In an op-ed for The Dispatch, Johns Hopkins Professor Marty Makary slammed the FDA for "holding up COVID-19 vaccine approval" and dubbed the agency's progress "Operation Turtle Speed."
"Pfizer (NYSE:PFE) submitted data detailing the safety and effectiveness of its vaccine on Nov. 22. But rather than immediately convening experts, the FDA scheduled a review meeting on Dec. 10, almost three weeks later. As Pfizer's application sits on the shelf at the FDA awaiting authorization, about 27,000 Americans will have died. So what is the FDA doing for three weeks?"
"Contrary to popular belief, the FDA process is not hands-on - it does not interview vaccine trial patients or look under a microscope at the immune cells. It's doing a statistical analysis and looking at data. For the vaccine trial, the data set is small and straightforward. If my research team, normally tasked with analyzing data on millions of patients, was asked to review the smaller Pfizer vaccine study of 43,000 patients, it would take about one hour."
"The FDA also reviews manufacturing data from Pfizer on how they made the drug. But not only can that data be reviewed in a few hours, it should have been done months ago when it was available. While the FDA was waiting for Pfizer’s long-term vaccine results to come in, the agency should have anticipated this step and done it early."
"The final step of the FDA review is to look at the outcomes of the study volunteers, including rates and severity of infection and side effects in the vaccine and placebo groups. Again, there is no plausible reason why this basic analysis cannot be done in 24 hours. The FDA and external scientists have a simple task: confirm or reject the review already conducted by the trial’s independent data safety monitoring board before FDA submission."
Note: Bahrain became the second country to grant emergency-use authorization to Pfizer and BioNTech's (NASDAQ:BNTX) vaccine on Friday. The U.K. became the first this past Wednesday.
Ten patients with severe forms of the blood disorders sickle cell or beta thalassemia have all seen their condition improve after receiving an experimental treatment that genetically modified their cells, marking the latest step forward for a landmark technology known as CRISPR that won the Nobel Prize in Chemistry this year.
The results, showcased at a virtual meeting of the American Society of Hematology on Saturday, come from two pioneering early-stage trials of a treatment called CTX001 and developed by biotech partners CRISPR Therapeutics and Vertex.
In one study, seven beta thalassemia patients no longer needed blood transfusions for at least three months and as long as 20.5 months post-treatment. Those patients used a median of 15 infusions per year before receiving CTX001, according to a presentation of the results.
In the other trial, three sickle cell patients treated with CTX001 haven't experienced any instances of theexcruciating pain episodes known as a vaso-occlusive crisis for at least three months and as long as about 17. Before joining the trial, they had a median of seven per year.
All 10 patients are also now producing "normal to near-normal" total levels of the oxygen-carrying protein hemoglobin, which is either missing or warped in people with severe beta thalassemia or sickle cell disease.
So far, the effects of treatment haven't diminished, the companies said, and results were consistent regardless of each patient's disease severity or underlying genetic characteristics. A report on the first two patients treated was published in the New England Journal of Medicine on Saturday.
"We have great hope that this can be a one-time treatment that's curative for life," said CRISPR Therapeutics CEO Sam Kulkarni, in an interview.
For many of the patients, results are still early, with follow-up for only three to six months. Whether the positive effects will wear off in some is uncertain, although the companies presented data to suggest benefit will be long-lasting.
Study participants will also continue to be watched for any safety problems.CRISPR and Vertex did report one serious side effect possibly related to CTX001, a case of a potentially life-threatening immune reaction known as hemophagocytic lymphohistiocytosis, whichappeared associated with several other side effects.
The condition, which can occur following bone marrow transplants, resolved and Kulkarni said the patient is "doing fine."
"I would expect that it's unlikely that we see that [moving forward] or that it's related to the therapy," he contended. Most other adverse events were deemed mild to moderate and related to the chemotherapy regimen needed to prepare patients for treatment.
While CRISPR Therapeutics and Vertex are reporting data on 10 patients at ASH, nine others have now been treated with CTX001. The partner companies aim to seek approval once they've treated enough patients in the two ongoing studies, according to Kulkarni. The exact number needed to satisfy regulators is still being determined, but "we could make a strong case that we don't need a large number of patients" given the degree of benefit observed so far, he added.
Typically, new drugs require testing in hundreds or thousands of patients across several phases of study. With gene editing and gene therapy, however, the "effect size" of treatment can be quite large, and the biological rationale of treatment particularly clear, meaning companies can make a convincing case to regulators with far few patients treated.
The two gene therapies approved to date in the U.S., for example, were cleared following study in 41 and 36 patients, respectively.
Both of those treatments, called Luxturna and Zolgensma, use a different technology than CTX001, replacing rather than editing genes. The Food and Drug Administration hasn't yet specifically outlined the approval requirements for a CRISPR-based medicine, meaning CRISPR Therapeutics and Vertex are forging the path forward as they go.
CRISPR gene editing involves a two-part biological system that can delete or later DNA sequences with a precise cut. Its potential use as a medicine, diagnostic and drug discovery tool is enormous, and has led to the creation of a wave of new biotech companies. CRISPR Therapeutics, Editas Medicine and Intellia Therapeutics, for example, were each formed to advance CRISPR-based therapies.
In October, CRISPR Therapeutics co-founder Emmanuelle Charpentier and Intellia co-founder Jennifer Doudna were awarded a Nobel Prize for their work developing the gene editing method.
All three companies now have CRISPR-based therapies in human testing, but CTX001 is the first to produce results in a clinical trial.
Both beta thalassemia and sickle cell are caused by mutations in a gene that encodes for beta-globin, a protein that forms part of adult hemoglobin. With the gene damaged, individuals with either disease can't produce healthy hemoglobin, leading to anemia and a host of other health problems.
Many beta thalassemia patients depend on chronic blood transfusions, for example, but there are complications associated with that treatment, too. Sickle cell patients, meanwhile, have crescent-shaped blood cells that get lodged in blood vessels, triggering painful episodes that can require hospitalization.
The aim of CTX001 is to help patients make enough hemoglobin to free them from blood transfusions and pain crises. CTX001 consists of stem cells collected from a patient's bone marrow, which are then altered using CRISPR to encourage production of a form of hemoglobin that's present at birth but fades with age. The cells are then infused back into the body, where they take hold in the bone marrow and, in theory, churn out enough so-called fetal hemoglobin to change the course of the disease.
So far, that appears to be the case. Patients in both groups are producing meaningful levels of fetal hemoglobin, even in those individuals with genetic make-ups that make their disease more severe.
One patient with very severe beta thalassemia, for example, was producing 11.5 g/dL of hemoglobin — within the range of normal — as of a four-month follow-up evaluation conducted recently, said study investigator Haydar Frangoul, medical director of pediatric hematology and oncology at Sarah Cannon Research Institute, in a presentation.
CRISPR Therapeutics and Vertex's findings could put pressure on Bluebird bio, which won European approval for its gene therapy Zynteglo in beta thalassemia, but has hit multiple delays in the U.S. Bluebird's gene therapies have shown promise for both diseases, although the company has had to fine tune its approach in sickle cell. In beta thalassemia, Bluebird has the most data in patients with a less severe form.
The results also appear more varied from patient to patient. CRISPR Therapeutics and Vertex hope that gene editing may be more predictable and durable than gene replacement, but that hasn't been proven.
"I think gene editing will be looked at as a different class of medicines by regulators and by investigators," Kulkarni said.
An experimental cell therapy developed by Johnson & Johnson reduced or eliminated signs of multiple myeloma in nearly all of 94 patients treated in a mid-stage study, according to results presented Saturday at a virtual meeting of the American Society of Hematology.
After one year, 89% of patients were still alive and roughly three quarters were still responding to treatment, the data showed. The study presentation at ASH strengthens earlier findings, which were similarly encouraging.
The new data are from patients enrolled into an expansion portion of J&J's trial, called CARTITUDE-1. Participants were very sick, with their cancer having progressed following a median of six different treatments. While J&J's therapy appears strongly effective, side effects were common and one patient died from an adverse reaction typical of cell therapy treatment.
J&J's cell therapy is one of a broad slate of new medicines being developed for multiple myeloma that target a protein called BCMA, which is commonly found on diseased blood cells.
Blenrep, an antibody drug from GlaxoSmithKline, became the first such drug approved when the Food and Drug Administration cleared it in August.
Cell therapies like J&J's and another from Bristol Myers Squibb called ide-cel appear much more potent, inducing strong response rates. Both are CAR-T cell therapies, which involve the re-engineering of patients' own immune cells to seek out BCMA-expressing cancers.
Three CAR-T therapies are approved to treat leukemia and lymphoma, but ide-cel would be the first in multiple myeloma if approved as expected by March of next year.
The catch with CAR-T therapies is the process by which they are made is complicated, expensive and can sometimes fail. Both ide-cel and J&J's cell therapy, which the pharma licensed from China-based Legend Biotech, could face competition from other BCMA-targeting antibody drugs, a handful of which are in early- to mid-stage testing.
In CARTITUDE-1, treatment with J&J's therapy, cilta-cel, led to responses in 94 of 97 enrolled patients. Sixty-seven percent met the criteria for a "stringent complete response," meaning no signs of diseases cells were observed in the bone marrow.
At 12 months, 77% of patients showed no sign of their cancer progressing, and 89% were still alive.
J&J plans to submit the data from its trial to the Food and Drug Administration in the next weeks. But CARTITUDE-1 is only a first step in the drugmaker's planned work with cilta-cel in multiple myeloma, said Mark Wildgust, vice president of oncology global medical affairs with J&J.
"I think the CARTITUDE-1 data gives us confidence in [cilta-cel as a] single agent," he said in an interview. "Now we have to see if we can do it earlier in the disease."
One significant concern with CAR-T therapy is its side effect profile. Treatment is commonly associated with an immune system response known as "cytokine release syndrome," or CRS, and neurotoxicity.
Nearly all patients in J&J's trial experienced CRS, and one patient died from the condition. Ninety-five percent of those affected had symptoms mild enough to be managed with fever-reducing medications or, if hospital care was necessary, responded quickly to treatment, J&J said.
The company hopes cilta-cel could potentially be given as an outpatient treatment, a goal of many CAR-T drug developers. Whether J&J's data supports that approach will be up to Food and Drug Administration officials, who will look closely at the risks of allowing patients to go home after treatment.
A neurological side effect associated with CRS occurred in 16 patients, but subsided, at the median, four days following onset. Twelve other cases of neurotoxicity were also observed, with one being fatal, J&J said.
Also at ASH, Bristol Myers disclosed updated data from Phase 1 and 2 trials of ide-cel. In the early study, which included four different doses tested, results showed three-quarters of patients responded to treatment.
Median progression-free survival in that study stretched to nine months, and participants lived a median of 34 months post treatment.
An analysis of the later, pivotal KarMMa study showed more than two thirds of patients responded to treatment in most high-risk groups, Bristol Myers said, while median progression-free survival was longer than seven months.
Results presented earlier this year showed, across the entire KarMMa study, an overall response rate of 73%, which rose to 82% among those given the highest dose.
We seem to be heading for a world with multiple coronavirus vaccines in it, and right off, I have to say that that this is a very good situation. But it has its complications, and one that I know many people have been wondering about is, what if you get two different ones? That could happen in several ways, of course, with the different vaccines themselves, the order in which a person is exposed to them, the total number of vaccinations involved, etc. And honestly, it’s not possible to be completely sure about the answer until this is actually tried (immunology!) But we can look back over previous vaccines and made some educated guesses.
The best outcome is that you get even stronger immunity. That seems to be what happens when people who received the oral (Sabin) polio vaccine were then given the injectable (Salk) form. The first is an attenuated live virus, and the second is a completely inactivated one. The Salk vaccine is better at producing humoral immunity (antibody and T-cell response), and the Sabin vaccine needs multiple doses to be effective. But it is better at producing mucosal immunity in the gut, which has a better chance of interrupting the spread of the disease in children. The choice about which one to use has always been a matter of argument. But the study linked above showed that in children who had already had the oral Sabin vaccine, that an injection of the Salk vaccine boosted their intestinal immunity better than another round of the oral vaccine. Again, you wouldn’t necessarily have predicted that – if it had come out that the injected dose didn’t seem to do much for mucosal immunity, it would have been easy to rationalize that as well.
There are other cases where multiple vaccines are available for the same pathogen, and where a mix-and-match approach doesn’t seem to make a difference either way. An example is hepatitis A, where there are several inactivated-virus options. In that case, it appears that the vaccines are basically interchangeable: the booster-shot schedule can be completed any way you like. The same goes for the two monovalent vaccines for hepatitis B, and for the three vaccines that target meningococcus group A, C, W, and Y. (Here’s an overview of vaccine interchangeability).
That said, all of those vaccines in each of those cases are rather similar to each other, and we now have the unusual – very, very unusual – situation of several different vaccine platforms coming into potential use against the same virus at almost the same time. By the spring we may well have two mRNA vaccines (Pfizer/BioNTech and Moderna), two different adenovirus vaccines (Oxford/AZ and J&J), and a recombinant protein vaccine (Novavax). We don’t have efficacy data on the J&J and Novavax candidates yet (numbers are on the way), and we can argue about the data for Oxford/AZ, but it’s certainly possible that all of them will be out there simultaneously. Putting one of these on top of the other is a step into the unknown.
And there are examples of vaccines for the same pathogen having some interference. Several vaccines for bacterial diseases are in the “conjugate vaccine” category: they have a bacterial polysaccharide fused to a carrier protein, which can give a more useful immune response than just dosing the polysaccharide by itself. But for pneumococcal vaccines, both types are given (with a different range of immune response to cover a variety of bacterial serotypes). It’s been found that if you give a pneumococcal polysaccharide vaccine (PPSV) followed by a pneumococcal polysaccharide conjugate vaccine (PCV), there’s a lower antibody responses for some serotypes targeted by the conjugate vaccine than there is if you give them in the opposite order. So the rule in this area is to give both for maximum protection, but to always give the conjugate vaccine first. Another tricky part is that the use of the same sorts of carrier proteins in different vaccines – you could imagine a situation where an immune response against the carrier protein causes a later vaccine to be less effective.
That last problem is similar to what we’re talking about with the immune response to adenovirus vectors and booster-shot dosing regimens with the same vaccine. But the Oxford/AZ vaccine is a chimpanzee adenovirus and the J&J one is Ad26, so that’s a different situation, and I have no idea of what would happen if you mixed those two. (The Russian vaccine is, in fact, a mixture of two different adenovirus vectors, one in the original shot and one in the booster). I also don’t know what happens if you take both an mRNA vaccine and one of the other types.
Overall, though, I would tend to think that it would work out. All of the coronavirus vaccines we’re talking about target the Spike protein, after all, and they are, by different means, presumably raising a pretty similar suite of antibodies (with perhaps more differences in T-cell response, which remains to be seen in detail). So the chances are that the immune response will be similar (as with the hepatitis vaccines) or perhaps even a bit better (as with mixing the polio vaccines), rather than worse. But we haven’t proven anything like that in the clinic yet, and educated guesses will only take you so far. I would assume that there will be people who end up taking both types, for all sorts of reasons, and I hope that we collect as much data from those cases as we can.
As human lifespans have gotten longer, certain proteins in our bodies are increasingly prone to take on alternative shapes. These misfolded proteins can ultimately trigger neurodegenerative diseases such as Alzheimer's, Parkinson's and Lou Gehrig's disease, formally known as amyotrophic lateral sclerosis (ALS).
Meredith Jackrel, assistant professor of chemistry in Arts & Sciences at Washington University in St. Louis, and her lab group studyproteinmisfolding disorders. They are especially interested in how protein misfolding occurs, how it leads to disease and how scientists might be able to prevent or even reverse protein misfolding. Their work promises applications in flu vaccines as well as in the currentcoronaviruspandemic.
In this Q&A, Jackrel describes how her lab's expertise in protein misfolding and neurodegenerative diseases has made them uniquely qualified to work on developing new amyloid-inspired vaccine technologies aimed at elderly populations.
How does your research relate to the current pandemic?
We are working on the development of new vaccine technologies specifically tailored to elderly populations. We originally initiated this project to evaluate new flu vaccine technologies, but this approach could also be relevant to COVID-19 since seniors are particularly susceptible to its severe complications.
A general problem with vaccination of elderly individuals is immuosenescence, or age-related dysfunction of the immune system. Immunosenescence is typically overcome by the addition of adjuvants to improve immune response and efficacy. However, adjuvants create local inflammation, which obstructs the immune system and makes vaccines less effective.
A colleague at WashU in biomedical engineering, Jai Rudra, studies self-assembling peptides as materials for developing novel vaccines that do not require the use of adjuvants. These self-adjuvanting peptide nanofibers are hypothesized to trigger the autophagy pathway, a kind of cellular recycling that can also promote good immunological functions, which has emerged as a potential vaccine target. Administration of these peptide nanofibers leads to robust, high-affinity, and neutralizing antibody responses without local reactions, making them attractive for vaccine delivery in the elderly.
To further pursue application of these nanofibers, we must now investigate the toxicity and clearance mechanism of these materials.
How are you using your expertise in protein folding/misfolding in your work on vaccine technology?
Peptide nanofiber materials rapidly assemble into configurations that closely resemble the underlying causes of neurodegenerative disorders. These amyloids are recognized as clumps of proteins that accumulate in patients with Alzheimer's, Huntington's and Parkinson's disease.
While there are key differences that we anticipate will not make use of these materials problematic, it is nonetheless essential that the safety and clearance mechanism of the peptide nanofiber vaccines be thoroughly tested. My lab's expertise in the development of model systems to study the toxicity and mechanism of disease-associated amyloid proteins is therefore highly relevant to this project.
Furthermore, due to the complexities of studying the peptide nanofibers in mammalian cells, my lab's expertise in the use of Baker's yeast as a model system is proving highly relevant for studying the mechanism of clearance of these new materials.
What are your specific goals in this project?
The primary goals for my lab are to determine the toxicity and mechanism of clearance of the peptide nanofiber vaccines in a yeast model system. We aim to compare the toxicity of the nanofibers to the toxicity of disease-associated proteins. We will also employ autophagy-deficient yeast models to establish the mechanism of clearance of the nanofibers. The Rudra lab then aims to assess the efficacy of the nanofiber-based vaccines in aged mice.
Where does the project stand now? What are the next steps?
We have established a yeast model system of these peptide nanofibers and completed much of the preliminary work. Excitingly, we have confirmed that these peptide nanofibers are not toxic in yeast and have made some new insights into their mechanism of clearance. We aim to complete the early stage of this project shortly, and the Rudra lab has begun work in animal systems. Once we complete work with the nanofibers alone, we will begin to test conjugates to various vaccine targets, notably those that underpin COVID-19.
Two COVID-19 vaccines are on the verge of approval in the United States, with pharmaceutical companies promising that millions of doses will be available to the first wave of recipients within a matter of weeks.
Creating two vaccines in less than a year is an astonishing achievement, experts say, but the next task could prove even more difficult—convincing Americans that it's safe to take vaccines developed at such a breakneck pace.
Average folks can take comfort from the safety data that's already been gathered in clinical trials, and additional data expected to pour in from millions more people participating in the earliest waves of COVID-19 vaccine distribution, said Dr. Paul Offit. He's director of the Vaccine Education Center at the Children's Hospital of Philadelphia.
"For people who are worried about safety, we are essentially, by necessity, testing the water with one foot," Offit said. "We'll have tens of millions of people who will be getting this vaccine before the general population gets it, so you'll have a much bigger safety profile than you have when it initially rolls out."
Offit is a member of the U.S. Food and Drug Administration advisory board that will review the clinical trial data for both the Pfizer and Moderna vaccines within the next two weeks.
In fact, Offit has already started to go over the data on the Pfizer vaccine, which will be considered at the advisory board's Dec. 10 meeting.
'Very reassured'
Offit said skyscraper-high reams of documents tend to be generated during clinical trials, and the FDA advisory board painstakingly reviews all that data before recommending vaccine approval.
"You don't want us [only] to look at the press release and say these data look great and just say, 'Let's go,'" Offit said. "You've seen the tip of the iceberg. We're going to look at the base of the iceberg and make sure there's nothing at the base that's cracking, that makes us wonder about whether the tip is really true."
Vaccine makers are not involved at all in this review process, Dr. Anthony Fauci, director of the U.S. National Institute of Allergy and Infectious Diseases, stressed last week during an HD Live! interview.
"You separate them so that the Data and Safety Monitoring Board does [its review] independently," Fauci explained.
Only after committees from both the FDA and the U.S. Centers for Disease Control and Prevention agree on the data would FDA officials decide that "'we're going to do an EUA'—we are going to have an Emergency Use Authorization," he said.
Offit and other infectious disease experts said they do have early confidence in the safety of the two COVID-19 vaccines, given what's been reported so far.
"We really see vaccine side effects in the first week after vaccine, and sometimes in the first month to two months of the vaccine," said Dr. Buddy Creech, director of the Vanderbilt Vaccine Research Program, in Nashville, Tenn. "We've been very reassured that we haven't seen a number of cases of things that we would not expect."
Offit added, "What you're going to be able to say now, when these vaccines roll out, is you're going to be able to say that, at least in tens of thousands of people, there were no uncommon serious side effects that were seen within two months of getting a dose."
Long-term tracking
Rare side effects remain a concern, however, and the limited number of people involved in a clinical trial means that these problems won't necessarily crop up in initial testing.
For example, polio-like Guillain-Barre syndrome is known to happen in about one out of every million people who get a flu shot, Offit noted. Researchers only learned of that after hundreds of millions of flu shots were handed out over the years.
"Usually if you have a serious side effect, you'll find it out quickly," Offit said. "That said, 20,000 people isn't 20 million people. You're only going to find a rare serious adverse event post-approval. That's always true."
That's why participants in the vaccine clinical trials will continue to be tracked for at least two years, serving as "canaries in the coal mine" for longer-term safety problems, the experts said.
According to Dr. Kathleen Neuzil, director of the Center for Vaccine Development and Global Health at the University of Maryland School of Medicine, in Baltimore, "If there is any safety signal in that gold-standard trial, those people will be five to six months ahead of where we are with vaccinating the public."
Researchers will also be tracking safety data from the first waves of people who get the vaccine, starting with about 21 million people in the health care industry and 3 million folks living or working at long-term care facilities.
Ongoing tracking data will also assess how long the vaccines will protect against COVID-19, Offit added.
As a general rule, he noted, coronaviruses induce immunity that is short-lived and incomplete.
"By short-lived, I mean years, not decades," Offit said. "By incomplete, I mean not sterilizing immunity—protection against moderate to severe disease, but not necessarily mild disease or asymptomatic infection."
Reducing hospitalizations
But even immunity that is temporary and incomplete will help during this pandemic, Offit explained.
"All you want to do is keep people out of the hospital and keep them out of the morgue, and I think this vaccine certainly can do that," he said.
But the fact that these are the first two broadly available vaccines based on bits of genetic material called messenger RNA will prompt additional scrutiny regarding long-term effects, experts added.
The vaccines work by delivering mRNA into a person's cells, supplying genetic instructions that prompt the cells to produce the specific "spike" protein that the novel coronavirus uses to bind with and enter cells. The immune system recognizes the protein as a potential threat and stimulates a response, creating antibodies that would ward off any subsequent attack by the actual coronavirus.
"What turns it off? What makes it so it is no longer making coronavirus spike protein anymore?" Offit said. "If you look at the animal model studies, mice for example, you would assume that goes on for about 10 days. But what happens in humans, I don't know. We will find that out, I think, over time."
Neuzil noted that RNA is very unstable, which is why these vaccines need to be kept frozen during distribution. But with that cold storage in place, she's confident the vaccine will do its job and then flush from the body.
"We can be assured that they don't integrate into our cells," Neuzil said. "They share that code, they do their job and then they are naturally taken away by enzymes in our bodies. We wouldn't expect any prolonged side effects from these mRNA vaccines."
Imagine swabbing your nostrils, putting the swab in a device, and getting a read-out on your phone in 15 to 30 minutes that tells you if you are infected with the COVID-19 virus. This has been the vision for a team of scientists at Gladstone Institutes, University of California, Berkeley (UC Berkeley), and University of California, San Francisco (UCSF). And now, they report a scientific breakthrough that brings them closer to making this vision a reality.
One of the major hurdles to combating the COVID-19 pandemic and fully reopening communities across the country is the availability of mass rapid testing. Knowing who is infected would provide valuable insights about the potential spread and threat of the virus for policymakers and citizens alike.
Yet, people must often wait several days for their results, or even longer when there is a backlog in processing lab tests. And, the situation is worsened by the fact that most infected people have mild or no symptoms, yet still carry and spread the virus.
In a new study published in the scientific journal Cell, the team from Gladstone, UC Berkeley, and UCSF has outlined the technology for a CRISPR-based test for COVID-19 that uses a smartphone camera to provide accurate results in under 30 minutes.
"It has been an urgent task for the scientific community to not only increase testing, but also to provide new testing options," says Melanie Ott, MD, Ph.D., director of the Gladstone Institute of Virology and one of the leaders of the study. "The assay we developed could provide rapid, low-cost testing to help control the spread of COVID-19."
The technique was designed in collaboration with UC Berkeley bioengineer Daniel Fletcher, Ph.D., as well as Jennifer Doudna, Ph.D., who is a senior investigator at Gladstone, a professor at UC Berkeley, president of the Innovative Genomics Institute, and an investigator of the Howard Hughes Medical Institute. Doudna recently won the 2020 Nobel Prize in Chemistry for co-discovering CRISPR-Cas genome editing, the technology that underlies this work.
Not only can their new diagnostic test generate a positive or negative result, it also measures the viral load (or the concentration of SARS-CoV-2, the virus that causes COVID-19) in a given sample.
"When coupled with repeated testing, measuring viral load could help determine whether an infection is increasing or decreasing," says Fletcher, who is also a Chan Zuckerberg Biohub Investigator. "Monitoring the course of a patient's infection could help health care professionals estimate the stage of infection and predict, in real time, how long is likely needed for recovery."
A photo of a device attached to an ordinary smartphone that can detect the presence of SARS-CoV-2 in a nasal swab. Credit: Daniel Fletcher and Melanie Ott
A Simpler Test through Direct Detection
Current COVID-19 tests use a method called quantitative PCR—the gold standard of testing. However, one of the issues with using this technique to test for SARS-CoV-2 is that it requires DNA. Coronavirus is an RNA virus, which means that to use the PCR approach, the viral RNA must first be converted to DNA. In addition, this technique relies on a two-step chemical reaction, including an amplification step to provide enough of the DNA to make it detectable. So, current tests typically need trained users, specialized reagents, and cumbersome lab equipment, which severely limits where testing can occur and causes delays in receiving results.
As an alternative to PCR, scientists are developing testing strategies based on the gene-editing technology CRISPR, which excels at specifically identifying genetic material.
All CRISPR diagnostics to date have required that the viral RNA be converted to DNA and amplified before it can be detected, adding time and complexity. In contrast, the novel approach described in this recent study skips all the conversion and amplification steps, using CRISPR to directly detect the viral RNA.
"One reason we're excited about CRISPR-based diagnostics is the potential for quick, accurate results at the point of need," says Doudna. "This is especially helpful in places with limited access to testing, or when frequent, rapid testing is needed. It could eliminate a lot of the bottlenecks we've seen with COVID-19."
Parinaz Fozouni, a UCSF graduate student working in Ott's lab at Gladstone, had been working on an RNA detection system for HIV for the past few years. But in January 2020, when it became clear that the coronavirus was becoming a bigger issue globally and that testing was a potential pitfall, she and her colleagues decided to shift their focus to COVID-19.
"We knew the assay we were developing would be a logical fit to help the crisis by allowing rapid testing with minimal resources," says Fozouni, who is co-first author of the paper, along with Sungmin Son and María Díaz de León Derby from Fletcher's team at UC Berkeley. "Instead of the well-known CRISPR protein called Cas9, which recognizes and cleaves DNA, we used Cas13, which cleaves RNA."
In the new test, the Cas13 protein is combined with a reporter molecule that becomes fluorescent when cut, and then mixed with a patient sample from a nasal swab. The sample is placed in a device that attaches to a smartphone. If the sample contains RNA from SARS-CoV-2, Cas13 will be activated and will cut the reporter molecule, causing the emission of a fluorescent signal. Then, the smartphone camera, essentially converted into a microscope, can detect the fluorescence and report that a swab tested positive for the virus.
"What really makes this test unique is that it uses a one-step reaction to directly test the viral RNA, as opposed to the two-step process in traditional PCR tests," says Ott, who is also a professor in the Department of Medicine at UCSF. "The simpler chemistry, paired with the smartphone camera, cuts down detection time and doesn't require complex lab equipment. It also allows the test to yield quantitative measurements rather than simply a positive or negative result."
The researchers also say that their assay could be adapted to a variety of mobile phones, making the technology easily accessible.
"We chose to use mobile phones as the basis for our detection device since they have intuitive user interfaces and highly sensitive cameras that we can use to detect fluorescence," explains Fletcher. "Mobile phones are also mass-produced and cost-effective, demonstrating that specialized lab instruments aren't necessary for this assay."
Accurate and Quick Results to Limit the Pandemic
When the scientists tested their device using patient samples, they confirmed that it could provide a very fast turnaround time of results for samples with clinically relevant viral loads. In fact, the device accurately detected a set of positive samples in under 5 minutes. For samples with a low viral load, the device required up to 30 minutes to distinguish it from a negative test.
"Recent models of SARS-CoV-2 suggest that frequent testing with a fast turnaround time is what we need to overcome the current pandemic," says Ott. "We hope that with increased testing, we can avoid lockdowns and protect the most vulnerable populations."
Not only does the new CRISPR-based test offer a promising option for rapid testing, but by using a smartphone and avoiding the need for bulky lab equipment, it has the potential to become portable and eventually be made available for point-of-care or even at-home use. And, it could also be expanded to diagnose other respiratory viruses beyond SARS-CoV-2.
In addition, the high sensitivity of smartphone cameras, together with their connectivity, GPS, and data-processing capabilities, have made them attractive tools for diagnosing disease in low-resource regions.
"We hope to develop our test into a device that could instantly upload results into cloud-based systems while maintaining patient privacy, which would be important for contact tracing and epidemiologic studies," Ott says. "This type of smartphone-based diagnostic test could play a crucial role in controlling the current and future pandemics."
More information: Parinaz Fozouni et al, Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy, Cell (2020). DOI: 10.1016/j.cell.2020.12.001
