Virus World

A new study highlights both the promise and the limitations of gene editing, as a highly lethal form of avian influenza continues to spread around the world.

A research team led by gene editing pioneer David R. Liu, PhD, reports the application of base editing technology to develop a one-time treatment for spinal muscular atrophy (SMA), showing promising effectiveness in cell and mouse models. Liu, a professor at Harvard University, the Broad Institute, and an investigator with the Howard Hughes Medical Institute, together with 16 co-authors, published their findings today in Science, in a report entitled, “Base editing rescue of spinal muscular atrophy in cells and in mice.” The paper is the first one published in a peer-reviewed journal that details a base editing approach to treating SMA—the leading genetic cause of infant mortality. Two months ago, Benjamin P. Kleinstiver, PhD, assistant professor in the Center for Genomic Medicine at Massachusetts General Hospital (MGH) and Harvard Medical School, and 12 co-authors posted a related preprint on bioRxiv detailing successful results from their study to optimize and implement a base editing treatment for SMA. In SMA, patients have either a loss of, or mutations in, the SMN1 gene. The result is a lack of its corresponding protein SMN, which is critical to cellular functions that include those required for embryo development. Liu and co-authors detailed how they restored SMN protein abundance to normal levels and rescued disease phenotypes in cell and mouse models of SMA by developing one-time genome editing approaches targeting another gene, called SMN2.

SMN2 differs from SMN1 at just one position—a single C-to-T mutation in exon 7. The result is a defective truncated SMN protein that fails to fully compensate for the loss of SMN1, but which provides just enough SMN protein activity to allow babies to be born with SMA. In the most common SMA cases (type 1), those babies typically live less than two years without treatment. Liu and colleagues developed a customized base editor (out of more than 40 tested) that directly converts SMN2 to the same sequence as SMN1 by reversing the C-to-T mutation that occurred during the duplication of SMN1 to SMN2. The investigators reported that their strategy converted SMN2 to SMN1 with near-ideal editing efficiency of 99% in cells, fully restoring SMN protein levels ~40-fold to normal levels, while generating minimal off-target edits. “Because all SMA patients have SMN2—otherwise loss of SMN1 would not allow them to be born—this base editing approach to treating SMA also should be applicable to all SMA patients, regardless of the specific mutation that caused their SMN1 loss,” Liu said.

Phages probably picked up DNA-cutting systems from microbial hosts, and might use them to fight other viruses. A systematic sweep of viral genomes has revealed a trove of potential CRISPR-based genome-editing tools. CRISPR–Cas systems are common in the microbial world of bacteria and archaea, where they often help cells to fend off viruses. But an analysis1 published on 23 November in Cell finds CRISPR–Cas systems in 0.4% of publicly available genome sequences from viruses that can infect these microbes. Researchers think that the viruses use CRISPR–Cas to compete with one another — and potentially also to manipulate gene activity in their host to their advantage. Some of these viral systems were capable of editing plant and mammalian genomes, and possess features — such as a compact structure and efficient editing — that could make them useful in the laboratory. “This is a significant step forward in the discovery of the enormous diversity of CRISPR–Cas systems,” says computational biologist Kira Makarova at the US National Center for Biotechnology Information in Bethesda, Maryland. “There is a lot of novelty discovered here.”

DNA-cutting defences

Although best known as a tool used to alter genomes in the laboratory, CRISPR–Cas can function in nature as a rudimentary immune system. About 40% of sampled bacteria and 85% of sampled archaea have CRISPR–Cas systems. Often, these microbes can capture pieces of an invading virus’s genome, and store the sequences in a region of their own genome, called a CRISPR array. CRISPR arrays then serve as templates to generate RNAs that direct CRISPR-associated (Cas) enzymes to cut the corresponding DNA. This can allow microbes carrying the array to slice up the viral genome and potentially stop viral infections. Viruses sometimes pick up snippets of their hosts’ genomes, and researchers had previously found isolated examples of CRISPR–Cas in viral genomes. If those stolen bits of DNA give the virus a competitive advantage, they could be retained and gradually modified to better serve the viral lifestyle. For example, a virus that infects the bacterium Vibrio cholera uses CRISPR–Cas to slice up and disable DNA in the bacterium that encodes antiviral defences2. Molecular biologist Jennifer Doudna and microbiologist Jillian Banfield at the University of California, Berkeley, and their colleagues decided to do a more comprehensive search for CRISPR–Cas systems in viruses that infect bacteria and archaea, known as phages. To their surprise, they found about 6,000 of them, including representatives of every known type of CRISPR–Cas system. “Evidence would suggest that these are systems that are useful to phages,” says Doudna. The team found a wide range of variations on the usual CRISPR–Cas structure, with some systems missing components and others unusually compact. “Even if phage-encoded CRISPR–Cas systems are rare, they are highly diverse and widely distributed,” says Anne Chevallereau, who studies phage ecology and evolution at the French National Centre for Scientific Research in Paris. “Nature is full of surprises.”

Small but efficient

Viral genomes tend to be compact, and some of the viral Cas enzymes were remarkably small. This could offer a particular advantage for genome-editing applications, because smaller enzymes are easier to shuttle into cells. Doudna and her colleagues focused on a particular cluster of small Cas enzymes called Casλ, and found that some of them could be used to edit the genomes of lab-grown cells from thale cress (Arabidopsis thaliana), wheat, as well as human kidney cells. The results suggest that viral Cas enzymes could join a growing collection of gene-editing tools discovered in microbes. Although researchers have uncovered other small Cas enzymes in nature, many of those have so far been relatively inefficient for genome-editing applications, says Doudna. By contrast, some of the viral Casλ enzymes combine both small size and high efficiency. In the meantime, researchers will continue to search microbes for potential improvements to known CRISPR–Cas systems. Makarova anticipates that scientists will also be looking for CRISPR–Cas systems that have been picked up by plasmids — bits of DNA that can be transferred from microbe to microbe. “Each year we have thousands of new genomes becoming available, and some of them are from very distinct environments,” she says. “So it’s really going to be interesting.”

Published in Nature (Nov. 23, 2022): https://doi.org/10.1038/d41586-022-03837-8

A new paradigm for HIV treatment is on the horizon as FDA gives nod for startup to begin trials of CRISPR-based gene therapy as a HIV cure.  The US Food and Drug Administration (FDA) has given the nod for Excision BioTherapeutics to begin trials testing CRISPR gene editing as a treatment for HIV. EBT-101 will be a first-in-human, CRISPR-based one-time gene therapy to be evaluated in individuals with HIV. On 15 September Excision announced that the FDA had accepted its Investigational New Drug (IND) application for EBT-101 as a potential functional cure for chronic HIV. The IND clearance will allow the firm to start a first-in-human Phase I/II trial to evaluate the safety, tolerability and efficacy of EBT-101 in healthy individuals living with HIV. “The clearance of our IND application for EBT-101 represents an important milestone for Excision and is the result of years of commitment to developing a functional cure for individuals living with HIV,” said Excision CEO Daniel Dornbusch. “Although antiviral treatments can manage HIV infection, they require life-long treatment, cause side effects, and do not provide the possibility of a functional cure. We are grateful for the FDA’s engaged review and acceptance of the IND for EBT-101 and look forward to initiating the Phase I/II clinical trial later this year.” EBT-101 uses CRISPR to cut out or excise HIV that has wrapped around the DNA in cells. It has been HIV’s ability to coil itself into DNA that has made it so difficult to treat and largely the reason that past curative efforts have fallen flat. The therapy harnesses an adeno-associated virus (AAV) at a relatively low dose to deliver the one-time treatment.  Excision said that the investigational programme employs CRISPR-Cas9 and two guide RNAs that target three sites within the HIV genome, “thereby excising large portions of the HIV genome and minimizing potential viral escape”.

The upcoming trial will be one of the first attempts to directly extract the latent virus from DNA. In preclinical studies, the therapy demonstrated the ability to excise HIV proviral DNA in human primary cells as well as multiple animal models including non-human primates. In later studies, after evaluating the safety and tolerability of the therapy, Excision hopes to take patients off their regular antiviral meds to test EBT-101 as a cure. “EBT-101 has demonstrated removal of proviral DNA in multiple animal models and offers an opportunity for individuals living with HIV to potentially cease life-long therapies,” said Excision’s CMO Lisa Danzig. “The Excision team looks forward to this important collaboration with our principal investigators, scientific advisors and regulators, to conduct a safe and informative trial with this first-in-class approach to a viral disease target previously considered to be incurable.” The news of the trial comes just weeks after it was announced that J&J’s recent efforts at an HIV vaccine had failed and news broke that Moderna is set to begin trials of its HIV vaccine based on mRNA technology. HIV can now be well managed with antiretroviral medications, but these are a lifelong commitment and cause side effects. For the last few decades, an elusive HIV vaccine has been considered ‘the holy grail’ in stopping the spread of the highly stigmatised disease, but CRISPR-based gene editing would represent a brand-new therapeutic approach.

Excision BioTherapeutics, Inc. (Excision) has secured $60 million in investor financing to advance CRISPR-based gene editing constructs developed by Kamel Khalili, PhD, Laura H. Carnell Professor and Chair of the Department of Neuroscience, Director of the Center for Neurovirology, and Director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine at Temple University (LKSOM). The funding will allow these treatments to be advanced into a Phase 1/2 clinical trial in patients with chronic HIV infection. The funding secured by Excision will also provide Temple scientists and Excision the ability to research CRISPR technology on other viral diseases at LKSOM including JC Virus for Progressive Multifocal Leukoencephalopathy, Herpes Simplex Virus and other viral diseases. Those additional studies will be led by Ilker K. Sariyer, DVM, PhD, Associate Professor of Neuroscience and Neurovirology at LKSOM. “The funding secured is a momentous occasion for Excision and Temple and the many individuals living with HIV and other viral diseases,” said John M. Daly, MD, FACS, Interim Dean & Dean Emeritus and Harry C. Donahoo Professor of Surgery at LKSOM. “These continued research and clinical trials bring us one step closer to having new technology available to suppress and eradicate multiple viral diseases.”

In research recently published by Nature Communications, Dr. Khalili along with Tricia H. Burdo, PhD, Associate Professor and Associate Chair of Education in the Department of Neuroscience at LKSOM, who is an expert on the utilization of the SIV (simian immunodeficiency virus)-infected antiretroviral therapy (ART)-treated rhesus macaque model for HIV pathogenesis and cure studies; Dr. Sariyer; Rafal Kaminski, PhD, Assistant Professor of Neuroscience and Assistant Professor in the Center for Neurovirology at LKSOM; Pietro Mancuso, PhD, an Assistant Scientist in Dr. Khalili’s laboratory; Chen Chen, a Research Technician in Dr. Khalili’s laboratory; other scientists in Dr. Khalili's, Dr. Burdo’s, Dr. Sariyer’s and Dr. Kaminski’s laboratories; and colleagues demonstrated success in editing the genome of simian immunodeficiency virus (SIV), a virus closely related to HIV, the cause of AIDS, from non-human primates. Dr. Khalili and Dr. Burdo were senior co-investigators on the study and Dr. Mancuso was first author.  The success of Dr. Khalili’s HIV discoveries has led Temple into a partnership with Excision to support the continued development of the CRISPR technology. In this partnership Dr. Khalili serves as Co-Founder and Chief Scientific Consultant, and holds equity in Excision, which has licensed the viral gene editing technology from Temple University. Dr. Burdo contributes to preclinical research and development, serves on the Scientific Advisory Board and holds equity in Excision. “This transformative financing will accelerate and support the research we have developed over the past decade,” said Dr. Khalili. “We have proven the technology and candidate programs in vitro and in vivo in both small animal models and primate models. We are optimistic that ongoing research will demonstrate the potential for a future therapeutic to generate functional cures for viral infectious diseases.”

Editor’s Note: Kamel Khalili is Co-Founder and Chief Scientific Consultant, and holds equity in Excision BioTherapeutics, which has licensed the viral gene editing technology from Temple University. Kamel Khalili and Rafal Kaminski are named inventors on patents that cover the viral gene editing technology. Tricia Burdo is a consultant for and holds equity in Excision BioTherapeutics. Ilker Sariyer is a consultant for Excision. Dr. Khalili , Dr. Burdo, Dr. Sariyer and Dr. Kaminski are employed by Temple University, and both they and LKSOM faculty within the department conduct research activities sponsored by the company. Questions regarding their affiliations with Temple University may be directed to coisom@temple.edu.  In addition to owning the viral gene editing technology that Excision is licensing, Temple University also holds an equity interest in Excision. As a result of these interests, Temple University could ultimately potentially benefit financially from the outcome of this research. These interests have been reviewed and approved by Temple University in accordance with its Institutional Conflict of Interest policy. Questions about this can be directed to coitemple@temple.edu.

Instead of addressing genetic mutations, the Crispr machinery prompted cells to lose entire chromosomes. A powerful gene-editing tool called Crispr-Cas9, which this month nabbed the Nobel Prize in Chemistry for two female scientists, can cause serious side effects in the cells of human embryos, prompting them to discard large chunks of their genetic material, a new study has found. Administered to cells to repair a mutation that can cause hereditary blindness, the Crispr-Cas9 technology appeared to wreak genetic havoc in about half the specimens that the researchers examined, according to a study published in the journal Cell on Thursday. The consequences of these errors can be quite serious in some cases, said Dieter Egli, a geneticist at Columbia University and an author of the study. Some cells were so flummoxed by the alterations that they simply gave up on trying to fix them, jettisoning entire chromosomes, the units into which human DNA is packaged, Dr. Egli said. “We’re often used to hearing about papers where Crispr is very successful,” said Nicole Kaplan, a geneticist at New York University who was not involved in the study. “But with the amount of power we hold” with this tool, Dr. Kaplan said, it is crucial “to understand consequences we didn’t intend.”

Crispr-Cas9, a scissorslike chemical tool that can precisely cut and customize stretches of genetic material, such as human DNA, stoked international controversy in 2018 when He Jiankui, a Chinese scientist, used the technology to yield the world’s first gene-edited infants. The experiment was widely condemned as irresponsible and dangerous — in large part because many of the ways in which Crispr-Cas9 can affect cells remain poorly understood. Dr. He was found guilty of conducting illegal medical practices in China and sentenced to three years in prison. The new paper’s findings further underscore that “it’s really too soon to be applying Crispr to reproductive genetics,” said Nita Farahany, a bioethicist at Duke University who was not involved in the study.  Crispr-Cas9 treatments have already been given directly to people to treat conditions like blindness — a potential cure that affects that patient, and that patient only. But modifications made to sperm, eggs and embryos can be passed to future generations, raising the stakes for any mistakes made along the way Although scientists have been tinkering with genomes for decades, Crispr-Cas9 can accomplish a precise type of genetic surgery that other tools cannot. Scientists can use Crispr-Cas9 to home in on a specific region of the genome and snip it in two. Sensing trouble, the cell rushes to heal its genetic wound, sometimes using a similar-looking stretch of nearby, intact DNA as a template as it stitches the pieces back together. This gives researchers an opportunity to splice in a tailor-made template of their own, in the hopes that the cell will incorporate the intended change....

Original Study Published in Cell (Oct. 29):

https://doi.org/10.1016/j.cell.2020.10.025

The COVID-19 pandemic has claimed the lives of more than one million people worldwide. The causative agent, SARS-CoV-2, is a member of the Coronaviridae family, which are viruses that cause respiratory infections of varying severity. The cellular host factors and pathways co-opted by SARS-CoV-2 and other coronaviruses in the execution of their life cycles remain ill-defined. To develop an extensive compendium of host factors required for infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E), we performed parallel genome-scale CRISPR knockout screens.

These screens uncovered multiple host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, SREBP signaling, and glycosylphosphatidylinositol biosynthesis, as well as an unexpected requirement for several poorly characterized proteins. We identified an absolute requirement for the VTT-domain containing protein TMEM41B for infection by SARS-CoV-2 and all other coronaviruses. This human Coronaviridae host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus spillover events.

Preprint available in bioRxiv (October 8, 2020):

https://doi.org/10.1101/2020.10.07.326462

One of the keys to bringing a viral outbreak under control is rapid detection and diagnosis, which depend on the availability of fast, low-cost, easy-to-use tests that don't require labs or expensive equipment to process them. Scientists at the Broad Institute of MIT and Harvard and collaborators in the United States, Nigeria, and Sierra Leone have now validated such tests for Ebola and Lassa—two of the deadliest and most transmissible human viruses—in settings with limited infrastructure. The work appears in Nature Communications. The diagnostic tests use the CRISPR-based SHERLOCK assay to detect low levels of virus in patient samples and generate either a fluorescent readout or a result on a paper strip. The test can be tailored to detect specific viruses from certain regions, requires only a simple heat block and basic supplies to run, costs less than US$1 per sample, could be used on saliva or urine—eliminating the need for blood draws—and can return results in less than an hour. The tests also use a rapid chemical and heat treatment called HUDSON to inactive the virus in patient samples. HUDSON makes the patient samples safer for clinical staff to handle in a low-tech environment, and eliminates the need to extract a virus's genetic material from the samples before analyzing. The research team was led at Broad by Kayla Barnes, an NIH Fogarty K fellow at the Harvard School of Public Health and Broad Institute, Anna Lachenauer, a medical student at Stanford University School of Medicine, and institute member Pardis Sabeti, a professor at Harvard University and investigator with the Howard Hughes Medical Institute.

To showcase SHERLOCK's field utility, team members led by Christian Happi at Redeemer's University in Nigeria deployed a Lassa-specific assay during a recent Lassa fever outbreak—the first use of SHERLOCK in a low/middle income country. The team also compared the diagnostic against a standard RT-qPCR assay for Lassa. Collaborators at Kenema Government Hospital in Sierra Leone and at the US Army Medical Research Institute of Infectious Diseases benchmarked an Ebola-specific version of the SHERLOCK assay, using samples collected during the 2014-16 outbreak in Sierra Leone and more recent outbreaks in the Democratic Republic of the Congo. The NIH Integrated Research Facility also validated HUDSON's ability to heat-inactivate Ebola virus in their BL4 facilities, further establishing the safety and efficiency of this step. The SHERLOCK assays performed as consistently as, or better than, other diagnostics in these validations—demonstrating the platform's potential for clinical use in the future in resource-limited areas. The team also developed a mobile phone app called HandLens, spearheaded by Andres Colubri, assistant professor in the Bioinformatics and Integrative Biology program at the University of Massachusetts Medical School, that can read and immediately report paper strip SHERLOCK results. The tool can aid in situations where the paper strip gives a faint signal that is challenging for a clinician to interpret. This app can be adapted for use on any smartphone or tablet, according to the researchers, allowing a clear, unbiased diagnostic readout.

Published in Nature Communications (August 17, 2020):

https://doi.org/10.1038/s41467-020-17994-9

The kit has been granted approval under ‘emergency use’ provisions, and should help to ease testing backlogs in the country. The US drug regulator has granted its first emergency-use approval for a new coronavirus test that takes advantage of the gene-editing technology CRISPR. 

The US Food and Drug Administration’s (FDA) emergency-use authority allows it to make tests and drugs available faster than usual in a public-health emergency. The new diagnostic kit is based on an approach co-developed by CRISPR pioneer Feng Zhang at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts. It will be used to test for the novel coronavirus behind the ongoing pandemic, SARS-CoV-2, in laboratories that are certified to provide clinical-test results. Although the United States has ramped up testing in the past week — averaging nearly 250,000 tests per day, according to the non-profit organization The COVID Tracking Project — there are test shortages in some places. Widespread use of the new FDA-approved kit could help to alleviate backlogs and increase testing, says Mitchell O’Connell, a biochemist at the University of Rochester in New York, who was not involved in developing the test. But O’Connell cautions that it remains to be seen how well the test performs in real-world conditions, such as hospitals, compared with standard tests.

The CRISPR-based diagnostic kit has been developed by Sherlock Biosciences, a biotechnology company also based in Cambridge. It works by programming the CRISPR machinery, which has the ability to home in on certain genetic sequences, to detect a snippet of SARS-CoV-2 genetic material in a nose, mouth or throat swab, or in fluid from the lungs. If the virus’s genetic material is found, a CRISPR enzyme generates a fluorescent glow. The test can return results in about an hour, according to the company....

CRISPR gene editing has created chickens that resist a common virus. It may be possible to use the same technique to make poultry resistant to bird flu too. The altered chickens showed no signs of disease even when exposed to high doses of the avian leukosis virus (ALV). The virus is a problem for poultry farmers around the world, says Jiri Hejnar at the Czech Academy of Sciences. Infected birds become ill, emaciated and depressed, and often develop tumours. The virus gets into cells by binding to a protein called chicken NHE-1 (chNHE-1). Hejnar’s team has previously shown that deleting three DNA letters from the chNHE-1 gene that makes this protein prevents ALV from infecting chicken cells.

The challenge was to make this change in entire animals rather than just in a few cells. No strains of chickens naturally have this mutation, so it can’t be done by breeding alone. But genetically modifying chickens is more difficult than modifying other animals such as pigs. The conventional method is to extract so-called primordial germ cells, alter them outside the body and then add the modified cells to embryos inside freshly laid eggs. This approach was used to create CRISPR chickens in 2016, but the success rate is extremely low. In 2017, Hejnar developed a better method: using altered germ cells to restore semen production in sterilised cockerels. His team then went on to create a cockerel with sperm that have the precise deletion in the chNHE-1 gene. By crossing its offspring, they have produced a flock of white leghorn chickens that have this deletion in both copies of the gene.

A company called Biopharm is now in discussion with poultry producers in Vietnam and China about introducing this change into commercial breeds. “It’s quite simple to do,” says Hejnar. Hejnar also plans to use CRISPR to make chickens resistant to other viruses, such as bird flu. This would make us all safer: bird flu viruses sometime kill people and there are fears a mutant strain could cause a deadly global pandemic. However, there is still opposition to GM foods in many countries. It remains to be seen whether consumers will find CRISPR chickens to their taste.

Published in PNAS:

https://doi.org/10.1073/pnas.1913827117

A new genetic engineering technology could help eliminate malaria and stave off extinctions — if humanity decides to unleash it. One early summer evening in 2018, the biologist Anthony James drove from his office at the University of California, Irvine, to the headquarters of the Creative Artists Agency, a sleek glass-and-steel high-rise in Los Angeles. There, roughly 200 writers, directors and producers — many of them involved in the making of science-and-technology thrillers — were gathered for an event called Science Speed Dating, where James and other scientists would explain their work. The sessions were organized, James told me, “in hopes of getting the facts at least somewhat straight.” Attendees were assigned to different groups, so each scientist had just seven minutes to describe his or her work to one group before running to the next room and starting over. “There were a lot of stairs, so I would get really out of breath,” James recalled. “I would arrive panting.” He also felt a bit overwhelmed. There were executives in expensive suits, young men and women looking unaccountably dressy in ripped jeans and, according to James, a disconcerting number of people wearing hats. Few, if any, had a deep knowledge of genetics; one participant in particular kept referring to “the dark genome,” as though that were a thing. “I had to tell him, ‘Real geneticists don’t usually talk that way,’ ” James said.

James began his presentation with a brief overview of mosquito-borne diseases like malaria and Zika. Then he turned cautiously to talking about his own area of scientific expertise: an obscure but powerful invention known as a gene drive. James began by noting that two brown-eyed human parents can sometimes produce a blue-eyed child, though only if both parents carry a copy of the recessive gene. A gene drive, he explained, was a tool that in some species could turn such events into a near certainty. For one thing, it guaranteed that a particular gene would be inherited, even if only one parent had it. And it would automatically insert the chosen gene into both copies of the offspring’s DNA, effectively turning a recessive trait into a dominant one. That alone, James explained, “lets you change the odds, so you get blue eyes 99 percent of the time.”

What made the gene drive truly strange and remarkable, though, was that it didn’t stop with one set of offspring. Generation after generation, it would relentlessly copy and paste the gene it carried, until it was present in every descendant. “For most of the people in the room, you could tell it was the first they’d heard of this,” James recalled. “You could see their eyes getting big.” This mattered, James explained, because it allowed you to change not just a single creature but — potentially — an entire population, and quickly. A few months after the technique was discovered in 2014, James engineered two mosquitoes to carry a gene drive that was tied to a gene for red fluorescent color that would target the mosquitoes’ eyes. He then put each into a box with 30 ordinary purple-eyed mosquitoes. As the mosquitoes bred, they produced offspring: roughly 3,900 after two generations. (Mosquitoes lay a lot of eggs.) Under the normal rules of inheritance, there should have been an equal number of red-eyed and purple-eyed mosquitoes. Instead, when James opened the boxes to check on the offspring, all but 25 of the 3,900 mosquitoes had red eyes....

The herpes simplex virus, commonly known as the cold sore virus, is a devious microbe. It enters the body through regions lined with mucous membranes—mouth, nose and genitals—but quickly establishes lifelong viral hideouts inside nerve cells. After initial infection, the virus lurks dormant only to be reawakened periodically to cause outbreaks marked by the eruption of cold sores or blisters. In a handful of people, the consequences of viral re-awaking can be devastating, including blindness and brain inflammation. Antiviral medications can prevent recurrent outbreaks, but they are not always effective, so for decades, researchers have sought a solution that would quiet the virus for good. Now, using human fibroblast cells infected with herpes simplex virus (HSV), researchers at Harvard Medical School have successfully used CRISPR-Cas9 gene editing to disrupt not only actively replicating virus but also the far-harder to reach dormant pools of the virus, demonstrating a possible strategy for achieving permanent viral control. The team's findings are described Dec. 2 in eLife.

"This is an exciting first step—one that suggests it is possible to permanently silence lifelong infections—but much more work remains to be done," said study lead investigator David Knipe, the Higgins Professor of Microbiology and Molecular Genetics in the Blavatnik Institute at Harvard Medical School. Notably, the research represents the first successful instance of disrupting latent viral reservoirs through gene editing. Latent reservoirs are notoriously impervious to antiviral medications and have also proven hard to gene-edit. The experiments also identify the mechanisms by which actively replicating virus becomes uniquely vulnerable to gene editing. These very mechanisms may also explain why latent forms of the virus are less amenable to this technique.

Specifically, the experiments reveal that the DNA of an actively replicating virus is more exposed to the Cas9 enzyme—the molecular scissors in the CRISPR-Cas9 gene-editing system. This is because actively replicating viruses have fewer protective histones that wrap around their DNA to shield it. "The absence of protective histones makes the DNA more accessible and easier to cut, so it's essentially identified HSV's Achilles heel," Knipe said. The new findings offer a model system for using gene editing in a localized way to disrupt active replication in specific sites. However, Knipe cautions, the arch-challenge of delivering gene-editing therapy to neurons—where the virus hides and enters a state of dormancy—remains to be solved, Knipe added...

Published in eLife (December 2, 2019):

https://doi.org/10.7554/eLife.51662

At age 45, Dr. Lakiea Bailey said, she was the oldest person with sickle cell anemia that she knew. The executive director of the nonprofit patient advocacy group the Sickle Cell Consortium was diagnosed with sickle cell disease at age 3. Because of it, she’s had heart problems, had her hips replaced, and experienced serious pain all her life. Bailey told the US Food and Drug Administration’s independent advisory committee Tuesday that she believes a cutting-edge therapy that is currently under review offers the sickle cell community something many haven’t ever had before: hope. “Hope is on the horizon, and we are looking toward this hope for a change of the lives that we are living of excruciating pain,” Bailey told the FDA committee on Tuesday. The independent committee is helping the FDA think through how it should evaluate a treatment called exa-cel that could potentially cure people of sickle cell disease, a painful and deadly disease with no universally successful treatment. This was an ongoing discussion with no vote or decision about the therapy, but the discussion likely moves the US one step closer to approving a groundbreaking new treatment that uses gene editing. If approved, exa-cel, made by Boston-based Vertex Pharmaceuticals and the Swiss company CRISPR Therapeutics, would be the first FDA-approved treatment that uses genetic modification called CRISPR. CRISPR, or clustered regularly interspaced short palindromic repeats, is a technology researchers use to selectively modify DNA, the carrier of genetic information that the body uses to function and develop. The FDA said treatment for severe sickle cell is an “unmet medical need.” When someone has sickle cell disease their red blood cells don’t function the way they should. Red blood cells are the helper cells that carry oxygen from the lungs to the body’s tissues, which use this oxygen to produce energy. The process also generates waste in the form of carbon dioxide that the red blood cells take to the lungs to be exhaled out.

With sickle cell disease — also called sickle cell anemia — red blood cells take on a folded or sickle shape that can clog tiny blood vessels and cause progressive organ damage and pain, and can lead to organ failure. The sickle cells also die earlier than they should, which means the person is constantly short red blood cells. One person with sickle cell who testified said she had been hospitalized 100 times just last year. Median life expectancy is only 45 years. Sickle cell is rare, and it disproportionately impacts African Americans. About 100,000 people in the US are diagnosed with sickle cell and, of those, 20,000 have what’s considered severe disease. Up until now, the only real treatment has been a stem cell or bone marrow transplant. For stem cells, fewer than 20% of patients have an appropriately matched donor, the FDA said, and the transplants are risky and may not work. Sometimes a transplant can kill the patient. The new exa-cel treatment under FDA consideration can use the patient’s own stem cells. Doctors would alter them with CRISPR to fix the genetic problems that cause sickle cell, and then the altered stem cells are given back to the patient in a one-time infusion. In company studies, the treatment was considered safe, and it had a “highly positive benefit-risk for patients with severe sickle cell disease,” Dr. Stephanie Krogmeier, vice president for global regulatory affairs with Vertex Pharmaceuticals Incorporated, told the panel. Thirty-nine of the 40 people tested with the treatment did not have a single vaso-occlusive crisis, which means the misshapen red blood cells block normal circulation and can cause moderate to severe pain. It’s the top reason patients with sickle cell go to the emergency room or are hospitalized. Before the treatment, patients experienced about four of these painful crises a year, resulting in about two weeks in the hospital. The FDA sought the independent panel’s advice, in part, because this would be the first time the FDA would approve a treatment that uses CRISPR technology, but Dr. Fyodor Urnov, a professor in the Department of Molecular and Cell Biology at the University of California, Berkeley, reminded the committee CRISPR has been around for 30 years and, in that time, scientists have learned a lot about how to use it safely. “The technology is, in fact, ready for primetime,” Urnov said. With this kind of genetic editing, scientists could inadvertently make a change to a patient’s DNA that is off-target, and the therapy could harm the patient. The FDA wanted the experts’ advice so it could understand what criteria it should use to evaluate the treatment and determine how to evaluate long-term safety issues.

An FDA presentation to the panel suggested the agency may have some questions about the data. It called a lack of confirmatory testing “concerning.” It also noted the study’s small size. In a discussion of the company’s methodology, several panel experts said that they believed the data the companies have submitted for FDA approval were reasonable. Committee member Dr. Gil Wolfe, a distinguished professor in the department of neurology at University at Buffalo Jacobs School of Medicine and Biomedical Sciences, said he liked that the company promised to monitor patients for 15 years to see if there were any problems down the road. He said, generally, it was “exciting” to see how many patients have been treated and how positive the results have been so far. As far as any concern for what’s called “off-target effects,” meaning the potential unwanted or adverse alterations to the genome that could accidentally happen in this process and cause cancer or other problems down the road, Dr. Daniel Bauer, principal investigator and staff physician at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center, Boston Children’s Hospital, told the panel the risk is “relatively small.” Wolfe thought the depth of analysis the companies used should be good enough to detect any potential problems down the road. “We want to be careful to not let the perfect be the enemy of the good,” Wolfe said. “At some point, you have to just try things out.” “I think, in this case, that there’s a huge unmet need for individuals with sickle cell disease, and it’s important we think about how we can advance therapies that could potentially help them, and I certainly think this is one of them,” Wolfe added. Asked by the committee how he would advise patients how to evaluate the risks with this treatment, Bauer said he would be honest that there is some uncertainty, but most of the human genome is non-coding, meaning it doesn’t provide instructions to the cells to act a certain way. “It might be that many places in the human genome can tolerate an off-target edit and not have a functional consequence,” Bauer told the committee. In other words, if they made an editing error, it might not matter, and may not harm the patient. “My guess is it’s a relatively small risk in the scheme of this risk benefit. But it’s new, it’s unknown,” Bauer said. “We need to be humble and open to learning from these brave patients participating.” The FDA is expected to make an approval decision by December 8.

The lone volunteer in a gene-editing study targeting a rare form of Duchenne muscular dystrophy likely died after having a reaction to the virus that delivered the therapy in his body, researchers concluded in an early study. The lone volunteer in a gene-editing study targeting a rare form of Duchenne muscular dystrophy likely died after having a reaction to the virus that delivered the therapy in his body, researchers concluded in an early study. Terry Horgan, 27, of Montour Falls, New York, died last year during one of the first tests of a gene-editing treatment designed for one person. Some scientists wondered if the gene-editing tool CRISPR played a part in his death. The tool has transformed genetic research, sparked the development of dozens of experimental drugs, and won its inventors the Nobel Prize in 2020. But researchers said the virus—one used to carry treatment into the body because it doesn't usually make people sick—combined with his condition, triggered the problems that ultimately killed him. Horgan appears to have had a more severe immune reaction "than others receiving similar or slightly higher doses" of the virus, the authors wrote in the study, which has not yet been peer-reviewed. Horgan was enrolled in an early-stage safety trial approved by the Food and Drug Administration. It was sponsored by Cure Rare Disease, a Connecticut-based nonprofit founded by his brother, Rich, to try and save him from the muscle-wasting disease caused by a mutation in the gene needed to produce a protein called dystrophin.

In a statement, Rich Horgan thanked the research team led by the University of Massachusetts Chan Medical School and Yale University for a "thorough, comprehensive" investigation that provided valuable insights. He added, "On a personal note, this study is another important step toward honoring Terry's legacy and his commitment, as well as our entire family's, to the rare disease community." The therapy Horgan got aimed to use CRISPR to increase a form of the dystrophin protein. The process began with suppressing Horgan's immune system to prepare his body for the therapy, which was delivered by IV with "a high dose" of what's known as an adeno-associated viral vector, or AAV, according to Cure Rare Disease. But Horgan soon began experiencing problems, went into cardiac arrest six days after the treatment and died two days later from organ failure and brain damage. Because of the timing of symptoms, and the fact researchers could find little of a gene-editing enzyme in his body, they concluded that the therapy hadn't been activated yet. This isn't the first time viral vectors have been implicated in a gene therapy trial death. In a major setback for the field, 18-year-old Jesse Gelsinger died in 1999 during a study aimed at combatting his rare metabolic disease. Scientists later learned that his immune system overreacted to the virus used to carry the treatment. The virus used in Horgan's trial is considered safer but it is not without problems. "People have been trying to make safer vectors … but they still remain challenging," said Arthur Caplan, a medical ethicist at New York University who was not involved in the study but has followed the case closely. "We don't really understand why some people run into trouble and others don't. We don't know whether it's their underlying disease, some co-morbidity, or some strange immunology." Rich Horgan said they plan to submit the study to a peer-reviewed journal. Meanwhile, Cure Rare Disease said it will use alternative viruses for the other treatments it is trying to develop. Dr. Terence Flotte, dean of the UMass medical school and senior author of the study, said he hopes it leads "to further research into how to identify subsets of patients who might be prone to severe, unexpected reactions like this."

Cited research available in  medRxiv (May 30, 2023):

 https://doi.org/10.1101/2023.05.16.23289881 

A study employing CRISPR/Cas9 to explore the evolutionary beginnings of some giant viruses finds evidence their large genomes arose from gene duplications.  Most viruses are small and carry minimalist genomes. Even one of the largest small viruses, Vaccinia, measures merely one-fiftieth the size of a pollen grain and contains only 270 genes. Giant viruses flout these rules. With sizes that rival small bacteria and genomes that contain thousands of genes, their complexity emulates that of cellular life. How these viruses came to be so large has been the subject of much debate. Now, scientists are finally poised to unravel the mystery of their evolutionary origins, thanks to a suite of CRISPR/Cas9-based tools described in a Nature Communications paper from January. “It was by chance that we encountered the first giant virus,” says Chantal Abergel, a virologist at Aix-Marseille University in France. “It was Mimivirus, and it was actually mistaken for a bacterium.” In the 20 years since that discovery, virologists have prioritized exploring the diversity of giant viruses. Now that they’ve found a fair few, the focus has shifted towards studying their evolution in more detail with molecular biology techniques. Evolutionary biologists have grappled over two possible origins of giant viruses. One possibility is that they were once cellular organisms that shrunk physically and genetically over time. But most virologists now suspect giant viruses grew out of much smaller ones—though the evidence supporting either hypothesis is scant.

To begin addressing this origin question, Abergel decided to examine how the essential genes in the Pandoravirus genome are distributed. In cellular organisms, essential genes are scattered throughout the genome—so if giant viruses are essentially reduced cells, one would expect a similar pattern. Alternatively, if the genes are clumped, that could indicate the viruses’ large genomes started out in a more compact form. One way to locate a virus’s essential genes is to knock out genes one at a time to find the ones that are needed for virus production. But to do that with a giant virus, Abergel needed a gene-editing system that worked in members of the group. With the help of Hugo Bisio, a postdoctoral researcher in Abergel’s lab, and colleagues at Aix–Marseille University, Abergel used a CRISPR/Cas9-based gene-editing system to modify the genome of the amoeba Acanthamoeba castellanii and the giant virus Pandoravirus neocaledonia, which infects it. The CRISPR/Cas9 system was designed to delete specific genes and consists of two guide RNAs and a Cas9 scission enzyme. Similar to other CRISPR/Cas9 systems, each guide RNA contains 17 to 20 bases designed to bind to one specific location on the genome of the giant virus or the amoeba, allowing the Cas9 scission enzyme to cut the genome at that site. The amoeba A. castellanii contains 25 copies of each chromosome, making it difficult to design an efficient CRISPR/Cas9 system that could delete each gene copy. To overcome this issue, the researchers modified their CRISPR/Cas9 system to generate a chain reaction. Each time DNA was cut to remove a gene, a DNA segment encoding the Cas9 enzyme and the guide RNAs responsible for the cut would take the place of the missing gene in the genome. This allowed gene deletions to repeat and propagate until all copies were removed.

Once they optimized their CRISPR/Cas9 system, the team deleted each gene separately from the Pandoravirus genome and measured the resulting change in virus production, in order to determine how important each gene is to the virus’s lifecycle. They found that essential genes clustered together at one end of the genome and were segregated from nonessential genes at the other end. This level of gene orderliness has not been seen in viruses, according to Bisio. Even bacterial genomes aren’t quite so tidy: While they do group genes with linked functions together into gene clusters known as operons, these tend to be dispersed throughout the genome rather than grouped all together in one spot. Bisio says the cluster of essential genes may echo a smaller “core genome” of an ancient virus. This genome could have become elongated through multiple rounds of gene duplication that were biased in one direction to produce an additional set of spare nonessential genes. This could explain how modern-day giant viruses came to possess thousands of genes. “Our data indicate that complex viruses arose from smaller and simpler ones,” Bisio tells The Scientist in an email—noting that it will take further research to determine whether that’s true of all giant viruses or just Pandoravirus. Other studies found that some genes in giant viruses were usurped from their amoeba hosts, suggesting gene exchange is another way giant viruses increased in size. The team then set their sights on one of the many evolutionary mysteries of Pandoravirus: its lack of a capsid. Small viruses package their genomes into capsids made of viral proteins. While some giant viruses, such as Mimivirus, continue this tradition, others, including Pandoravirus, do not. If giant viruses did indeed evolve from smaller ones, there could be traces of capsid proteins hiding in their genomes. So, the researchers set out to study the function of potential capsid protein remnants in a close cousin of Pandoravirus, the smaller Mollivirus, which can also infect A. castellanii.

Researchers have suspected that a Mollivirus protein called ml_347 evolved from a capsid gene based on its gene’s sequence and predicted 3D shape. So, the team investigated its function by deleting the gene using their CRISPR/Cas9 system. They found that the gene is important for Mollivirus assembly, which the authors say is intriguing given its possible capsid ancestry. It’s possible that, as capsids were lost in giant virus evolution, obsolete capsid genes were adapted for new assembly functions. Frederik Schulz, an evolutionary biologist with the DOE Joint Genome Institute in California who wasn’t involved with the study but who has worked with Chantal Abergel in the past, tells The Scientist that the findings align with recent discoveries. “There was a debate for a long time [about] how giant virus[es] evolved,” he says. “The working hypothesis in the previous years was that they evolved from smaller viruses, and that’s exactly what Chantal and her team could show and confirm using their CRISPR/Cas9 gene-editing approach.” Schulz notes that it will be exciting to see the CRISPR/Cas9 technology introduced into other host species, such as algae, which would allow researchers to expand research into a greater variety of giant viruses. He also points out that the system only works for viruses that replicate in a host cell’s nucleus, while most giant viruses replicate in cytoplasmic structures called viral factories, which the Cas9 enzyme and guide RNAs can’t penetrate. Still, Bisio says there’s much left to discover in Pandoravirus. “[This CRISPR/Cas9 technology is] a goldmine to find new functions,” he says—one that he and his colleagues are eager to employ to tease apart what all the virus’s genes do. 

Cited research published in Nat. Communications (Jan. 26, 2023):

https://doi.org/10.1038/s41467-023-36145-4 

An experiment tests whether the gene-editing technology can stop the virus from replicating, which would ultimately wipe out the infection. 

IN JULY, AN HIV-positive man became the first volunteer in a clinical trial aimed at using Crispr gene editing to snip the AIDS-causing virus out of his cells. For an hour, he was hooked up to an IV bag that pumped the experimental treatment directly into his bloodstream. The one-time infusion is designed to carry the gene-editing tools to the man’s infected cells to clear the virus. Later this month, the volunteer will stop taking the antiretroviral drugs he’s been on to keep the virus at undetectable levels. Then, investigators will wait 12 weeks to see if the virus rebounds. If not, they’ll consider the experiment a success. “What we’re trying to do is return the cell to a near-normal state,” says Daniel Dornbusch, CEO of Excision BioTherapeutics, the San Francisco-based biotech company that’s running the trial. HIV attacks immune cells in the body called CD4 cells and hijacks their machinery to make copies of itself. But some HIV-infected cells can go dormant—sometimes for years—and not actively produce new virus copies. These so-called reservoirs are a major barrier to curing HIV. “HIV is a tough foe to fight because it’s able to insert itself into our own DNA, and it’s also able to become silent and reactivate at different points in a person’s life,” says Jonathan Li, a physician at Brigham and Women’s Hospital and HIV researcher at Harvard University who’s not involved with the Crispr trial. Figuring out how to target these reservoirs—and doing it without harming vital CD4 cells—has proven challenging, Li says. While antiretroviral drugs can halt viral replication and clear the virus from the blood, they can’t reach these reservoirs, so people have to take medication every day for the rest of their lives. But Excision BioTherapeutics is hoping that Crispr will remove HIV for good.

Crispr is being used in several other studies to treat a handful of conditions that arise from genetic mutations. In those cases, scientists are using Crispr to edit peoples’ own cells. But for the HIV trial, Excision researchers are turning the gene-editing tool against the virus. The Crispr infusion contains gene-editing molecules that target two regions in the HIV genome important for viral replication. The virus can only reproduce if it’s fully intact, so Crispr disrupts that process by cutting out chunks of the genome. In 2019, researchers at Temple University and the University of Nebraska found that using Crispr to delete those regions eliminated HIV from the genomes of rats and mice. A year later, the Temple group also showed that the approach safely removed viral DNA from macaques with SIV, the monkey version of HIV. That was an important step toward testing the treatment in people, says Kamel Khalili, a professor of microbiology at Temple University who led the work and is a cofounder of Excision Biotherapeutics. “You don’t want to eliminate the viral genome but at the same time cause any disruption in another part of the human genome and then create another set of problems for the patients,” he says. “We had to make sure that we identified a region within HIV that did not overlap with the human genome.”  Dornbusch thinks this strategy will spare patients from serious side effects and “off-target” edits—unintentional cuts elsewhere in the genome that could cause problems such as cancer. The regions targeted by the company’s Crispr therapy are also in a part of the genome that tends to stay the same even when HIV evolves. That’s important because the virus mutates rapidly, and the researchers don’t want a moving target. This isn’t the first time scientists have tried to use gene editing in the hope of curing people with HIV, but other efforts have focused on a protective mutation in a gene called CCR5. In the 1990s, scientists found that people with this naturally occurring mutation didn’t get HIV when exposed to it. The mutation—known as delta 32—thwarts the virus’s ability to get inside immune cells. In 2009, California-based Sangamo Therapeutics used an older editing technology called zinc finger nucleases to add that protective mutation into patients’ T cells—an important part of the immune system. Those trials have had limited success.

In 2017, Chinese scientists combined Crispr with a bone marrow transplant in an attempt to cure a patient with HIV and leukemia. In a typical transplant, donor stem cells are transferred to a recipient to replace their cancerous blood cells. These cells go on to form new, healthy blood cells. To also address the patient’s HIV, researchers edited the donor stem cells with Crispr to disable CCR5. But after the transplant, only a small percentage of the patient’s bone marrow cells ended up with the desired edit. Then in 2018, Chinese scientist He Jiankui used Crispr to edit the CCR5 mutation into the genomes of twin baby girls to make them resistant to HIV. Fraught with ethical violations, the experiment was widely condemned by scientists. He’s research was suspended by the Chinese government, and he served a three-year prison sentence. While the twins were born healthy, only some of their cells were successfully edited, meaning the girls might in fact not be immune to HIV. As of 2022, two people have now been cured of HIV after receiving bone marrow transplants from donors with the CCR5. Known as the Berlin patient and the London patient, both had cancer and received transplants to treat their disease. But these transplants aren’t a viable option for most people—they’re highly risky, and donors with the delta 32 mutation are scarce. But a third person was declared cured of HIV earlier this year after she received a new type of transplant involving umbilical cord blood.

The Excision trial will eventually enroll nine participants and test three dosage amounts to determine which is most effective. Investigators will measure each person’s viral load and CD4 count before receiving the therapy and after they stop taking antiretroviral drugs. The ultimate goal is to get viral loads down to an undetectable level—that is, less than 200 copies of HIV per milliliter of blood. At this level, HIV can’t be passed on through sex. The challenge for Excision will be getting Crispr to enough cells to bring HIV down to undetectable levels. The company is using an engineered virus to shuttle the gene-editing components to patients’ HIV-infected CD4 cells. But so far, there’s little human data on how well Crispr works when it’s delivered directly to the body. “It’s possible that you get the virus to such low levels that if a person’s immune system were intact, they might be able to keep the virus at bay such that they don’t have to take antiretroviral therapy anymore,” says Rowena Johnston, vice president and director of research for amfAR, the Foundation for AIDS Research.

And even though these drugs are very effective, Johnston says, many people would rather be completely free of the virus. A single Crispr infusion—if it works—would eliminate the need for daily pills. “People with HIV still live with a lot of stigma and internalized shame,” she says. “I think a cure is something that addresses that much better than lifelong therapy, regardless of how easy that therapy becomes.”

Novel treatment using messenger RNA sharply cuts production of mutant liver protein, although it’s too early to show patients with rare condition benefit. The gene editor CRISPR excels at fixing disease mutations in lab-grown cells. But using CRISPR to treat most people with genetic disorders requires clearing an enormous hurdle: getting the molecular scissors into the body and having it slice DNA in the tissues where it’s needed. Now, in a medical first, researchers have injected a CRISPR drug into the blood of people born with a disease that causes fatal nerve and heart disease and shown that in three of them it nearly shut off production of toxic protein by their livers.  Although it’s too soon to know whether the CRISPR treatment will ease the symptoms of the disease, known as transthyretin amyloidosis, the preliminary data reported today are generating excitement about what could be a one-time, lifelong treatment. “These are stunning results,” says gene editing researcher and cardiologist Kiran Musunuru of the University of Pennsylvania, who was not involved in the trial. “It exceeds all my expectations.” The work also marks a milestone for the race to develop treatments based on messenger RNA (mRNA), the protein-building instructions naturally made by cells. Synthetic mRNAs power two COVID-19 vaccines being given to millions of people to fight the coronavirus pandemic, and many companies are working on other mRNA vaccines and drugs. The new treatment, which includes an mRNA encoding one of CRISPR’s two components, “begins the convergence of the fields of CRISPR and mRNA,” says cardiovascular researcher Kenneth Chien of the Karolinska Institute, a co-founder of Moderna, which makes one of the COVID-19 vaccines and is also developing mRNA drugs. The CRISPR clinical trial aims to deactivate a mutated gene that causes liver cells to churn out misfolded forms of a protein called transthyretin (TTR), which build up on nerves and the heart and lead to pain, numbness, and heart disease. The resulting condition is relatively rare, and an approved drug, patisiran, can stabilize it. But researchers at veteran biotech Regeneron Pharmaceuticals and startup Intellia Therapeutics saw it as a good proof of principle for the injectable CRISPR treatment they were developing. 

Last year, researchers used CRISPR to turn on a fetal form of hemoglobin to correct sickle cell disease or a related disease in several people. The treatment required removing a patient’s diseased blood stem cells, modifying them with CRISPR in a dish, and then infusing them back into the body. A trial testing a direct injection of a virus encoding CRISPR’s components into the eye to treat a condition that causes blindness is also underway. But treating most other diseases means somehow injecting CRISPR’s components, or genetic instructions for them, into the blood and having the therapy target an organ or tissue—a huge challenge, but potentially easier in the liver because it sops up foreign particles. In the CRISPR trial, four men and two women with transthyretin amyloidosis between ages 46 and 64 were injected with a lipid particle carrying two different RNAs: an mRNA encoding the protein Cas, the CRISPR component that snips DNA, and a guide RNA to direct it to the gene for TTR. After Cas makes its cut, the cell’s DNA repair machinery heals the break, but imperfectly, knocking out the activity of the gene. After 28 days, three men given the higher of two doses of the treatment had an 80% to 96% drop in TTR levels, on par or better than the average of 81% with patisiran, the team reports today in The New England Journal of Medicine. “The data are extremely encouraging,” says trial leader Julian Gillmore of University College London, who also presented the study today at the online annual meeting of the Peripheral Nerve Society. “It could be potentially the first curative treatment for this hereditary disabling and life-threatening disease,” says neurologist David Adams of the University of Paris-Saclay, who led trials for patisiran. (That drug is a kind of RNA that silences TTR's production temporarily, meaning it must be injected on a regular basis.)

It may take months for patients receiving the CRISPR treatment to see their symptoms lessen, but they reported few short-term side effects. Problems could surface over time: CRISPR could potentially make cuts in the wrong DNA location (and in non-liver cells), triggering cancer or other problems. But the lipid-encased mRNA approach is potentially safer than using viruses to ferry genetic instruction for encoding an editing protein and guide RNA into cells, a tried-and-true approach others are pursuing for systemic treatments. Those genes can persist in cells, continuing to make the gene editor long after it has done its job. In contrast, “The beauty of mRNA is that it is gone afterwards,” Chien says. The study paves the way for treating other liver diseases with CRISPR, either by knocking out a gene or—more challenging—modifying it with the help of a DNA template. The latter approach could also be used to turn the liver into a factory for making an enzyme needed elsewhere in body. Jennifer Doudna of the University of California, Berkeley, who shared a Nobel Prize last year for discovering CRISPR and co-founded Intellia, sees even bigger prospects. The new work, she says, is “a critical first step in being able to inactivate, repair, or replace any gene that causes disease, anywhere in the body.”

See also NEJM (June 26, 2021):

https://www.nejm.org/doi/full/10.1056/NEJMoa2107454 

Using gene-editing to remove a retrovirus is a step towards a cure for HIV infection in humans. Scientists have used Crispr gene-editing to remove an HIV-like virus from monkey DNA, a major step towards a cure for HIV infection in humans. In a study led by neurovirologist Kamel Khalili of Temple University in Philadelphia, researchers constructed a modified adenovirus containing a Crispr-Cas9 gene-editing system. That 'construct' (called 'AAV9-CRISPR-Cas9') was then injected into rhesus macaque monkeys to deliver the Crispr system into cells. The monkey cells were infected with SIV (Simian Immunodeficiency Virus), a close relative of HIV (Human Immunodeficiency Virus). Both are retroviruses — viral parasites that cut-and-paste their genetic material into a host's DNA. SIV infects macaques and other non-human primates in the same way that HIV infects people, making it a good model for studying retroviral infection — and testing how to remove those viruses from the human genome.

The gene-editing construct was designed to target specific sites where the retrovirus was integrated into the macaque genome. It was able to reach tissues where viruses like SIV and HIV can hide for years without being detected, known as reservoirs, such as bone marrow, lymph nodes, T cells of the immune system and the brain. According to the study, the construct was precise and has a low risk of cutting the wrong places in DNA ('off-target' sites). The research has obvious implications for preventing or treating AIDS (Acquired Immunodeficiency Syndrome) in humans by curing a patient of HIV infection.

Original study published in Nature Comm. (November 27, 2020):

https://doi.org/10.1038/s41467-020-19821-7

CRISPR-based approach is much faster than current diagnostics, but does have some limitation. CRISPR diagnostics are just one way researchers are trying to speed coronavirus testing. The new test is the fastest CRISPR-based diagnostic yet. In May, for example, two teams reported creating CRISPR-based coronavirus tests that could detect the virus in about an hour, much faster than the 24 hours needed for conventional coronavirus diagnostic tests.  CRISPR tests work by identifying a sequence of RNA—about 20 RNA bases long—that is unique to SARS-CoV-2. They do so by creating a “guide” RNA that is complementary to the target RNA sequence and, thus, will bind to it in solution. When the guide binds to its target, the CRISPR tool’s Cas13 “scissors” enzyme turns on and cuts apart any nearby single-stranded RNA. These cuts release a separately introduced fluorescent particle in the test solution. When the sample is then hit with a burst of laser light, the released fluorescent particles light up, signaling the presence of the virus. These initial CRISPR tests, however, required researchers to first amplify any potential viral RNA before running it through the diagnostic to increase their odds of spotting a signal. That added complexity, cost, and time, and put a strain on scarce chemical reagents.

Now, researchers led by Jennifer Doudna, who won a share of this year’s Nobel Prize in Chemistry yesterday for her co-discovery of CRISPR, report creating a novel CRISPR diagnostic that doesn’t amplify coronavirus RNA. Instead, Doudna and her colleagues spent months testing hundreds of guide RNAs to find multiple guides that work in tandem to increase the sensitivity of the test. 

In a new preprint, the researchers report that with a single guide RNA, they could detect as few as 100,000 viruses per microliter of solution. And if they add a second guide RNA, they can detect as few as 100 viruses per microliter.  That’s still not as good as the conventional coronavirus diagnostic setup, which uses expensive lab-based machines to track the virus down to one virus per microliter, says Melanie Ott, a virologist at UC San Francisco who helped lead the project with Doudna. However, she says, the new setup was able to accurately identify a batch of five positive clinical samples with perfect accuracy in just 5 minutes per test, whereas the standard test can take 1 day or more to return results.

Emmanuelle Charpentier and Jennifer A. Doudna developed the Crispr tool, which can change the DNA of animals, plants and microorganisms with high precision.  The Nobel Prize in Chemistry was jointly awarded on Wednesday to Emmanuelle Charpentier and Jennifer A. Doudna for their 2012 work on Crispr-Cas9, a method to edit DNA. The announcement marks the first time the award has gone to two women. “This year’s prize is about rewriting the code of life,” Goran K. Hansson, the secretary-general of the Royal Swedish Academy of Sciences, said as he announced the names of the laureates. Dr. Charpentier and Dr. Doudna, only the sixth and seventh women in history to win a chemistry prize, did much of the pioneering work to turn molecules made by microbes into a tool for customizing genes — whether in microbes, plants, animals or even humans.“I’m over the moon, I’m in shock,” Dr. Doudna, a professor at the University of California, Berkeley, said at a news conference on Wednesday. It has been only eight years since Dr. Doudna and Dr. Charpentier — now the director of the Max Planck Unit for the Science of Pathogens in Berlin — co-authored their first paper demonstrating the power of Crispr-Cas9. Since then, the technology has exploded. Doctors are testing it as a cure for genetic disorders such as sickle cell disease and hereditary blindness. Plant scientists are using it to create new crops. Some researchers are even trying to use Crispr to bring species back from extinction.

A trawl through a monkey genome using the CRISPR–Cas9 genome-editing system has identified a handful of genes that might help the new coronavirus to infect its hosts.

The discovery of host genes that aid viral activity could aid the development of new therapies, and reveal why some people are more susceptible to COVID-19 than others. John Doench at the Broad Institute of MIT and Harvard in Cambridge, Massachusetts, Craig Wilen at Yale School of Medicine in New Haven, Connecticut, and their colleagues used CRISPR–Cas9 to alter genes in cultured monkey cells. They then looked for those genes that influenced viral infection and host-cell death (J. Wei et al. Preprint at bioRxiv http://doi.org/dzz3; 2020).

The team’s survey found genes that code for several proteins not known to assist the coronavirus. Among them are proteins in the TGF-β signalling pathway, which is involved in cell growth and death. Chemicals that inhibit this pathway also prevented coronavirus-induced cell death. The findings have not yet been peer reviewed.

Preprint of the Original Study available at bioRxiv (June 17, 2020)"

https://doi.org/10.1101/2020.06.16.155101