharmaceutical uses of rna look set to come into their own, as well. The way molecular biology is applied to medicine seems to be in the throes of revolution.

Incarnation incarnate

The great unifying truth of molecular biology, uncovered during the intellectual revolution which followed the discovery of dna’s double-helix structure, is the way in which the worlds of shape and sequence are linked. The shape of a protein depends on the intricate way in which the chain of amino acids of which it consists is folded up. That depends in turn on the order in which amino acids of different types are strung together on that chain. And the order of the amino acids is a crucial part of the genetic information stored in the dna sequences of the cell’s genome.

The transfer of information from the staid archival form it takes in the genome to its active physical instantiation in the machineries of the cell depends on rna, a molecule in which both sequence and shape play crucial roles. The gene sequence is first copied from dna to rna; that rna transcript is then edited to form a molecule called a messenger rna, or mrna (see diagram).

The end of the mrna molecule is formatted into a distinctive shape which is recognised by ribosomes, complex pieces of machinery composed of dozens of proteins draped around another set of rna molecules. With the help of yet more rna molecules—little ones called trnas which stick to the mrna sequence three letters at a time—the ribosome translates the genetic message into the protein it refers to by creating a chain of amino acids as it moves along the message.

This is the mechanism exploited by the rna vaccines developed by BioNTech, a German biotechnology company based in Mainz, and Moderna, an American one from Cambridge, Massachusetts, against sars-cov-2, the virus which causes covid-19. The companies mass produce the rna sequence describing the distinctive “spike” protein, which studs the outer membrane of the virus, formatted so as to look like a natural mrna. These rna molecules, wrapped in little fatty bubbles called liposomes are injected into patients, where the liposomes smuggle the mrna into cells. Ribosomes pick up on the mrna format and read the sequence, thus producing the spike protein. The immune system learns to recognise the spike which the vaccinated cells are producing and stores away the memory of how to do so. This allows it to mount a swift response if it later comes across the same protein on the surfaces of viral particles and infected cells.

This ability to get cells to churn out proteins for which their dna contains no genes is, in itself, enough to open up swathes of new therapeutic territory. But it is not the whole story. Cells make vast amounts of rna that does not describe proteins. Its ability to recognise specific genetic sequences makes it useful for all sorts of processes, including turning the translation of genes on and off. Its ability to fold itself into particular forms—hairpins, loops and the like—makes it good at interacting with proteins.

This alphabet soup of rnas (see table) seems to function a bit like a computer’s operating system, mediating the relationship between the cell’s hardware and its software. Many of the details of how this works remain obscure. But some are understood well enough for a lot of brainpower and money to have been poured into attempts to hack the operating system for therapeutic purposes.

These abilities should enable drugmakers to head upstream from the proteins whose shapes they have long studied into the realms of sequence. Where previously they targeted proteins which were already present, now they can in principle target the processes which control which proteins get made in the first place, adding helpful new ones to the roster and crossing harmful old ones off. There are rna-based drugs in clinical trials for the treatment of cancer, heart disease and numerous inherited disorders—as well as brain diseases such as Alzheimer’s and Parkinson’s.

Moreover, rna’s mixture of sequence and shape means that in many of these areas the once-haphazard process of drug discovery, long dependent on matching the shape of small synthetic molecules to the crannies and crevices of the proteins they targeted, can itself be systematised. A sequence which recognises, or forms a part of, one gene can be switched out for a sequence tailored to another. When what an rna drug does depends on its sequence, its target and action can be modified by the click of a mouse.

The medicine is the message

Both the firms with mrna vaccines on sale had other vaccines in the pipeline before covid-19 struck. It is part of the appeal of the technology that they were able to turn on a sixpence and refocus their efforts on sars-cov-2 as soon as the sequence for its spike gene was released last January. Now they are both getting on with what they had planned beforehand. Moderna is looking at vaccines to fend off infection by cytomegalovirus (a herpes virus which causes neurological problems in newborns), three lung viruses which cause respiratory disease in young children and Zika, a mosquito-borne virus found mainly in the tropics. BioNTech is focusing more on developing vaccines, and other treatments, with which to treat a wide range of cancers.

Cancer cells tend to have peculiar constellations of proteins on their surfaces, including both normal ones that are overexpressed and, more intriguingly, mutant forms peculiar to the development of that tumour. Comparing the genes expressed in a patient’s healthy cells with those used by their tumour cells reveals which mutant proteins the cancers are producing; mrnas for those proteins can then be incorporated into a vaccine.

Produced as a result of vaccination, the proteins can engender a vigorous immune response the cancer itself does not—part of being a successful tumour is deploying mechanisms that stop the immune system from coming to grips with you. According to Ozlem Tureci, BioNTech’s co-founder, the firm has 500 patients enrolled in clinical trials for cancer. Moderna is pursuing similar ideas.

BioNTech is also testing mrna vaccines aimed at overexpressed but unmutated proteins. Moderna, meanwhile, is looking into vaccines that train the immune system to recognise proteins created by common mutations in kras, a gene implicated in about 20% of human cancers. CureVac, based in Tübingen, an mrna firm which also has a sars-cov-2 vaccine in trials, is conducting trials of a vaccine for non-small-cell lung cancer.

Vaccination is not the only way that mrna injection might fight viruses and tumours. The technique could also be used to get cells to produce therapeutic proteins that are currently administered through injection or infusion: interleukins and antibodies. Designer antibodies are a massive faff to make in industrial quantities; getting patients’ cells to take on the manufacturing duties instead would be a great step forward if it proved practical.

There are many other sorts of proteins which can be stimulated to therapeutic effect. A project on which Moderna is collaborating with AstraZeneca, a pharmaceutical giant, delivers the mrna for a protein which encourages the regrowth of blood vessels. The idea is that the therapy, now in phase 2 clinical trials, could stimulate the growth of new cardiac blood vessels after heart attacks.

Getting the body to produce a protein it needs just for a short while—an antibody, say, or a growth factor—is one thing. But what about a protein that it needs on an everyday basis, but lacks the gene for? Such genetic diseases have always been the most obvious targets for gene therapy—treatments which add a missing gene to a patient’s cells, or repair a broken one, thus allowing them to make a protein they have hitherto lacked. But at least some such conditions might instead be treated with mrna. Inserting a gene might be more elegant—but getting it in the right place and regulated in the right way is challenging. If mrna treatments get the job done, they might offer a nice alternative.

There are thus mrna treatments being studied for phenylketonuria, a metabolic disorder which requires sufferers to restrict their diets for their entire lives; glycogen-storage disease, which enlarges the liver and kidneys and stunts children’s growth; and propionic and methylmalonic acidemias, two illnesses in which the body cannot properly break down proteins and fats. All are conditions that gene therapists are looking at, too.

That BioNTech, Curevac, Moderna and some others now have all these projects on the go is largely down to the fact that they have spent many years developing the basics of their platforms. Many hurdles had to be crossed before they could get cells to accept and act on messages from beyond; the rna had to be subtly toughened up so that it would not itself fall prey to the immune system or get dismantled inside cells; the right lipids had to be found for delivery, sometimes tailored to particular tissues like those of the liver or lymph nodes. The potential inherent in the idea meant that their work was not completely ignored; in 2018 Moderna’s ipo valued the company at $7.5bn, a record for the biotech sector. But biotechnology has a long history of proving biology to be messier and more contrary than those seeking to exploit its loopholes expect.

Stop making sense

Scepticism was also warranted, it seemed, by the fact that messing around with rna had been through bursts of popularity before. One of the very oldest companies in the field, Ionis Pharmaceutials (known as Isis until that name was appropriated by a would-be caliphate) was founded in 1989. Its intention, then and now, was not to make use of mrna, but to hobble it.

The sequence of an mrna molecule carries the same information as can be found in the gene which served as its template; but thanks to the way rna is made it carries it in a complementary way. Where the dna has a letter called C for cytosine, the rna will have G for guanine; where the rna has a C the dna will have a G, and so on. Complementary strands stick together; that is what keeps dna molecules paired up in double helices. If you introduce an mrna to a molecule with a complementary sequence the two will stick together, too, rendering the mrna useless (see bottom deck of diagram above).

Again, getting the neat idea to work in ways that helped proved hard. It took Ionis a quarter century to start getting its “antisense” drugs to market on a regular basis. It now has three: nusinersen, approved in America in 2016 and Europe in 2017 for use against childhood spinal muscular atrophy, a muscle-wasting illness; inotersen, approved in 2018 for hereditary transthyretin-mediated amyloidosis (hattr), which damages the peripheral nervous system; and volanesorsen, approved in Europe in 2019, which lowers levels of triglyceride fats in the blood of people with a metabolic error that makes them far too high.

Ionis currently has a further 37 antisense molecules in clinical trials for conditions including Huntington’s disease (a study being carried out in collaboration with Roche, a large Swiss pharma company); amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease (in collaborations with Biogen, a specialist in treatments for neurological disease); beta thalassaemia, a blood disorder similar to sickle-cell anaemia; and cystic fibrosis.

The firm is also developing, in collaboration with Novartis, another Swiss company, a way of reducing levels of lipoprotein(a), a particularly damaging form of low-density-lipoprotein (ldl) cholesterol. Lipoprotein(a) levels are untreatable with existing medicines; Pelacarsen, as the drug is known, is in phase 3 clinical trials to see if it can change that.

Unlike molecules of mrna, which can tolerate only a small amount of chemical tinkering before becoming ribosome-unfriendly, antisense molecules can be tweaked quite a bit, and thus made long-lasting. Ionis’s researchers have worked out how to stabilise them so that they will hang around inside cells for months. This is important because most of Ionis’s targets are chronic diseases that require continuous treatment. The fewer injections per year the better.

While biotech companies were beavering away at antisense molecules in the 1990s, researchers elsewhere discovered that nature had a similar technology of its own: gene silencing, a process guided by small interfering rnas (sirnas). The early 2000s saw a gene-silencing biotech boom led by Alnylam, founded in Cambridge, Massachusetts in 2002, and Sirna Therapeutics, which got going in San Francisco the following year.

Established pharma companies, including Abbott, Merck, Novartis, Pfizer, Roche and Takeda, waded in, too, with Merck buying Sirna for more than $1bn in 2006. For almost a decade, attention and money were showered on the field. But though there were many promising leads, they failed to turn into drugs. By the early 2010s it seemed that the party was over.

Nonlinear, nonvisual and inclusive

Alnylam, though, kept dancing. In 2014 it bought what was left of Sirna from Merck for a knock-down price. It launched its first product, patisiran, a treatment for hattr, in 2018. It now has two others, givosiran and lumasiran, which also address rare genetic disorders.

A fourth substance developed using its technology has broader appeal. This is inclisiran, developed to treat an inherited disorder that pushes the concentration of ldl cholesterol in the blood to dangerous levels; around 30m people worldwide suffer from it. A firm called the Medicines Company licensed Inclisiran from Alnylam to bring it to market. With approval looking likely (it was given in Europe late last year) Novartis bought the Medicines Company for $9.7bn in January 2020.

According to Akshay Vaishnaw, Alnylam’s head of r&d, the firm has another 14 sirna drugs in clinical trials. These including potential treatments for haemophilia, hepatitis B and recurrent kidney stones. Arrowhead Pharmaceuticals, of Pasadena, California, has eight potential sirna drugs in trials, including one directed at cystic fibrosis. Dicerna Pharmaceuticals, of Lexington, Massachusetts, has three.

These sirnas work by straddling the worlds of shape and sequence. Their shape fits them into a group of proteins called an rna-induced silencing complex (risc). But a bit of the sirna is left sticking out of this complex; this tail contains a sequence complementary to that of the rna to be silenced. When sirna and mrna meet, the proteins in the risc chop the messenger to pieces. (A conceptually similar mechanism for the rna-guided protein-executed chopping up of genes found in bacteria is the basis of the crispr tools now revolutionising gene editing.)

In plants and invertebrates the natural function of the sirna mechanism is clear: cutting up mrnas associated with viruses. They do not seem to serve that function in vertebrates, and no one is quite sure what they do instead. But that does not stop them from looking like promising drugs.

So do another set of rnas associated with riscs: micro-rnas, which use their complementary sequences not to destroy mrnas but to regulate them. The human genome seems to contain about 2,600 of these mirnas, and they are thought to be involved in regulating the rate at which about 60% of the genes describing proteins get transcribed. Several look like promising therapeutic targets.

Since the active bit of an mirna is a single-stranded sequence-specific tail, the obvious way to target them is with antisense. Regulus Pharmaceuticals, a firm that started life as a collaboration between Ionis and Alnylam, is trying to develop antisense molecules aimed at mirna-21 to treat two kidney-related genetic conditions in which that mirna plays a role. When you start targeting mirnas, though, things get positively baroque. Santaris Pharma, a Danish firm, has developed Miravirsen, an antisense suppressor for mirna-122 which the hepatitis C virus uses for its own unhelpful ends. The drug has now been taken on by Roche.

The innovation continues. Mina Therapeutics, a startup in London, is working on the potential of sarnas, which activate genes which otherwise stay silent. Others are investigating systems for “self-amplifying” mrna drugs. These mrnas would inveigle a cell’s ribosomes into producing not just the protein that was meant to be delivered, but also a second protein, called rna-replicase, which would make more of the mrna, thus leading to even more protein being expressed. There is surely further cleverness to come.

Could be so exciting

Even if only a fraction of these possibilities pan out it looks certain that, in popularity contests to come as in stockmarkets today (see chart), more people will be plumping for rna. Their support will be welcomed by the small band of biologists with an interest in the very earliest history of life that has long formed the discerning core of the molecule’s following. Life needs both a way of doing things in the now—catalysing the reactions on which its metabolism depends—and of passing information into the future. Of the molecules known today only rna, in its shape-and-sequence versatility, can do both those things, dealing with the needs of the everyday at the same time as encoding instructions for its own reproduction in the form of a legible sequence. This suggests to many that early life spent some time in an “rna world” before the division of labour allocated doing things to the proteins and storing data to dna, reducing rna to a supporting role in the world it had created.

The application of rna has met many obstacles over past decades, and the fact that it has proved itself in vaccines does not mean it will not meet more in the future. But it does seem that medicine now has a way to target drugs not just at proteins, but at the processes that make them, and that opens up new realms of possibility. The next rna world awaits.

Testing and tracing could have worked better against covid-19


The first outbreak of a novel disease is the opening scene of a whodunnit. In 1976, when more than two dozen members of the American Legion died after a convention in Philadelphia, public-health officials spent months scouring the hotel they had met in before finally tracking down the culprit in the water tank on the roof: a new bacterium which, having caused the first known cases of Legionnaires’ disease, was named Legionella. In the 1980s it took years of hard work and acrimonious argument among epidemiologists and virologists to blame the terrible and varied symptoms of aids on hiv, a virus of a type never previously seen in humans.Listen to this story

For covid-19, the mystery was solved almost as soon as it had begun. The novel pneumonia that doctors in Wuhan noticed in December 2019 immediately brought to mind Severe Acute Respiratory Syndrome (sars), a disease caused by a coronavirus which broke out in 2002. As a result of sars and the subsequent outbreak in 2012 of Middle East Respiratory Syndrome (mers), also caused by a coronavirus, there were already established protocols for growing cells from the lining of the nose, throat and lungs in order to look for coronavirus infection. They were soon put to use.

To identify the possible coronavirus responsible meant producing a sequence of its genome. The first step in this process was to extract rna—the molecule on which coronavirus genomes are written—from the cell cultures. The genetic sequences in these rna molecules then had to be transcribed into complementary bits of dna, because that is what automated sequencing machines work with (see part A of diagram).

Computer programs assembled the sequences those machines produced into a recognisable, if gappy, coronavirus genome. Researchers used this to make dna “primers” with which to fish out the not-yet-sequenced bits of the genome. Finished sequences were published less than two weeks after the process had begun. On January 12th 2020 the world knew its enemy—soon thereafter named sars-cov-2—down to the last letter of its genome.

In terms of the science done, this was all routine; the appropriate use of standard laboratory techniques. In terms of its impact, it was enormous. Knowing the viral sequence was fundamental to vaccination efforts, made it possible to track the virus’s evolution and, most immediately, made it possible to test people with a cough and see if they were infected. The first aids tests were not available until four years after medical science became aware of the condition they tested for. For sars it took six months. Procedures for testing swabs from the nose and throat for rna from sars-cov-2 were published 11 days after the genome sequence, on January 23rd.

There was, however, a drawback to the tests. They required suitably equipped laboratories. Most countries did not have nearly enough of the relevant lab capacity; in others, much of it was being used for different things. “Prior to March 23rd my lab had never performed a viral diagnostic,” says Stacey Gabriel, who runs genetic-sequencing operations at the Broad Institute in Cambridge, Massachusetts. But with public institutions swamped she and her colleagues created one of the largest testing shops on America’s east coast from scratch, reconfiguring the specialised robots that populate one of the world’s most advanced cancer-genetics labs to do the grunt work.

Dr Gabriel learned two lessons in the process. The first is that uniformity matters a lot. The Broad started off testing samples from Massachusetts nursing homes which came in containers of varying size and with various amounts of liquid, some accompanied by handwritten forms, some by barcodes. Dealing with such messiness is no task for a robot, and so to begin with just a few thousand samples a day passed through machines capable of handling far more. The second is that you need software to track the whole process. Commercial software, dry swabs and barcoding soon had the lab firing on all cylinders. By March 2021 it could handle 200,000 tests a day and was serving customers as far afield as New York.

The data such labs produce are not just for patients and doctors. In most countries covid-19 is a notifiable disease; the authorities have a legal right to know who has been found to be infected. When a sample tests positive the lab has to pass the identity of the person it came from on to public-health officials. At that point a new sort of detective work begins: where did that person—the index case, in public-health speak—pick up the virus? To whom might they have given it?

Without a trace

Several East Asian countries demonstrated that, if started in the earliest days of an epidemic and pursued with vigour and persistence, such contact tracing can be a powerful tool. Some, such as Singapore and Taiwan, benefited in this from their experience with sars in the mid-2000s; tracing systems set in place back then were put to use with an urgency born of experience. A level of invasiveness from which most Western authorities shied away was often employed. In South Korea, for instance, contact tracers were able to download a list of all financial transactions made by those who tested positive; they could then obtain cctv footage from shops the index case had visited in search of other customers to check up on. “[Such snooping] has come up with discussions I’ve had with policymakers,” says Christophe Fraser, a digital epidemiologist at the University of Oxford. “We got very hung up on the idea of contact tracing disrupting people’s lives.”

Western governments acted in a slower, less thoroughgoing way. They failed to track the initial spread, in part because of insufficient testing capacity (and, in America, dud tests from the Centres for Disease Control and Prevention). They ended up with much less impressive systems. Tom Frieden, a former director of the cdc, says he thinks that, at its current level of effort, America could plausibly trace about 15,000 cases a day—a level that has been handsomely exceeded every day since April 2nd 2020. Britain has earmarked £37bn for testing and tracing over the 2020 and 2021 financial years, and though it may well spend less, the shoddiness that has dogged some elements of the campaign, such as a database cock-up which lost thousands of results in September 2020, will be remembered.

A basic problem is that contact tracing is a lot of work. Theoretical assessments based on analysing social networks and experience in Asia both suggest that some 30 contacts need to be identified for each index case. Digital tools can lessen the load. Resolve to Save Lives, a campaign run by Vital Strategies, an ngo, developed Locator, which taps into credit data to help tracers track down people who may have caught the virus from a specific index case. But the amount of work required remained enormous.

Apps and antibodies

A much discussed alternative to such programmes was to use the world’s most ubiquitous tracking devices: smartphones. Google and Apple worked together to develop a system which enabled phones to keep a list of occasions when they were near another phone for a significant period of time, and the identity of that second phone. When someone tests positive for sars-cov-2 they are asked to send a message from their phone which is used to notify all the other phones they had been near within a particular window of time. But everything is peer-to-peer; neither the big tech companies nor the public-health authorities get a list of contacts.

Unfortunately this built-in privacy makes it hard to assess the technology’s efficacy. British and Swiss studies suggest such apps do reduce spread, but not enough to make them more than an also-ran technology. None of the successful contact-tracing systems in East Asia relies on such things to any significant extent.

Even without good tracing, self isolation of those who tested positive helped slow the spread of the disease. But over time the shortcomings of the initial testing technology, reverse-transcriptase polymerase chain reaction (rt-pcr), became ever more apparent. It is conceptually elegant (see diagram) and easy for labs to use. But despite its familiarity, reliability and sensitivity, it has real disadvantages.

One is that it needs labs, and is carried out most efficiently in big ones. This means samples may have to travel a long way. It also means that they can get held in queues. The Broad runs its rt-pcr tests in just three and a half hours; but the average sample takes 15 hours to process. Results from rt-pcr tests normally come in days not minutes.

Another problem is that though the presence of viral rna clearly shows that a person has been infected, it says very little about where they stand in the course of the disease; rna is detectible from very soon after infection to long after the disease has run its course. In public-health terms what is needed is a test that spots people who are actually infectious—people with cells in their noses and throats actively churning out virus particles.

To look for the sars-cov-2 particles themselves means looking for their distinctive protein components, not for the rna that tells cells how to make them. The most detectible such component is the spike protein which studs the particles’ outer membranes. And one of the basic rules of modern biotechnology is that when you want to find a protein, use an antibody.

Antibodies are large molecules that come in millions of varieties, each of which sticks to one target—known as that antibody’s “antigen”—and one target only. A handy technology called lateral-flow testing makes use of that specificity. A sample is placed at one end of a porous membrane and, as it seeps along to the other, encounters a line of antibodies designed to recognise it. When the sample is urine and the antigen is a hormone found in expectant women, you have a pregnancy test. When the sample is mucus from a swab and the antigen is the spike protein, it is a covid-19 test—a cheap, convenient one which can provide results with in half an hour. Such tests may not pick up all the people in whom rt-pcr might detect a trace of the virus. But if their nose and throat cells are not producing enough antigen for the test to detect, they are probably not producing enough to be infectious, either.

At the beginning of the epidemic “the supply chain for the lateral-flow tests wasn’t there,” says Dr Gabriel. Chris Hand, the chairman of Abingdon Health, a British contract manufacturer of lateral-flow tests, says the main bottleneck was the speciality membranes that are part of every test kit. “They come on large reels of 100 metres plus, which go through automated equipment to add biochemicals by spraying them at low volumes,” he says. But once the biochemicals—the bespoke antibodies and some more generic bits and bobs—are ready, the production processes in place and the packaging sorted, the tests could be churned out by the million.

New technologies now reaching the market will further change the dynamics of test and trace. Quantumdx is one of a number of companies developing automated pcr-in-a-box systems that provide results within a couple of hours. Jonathan O’Halloran, the firm’s boss, says the British company has been relying on its own testing system for the past 22 weeks, testing its 90 staff members every morning (they are free to decline). About once a week lunchtime brings the news that someone has tested positive; they are immediately sent home to isolate. When things are done this fast, the fact that rna is detectable before people are infectious is a plus; isolating on the basis of an early pcr test means no one ever turns up to work infectious. The company claims not to have lost a single day to infection.

A combination of local, automated pcr and lateral-flow tests could be the basis of an ideal testing system—one which has a chance of keeping ahead of, and containing, a low level of disease rather than lagging behind one that is shooting up. Antigen tests would be used to scan the population for new infections. Those found would be referred to contact tracers; contacts who might have been infected could then be pcr-tested to find out which of them actually were.

Great for public health. No benefit, in itself, to the index cases who would wait, isolated, to see what fate the virus and their immune systems had in store for them—painfully aware that their next encounter with the wonders of modern medical technology could be in a hospital bed. 

The fast-spreading coronavirus variant is turning up in US sewers

A hyper-transmissible form of the coronavirus that causes covid-19 has been found in US sewer systems in California and Florida, confirming its widening presence in the US.

Buckets of dirty water drawn from sewer pipes near Los Angeles and outside Orlando starting in late January are among those in which genetic mutations shared by a so-called UK variant have been detected.

The UK strain B.1.1.7, a mutated form of the coronavirus  first discovered in southeast England in December, doesn’t seem to resist vaccines, but it does appear to spread more easily and has already taken over in countries including Israel, where it’s now responsible for 80% of cases. Some researchers have warned that if the variant takes hold in the US, it could become the dominant form by March.

The new sewage data is consistent with other estimates that the variant is increasing its reach. On January 7, the company Helix and researchers in California used patient test results to estimate that the variant is now responsible for 1% to 2% of cases in the US as a whole and 4% in Florida, about four times the proportion found in early January.

In the sewers of Altamonte Springs, near Orlando, tests on waste water suggest that 4% of those infected have the new variant. “I was thinking it would be in Miami or larger areas. But that was wishful thinking,” says Ed Torres, director of public works and utilities in Altamonte Springs, who oversees the sewage testing program.

A strain of covid-19 that appears to spread faster is colliding with the campaign to vaccinate Americans.

Local spread

Sewer tests are now providing a direct glimpse of just how many people are infected with the variant in some cities. Torres says a model he works with indicates that more than 200 people are infected with the variant just in his wastewater collection region, an exurb of 77,500 people.

Health officials in central Florida initially blamed their B.1.1.7 cases on visitors who tested positive, but the sewer tests in Altamonte indicate that the variant is spreading locally, too. Florida remains largely open for business, including theme parks, which are operating with the use of masks and physical distancing.

According to the US Centers for Disease Control and Prevention, only 611 cases of B.1.1.7 have been directly confirmed nationally via genetic sequencing of the viral samples collected in patient nose swabs, with many of the positives coming from California and Florida. Because only a small percentage of hospital swabs are ever analyzed for what form of the virus is present, the true number of B.1.1.7 cases is certainly much larger.

The US has less ability than some European countries to track variants of the virus because test swabs are not subjected to complete genomic sequencing as often, a situation some experts have likened to “flying blind” in the face of a changing pandemic.

According to the New York Times, as of January about 1.4 million people were testing positive for the coronavirus every seven days, but fewer than 3,000 of those clinical samples were being sequenced letter for letter, a step that’s usually necessary to see what mutations the virus has acquired.

Sewage surveillance

Wastewater offers a chance to monitor the variant more widely, and at lower expense. A single liter of dirty water carries the remains of viruses shed into toilets by everyone who shares a sewer system, offering a readout on the health of thousands, even millions, of people.

Since last spring, some cities have used molecular tests on sewage as an early warning system, since the amount of coronavirus in sewage can predict how many people will turn up in hospitals a week to 10 days later. The reason sewage results go up or down before official case numbers do is that people seem to start shedding the virus into toilets a day or two before they feel ill, and it often takes even more time to receive a test result.

“You can see the post-Thanksgiving spike, and the after-Christmas boom,” says Raul Gonzalez, who carries out tests on sewer water for a utility in Virginia Beach.

The dashboard COVIDPoops19, maintained by the University of California, Merced, tracks readings from over 1,000 sites in 47 countries. Testing experts say the virus or virus fragments in sewage aren’t alive or dangerous.

Initial results

Sewage tests use a sensitive version of PCR, the testing technology employed in hospital tests. Called digital PCR, it is also employed in so-called liquid biopsies to spot signs of cancer in a blood draw.

When the first reports of the B.1.1.7 variant emerged in December, GT Molecular, a company in Fort Collins, Colorado, was the first to reformat its sewage test to search for the variant. That involves checking sewage for two mutations characteristic of the B.1.1.7 strain.

The CDC ordered software that was meant to manage the vaccine rollout. Instead, it has been plagued by problems and abandoned by most states.

“We measure the amount of the virus that has the parental sequence and the amount of virus with the mutant sequences,” says Rose Nash, director of R&D at the company. “Most samples are not coming back positive, but what we are seeing across samples that do come back positive is about 5% levels of the variant. We are expecting that to increase.”

Because the sewage test has only been in use for a few weeks, it’s too soon to say on the basis of that evidence alone if variant levels are rising or falling. “There is no distinct trend yet in the variant,” says Mike Shaffer, who manages data for Oxnard, California, a beach city northwest of LA, which found low levels of the variant in January.

Shaffer says he also wants to confirm that the test is really finding the UK variant, not some look-alike strain, and says the Oxnard sewer samples were sent to Stanford University for sequencing. Christopher McKee, CEO of GT Molecular, agrees that the science behind the variant test “is still pretty nascent.”

According to GT Molecular, several other districts in California and Florida have positive results but haven’t announced them. “To me it’s worthless if you don’t put the information out there,” says Torres. “We need to communicate it to people who can do something about it.”

Soo far, however, news of the variant in sewage or its spread has not led to any major change in public policy. As yet, there is no national plan to deal with the threat that the variant will spread quickly and cause case numbers to rise.

Robert Levin, the public health officer for Ventura County, where Oxnard is located, presented the sewage findings to a meeting of a supervisory board last week. “They found a tiny amount of the variants that are considered hyper-transmissible,” Levin said. “The impact that this will have on our county cannot be predicted. This is uncharted territory.”

How vaccines are made, and why it is hard

Nine vaccines against covid-19 have already been approved in one jurisdiction or another, with many more in various stages of preparation. That this has happened within a year of the illness coming to the world’s attention is remarkable. But it is one thing to design and test vaccines. It is another to make them at sufficient scale to generate the billions of doses needed to vaccinate the world’s population, and to do so at such speed that the rate of inoculation can outpace the spread and possible mutation of the virus.Listen to this story

Broadly, there are two ways of making antiviral vaccines. One, tried and trusted, involves growing, in tanks called bioreactors, cell cultures that act as hosts for viruses which are then used in one way or another to make the vaccine in question. Cells grown this way can be of many types—insect, human kidney, monkey kidney, hamster ovary—as can the resulting vaccines. These may be weakened or killed versions of the virus to be protected against, or live viruses of a different and less-dangerous sort that carry a gene or two abstracted from the target virus, or even just isolated target-viral proteins. The point is that the vaccine should introduce into the body, or induce that body to make, something which the immune system can learn to recognise and attack if the real target virus should ever turn up.

In with the new

The alternative method, developed recently and employed to make the mrna vaccines, such as those of Moderna and Pfizer, that the pandemic has stimulated the invention of, requires culturing cells only at the beginning of the process. mrna is the substance that carries instructions about how to make a protein from a cell’s dna to the molecular factories, known as ribosomes, which do the actual manufacturing. In the case of covid-19, the instructions in question generate spike, a protein found on the surfaces of particles of sars-cov-2, the virus that causes this illness. Suitably packaged and delivered, such mrna can induce some of the body cells of the inoculee to turn out spike, which the immune system then learns to recognise. To make this type of vaccine you therefore have to generate lots of the relevant mrna.

That process does indeed start with cells, though they are bacterial cells, rather than those of animals. But it does not end with them. The bacteria used, normally a well-understood species called E coli, have spliced into them a dna version of the part of the sars-cov-2 genome which describes spike. (Confusingly, as is true of many viruses, sars-cov-2’s actual genes are made of rna.) The bacteria are then allowed to multiply for a few days before being broken open, their dna filtered out, and the dna versions of the spike gene extracted as what is known as a dna template.

Once purified, this template is mixed with a soup of pertinent enzymes and fed molecules called nucleotides, the chemical “letters” of which rna is composed. Thus supplied, the enzymes use the templates to run off appropriate mrnas by the zillion. These are extracted and packaged into tiny, fatty bubbles to form the vaccine.

Both the cell-culture and the mrna approaches have benefits and drawbacks. The former has the advantage of being well established. Versions of it go back to vaccine-making’s origins. But keeping cultured animal cells alive and healthy is a tricky business. A whole subfield of bioengineering is dedicated to this task. Vaccine-makers who rely on live cultures constantly struggle with yields. Using this method to make a lot of vaccine, fast, is hard.

It was difficulties of this sort that Pascal Soriot, boss of AstraZeneca, cited on January 26th in defence of his firm’s failure to provide vaccine supplies which the European Union claimed it had been promised. AstraZeneca is an Anglo-Swedish company that, in collaboration with Oxford University, created one of the first vaccines to be approved. As Mr Soriot told La Repubblica, an Italian newspaper, “You have glitches, you have scale-up problems. The best site we have produces three times more vaccine out of a batch than the lowest-producing site.”

De-necking the bottles

Maximising a bioreactor’s yield is as much an art as a science. The underlying health of the cells involved matters. So do environmental conditions at the manufacturing site. That AstraZeneca has not been able to meet its own production targets shows how hard it is to predict when the right balance of biology will be found. The company says it can take six to nine months to start a production site up from scratch, and that even this timetable is possible only by working with experienced partners and at an accelerated pace. At the moment, AstraZeneca is working with 25 manufacturing organisations in 15 countries to make its vaccine.

Producing mrna vaccines at scale has problems, too. The biggest is how to protect the mrna molecules both from the environment they must travel through in order to reach the arm of their recipient, and from the recipient’s own body, which will attack them as they journey to the ribosomes which will transcribe them.

Protection from the environment is mainly a matter of having a strategically located set of refrigerators, known as a cold chain. Protection from the body, though, is where the fatty bubbles come in.

Production of these bubbles was a cottage industry before the pandemic. A small Austrian firm, Polymun Scientific, is one of just a handful that can make them. Their main previous use was in niche cancer treatments. Scaling up their production, which is happening right now, has never been done before and adds uncertainty to the continued supply of mrna vaccine.

There are other bottlenecks, too. In particular, the factories in which vaccines are made must be built to a high standard, known as gmp, for “Good Manufacturing Practice”. There is currently a shortage of gmp facilities. Andrey Zarur, boss of GreenLight Biosciences, a firm in Boston that is developing an mrna vaccine, says his company has employees whose entire job, at present, is to work the phones trying to find gmp facilities in which to make their vaccine. There is, though, nothing available. He is therefore looking to buy firms whose vaccine candidates have turned out not to work, simply in order to acquire the facilities in question.

Supplies of raw materials such as nucleotides are also tight. According to Dr Zarur, Thermo Fisher, an American chemical-supplies company, has spent $200m on a new facility in Lithuania to make these molecules, though the firm itself would not confirm this.

On top of all this, the transport and distribution of vaccines once they have been made presents yet further challenges, and concomitant potential for hold ups. Vaccines must be stored in special non-reactive glass vials. Some, such as the current version of Pfizer’s mrna vaccine, must also be kept at extremely low temperatures, though that problem may go away soon. Drew Weissman, one of the inventors of mrna-vaccine technology, says producers are now testing shots which are stable for three months when kept at 4°C.

Once supply chains for both cell-culture and mrna vaccines have been scaled up, and bottlenecks unblocked, the manufacturing processes may face a different test—how quickly they can produce new vaccines to deal with new viral variants as these emerge. The continued efficacy of approved vaccines against such variants is not guaranteed, and it may be necessary to make others (see article).

Here, the mrna approach may have an advantage. Its production systems will require a simple tweak—the dropping in at the start of a dna template describing the new variant’s spike protein. Cell-culture systems, by contrast, will have to be rebuilt to some degree for every new variant they aim to vaccinate against.

Scale models

Producers, such as those in China, who use older-fashioned cell-culture techniques, will have to recalibrate their entire operations. Newer systems, like AstraZeneca’s, which use cells specially designed so as not to be influenced by the new version of the spike gene in the viruses they are carrying, should be able to get on track in the time it takes to start a culture from scratch—about a month. For mrna systems, Drs Weissman and Zarur say it would take a couple of months to go from new variant to large-scale vaccine production. If variants resistant to the current crop of vaccines do evolve, then that speed and certainty in making new vaccines to combat them will be essential. 

Growing Pains for Field of Epigenetics as Some Call for Overhaul

Our genes are not just naked stretches of DNA.

They’re coiled into intricate three-dimensional tangles, their lengths decorated with tiny molecular “caps.” These so-called epigenetic marks are crucial to the workings of the genome: They can silence some genes and activate others.

Epigenetic marks are crucial for our development. Among other functions, they direct a single egg to produce the many cell types, including blood and brain cells, in our bodies. But some high-profile studies have recently suggested something more: that the environment can change your epigenetic marks later in life, and that those changes can have long-lasting effects on health.

In May, Duke University researchers claimed that epigenetics could explain why people who grow up poor are at greater risk of depression as adults. Even more provocative studies suggest that when epigenetic marks change, people can pass them to their children, reprogramming their genes.

But criticism of these studies has been growing. Some researchers argue that the experiments have been weakly designed: Very often, they say, it’s impossible for scientists to confirm that epigenetics is responsible for the effects they see.

Three prominent researchers recently outlined their skepticism in detail in the journal PLoS Genetics. The field, they say, needs an overhaul.

“We need to get drunk, go home, have a bit of a cry, and then do something about it tomorrow,” said John M. Greally, one of the authors and an epigenetics expert at the Albert Einstein College of Medicine in New York.

Among other criticisms, he and his co-authors — Ewan Birney of the European Bioinformatics Institute and George Davey Smith of the MRC Integrative Epidemiology Unit at the University of Bristol in England — argue that in some cases, changes to epigenetic marks don’t cause disease, but are merely consequences of disease.

Some studies, for example, have found that people with a high body mass index have unusual epigenetic marks on a gene called HIF3A. Some researchers have suggested that those marks change how HIF3A functions, perhaps reprogramming fat cells to store more fat.

If that were true, then drugs that reverse these changes might be able to help obese people lose weight. But Dr. Smith and his colleagues have found that overweight subjects experienced epigenetic changes to HIF3A only after they put on weight.

But these experiments are especially hard to set up, he noted, because scientists have to gather blood or other genetic samples from healthy people and then wait years for some of them to get sick.

In other cases, apparent changes in epigenetic marks may actually be the result of different kinds of cells becoming more or less common in people, Dr. Greally and his colleagues also warned. “That’s where things get hairy,” Dr. Greally said.

Smoking, for example, triggers a boom in immature blood cells, which carry epigenetic marks different from those of other cell types in the blood.

Rafael A. Irizarry, an applied statistician at Dana-Farber Cancer Center and the Harvard School of Public Health, said new methods could help researchers steer clear of this confusion.

Scientists can sort cells into different types before looking at their epigenetic marks, he said. It’s even becoming possible to look at the epigenetics of one cell at a time.

“But it makes the process way more expensive,” Dr. Irizarry said.

Dr. Greally and his colleagues note another source of confusion: Normal genetic variation leads some people to produce different epigenetic marks than others.

If researchers were to find that alcoholics carry an unusual epigenetic mark, for instance, that wouldn’t necessarily mean that it resulted from heavy drinking. These people may have a genetic variation that puts them at risk of alcoholism and, perhaps coincidentally, creates an unusual epigenetic mark on their DNA.

Dr. Greally said these possibilities have been neglected because scientists have been so captivated by the idea that epigenetic marks can reprogram cells.

“Since you don’t talk about anything else, you interpret the results solely through that little sliver of possibility,” he said.

He and his colleagues go so far as to claim that no published results on the links between epigenetic marks and disease “can be said to be fully interpretable.”

Other experts feel that such an indictment is a bit too broad. Dr. Flanagan pointed to several recent studies in which scientists confronted the very challenges that Dr. Greally and his colleagues wrote about. Last year, for example, a team of European scientists investigated how smoking causes lung cancer. They took advantage of large-scale studies in Australia, Norway and Sweden that collected blood from tens of thousands of people and tracked their health for years.

The scientists found that smokers who got lung cancer tended to lose the same epigenetic marks on a pair of genes.

Dr. Greally said that genetic variations the smokers were born with might account for the results. “That’s not tested in the study,” he said. “It could definitely be the case.”

Nevertheless, he added, these reports offer some good starting points for bigger studies in the future.

“There’s nothing wrong with an exploratory study, but call it an exploratory study and acknowledge the fact that it may merely be reporting noise,” Dr. Greally said.

“If you say, ‘Look, I’m finding something that’s intriguing here,’ that’s legit.”


This article was originally published in The New York Times. Read the original article.

DNA Gets a New — and Bigger — Genetic Alphabet

In 1985, the chemist Steven A. Benner sat down with some colleagues and a notebook and sketched out a way to expand the alphabet of DNA. He has been trying to make those sketches real ever since.

On Thursday, Dr. Benner and a team of scientists reported success: in a paper, published in Science, they said they have in effect doubled the genetic alphabet.

Natural DNA is spelled out with four different letters known as bases — A, C, G and T. Dr. Benner and his colleagues have built DNA with eight bases — four natural, and four unnatural. They named their new system Hachimoji DNA (hachi is Japanese for eight, moji for letter).

Crafting the four new bases that don’t exist in nature was a chemical tour-de-force. They fit neatly into DNA’s double helix, and enzymes can read them as easily as natural bases, in order to make molecules.

“We can do everything here that is necessary for life,” said Dr. Benner, now a distinguished fellow at the Foundation for Applied Molecular Evolution in Florida.

Hachimoji DNA could have many applications, including a far more durable way to store digital data that could last for centuries. “This could be huge that way,” said Dr. Nicholas V. Hud, a biochemist at Georgia Institute of Technology who was not involved in research.

It also raises a profound question about the nature of life elsewhere in the universe, offering the possibility that the four-base DNA we are familiar with may not be the only chemistry that could support life.

The four natural bases of DNA are all anchored to molecular backbones. A pair of backbones can join into a double helix because their bases are attracted to each other. The bases form a bond with their hydrogen atoms.

But bases don’t stick together at random. C can only bond to G, and A can only bond to T. These strict rules help ensure that DNA strands don’t clump together into a jumble. No matter what sequence of bases are contained in natural DNA, it still keeps its shape.

Working at the Swiss university ETH Zurich at the time, Dr. Benner tried to make some of those imaginary bases real.

“Of course, the first thing you discover is your design theory is not terribly good,” said Dr. Benner.

Once Dr. Benner and his colleagues combined real atoms, according to his designs, the artificial bases didn’t work as he had hoped.

Nevertheless, Dr. Benner’s initial forays impressed other chemists. “His work was a real inspiration for me,” said Floyd E. Romesberg, now of the Scripps Research Institute in San Diego. Reading about Dr. Benner’s early experiments, Dr. Romesberg decided to try to create his own bases.

Dr. Romesberg chose not to make bases that linked together with hydrogen bonds; instead, he fashioned a pair of oily compounds that repelled water. That chemistry brought his unnatural pair of bases together. “Oil doesn’t like to mix with water, but it does like to mix with oil,” said Dr. Romesberg.

In the years that followed, Dr. Romesberg and his colleagues fashioned enzymes that could copy DNA made from both natural bases and unnatural, oily ones. In 2014, the scientists engineered bacteria that could make new copies of these hybrid genes.

In recent years, Dr. Romesberg’s team has begun making unnatural proteins from these unnatural genes. He founded a company, Synthorx, to develop some of these proteins as cancer drugs.

At the same time, Dr. Benner continued with his own experiments. He and his colleagues succeeded in creating one pair of new bases.

Like Dr. Romesberg, they found an application for their unnatural DNA. Their six-base DNA became the basis of a new, sensitive test for viruses in blood samples.

They then went on to create a second pair of new bases. Now with eight bases to play with, the researchers started building DNA molecules with a variety of different sequences. The researchers found that no matter which sequence they created, the molecules still formed the standard double helix.

Because Hachimoji DNA held onto this shape, it could act like regular DNA: it could store information, and that information could be read to make a molecule.

For a cell, the first step in making a molecule is to read a gene using special enzymes. They make a copy of the gene in a single-stranded version of DNA, called RNA.

Depending on the gene, the cell will then do one of two things with that RNA. In some cases, it will use the RNA as a guide to build a protein. But in other cases, the RNA molecule floats off to do a job of its own.

Dr. Benner and his colleagues created a Hachimoji gene for an RNA molecule. They predicted that the RNA molecule would be able to grab a molecule called a fluorophore. Cradled by the RNA molecule, the fluorophore would absorb light and release it as a green flash.

Andrew Ellington, an evolutionary engineer at the University of Texas, led the effort to find an enzyme that could read Hachimoji DNA. He and his colleagues found a promising one made by a virus, and they tinkered with it until the enzyme could easily read all eight bases.

They mixed the enzyme in test tubes with the Hachimoji gene. As they had hoped, their test tubes began glowing green.

“Here you have it from start to finish,” said Dr. Benner. “We can store information, we can transfer it to another molecule and that other molecule has a function — and here it is, glowing.”

In the future, Hachimoji DNA may store information of a radically different sort. It might someday encode a movie or a spreadsheet.

Today, movies, spreadsheets and other digital files are typically stored on silicon chips or magnetic tapes. But those kinds of storage have serious shortcomings. For one thing, they can deteriorate in just years.

DNA, by contrast, can remain intact for centuries. Last year, researchers at Microsoft and the University of Washington managed to encode 35 songs, videos, documents, and other files, totaling 200 megabytes, in a batch of DNA molecules.

With eight bases instead of four, Hachimoji DNA could potentially encode far more information. “DNA capable of twice as much storage? That’s pretty amazing in my view,” said Dr. Ellington.

Beyond our current need for storage, Hachimoji DNA also offers some clues about life itself. Scientists have long wondered if our DNA evolved only four bases because they’re the only ones that can work in genes. Could life have taken a different path?

“Steve’s work goes a long way to say that it could have — it just didn’t,” said Dr. Romesberg.


This article was originally published in The New York Times. Read the original article.

Should Scientists Toy With the Secret to Life?

Scientists quickly condemned the Chinese researcher who altered the DNA of at least two embryos to create the world’s first genetically edited babies, defying a broad consensus against hereditary tinkering.

But as The Times reported last week, the global scientific community is divided over what to do next. Should researchers agree to a moratorium on any human genome editing that can be passed down to future generations? Or should they simply tighten existing criteria?

It’s good that the National Academies of Sciences, Engineering and Medicine are planning a global forum to address these questions. But it will be crucial for biologists to seek substantial input from policymakers, ethicists, social scientists and others.

Crispr, the gene editing technique that the Chinese scientist, He Jiankui, used, has enabled scientists to alter human DNA with far greater ease than ever before. It has the potential to remake life as we know it — by preventing devastating diseases, among many other possibilities — and decisions about its future use should be driven by as inclusive and global a dialogue as possible.

Fortunately, there are several ways to broaden the conversation.

Diversify the deciders. Science is a noble endeavor, but it is not entirely pure. Patents and profits and the race against competitors influence individual researchers as well as entire scientific programs. (The Crispr patent, which is currently the subject of a fierce legal battle, is expected to be worth $1 billion at least.) Those influences are not necessarily corrupting, but money and ego have a way of skewing priorities. Dr. He, for example, is said to have gone rogue partly out of a desire to be the first to create “Crispr babies.”

As gene-editing technology advances toward the clinic, scientists will need to do more than listen to the concerns of bioethicists, legal scholars and social scientists. They will have to let these other voices help set priorities — decide what questions and issues need to be resolved — before theory becomes practice. That may mean allowing questions over societal risks and benefits to trump ones about scientific feasibility.

As several scholars have suggested, a “global observatory” — an international consortium of experts from many different fields in many different countries — would go a long way toward making this shift.

Engage the public. Obvious though this may sound, it’s not a given. “There’s a lot of skepticism about the value of public involvement in science and technology decisions,” says Simon Burall, a senior associate with Involve, a British nonprofit dedicated to increasing public engagement in science. That’s too bad. There’s plenty of evidence that having citizens weigh in on proposed policies makes them better and more sustainable. There are also far too many examples of the converse: Leaving the public out of the conversation invites suspicion and mistrust that can be difficult to overcome. It’s easy to dismiss concerns over new technology as the product of ignorance. It’s also a mistake.

Surveys show that most people already support genome editing, as long as it’s directed at intractable diseases and not at the creation of genetically enhanced “designer babies.” Scientists and policymakers stand a better chance of preserving that good will, especially in the face of the He baby scandal, if they give the concerns that do arise a fair hearing. Social media offers an unprecedented platform for doing just that. Crispr’s proponents should start by using that platform to clarify the following:

Scientists are nowhere near being able to make “designer babies.” They have barely figured out the genetic determinants of height; there’s no telling how long it will take them to understand more complex traits, like intelligence, beauty and athleticism. What they are close to doing is using tools like Crispr to repair faulty genes that cause serious diseases. Clinical trials are already underway for hemophilia and sickle cell disease. And these trials involve editing DNA in adult study participants, not in sperm, eggs or embryos; so the results, good or bad, can’t be passed on to offspring.

The bluntest of these tools — legal prohibition — is already being used in the United States, where doctors and scientists are barred from editing human embryos. While such stringent policies may help avoid the muddiness that led to the He scandal, they have a clear downside: They also block the use of less questionable technology. For some desperate families, mitochondrial gene transfer offers the only hope for preventing horrific diseases. But because federal regulators have grouped it with other forms of embryo editing, it’s prohibited in the United States.

There are better, subtler ways to move forward. Lawmakers, regulatory agencies, patent holders, ethics review boards, funding foundations and professional journals all hold sway over how a technology is developed and used. By working together to limit what is funded, permitted or published, they might create a dynamic and flexible process for safeguarding the public while still allowing promising work to progress. It may be impossible to prevent truly rogue actors, but it is possible to slow them down without stopping everyone else.


This article was originally published in The New York Times. Read the original article.

Searching for the Genetic Underpinnings of Morning Persons and Night Owls

Early to bed and early to rise is a maxim that’s easy to follow for some people, and devilishly hard for others.

Now, in a study published Tuesday in Nature Communications, researchers curious about the genetic underpinnings of chronotype — whether you are a morning person, a night owl or somewhere in between — looked at about 700,000 people’s genomes. They identified 351 variations that may be connected to when people go to bed. While these variants are just the beginning of exploring the differences in chronotypes, the study goes on to suggest tantalizing links between chronotype and mental health.

The researchers drew on data from 23andMe, the genetic testing company, and the UK Biobank, which tracks hundreds of thousands of volunteer subjects in Britain, about 85,000 of whom wear activity monitors that record their movements.

Those data were key, said Michael Weedon, a bioinformaticist at University of Exeter in England and an author of the new paper; earlier studies had relied only on people’s subjective opinions of whether they were morning people. Using the activity monitors, however, the team was able to confirm that self-reported morning people did go to sleep earlier — and people with the most morning-linked gene variants went to bed 25 minutes earlier than people with the fewest. Morning people did not sleep longer or better than night people; all that differed was the time that they went to sleep.

The genes flagged in the study play a wide variety of roles in the body.

Many seem to play a role in brain tissues, and others are already known to be central to the body’s circadian rhythm. A few were active mainly in the retina, and the people who possessed an uncommon version of one of these genes had an increased chance of being night owls, said Samuel Jones, a researcher at the University of Exeter and the study’s lead author. That could imply a potential a connection between how the eye responds to sunlight and when a person sleeps.

Another gene was involved in the body’s processing of caffeine and nicotine, two of our species’ favorite stimulants. Continued study of these and the other genes could provide leads for future work on the biology of sleep timing.

“The most interesting ones are the ones where we don’t know what it is,” said Dr. Weedon.

When the researchers crunched the numbers on chronotype’s connection to mental health, they also found that self-identified morning people reported a higher level of general well-being. People in this group also were less likely to report having depression or schizophrenia, in line with epidemiological studies suggesting that evening people struggle with mental health.

The researchers wonder whether having a lifestyle that aligns with one’s chronotype may be more important in mental and physical health than whether you are merely a morning or night person. In future work, they are hoping to see whether morning people who are required to stay up late for their jobs or other commitments — perhaps similar to night owls who must rise early for 9-to-5- jobs — show higher levels of mental disorders than their well-aligned counterparts.

“Perhaps evening people are constantly fighting their natural clock, which might have unintended consequences farther down the line,” said Dr. Jones.

This article was originally published in The New York Times. Read the original article.