Image Courtesy of Sophia Zhao.
Amid the COVID-19 pandemic, a figure crawls from the darkness. Born from the collaborative efforts of investigators at the Yale School of Medicine, he represents a crucial scientific weapon for COVID-19 researchers –– a bridge between understanding the disease and effectively treating it. He is a hero that wears no cape, and his name…is Mr. G.
Okay, his full name is MISTRG6.
And he is a mouse.
Who is Mr. G?
Mr. G is a genetically engineered mouse with a human-like immune response to COVID-19: through him (and mice like him), researchers may be able to better test both existing and new potential treatments against the virus. Mouse models like Mr. G can be crucial to answering key questions about how the virus works and how we can combat it.
Over four hundred million cumulative cases of COVID-19 have been recorded in the past six months. Roughly eighty percent of them have been classified as “mild”. The remaining twenty percent of cases are “severe,” with symptoms including respiratory failure, blood clotting, and multi-organ dysfunction.
Why do some people experience only mild cases while others face life-threatening ones? Through Mr. G, Yale School of Medicine Sterling Professor of Immunobiology Richard Flavell and Esen Sefik, a post-doctoral fellow in his lab, aimed to find out.
“Some [COVID-19 treatments] worked in a subset of patients, but not all of them,” Sefik said. “There were a lot of unknowns at the time, and we thought that if we had a model, we could help.”
The challenges of an animal model
Scientists have traditionally relied on animal models to evaluate the safety and efficacy of vaccines and antiviral candidates. However, while a plethora of animals – ranging from rabbits to primates – have been studied for their immune response to SARS-CoV-2, no standard laboratory animals have developed severe respiratory failure, organ failure, or cytokine storms, which are intense inflammatory processes, seen in severe human cases. Some animals barely show any symptoms.
But the lack of symptom overlap with humans does not mean that these animal models lack usefulness as a starting point for study. Animals are affected by SARS-CoV-2; the difference merely lies in how they respond. With this in mind, if researchers could alter the response of a COVID-infectable species to match the human immune response, they could create a suitable animal model to study the disease.
Of the animal species that do get infected, mice stand out as the most promising for this type of study. Mice have been used in biomedical research for nearly a century, and, as a result, scientists understand their physiology with near genomic-level precision. We also share about ninety-five percent of our DNA with mice, so our biological responses to disease are typically similar enough for findings to be translatable to humans. In addition, practically speaking, mice are small, easy to transport, and have a fast reproduction time with an accelerated lifespan, making them incredibly cost-effective and efficient for studying infectious disease processes.
However, the differences in the immune response to COVID-19 between humans and mice still represent a major obstacle for researchers. In humans, inhaled SARS-CoV-2 travels to the alveoli in the lungs, where the exchange of carbon dioxide for fresh oxygen in the blood occurs. There, the virus hooks onto a protein called the angiotensin-converting enzyme type 2 receptor (ACE2), which provides an entry point into the alveolar cell lining. Once taken in, the virus breaks the cell apart, releasing millions of new viral particles and proinflammatory cytokines. These cytokines cause plasma and immune cells in the blood to leak into the alveoli, blocking gas exchange and causing fluid buildup in the lungs.
However, unlike humans, standard laboratory mice that come into contact with SARS-CoV-2 do not show major signs of infection. This is partly because the ACE2 receptor in mice is structurally different from the ACE2 receptor in humans, enough so that SARS-CoV-2 generally cannot effectively bind to the mouse receptor, enter alveolar cells, and cause chronic infection. To address this difference, Flavell and Sefik turned to Akiko Iwasaki, the Waldemar Von Zedtwitz Professor in the Department of Immunology at Yale, who found a way to use gene therapy to induce mice to transiently express the human version of ACE2. By delivering the human-ACE2 gene through a mild adeno-associated virus (AAV) injected into the trachea, her team successfully transferred the gene into cells into the lung tissue of mice.
“Humanizing” a mouse
While mice with just the human-ACE2 gene get sick, they do not necessarily exhibit severe COVID-19 symptoms. The immune systems of mice and humans are just different enough that “humanized mice,” or mice adapted to have a human immune system, have become crucial tools in studying the clinical applications of anticancer and anti-HIV drugs. Thus, Flavell and Sefik teamed up with Iwasaki to develop a mouse with both the human receptor to SARS-CoV-2 and the human immune cells for disease response.
“Humanizing” the mouse immune system occurs by taking progenitors of human immune cells and injecting them into a mouse. This technology is decades-old, and Flavell, along with Markus Manz and Regeneron Pharmaceuticals, have been pioneering work in this field for years. To create Mr. G, Flavell’s lab took a variety of human hematopoietic stem cells from fetal liver, cord blood, and adult blood and injected them into the liver of an immunocompromised baby mouse. Once the mouse was eight weeks old, the stem cells had differentiated to yield a system of human immune cells.
Ordinarily, the mouse’s immune system would recognize these human stem cells as ‘foreign’ and reject them. To preemptively address this issue, the researchers first genetically modified the mice when they were still clumps of embryonic stem cells: several mouse genes were replaced with human genes coding for proteins that would support humanization. The names of these ‘humanization’ proteins — M-CSF, IL3, SIRPɑ, thrombopoietin, RAG2, and IL2RGamma — can be combined to form the acronym MISTRG, or, more concisely, the name of our hero, Mr. G.
Once the human immune cells were grafted, the human-ACE2 gene was injected into Mr. G’s neck so that his lungs would respond appropriately to COVID-19. And after 14 additional days of waiting, Mr. G’s “humanization” process was finally complete.
Mr. G on the battleground
While the concentration of infectious SARS-CoV-2 in normal mice is quite low, the viral concentrations in Mr. G are comparable with the high levels found in severe human cases. “It’s a good model to start with, and it is already telling us a lot about how we can go about treating the disease,” Sefik said. Physiologically, Mr. G exhibits the same COVID-19 symptoms as severely ill humans: fibrosis, weight loss, and a heightened, persistent inflammatory immune response that damages tissues. These responses are virtually unobserved in normal mouse models.
“As we learned more about [COVID-19] and the patient data kept coming, macrophages and monocytes seemed to be at the center of pathology,” Sefik said. “If you look at other humanized animal models, unfortunately, most of them lack these cells.”
Why does replacing mouse immune cells with human ones produce such adverse outcomes? Sefik hypothesized that human cells contribute in a unique way. “The way that human immune cells respond to the virus and produce antibodies results in delayed viral clearance, and so the virus also stays longer,” she said. In standard laboratory mice, COVID-19 infection peaks in two days and goes away after four. In Mr. G, the infection lasts over a month, making it a chronic infection.
To test the effectiveness of different vaccines and antiviral therapeutic agents, Flavell and Sefik treated Mr. G with human monoclonal antibodies collected from patients by Michel Nussenzweig, an immunologist at Rockefeller University. They found that administering human antibodies to Mr. G eight hours before infection blocked his excessive weight loss and reduced the amount of infectious SARS-CoV-2 to undetectable levels. However, when these anti-COVID-19 antibodies were administered after infection as a therapeutic practice, the effect was much less pronounced, and the mice still exhibited some, albeit milder, symptoms.
Finally, Flavell and Sefik tested the effect of dexamethasone, a potent immunosuppressive steroid currently used to treat patients with severe COVID-19 infection. They found that dexamethasone administration for mice like Mr. G, as in humans, worked best if delivered during a specific window of time when the immune system was activated for long enough to fight infection but not too long to cause infection.
Mr. G: more than a mouse
Flavell and Sefik’s research, done in collaboration with Iwasaki and several other researchers in and outside of Yale, is crucial in developing means to better understand and treat SARS-CoV-2 infection. Nevertheless, much work remains to be done. Mr. G’s effectiveness as a model organism is still limited. “We have a good representation of monocytes and macrophages [immune cells], which is great, but we don’t have all the cell types in place,” Sefik said. “We are not going to see all the pathologies that we need.” For instance, Mr. G does not exhibit blood clotting, a common symptom found in patients with severe COVID-19.
Regardless, Mr. G does bear many of the same viral and therapeutic responses to different COVID-19 variants as humans. His creation represents a major milestone for researchers aiming to understand and treat the virus. Mr. G will scurry down the path of SARS-CoV-2 infection, sniffing out vaccines and antiviral drugs to save human lives.
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Sefik, E., Israelow, B., Mirza, H., Zhao, J., Qu, R., Kaffe, E., Song, E., Halene, S., Meffre, E., Kluger, Y., Nussenzweig, M., Wilen, C. B., Iwasaki, A., & Flavell, R. A. (2021). A humanized mouse model of chronic covid-19. Nature Biotechnology. https://doi.org/10.1038/s41587-021-01155-4