Virus World

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.”

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.

He Jiankui’s work was also carried out on a third infant, according to China’s state media, in a new disclosure that is likely to add to the global uproar over such experiments. A court in China on Monday sentenced He Jiankui, the researcher who shocked the global scientific community when he claimed that he had created the world’s first genetically edited babies, to three years in prison for carrying out “illegal medical practices.” In a surprise announcement from a trial that was closed to the public, the court in the southern city of Shenzhen found Dr. He guilty of forging approval documents from ethics review boards to recruit couples in which the man had H.I.V. and the woman did not, Xinhua, China’s official news agency, reported. Dr. He had said he was trying to prevent H.I.V. infections in newborns, but the state media on Monday said he deceived the subjects and the medical authorities alike.

Dr. He, 35, sent the scientific world into an uproar last year when he announced at a conference in Hong Kong that he had created the world’s first genetically edited babies — twin girls. On Monday, China’s state media said his work had resulted in a third genetically edited baby, who had been previously undisclosed. Dr. He pleaded guilty and was also fined $430,000, according to Xinhua. In a brief trial, the court also handed down prison sentences to two other scientists who it said had “conspired” with him: Zhang Renli, who was sentenced to two years in prison, and Qin Jinzhou, who got a suspended sentence of one and a half years. The court held that the defendants, “in the pursuit of fame and profit, deliberately violated the relevant national regulations on scientific and medical research and crossed the bottom line on scientific and medical ethics,” Xinhua said.

Dr. He’s declaration made him a pariah among scientists, cast a harsh light on China’s scientific ambitions and embroiled other scientists in the United States who were connected to Dr. He. Though Dr. He offered no proof and did not share any evidence or data that definitively proved he had done it, his colleagues had said it was possible that he had succeeded. American scientists who knew of Dr. He’s plans are now under scrutiny. Dr. He’s former academic adviser, Stephen Quake, a star Stanford bioengineer and inventor, is facing a Stanford investigation into his interaction with his former student. Rice University has been investigating Michael Deem, Dr. He’s Ph.D. adviser, because of allegations that he was actively involved in the project. Dr. Quake has said he had nothing to do with Dr. He’s work. Mr. Deem has said he was present for parts of Dr. He’s research but his lawyers have denied that he was actively involved. During the Hong Kong conference, Dr. He said he used in vitro fertilization to create human embryos that were resistant to H.I.V., the virus that causes AIDS. He said he did it by using the Crispr-Cas9 editing technique to deliberately disable a gene, known as CCR₅, that is used to make a protein H.I.V. needs to enter cells. he international condemnation from the scientific community that followed Dr. He’s announcement came because many nations, including the United States, had banned such work, fearing it could be misused to create “designer babies” and alter everything from eye color to I.Q.

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.

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

For the first time, a patient got treated for HIV and cancer at the same time, with an infusion of gene-edited stem cells. The results? Mixed. Imagine you’re 27 years old and you start feeling ill. Ill enough that you go to the hospital, and after much poking and prodding and waiting for lab results you learn you’re HIV-positive. Two weeks later you find out that’s not even the worst of it. You’ve got leukemia too.

Under any circumstances it would be a lot to take in. Especially in China, where HIV/AIDSis highly stigmatized. But for one young man living there, who this happened to in the late spring of 2016, there was one small but significant silver lining to this double whammy of a diagnosis. He would be eligible to participate in the first-ever clinical trial to assess the safety of trying to cure both the cancer and the infection in a single procedure using the gene editing tool call Crispr. In July of 2017, doctors in Beijing blasted the patient with chemicals and radiation to wipe out his bone marrow, making space for millions of stem cells they then pumped into his body through an IV. These new stem cells, donated by a healthy fellow countryman, would replace the patient’s unhealthy ones, hopefully resolving his cancer. But unlike any other routine bone marrow transplant, this time researchers edited those stem cells with Crispr to cripple a gene called CCR5, without which HIV can’t infiltrate immune cells.

Now, more than two years later, the patient is in good health, his cancer in full remission, as researchers report today in the New England Journal of Medicine. The edited stem cells survived and are still keeping his body supplied with all the necessary blood and immune cells, and a small percentage of them continue to carry the protective CCR5 mutation. Not enough to have cured him of HIV, though—he remains infected and on antiretroviral drugs to keep the virus in check. Still, experts say the new case study shows this use of Crispr appears to be safe in humans and moves the field one step closer toward creating drug-free HIV treatments.

“The safety profile appears to be acceptable,” pioneering cancer researcher Carl June wrote in an accompanying editorial, noting that the editing appeared to be precise, and that the engineered stem cells didn’t provoke an immune response in the patient. June did offer a caveat that the study’s single patient offered only limited data to draw on. What he found more striking was how quickly the science has moved from the first reports of using Crispr to treat HIV infection in mice to trying it in humans: only two years. At the University of Pennsylvania, June has led work in  a ground-breaking cancer treatment called CAR-T, which involves genetically reprogramming immune cells into a clone army of tumor-targeting assassins. But it took him five years to go from studies in animals to trials involving humans. In this case, China’s more permissive biomedical research regulations might have expedited the work, or it could be that genetic engineering is lending new momentum to the race for an HIV/AIDS cure, he wrote. “In any case, the genie is out of the bottle with genome editing.”

This is the first time an HIV-positive patient has been treated with Crispr-edited cells. But scientists have been trying to find ways to genetically disable CCR5 for more than a decade now. It all started in 2007, when a German doctor took a 41-year-old man with HIV/AIDS and leukemia off of his antiretroviral drugs and hooked a thin tube up to a vein in his chest. Through it, the so-called Berlin Patient received blood cells from a bone marrow donor who had a naturally occurring mutation called CCR5 Δ32. He was missing a chunk of DNA that ultimately allows an HIV virus to enter immune cells. The patient survived his cancer and became the first (though no longer the only) person considered to be fully cured of HIV/AIDS....

The study was published on September 11, 2019 in the New England Journal of Medicine:

https://doi.org/10.1056/NEJMoa1817426