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Tag: National Institute of General Medical Sciences (NIGMS)

  • High School Students Learn the Basics of Base Editing to Cure “GFP-itis”

    High School Students Learn the Basics of Base Editing to Cure “GFP-itis”

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    Newswise — Genome editing is used to modify the genes of living organisms to elicit certain traits, such as climate-resilient crops or treating human disease at the genetic level. It has become increasingly popular in agriculture, medicine and basic science research over the past decade, and will continue to be relevant and utilized well into the future. Given this prevalence, researchers at the University of California San Diego have started an outreach program that introduces genome-editing technologies to high school students.

    Assistant Professor of Chemistry and Biochemistry Alexis Komor, and Ph.D. candidates Mallory Evanoff and Carlos Vasquez, designed the Genome Editing Technologies Program as a way to educate students on base-editing technologies, expose them to scientists from diverse backgrounds and invite questions about college, professional development, and the everyday life of a graduate student or faculty member within academia. The program is detailed in April 20 issue of The CRISPR Journal.

    Base editors enable scientists to introduce point mutations at targeted sites in the genome of living cells with high efficiency and precision and, thus, have the therapeutic potential to treat thousands of human genetic disorders. Proof-of-concept studies have already demonstrated this technology’s potential in cell therapies and in treating progeria, sickle cell disease and liver diseases.

    “As we were testing out some of these tools, we asked ourselves, how do we make base editors accessible to high schoolers? How do we make this process really visible?” said Evanoff.

    Komor’s team generated a base-editing reporter system using E. coli bacteria. In this system, base-editing activity results in the expression of green fluorescent protein (GFP). The team installed a mutation in the bacterium’s GFP gene to remove its fluorescence. To emphasize the connection to genetic diseases, this phenotype is called “GFP-itis,” and students are tasked with “curing” the bacteria. Using base-editing technology, students correct the mutation back to wild-type, resulting in bacterial cells that fluoresce green.

    The program happens over three days, creating a more meaningful partnership with the school and building a better foundation of trust with the students. “We wanted the students to get to know us better and feel comfortable asking questions about a career in STEM,” said Komor. “A popular question is simply, ‘How do I get into undergraduate research?’ One of the students in the first school we visited, Sage Creek High School, is actually an undergraduate researcher in our lab now.”

    That student is Preety Iyer, a first-year human biology major, who recalled Komor’s visit to her high school as “an amazing opportunity to get hands-on experience with gene-editing technology. It seemed like an intangible concept to me when I was learning about it in my biology classes. Being walked through the entire process and being able to do it myself strengthened my understanding of DNA and gene editing.” 

    Iyer plans to become a doctor working with patients who have rare genetic disorders, and she’s excited to gain more valuable hands-on experience in Komor’s lab: “I’ve been able to use equipment and practice techniques, like flow cytometry and plasmid preparation, that other students don’t get to use until later in their academic careers.” 

    So far, the Genome Editing Technologies Program has visited three local high schools. The schools have had well-developed science classes and much of the equipment needed to run the experiment. The majority of students had also heard of or learned about genome engineering before. Now that Komor’s team has run the program a few times and solicited feedback from students, they hope to expand to schools without such robust science programming. 

    “My high school background in science wasn’t strong in large part because of the lack of mentorship,” said Vasquez. “It’s important to us to reach students who may not have even considered a career in STEM or medicine. To look in their eyes and instill confidence, to show we believe in them — having someone like that when I was in high school would have made a world of difference.”

    The make the experiment as accessible as possible, the team has simplified the base-editing experiment and provides all the necessary equipment. Accessibility also means making the program available to other institutions that may want to implement something similar. Interested scientists or instructors can order plasmid materials from AddGene, a worldwide nonprofit plasmid repository. These plasmids are the DNA needed to make the GFP-itis cells, as well the plasmids needed to as “cure” GFP-itis.

    The goal of the program is not only to make base editing accessible to high school students, but also to encourage critical thinking and reflect on base editing in social and cultural contexts. Komor’s team asked students to think about the difference between a disease and a trait and to consider the implications of germline genome editing, in which edits are inherited by all future descendants of the edited individual, regardless of whether those descendants consent to the procedure.

    “The ethical discussion is what hits a home run with the students,” said Vasquez. “They’ll be responsible for future gene-editing policies. It’s interesting to see them thinking about the ethical side of science.”

    “We’ve had some really good discussions about what is a disease and what is a trait,” stated Evanoff. “If we have the ability to make genetic-disease corrections, who will be able to afford those treatments? Where does the equitability lie in this technology? We don’t have the answers to that. I say to students, ‘That’s going to be your job to figure out!’”

    This research was supported by the National Science Foundation (MCB-2048207), the National Institute of General Medical Sciences (T32 GM007240-41), the National Institute of Health (T32 GM112584), the Howard Hughes Medical Institute (GT13672 and the Gilliam Fellowship Program) and the National Academies of Sciences, Engineering, and Medicine Ford Foundation Predoctoral Fellowship Program.

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    University of California San Diego

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  • CHOP Researchers Identify Molecules that Optimize Immune Presentation of Antigens across the Human Population

    CHOP Researchers Identify Molecules that Optimize Immune Presentation of Antigens across the Human Population

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    Newswise — Philadelphia, February 24, 2023—Researchers at Children’s Hospital of Philadelphia (CHOP) have identified variants of a chaperone molecule that optimizes the binding and presentation of foreign antigens across the human population, which could open the door to numerous applications where robust presentation to the immune system is important, including cell therapy and immunization. The findings were published today in Science Advances. 

    Class I major histocompatibility complex (MHC-I) proteins are found on the surface of cells from all jawed vertebrates and play an essential role in the immune system. The MHC-I displays peptide fragments of proteins from within the cell on the cell surface, effectively “presenting” them to the immune system, which is constantly scanning the body for foreign or toxic antigens. When foreign peptides are identified, they trigger a cascade that allows cytotoxic T cells to eliminate intruders.

    For a peptide to be presented to the immune system, it needs to be loaded on a folded MHC-I protein. Several molecules facilitate this process, including proteins known as molecular chaperones, which assist with MHC-I folding. Tapasin and a similar molecule known as TAPBPR are both molecular chaperones that facilitate MHC-I folding and peptide loading. Because TAPBR functions independently outside of the peptide-loading complex, it is well-suited for clinical applications that involve peptide exchange, such as loading immunogenic peptides on MHC-I molecules and generating libraries to detect T-cells that recognize peptides or antigens from infected or cancerous cells.

    However, TAPBPR-mediated peptide exchange has thus far only worked for a limited set of common allotypes of human MHC-I, known as human leukocyte antigen (HLA), which has limited wider use of these technologies in biomedical applications. Over time, HLA subtypes, which include HLA-A, HLA-B, and HLA-C, have evolved such that not all alleles interact equally well with TAPBPR. This has been a roadblock in developing and enhancing novel therapies with the help of molecular chaperones, as some HLA allotypes do not interact with these molecules. 

    To solve this problem, the CHOP researchers analyzed three different TAPBPR proteins: one from humans, one from chickens, and one from mice. They found that unlike human TAPBPR, chicken TAPBPR co-evolved with its class I genes, so that it maintains high affinity across MHC-I allotypes. In their analysis, they found that chicken TAPBPR was able to react with multiple HLA allotypes, many of which were not able to bind to human TAPBPR. They also demonstrated that TAPBPR stabilizes the empty MHC-I groove in an “open” conformation, boosting its affinity for peptide loading.

    Simultaneously, in close collaboration with researchers at the University of Illinois led by Erik Procko, PhD, the research team used deep mutational scanning to characterize the effects from 100s of point mutations on human TAPBPR and found a variant that mimics the chicken sequence. Like the chicken TAPBPR, this variant enhanced peptide exchange across a broad range of HLA types.

    “Although the highly polymorphic nature of MHC-I molecules makes it challenging to engineer ‘universal’ chaperones, our research team demonstrated that both a chicken ortholog of TAPBPR and a human variant with minor adjustments could enhance peptide exchange across multiple disease-relevant HLAs,” said senior author Nikolaos G. Sgourakis, PhD, Associate Professor in the Center for Computational and Genomic Medicine at Children’s Hospital of Philadelphia. “These TAPBPR orthologs could be utilized in various cancer immunotherapeutic settings to narrow the peptide repertoire and increase immunogenicity. The knowledge gained by our studies can guide the design of engineered TAPBPR variants with tailored HLA specificity and catalytic efficiency for peptide exchange applications both in vitroand in vivo.”

    This research was supported by funding from the National Institute of Allergy and Infectious Diseases (5R01AI143997) and National Institute of General Medical Sciences (5R35GM125034). 

    Yi Sun, Georgia F. Papadaki, Christine A. Devlin, Julia N. Danon, Michael C. Young, Trenton J. Winters, George M. Burslem, Erik Procko, and Nikolaos G. Sgourakis. “Xeno-interactions between MHC-I proteins and molecular chaperones enable ligand exchange on a broad repertoire of HLA allotypes,” Science Advances, February 24, 2023, DOI: 10.1126/sciadv.ade7151 

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    About Children’s Hospital of Philadelphia: A non-profit, charitable organization, Children’s Hospital of Philadelphia was founded in 1855 as the nation’s first pediatric hospital. Through its long-standing commitment to providing exceptional patient care, training new generations of pediatric healthcare professionals, and pioneering major research initiatives, the 595-bed hospital has fostered many discoveries that have benefited children worldwide. Its pediatric research program is among the largest in the country. The institution has a well-established history of providing advanced pediatric care close to home through its CHOP Care Network, which includes more than 50 primary care practices, specialty care and surgical centers, urgent care centers, and community hospital alliances throughout Pennsylvania and New Jersey, as well as a new inpatient hospital with a dedicated pediatric emergency department in King of Prussia. In addition, its unique family-centered care and public service programs have brought Children’s Hospital of Philadelphia recognition as a leading advocate for children and adolescents. For more information, visit http://www.chop.edu.

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    Children’s Hospital of Philadelphia

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  • Dry eye disease alters how the eye’s cornea heals itself after injury

    Dry eye disease alters how the eye’s cornea heals itself after injury

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    Newswise — People with a condition known as dry eye disease are more likely than those with healthy eyes to suffer injuries to their corneas. Studying mice, researchers at Washington University School of Medicine in St. Louis have found that proteins made by stem cells that regenerate the cornea may be new targets for treating and preventing such injuries.

    The study is published online Jan. 2 in the Proceedings of the National Academy of Sciences.

    Dry eye disease occurs when the eye can’t provide adequate lubrication with natural tears. People with the common disorder use various types of drops to replace missing natural tears and keep the eyes lubricated, but when eyes are dry, the cornea is more susceptible to injury.

    “We have drugs, but they only work well in about 10% to 15% of patients,” said senior investigator Rajendra S. Apte, MD, PhD, the Paul A. Cibis Distinguished Professor in the John F. Hardesty, MD, Department of Ophthalmology & Visual Sciences. “In this study involving genes that are key to eye health, we identified potential targets for treatment that appear different in dry eyes than in healthy eyes. Tens of millions of people around the world — with an estimated 15 million in the United States alone — endure eye pain and blurred vision as a result of complications and injury associated with dry eye disease, and by targeting these proteins, we may be able to more successfully treat or even prevent those injuries.”

    The researchers analyzed genes expressed by the cornea in several mouse models — not only of dry eye disease, but also of diabetes and other conditions. They found that in mice with dry eye disease, the cornea activated expression of the gene SPARC. They also found that higher levels of SPARC protein were associated with better healing.

    “We conducted single-cell RNA sequencing to identify genes important to maintaining the health of the cornea, and we believe that a few of them, particularly SPARC, may provide potential therapeutic targets for treating dry eye disease and corneal injury,” said first author Joseph B. Lin, an MD/PhD student in Apte’s lab.

    “These stem cells are important and resilient and a key reason corneal transplantation works so well,” Apte explained. “If the proteins we’ve identified don’t pan out as therapies to activate these cells in people with dry eye syndrome, we may even be able to transplant engineered limbal stem cells to prevent corneal injury in patients with dry eyes.”

    Lin JB, Shen X, Pfeifer CW, Shiau F, Santeford A, Ruzycki PA, Clark BS, Liu Q, Huang AJW, Apte RS. Dry eye disease in mice activates adaptive corneal epithelial regeneration distinct from constitutive renewal in homeostasis. Proceedings of the National Academy of Sciences, Jan. 2, 2023.

    The study was funded with support from the National Eye Institute, the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of General Medical Sciences of the National Institutes of Health (NIH). Grant numbers: R01 EY019287, R01 EY027844, R01 EY024704, P30 EY02687, F30 DK130282, T32 GM07200 Additional funding provided by the Jeffrey T. Fort Innovation Fund, a Centene Corp. contract for the Washington University-Centene ARCH Personalized Medicine Initiative and Research to Prevent Blindness.

    About Washington University School of Medicine

    WashU Medicine is a global leader in academic medicine, including biomedical research, patient care and educational programs with 2,700 faculty. Its National Institutes of Health (NIH) research funding portfolio is the fourth largest among U.S. medical schools, has grown 54% in the last five years, and, together with institutional investment, WashU Medicine commits well over $1 billion annually to basic and clinical research innovation and training. Its faculty practice is consistently within the top five in the country, with more than 1,790 faculty physicians practicing at over 60 locations and who are also the medical staffs of Barnes-Jewish and St. Louis Children’s hospitals of BJC HealthCare. WashU Medicine has a storied history in MD/PhD training, recently dedicated $100 million to scholarships and curriculum renewal for its medical students, and is home to top-notch training programs in every medical subspecialty as well as physical therapy, occupational therapy, and audiology and communications sciences.

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    Washington University in St. Louis

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  • In some US zip codes, young men face more risk of firearm death than those deployed in recent wars

    In some US zip codes, young men face more risk of firearm death than those deployed in recent wars

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    Newswise — PROVIDENCE, R.I. [Brown University] — The risk of firearm death in the U.S. is on the rise: in 2020, firearms became the leading cause of death for children, adolescents and young adults. Yet the risk is far from even — young men in some U.S. zip codes face disproportionately higher risks of firearm-related injuries and deaths.

    To better understand the magnitude of the gun violence crisis and put it in perspective, researchers at Brown University and the University of Pennsylvania compared the risk of firearm-related death for young adult men living in the most violent areas in four major U.S. cities with the risks of combat death and injury faced by U.S. military personnel who served in Afghanistan and Iraq during active periods of war.

    The results were mixed: The study, published in JAMA Network Open, found that young men from zip codes with the most firearm violence in Chicago and Philadelphia faced a notably higher risk of firearm-related death than U.S. military personnel deployed to wartime service in Afghanistan and Iraq. But the opposite was true in two other cities: The most violent areas in New York and Los Angeles were associated with much less risk for young men than those in the two wars.

    In all zip codes studied, risks were overwhelmingly borne by young men from minority racial and ethnic groups, the study found.

    “These results are an urgent wake-up call for understanding, appreciating and responding to the risks and attendant traumas faced by this demographic of young men,” said Brandon del Pozo, an assistant professor of medicine (research) at Brown’s Warren Alpert Medical School and an assistant professor of health services, policy and practice (research) at the University’s School of Public Health.

    Del Pozo conducts research at the intersection of public health, public safety and justice, focusing on substance use, the overdose crisis, and violence. His recently released book, “The Police and the State: Security, Social Cooperation, and the Public Good,” is based on his academic research as well as his 23 years of experience as a police officer in New York City and as chief of police of Burlington, Vermont.

    “Working as a police officer, I witnessed the toll of gun violence, and how disruptive it was for families and communities,” del Pozo said. “It stood out to me that the burden was not distributed evenly by geography or demographic. Some communities felt the brunt of gun violence much more acutely than others. By analyzing publicly available data on firearm fatalities in cities and in war, we sought to place that burden in sharp relief.”

    At the same time, del Pozo said, he and the other study authors were responding to oft-repeated inflammatory claims about gun violence in American cities.  

    “We often hear opposing claims about gun violence that fall along partisan lines: One is that big cities are war zones that require a severe crackdown on crime, and the other is that our fears about homicides are greatly exaggerated and don’t require drastic action,” del Pozo said. “We wanted to use data to explore these claims — and it turns out both are wrong. While most city residents are relatively safe from gun violence, the risks are more severe than war for some demographics.”

    Illustrating the magnitude of the firearm crisis

    To conduct their analysis, the researchers obtained information on all fatal and nonfatal shootings of 18- to 29-year-old men recorded as crimes in 2020 and 2021 in Chicago; Los Angeles; New York; and Philadelphia — the four largest U.S. cities for which public data on those who were shot were available. For New York, Chicago and Philadelphia, they used shooting death and injury data sets made public by each city; for Los Angeles, they extracted firearm death and injury data from a larger public data set of recorded crimes. Data were aggregated to the zip code level and linked to corresponding demographic characteristics from the U.S. Census Bureau’s 2019 American Community Survey.

    The researchers acquired wartime combat-related mortality and injury counts for the conflicts in Iraq and Afghanistan from peer-reviewed analyses of U.S. military data covering the years 2001 to 2014 for the war in Afghanistan and 2003 to 2009 for the war in Iraq, both of which were periods of active combat. Because there is limited data about the risks of serving in different military units at different times during the Afghanistan and Iraq wars, the researchers considered the mortality and injury data of a single, de-identified Army brigade combat team engaged in combat during a 15-month period of the Iraq War that involved notably above-average combat death and injury rates at a time considered to be the height of the conflict.

    The analysis included 129,826 young men residing in the four cities considered in the study.

    The researchers found that compared to the risk of combat death faced by U.S. soldiers who were deployed to Afghanistan, the more dangerous of the two wars, young men living in the most violent zip code of Chicago (2,585 individuals) had a 3.23 times higher average risk of firearm-related homicide, and those in Philadelphia (2,448 people) faced a 1.9 times higher average risk of firearm-related homicide. Singling out the elevated dangers faced by the U.S. Army combat brigade in Iraq, the young men studied in Chicago still faced notably greater risks, and the ones faced in Philadelphia were comparable.

    However, these findings were not observed in the most violent zip codes of Los Angeles and New York, where young men faced a 70% to 91% lower risk than soldiers in the Afghanistan war across fatal and nonfatal categories.

    When the researchers looked at the demographics of the young men in the zip codes studied, they determined that the risk of violent death and injury observed in the zip codes studied was almost entirely borne by individuals from minority racial and ethnic groups: Black and Hispanic males represented 96.2% of those who were fatally shot and 97.3% of those who experienced nonfatal injury across all four cities.

    In the study, the researchers make the point that the risk of firearm death is not the only thing that young men living in violent U.S. zip codes have in common with young men at war.

    “Exposure to combat has been associated with stress-inducing hypervigilance and elevated rates of homelessness, alcohol use, mental illness and substance use, which, in turn, are associated with a steep discounting of future rewards,” they write. “Our findings — which show that young men in some of the communities we studied were subject to annual firearm homicide and violent injury rates in excess of 3.0% and as high as 5.8% — lend support to the hypothesis that beyond the deaths and injuries of firearm violence, ongoing exposure to these violent events and their risks are a significant contributor to other health problems and risk behaviors in many U.S. communities.”

    Del Pozo added that the health risks are likely even higher for people in cities, because they need to face their “battles” every day over a lifetime, as opposed to military personnel in a tour of duty in Afghanistan, which typically lasted 12 months. The study results, del Pozo said, help illustrate the magnitude of the firearms crisis, a necessary understanding to municipalities seeking to formulate an effective public health response.

    “The findings suggest that urban health strategies should prioritize violence reduction and take a trauma-informed approach to addressing the health needs of these communities,” del Pozo said.

    Other Brown contributors included Dr. Michael J. Mello, a physician and researcher at the Warren Alpert Medical School and the Injury Prevention Center at Rhode Island Hospital.

    The study was supported by the National Institute on Drug Abuse (K01DA056654) and the National Institute of General Medical Sciences (P20GM139664).

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    Brown University

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