Researchers from the University of Basel have developed a virtual reality app for smartphones to reduce fear of heights. Now, they have conducted a clinical trial to study its efficacy. Trial participants who spent a total of four hours training with the app at home showed an improvement in their ability to handle real height situations.
Fear of heights is a widespread phenomenon. Approximately 5% of the general population experiences a debilitating level of discomfort in height situations. However, the people affected rarely take advantage of the available treatment options, such as exposure therapy, which involves putting the person in the anxiety-causing situation under the guidance of a professional. On the one hand, people are reluctant to confront their fear of heights. On the other hand, it can be difficult to reproduce the right kinds of height situations in a therapy setting.
This motivated the interdisciplinary research team led by Professor Dominique de Quervain of the University of Basel to develop a smartphone-based virtual reality exposure therapy app called Easyheights. The app uses 360° images of real locations, which the researchers captured using a drone. People can use the app on their own smartphones together with a special virtual reality headset.
Gradually increasing the height
During the virtual experience, the user stands on a platform that is initially one meter above the ground. After allowing acclimatization to the situation for a certain interval, the platform automatically rises. In this way, the perceived distance above the ground increases slowly but steadily without an increase in the person’s level of fear.
The research team studied the efficacy of this approach in a randomized, controlled trial and published the results in the journal NPJ Digital Medicine. Fifty trial participants with a fear of heights either completed a four-hour height training program (one 60-minute session and six 30-minute sessions over the course of two weeks) using virtual reality, or were assigned to the control group, which did not complete these training sessions.
Before and after the training phase – or the same period of time without training – the trial participants ascended the Uetliberg lookout tower near Zurich as far as their fear of heights allowed them. The researchers recorded the height level reached by the participants along with their subjective fear level at each level of the tower. At the end of the trial, the researchers evaluated the results from 22 subjects who completed the Easyheights training and 25 from the control group.
The group that completed the training with the app exhibited less fear on the tower and was able to ascend further towards the top than they could before completing the training. The control group exhibited no positive changes. The efficacy of the Easyheights training proved comparable to that of conventional exposure therapy.
Therapy in your own living room
Researchers have already been studying the use of virtual reality for treating fear of heights for more than two decades. “What is new, however, is that smartphones can be used to produce the virtual scenarios that previously required a technically complicated type of treatment, and this makes it much more accessible,” explains Dr. Dorothée Bentz, lead author of the study.
The results from the study suggest that the repeated use of a smartphone-based virtual reality exposure therapy can greatly improve the behavior and subjective state of well-being in height situations. People who suffer from a mild fear of heights will soon be able to download the free app from major app stores and complete training sessions on their own. However, the researchers recommend that people who suffer from a serious fear of heights only use the app with the supervision of a professional.
The current study is one of several projects in progress at the Transfaculty Research Platform for Molecular and Cognitive Neurosciences, led by Professor Andreas Papassotiropoulos and Professor Dominique de Quervain. Their goal is to improve the treatment of mental disorders through the use of new technologies and to make these treatments widely available.
Featured image: In the virtual reality app, users gradually rise to greater heights and can indicate the degree of their fear at each level. (Image: Bentz et al., NPJ Digital Medicine 2021)
Large parts of the human genome do not contain protein-coding genes. Now, however, a research team with participation from the University of Basel has discovered the cause of a severe hereditary defect in such a “gene desert”. The study in the scientific journal Nature shows that a single genetic change in the “junk DNA” long thought to be useless can have serious consequences.
An interdisciplinary research team from Lausanne, Berlin and Basel has uncovered a new mechanism for inherited diseases. The findings, published in the journal Nature, have far-reaching implications for the entire field of medical genetics. The study involved researchers from the Institute of Molecular and Clinical Ophthalmology (IOB) at the University of Basel, the University and University Hospital of Lausanne (CHUV), the Max Planck Institute and the University of Berlin.
“When we realized the importance of our findings, we were really stunned,“ says Professor Andrea Superti-Furga from the University Lausanne and CHUV. Even the most sophisticated diagnostic tests, such as whole genome sequencing, provide an accurate diagnosis of the genetic defect in only half of the cases in which a genetic cause for a disease is suspected. The new results suggest that some of the undiagnosed cases may be due to changes in “empty” regions of the genome.
“Although we knew that some of these regions – originally believed to contain inessential sequences and referred to as “junk” DNA – could have a function, we never imagined that they could be responsible for important genetic diseases,” Superti-Furga states.
Puzzling hereditary defect
The study focused on severe limb malformations in four unrelated newborns, behind which a genetic defect was suspected. Surprisingly, no variant was found in any of the genes already identified in the human genome that could have explained the malformations.
In their search for the genetic cause, the team benefited from the technical expertise that Professor Carlo Rivolta and his team at the IOB of the University of Basel have accumulated on genetic diseases of the eye. “We identified the genomic event that was responsible for this invalidating condition according to the same bioinformatic and molecular protocols we would have used for a rare and recessive form of retinal degeneration, and we were successful”, Rivolta explains.
According to the study, the cause for the malformations lies in the loss of a small section in the genetic material that is located in the middle of a so-called “gene desert”, far away from the next known gene. A bioinformatic analysis of this apparently informationless section of the genome indicated that the missing DNA segment contained a so-called “long non-coding RNA” (lncRNA). This is a section of the genome that is transcribed to RNA, but the transcript is not used as a template for making a protein. Instead, the RNA molecule itself serves as an element involved in the regulation of cellular processes.
Further experiments revealed that this previously unknown lncRNA was indeed necessary to activate the nearest gene called EN1. Although the EN1 gene itself was intact, the lack of activation of this gene was responsible for the malformations.
Important information hidden in the “junk DNA”
Today, about 8000 different genetic disorders and diseases are known, and the discovery of a new one, while important for affected individuals and their families, is no longer exceptional. However, the current study shows that such a disease, triggered by a single genetic alteration, can be caused not only by defects in one of the approximately 20,000 known genes that lead to a protein product. The cause may also lie in alterations in elements that are far removed from a gene and yet important for its activation and regulation.
The majority of such elements are still unknown, as was the lncRNA now identified in a section of the genome considered “empty.” Thus, while the known protein-coding genes, which make up only 2 % of the human genome, are the basic functional elements for the life of a cell and an organism, single changes in the remaining 98 % of the genome may also have consequences for human health.
Featured image: Even in regions of the genome that do not code for proteins, changes in the DNA sequence can have far-reaching consequences. (Symbolic image: MIKI Yoshihito, flickr CC BY 2.0)
Osaka City University and IMSUT demonstrate fecal microbiota transplantation (FMT) success by revealing the coordinated effort of bacteriophages (phages) and their host bacteria in restoring human intestinal flora
In a study published in Gastroenterology – Researchers at Osaka City University and The Institute for Medical Science, The University of Tokyo(IMSUT), in collaboration with Brigham and Women’s Hospital in Boston, report the intestinal bacterial and viral metagenome information from the fecal samples of patients with recurrent Clostridioides difficile infection (rCDI). This comprehensive analysis reveals the bacteria and phages involved in pathogenesis in rCDI, and their remarkable pathways important for the recovery of intestinal flora function.
Clostridioides difficile infection (rCDI) occurs in the gut and is caused by the Gram-positive, spore-forming anaerobic bacterium, C. difficile when its spores attach to fecal matter and are transferred from hand to mouth by health care workers. Patients undergoing antibiotic treatment are especially susceptible as the microorganisms that maintain a healthy gut are greatly damaged by the antibiotics.
Treatment of rCDI involves withdrawing the causative antibiotics and initiating antibiotic therapy, although this can be very challenging. Fecal microbiota transplantation (FMT) is considered an effective alternative therapy as it addresses the issue from the ground up by replacing the damaged microflora with a healthy one through a stool transplant.
However, two deaths caused by antibiotic-resistant bacterial infections after FMT were reported in 2019, suggesting that a modification of FMT or alternatives are required to resolve safety concerns surrounding the treatment.
Researchers at Osaka City University and the Institute for Medical Science, University of Tokyo tackled this challenge head-on in a great study now published in Gastroenterology.
Using their original analysis pipeline reported in 2020, the researchers obtained intestinal bacterial and viral metagenome information from the fecal samples of nine rCDI patients from Brigham and Women’s Hospital in Boston who successfully had an FMT. They revealed the bacteria and phages involved in the pathogenesis of rCDI and the remarkable pathways important for the recovery of intestinal flora function.
By revealing how the bacteriome and virome in the intestine work together as an organ, the research team was able to show how FMT can be as safe as swapping out a bad organ with a good one.
“Intestinal microbiota should definitely be treated as an ‘organ’!” says principal investigator Professor Satoshi Uematsu, “FMT drastically changed the intestinal bacteriome and virome and is sure to restore the intestinal bacterial and viral functions.”
In the post-COVID-19 world, rCDI will become one of the more pressing international diseases. There is no doubt that FMT is an important therapeutic strategy for rCDI. “In addition to a variety of clinical surveys, comprehensive metagenomic analysis is very important in considering the safety of FMT.,” say Dr. Kosuke Fujimoto and Prof. Seiya Imoto.
This research was sponsored by the Takeda Science Foundation, the Canon Foundation and strategic programs for innovative research field 1 from Ministry of Education, Culture, Sports, Science and Technology of Japan, the Center of Innovation Program from Japan Science and Technology Agency.
Featured image: Graphical abstract by Fujimoto et al.
Reference: Kosuke Fujimoto Yasumasa Kimura, Jessica R Allegretti, Mako Yamamoto, Yao-zhong Zhang, Kotoe Katayama, Georg Tremmel, Yunosuke Kawaguchi, Masaki Shimohigoshi, Tetsuya Hayashi, Miho Uematsu, Kiyoshi Yamaguchi, Yoichi Furukawa, Yutaka Akiyama, Rui Yamaguchi, Sheila E. Crowe, Peter B. Ernst, Satoru Miyano, Hiroshi Kiyono, Seiya Imoto and Satoshi Uematsu, “Functional Restoration of Bacteriomes and Viromes by Fecal Microbiota Transplantation”, Gastroenterology (IF=17.373). DOI:10.1053/j.gastro.2021.02.013 URL：https://www.gastrojournal.org/article/S0016-5085(21)00400-5/fulltext
Long-term observations of two binary near-Earth asteroid systems have allowed researchers to constrain the long-term mutual orbital evolution of their components, and to derive physical and dynamical properties of the binary systems. Scientists including PSI’s Eva Lilly obtained thorough photometric observations of two binary near-Earth asteroids 66391 (1999 KW4) and (88710) 2001 SL9 taken from 2000 to 2019.
Lilly collected photometric observations for asteroid (88710) 2001 SL9 over three nights with the University of Hawaii 88-inch telescope and helped with the data reduction.
“Our results show the components of (88710) 2001 SL drift inwards (the semi-major axis decreases), while the components of (66391) 1999 KW4 drift outwards (semi-major axis is expanding). The only other well-known system studied before – (175706) 1996 FG3 has components in dynamical equilibrium. These three binary systems present examples of the three states of the mutual orbital evolution – equilibrium, expanding and contracting in the population of near-Earth binary asteroids, and provide observational confirmation that synchronous binary asteroids are in a state of stable equilibrium between binary YORP (BYORP) effect and mutual body tides.
The Yarkovsky–O’Keefe–Radzievskii–Paddack (YORP) effect is a result of sunlight being absorbed and re-radiated as heat on a small body, which creates a thermal torque capable of modifying the asteroid’s rotation rate and obliquity. The increase in spin rate can be so significant it could change a body’s shape and eventually lead to asteroid’s break-up. Essentially, the BYORP effect is a version of the YORP effect affecting orbital properties of a binary asteroid. It can either separate the binary components or cause them to collide.
Our understanding of the YORP and BYORP effects sheds light on the physical and dynamical evolution of small NEAs, and is very important for planning future space missions and for assessing the impact risk these bodies could pose to Earth.
Featured image:Three radar images from Arecibo Observatory unambiguously reveal the binary nature of near-Earth asteroid 66391 (1999 KW4). The images have resolution of 30 meters per pixel in the vertical direction and 0.3 Hz in the horizontal direction and are sums of all data collected at this resolution on each day. The primary component, the larger and wider body at the center of each frame, is more than one kilometer in diameter, while its satellite is about one-third the size of the primary. Credit: Arecibo/NASA/NSF
Reference: Scheirich, P. Pravec, P. KuÅ¡nirÃ¡k, K. Hornoch, J. McMahon, D.J. Scheeres, D. ÄŒapek, D.P. Pray, H. KuÄ¡kova, A. GalÃ¡d, J. VraÅ¡til, Yu N. Krugly, N. Moskovitz, L.D. Avner, B. Skiff, R.S. McMillan, J.A. Larsen, M.J. Brucker, A.F. Tubbiolo, W.R. Cooney, J. Gross, D. Terrell, O. Burkhonov, K.E. Ergashev, Sh.A. Ehgamberdiev, P. Fatka, R. Durkee, E. Lilly Schunova, R. Ya Inasaridze, V.R. Ayvazian, G. Kapanadze, N.M. Gaftonyuk, J.A. Sanchez, V. Reddy, L. McGraw, M.S. Kelley, I.E. Molotov, A satellite orbit drift in binary near-Earth asteroids (66391) 1999 KW4 and (88710) 2001 SL9 â€” Indication of the BYORP effect, Icarus, 2021, 114321, ISSN 0019-1035, https://doi.org/10.1016/j.icarus.2021.114321. (https://www.sciencedirect.com/science/article/pii/S0019103521000208)
Millie Hughes-Fulford, PhD, a UC San Francisco scientist who flew in June 1991 aboard the first space shuttle mission dedicated to biomedical studies, died Feb. 2 at the age of 75. She was the first woman to fly as a NASA payload specialist and was part of the first crew to include three women.
On that nine-day mission aboard the space shuttle Columbia, Hughes-Fulford helped complete more than 18 experiments, which included herself and fellow crew members as subjects, as well as rodents and jellyfish. The mission brought back more medical data than any previous NASA mission, including documenting how space flight and microgravity affected the human body, a topic that would remain a focus of Hughes-Fulford’s long scientific career.
After her space flight, she returned to UCSF and became director of the laboratory that bears her name at the San Francisco VA Health Care System, focusing on the impact of microgravity on human cells. She received a NASA award for Best Flight Experiment on STS-131 (launched in April 2010), contributed to over 120 scientific papers, and was a scientific adviser to the Under Secretary of the Department of Veterans Affairs for many years. In 2018, she helped found the UC Space Health program, based at UCSF. Ever enthusiastic about science, she continued her work even after she became ill with lymphoma.
The Hughes-Fulford Laboratory at the San Francisco Veterans Affairs Medical Center has studied the impaired growth of bone cells and immune cells in space, sending multiple experiments on eight separate missions aboard space shuttles and the International Space Station. Understanding the physiological effects of space flight is of critical importance for potential long-term space exploration, such as a Mars mission, and for longer residence in the space station. But space experiments also offer rare opportunities to illuminate the basic cellular mechanisms that affect health on Earth.
“When we go into spaceflight and we have microgravity, we have eliminated one variable. In mathematics, if you get rid of a variable, you can solve the equation, and we’re able to look at the immune system in a whole new way that has not been possible,” said Hughes-Fulford in a 2015 interview.
Her earlier work, beginning on her Columbia flight, focused on space osteoporosis, a continuous loss of calcium and bone due to the loss of mechanical stress in microgravity. Astronauts can lose approximately 1 percent of their bone per month in space. The loss is only partly ameliorated by daily exercise, suggesting that microgravity has additional molecular impacts on how bone cells generate.
Video: NASA’s Lori Meggs at the Marshall Space Flight Center speaks with Millie Hughes-Fulford about her research into fighting the suppression of the human immune system in space. Video by NASA
Hughes-Fulford then turned her research to immunosuppression in space, a phenomenon that had been observed in earlier space flights. On the Apollo missions, for example, half the astronauts reported bacterial or viral infection during their missions or within one week of returning to Earth. Hughes-Fulford’s lab revealed that microgravity altered gene expression and inhibited the activation of T-cells, a type of white blood cell that helps fight off infections. In June 2013, NASA honored that work as a top discovery on the International Space Station. Her most recent immunology experiment flew in January 2015 on a SpaceX mission to the space station.
“Millie launched the careers of many scientists, physicians and surgeons, and space explorers,” said Aenor Sawyer, MD, assistant professor of Orthopaedic Surgery, who co-founded the UC Space Health Program with Hughes-Fulford. “Her legacy will create an impact for many generations to come and her direct scientific contributions will reach far into the future.”
Sawyer said Hughes-Fulford had recently contributed to an upcoming manuscript on the health effects of space travel on women and had collaborated on a immunosenescence project set to launch on SpaceX mission later this year.
Millie launched the careers of many scientists, physicians and surgeons, and space explorers. Her legacy will create an impact for many generations to come and her direct scientific contributions will reach far into the future.
— Aenor Sawyer, MD, Assistant professor of Orthopaedic Surgery
“She was a person of many virtues, warm, kind and caring, painstakingly honest and straightforward, and possessed of a salty sense of humor,” said Thomas Lang, PhD, professor of Radiology and Biomedical Imaging, who was a collaborator and friend.
Millie Elizabeth Hughes was born Dec. 21, 1945, in Mineral Wells, Texas. She entered college at age 16, receiving a degree in Chemistry and Biology from Tarleton State University in 1968 and then a PhD from Texas Women’s University in 1972. She completed a postdoctoral fellowship studying cholesterol metabolism at what is now UT Southwestern School of Medicine in Dallas in the lab of Marvin Siperstein, who later recruited Hughes-Fulford when he moved his lab to the San Francisco VA.
A lover of science fiction and a “space buff” from childhood, Hughes-Fulford dreamed of being an astronaut even before space flight became a reality. In 1978, she took a step in realizing that dream by answering an ad in Family Circle magazine seeking candidates to become the first woman in space. Among 8,000 applicants, she made it to the top 20 before Sally Ride was chosen to fly on the Challenger in June 1983.
Although Hughes-Fulford was selected as a payload specialist by NASA in January 1983, the shuttle program was delayed after Challenger exploded shortly after liftoff in 1986. She would realize her childhood dreams five years later on Columbia.
In 1983, she married George Fulford, a United Airline pilot based in San Francisco. She is survived by her daughter Tori Herzog, and granddaughters Shoshana Herzog and Kira Herzog. The family requests that donations in her memory be given to Stand Up to Cancer, P.O. Box 843721, Los Angeles, CA 90084‐3721.
Featured image: Millie Hughes-Fulford, PhD, speak with Thomas Lang, PhD, in her San Francisco VA Medical Center laboratory in 2016. Photo by Noah Berger
Experiments Point to New Synergistic Treatment Possibilities
Many cancer patients might respond better to treatments with the help of a new prognostic indicator based on a distinctive pattern of gene activity within tumor cells, according to a new study of human cancer data and experiments on human cancer cell lines grown in the lab.
The new research, led by scientists from UC San Francisco and the Catalan Institute of Oncology, in Barcelona, Spain, shows that the newly identified pattern of gene activity is present in many cases of the most common cancers and could be used to predict who is most likely to benefit from “genotoxic” therapies, which include common treatments such as radiation therapy and long-standard chemotherapies.
“The test panel we developed should prove predictive of patient response to genotoxic therapy, and we expect it to be a key for optimizing treatment – either through escalation of the dose in patients with poorer anticipated sensitivity or by combining therapies synergistically that target mechanisms through which tumors resist treatment,” said UCSF’s Mary Helen Barcellos-Hoff, PhD, professor of radiation oncology, who is co-senior author of the new paper with Miquel Àngel Pujana, PhD, of the Catalan Institute.
Genotoxic treatments, whether chemotherapy or radiation therapy, cause DNA damage, and rapidly dividing or genetically unstable cancer cells are most susceptible to the damage induced by these treatments when they have defects in DNA repair pathways.
Many standard-of-care regimens for different cancers exploit these inherent defects in repair pathways, but patient treatments could be further optimized by knowing which pathway is defective in a given tumor. The new paper offers a method to identify a particular repair pathway that makes tumors most susceptible to genotoxic treatment. Use of the new method could help doctors choose the best therapy by identify which patients may get the greatest benefit.
A key component of the favorable prognostic profile the researchers identified in the new study is loss of activity normally triggered by a signaling protein called transforming growth factor-beta (TGF-b). For decades, Barcellos-Hoff’s research has focused on the way that TGF-b affects the response to radiation treatment.
“The adage in my lab is that TGF-b is the center of the universe, because it touches on just about everything that goes on within cells and tissues,” Barcellos-Hoff said. But despite its myriad of functions, Barcellos-Hoff nonetheless was surprised to discover more than a decade ago that TGF-b also influences DNA repair within cells.
When she subsequently learned from a colleague at a scientific meeting that human papilloma virus (HPV) suppresses TGF-b signaling, she became even more intrigued about how the role of TGF-b in DNA repair might affect cancer treatment responses. Head and neck cancers associated with HPV often respond well to genotoxic drugs. “The standard of care, chemotherapy with cisplatin along with radiation therapy, leads to 70 percent remission in HPV-positive head and neck cancers, but only 30 percent in HPV-negative head and neck cancers,” Barcellos-Hoff said. “We tied the loss of TGF-b signaling to a DNA repair shift that underlies this clinical observation.”
In the new study she and her colleagues demonstrated that a mechanism of DNA repair called alternative end-joining is the link between loss of TGF-b signaling and responsiveness to genotoxic cancer treatments. The researchers also found that the degree of TGF-b signaling, as measured by downstream gene activity, varied widely from tumor to tumor.
Normally, cells rely on two DNA repair mechanisms. The prime mechanism is homologous recombination, which corrects mistakes made during DNA replication during cell division. Cells use a second mechanism, non-homologous end-joining, to repair DNA damage from everyday wear and tear. TGF-b signaling keeps these two DNA-repair mechanisms at the forefront.
A third mechanism, alternative end-joining, is error-prone, so it is seldom used by normal cells, except for some components used by the immune system to randomly generate an immense number of distinct antibodies. TGF-b signaling suppresses this repair mechanism, which helps cells repair DNA well. In cancers in which TGF-β signaling is lost, the good repair mechanisms are decreased and error-prone repair by alternative end-joining increases. Together this makes cancer cells more likely to be killed by therapy.
The scientists identified a set of genes associated with TGF-b signaling, and another set of genes associated with alternative end-joining to interrogate The Cancer Genome Atlas, a public database with gene expression profiles from more than 10,000 primary cancers, as well as information on treatment outcomes. The researchers found that the gene signature for TGF-b signaling was anti-correlated with the expression of genes involved in alternative end-joining, the error-prone DNA repair mechanism, in every one of the 17 solid cancer types they evaluated, except for pancreatic cancer.
Consistent with error-prone repair, more of the genome was altered in tumors classified as both low TGF-b and high alternative end-joining, and, consistent with a greater killing of cancer cells by chemotherapy and radiation therapy, the corresponding patients had better outcomes in response to these treatments.
In addition, the researchers found that drugs that block TGF-b signaling increased the expression of genes associated with the normally suppressed alternative end-joining DNA-repair in human cancer cell lines.
Finally, TGF-b inhibition, when combined with genotoxic therapies, was effective in killing tumor cells that would not succumb to genotoxic treatment alone. These data suggest that adding TGF-b inhibition to cancer treatment could greatly increase the response to genotoxic therapy and expand the population of cancer patients who can benefit from these widely used therapies, said Barcellos-Hoff.
Drugs that target TGF-b are not yet clinically available, but they are under development.
“This feature of cancer reveals opportunities to improve the care of patients, from estimating prognosis to selecting therapeutic approaches that exploit this vulnerability,” Barcellos-Hoff said.
Authors: Barcellos-Hoff and Àngel Pujana are senior authors. First authors of the study are Qi Liu, now at the Institute of Biomedical Engineering, Shenzhen Bay Laboratory, in Guangdong, China, and Luis Palomero of the Catalan Institute of Oncology. They were joined by UCSF’s Jade Moore, Ines Guix, and John Murnane; Roderic Espin and Alvaro Aytés, also of the Catalan Institute; Jian-Hua Mao of the Lawrence Berkeley National Laboratory; Amanda G. Paulovich, Jeffrey R. Whiteaker, and Richard G. Ivey of the Fred Hutchinson Cancer Research Center; George Iliakis and Daxian Luo of the University of Duisberg-Essen in Germany, and Anthony J. Chalmers of the University of Glasgow.
Funding: Funding for the research came from the National Institutes of Health, the UCSF Department of Radiation Oncology, the Institut Català d’Oncologia and Departament de Salut, Generalitat de Catalunya.
Featured image: A breast cancer tumor and its microenvironment is shown in a live mouse model. Image by NIH
Reference: Qi Liu, Luis Palomero, Jade Moore, Ines Guix, Roderic Espín, Alvaro Aytés, Jian-Hua Mao, Amanda G. Paulovich, Jeffrey R. Whiteaker, Richard G. Ivey, George Iliakis, Daxian Luo, Anthony J. Chalmers, John Murnane, Miquel Angel Pujana, Mary Helen Barcellos-Hoff, “Loss of TGFβ signaling increases alternative end-joining DNA repair that sensitizes to genotoxic therapies across cancer types”, Science Translational Medicine 10 Feb 2021: Vol. 13, Issue 580, eabc4465 DOI: 10.1126/scitranslmed.abc4465
A broadly applicable AAV genome-coupled immunomodulation strategy helps cloak the AAV virus from unwanted immune responses, and offers important insights into ocular inflammation
In recent years, adeno-associated virus (AAV) has been recognized as the leading vehicle (vector) for in vivo delivery of therapeutic genes because it is non-pathogenic and efficiently targets many different cell and tissue types. The recent Federal Drug Administration (FDA) approvals of AAV-based gene-replacement therapies to treat spinal muscular atrophy and a form of inherited retinal dystrophy highlight the promise of this therapeutic modality.
A key challenge of in vivo gene therapies is their potential to cause immune reactions and inflammation, which can affect how well the therapies work or last, and in rare cases can even be life-threatening. The recently reported deaths of three children that received a high-dose systemically delivered AAV gene therapy in a trial to treat X-linked myotubular myopathy dramatically showed that AAV-mediated toxicity and immune responses are only incompletely understood and that current AAV delivery vectors still need to be further improved.
The AAV capsid and genome can both act as immunogenic components. Specifically, the vector genome, which encompasses the therapeutic gene, can activate a protein known as Toll-like receptor 9 (TLR9), a so-called pattern recognition receptor that senses foreign DNA in specialized immune cells. This sensing first triggers an immune response that results in inflammation (innate immunity), and subsequently more specific immune responses (adaptive immunity, in the form of cytotoxic T cells) against the AAV capsid, preventing the therapy from taking effect and posing a potential risk.
Now, an international collaboration of leading groups in gene therapy and vision science, including George Church’s group at Harvard’s Wyss Institute for Biologically Inspired Engineering and Harvard Medical School (HMS) and Constance Cepko’s group at HMS, developed a “coupled immunomodulation” strategy in which short TLR9-inhibitory sequences are incorporated directly into the much longer AAV genome containing therapeutic DNA sequences. Investigated in different tissues of mice, as well as ocular tissues of pigs and non-human primates, the approach showed broad anti-immunogenic potential. Importantly, the study also highlights that pathways other than TLR9 activation likely contribute to inflammation in the highly immunogenic model of intravitreal AAV injections in macaques. The work is published in Science Translational Medicine.
The project was initiated in George Church’s group at the Wyss Institute and HMS. Church, Ph.D., is a Core Faculty member at the Wyss Institute and leads the Institute’s Synthetic Biology platform. He also is Professor of Genetics at Harvard Medical School and of Health Sciences and Technology at Harvard and the Massachusetts Institutes of Technology (MIT).
Cloaking AAV using coupled immunomodulation
“We hypothesized that small snippets of DNA that bind and inhibit TLR9 activation, including DNA sequences from the ends of human chromosomes called telomeres, would be a way to cloak the AAV genome from this immune-surveillance mechanism when incorporated directly into it,” said first- and co-corresponding author Ying Kai Chan, Ph.D., formerly a Postdoctoral Fellow working with Church and Chief Scientific Officer at Ally Therapeutics, and currently a Visiting Scholar at the Wyss. Chan and Church are both co-founders of Wyss gene therapy startup company Ally Therapeutics.
The team started by generating a series of synthetic DNA “inflammation-inhibiting oligonucleotide” (IO) sequences that each carry a highly inflammatory portion linked to one of different TLR9-inhibitory sequences, and tested their effects on cultured cells. The presence of TLR9-inhibitory sequences dampened the inflammatory response by up to 95%. When directly incorporated as a tandem series into an AAV vector, the IOs dampened innate immune responses in primary human immune cells compared to an unmodified vector.
Every novel therapeutic modality that achieves initial success in the clinic has to grapple with emerging issues before it can be deployed broadly, and AAV gene therapy is no exception. Our work represents an important step in development of next generation AAV vectors that are safer and more effective.
— Ying Kai Chan
To test the strategy in AAV in vivo, the researchers administered AAVs as a systemic treatment or locally into muscle tissue of mice. Control viruses lacking IO sequences induced anti-viral interferon responses and the infiltration of innate immune cells in the animals’ livers, and led to infiltration and activation of cytotoxic T cells in muscle tissues. These effects were absent in mutant mice lacking a functional TLR9 pathway, showing that TLR9 was indeed a key regulator of AAV-induced inflammation. Importantly, the effects were blocked or much reduced in mice that received engineered AAVs containing IO sequences in their genomes, and the coupled immunomodulation strategy enhanced expression of the transgene that the virus delivered, indicative of potentially higher efficacy.
Investigating coupled immunomodulation in the eye
The eye is often described as an immune-privileged site because of the presence of a blood-retina barrier that limits entry of immune cells, and of immune-suppressive factors. However, multiple clinical trials have reported intraocular inflammation following delivery of therapeutically relevant doses of AAV into the eye, demonstrating a limit for immune privilege. Most AAV-based gene therapies in the eye are directly applied to the retina (subretinal injection). AAV delivery to the vitreous cavity (intravitreal injection) of the eye is highly desirable since it would be less invasive and potentially allows for targeting more cells, but it is unfortunately highly inflammatory.
Using in vivo imaging and immune cell characterization techniques after intravitreal injection of AAV virus in mice, the team demonstrated that the incorporation of IO sequences in the virus genome reduced the inflammation and numbers of infiltrating T cell populations in the eye compared to unmodified AAVs. This further coincided with a multifold boost in expression of the vector-encoded reporter gene in the retina.
Next, the team studied their coupled immunomodulation strategy in large animal models, first in pigs via subretinal injections, and then in macaque monkeys via intravitreal injections. “We found that the strategy ameliorated distinct pathologies triggered by control AAV viruses in pigs, including the shortening of photoreceptor cells essential for high-acuity vision,” said Sean Wang, M.D., who collaborated with Chan as a medical student in Cepko’s group. They also found the infiltration of the photoreceptor layer of the retina by immune cells, including microglia and T cells, to be substantially reversed.
“In macaques that received engineered and control AAVs via intravitreal injections, these immunosuppressive effects unfortunately were not as pronounced although we saw that the coupled immunomodulation approach delayed the clinical uveitis symptoms triggered by control virus, and allowed a two-fold increase in the expression of a therapeutic gene,” said Wang. Also, the use of prophylactic systemic immunosuppression was unable to prevent the observed uveitis, showing that immunogenicity challenges for this route are more complex.
“The results from the intravitreal toxicity induced by AAV, and the modest response to the TLR9 blocking sequence and to steroids, indicate that there is more than one mechanism leading to toxicity from this injection site. We can now go forward with this understanding and search for additional pathways,” said Cepko, Ph.D., the Bullard Professor of Genetics and Neuroscience in the Blavatnik Institute at HMS, and an Investigator of the Howard Hughes Medical Institute and member of the Harvard Stem Cell Institute.
“Every novel therapeutic modality that achieves initial success in the clinic has to grapple with emerging issues before it can be deployed broadly, and AAV gene therapy is no exception. Our work represents a critical step in development of next generation AAV vectors that are safer and more effective” said Chan.
“This important Wyss-initiated collaboration among leading experts in synthetic biology, vision science, and gene therapy opens up a new direction in AAV engineering that ultimately could help make gene therapies safer and more effective through targeted viral genome engineering,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
Other key authors with key contributions to the study were Andrew Dick at the University of Bristol, UK; Federico Mingozzi, at Sorbonne Université, Paris, and Genethon; Maureen McCall and Henry Kaplan at the University of Louisville, KY; Guangping Gao at University of Massachusetts Medical School, Worcester, MA; and additional collaborators and members of the above author’s groups and the Wyss Institute, including Colin Chu, David Copland, Alexander Letizia, Helena Costa Verdera, Jessica Chiang, Meher Sethi, May Wang, William Neidermyer Jr., Yingleong Chan, Elaine Lim, Amanda Graveline, Melinda Sanchez, Ryan Boyd, Thomas Vihtelic, Rolando Gian Carlo Inciong, Jared Slain, Priscilla Alphonse, Yunlu Xue, Lindsey R. Robinson-McCarthy, Jenny Tam, Maha Jabbar, Bhubanananda Sahu, Janelle Adeniran, Manish Muhuri, Phillip Tai, Jun Xie, Tyler Krause, Andyna Vernet, Matthew Pezone, Ru Xiao, Tina Liu, and Wei Wang. The study was funded by the Wyss Institute for Biologically Inspired Engineering at Harvard Unversity, National Institutes of Health under grant# RM1 HG008525 and EY026158, European Research Council under grant# 617432, National Eye Research Centre, UK, The Underwood Trust, and Ally Therapeutics.
Featured image: AAVs with TLR9-inhibitory sequences incorporated into their genomes allowed significantly higher expression of a linked fluorescent reporter gene in the mouse retina (right) than AAVs that were lacking the sequences (left) following intravitreal administration. Credit: David Copland/University of Bristol, UK
Reference: Ying Kai Chan, Sean K. Wang, Colin J. Chu, David A. Copland, Alexander J. Letizia, Helena Costa Verdera, Jessica J. Chiang, Meher Sethi, May K. Wang, William J. Neidermyer, Jr., Yingleong Chan, Elaine T. Lim, Amanda R. Graveline, Melinda Sanchez, Ryan F. Boyd, Thomas S. Vihtelic, Rolando Gian Carlo O. Inciong, Jared M. Slain, Priscilla J. Alphonse, Yunlu Xue, Lindsey R. Robinson-McCarthy, Jenny M. Tam, Maha H. Jabbar, Bhubanananda Sahu, Janelle F. Adeniran, Manish Muhuri, Phillip W. L. Tai, Jun Xie, Tyler B. Krause, Andyna Vernet, Matthew Pezone, Ru Xiao, Tina Liu, Wei Wang, Henry J. Kaplan, Guangping Gao, Andrew D. Dick, Federico Mingozzi, Maureen A. McCall, Constance L. Cepko, George M. Church, “Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses”, Science Translational Medicine 10 Feb 2021: Vol. 13, Issue 580, eabd3438 DOI: 10.1126/scitranslmed.abd3438
Machine learning study initiated at the Wyss Institute in collaboration with Google Research enables unprecedented AAV capsid diversification with potential for improving gene therapies
Adeno-associated viruses (AAVs) have become promising vehicles for delivering gene therapies to defective tissues in the human body because they are non-pathogenic and can transfer therapeutic DNA into target cells. However, while the first gene therapy products approved by the Federal Drug Administration (FDA) use AAV vectors and others are likely to follow, AAV vectors still have not reached their full potential to meet gene therapeutic challenges.
First, currently used AAV capsids – the spherical protein structures enveloping the virus’ single-stranded DNA genome which can be modified to encode therapeutic genes – are limited in their ability to specifically hone in on the tissue affected by a disease. And secondly, patients’ immune systems, after having been exposed to a similar AAV virus, can produce neutralizing antibodies that, even at low levels, can destroy AAVs upon re-exposure (neutralization), blocking the delivery of their therapeutic DNA payloads.
To overcome this neutralization problem, researchers are engineering enhanced AAV capsids that they hope will be able to evade the immune system. Currently used methods, including “directed evolution” strategies that fast-track the evolution of a protein in laboratory conditions, only can create a limited diversity of capsids with most of them still resembling the naturally occurring AAV variants known as serotypes. However, it remains difficult to generate sufficient diversity using this approach without losing other desired functions of the capsid, such as their stability or ability to bind to specific cell types.
Now, a new study initiated by Wyss Core Faculty member George Church’s Synthetic Biology team at Harvard’s Wyss Institute for Biologically Inspired Engineering, and driven by a collaboration with Google Research has applied a computational deep learning approach to design highly diverse capsid variants from the AAV2 serotype. The approach focused on a DNA sequences encoding a key protein segment that plays a role in immune-recognition as well as infection of target tissues. AAV2 is the most-studied serotype and has been used in the first FDA approved gene therapy, to treat a blinding disease.
Starting from a relatively small collection of capsid data, the team trained multiple machine learning methods and used them to design 200,000 virus variants. 110,689 of these variants produced viable AAVs. Between any two naturally occurring AAV serotypes, 12 amino acids within this segment are expected to differ. The team’s effort produced more than 57,000 variants that exhibited much higher diversity than this, some containing up to 29 combined substituted or additionally inserted amino acids. The findings are published in Nature Biotechnology.
“Our approach achieves the highest functional diversity of any capsid library thus far. It unlocks vast areas of functional but previously unreachable sequence space, with many potential applications for generating improved viral vectors, like AAVs with much reduced immunogenicity and much improved target tissue selectivity, and also for highly efficient gene therapies,” said co-corresponding author Eric Kelsic, Ph.D., who started the project with Church, Ph.D., and co-founded the startup Dyno Therapeutics where he is now CEO. Dyno Therapeutics’ mission is to develop advanced gene therapy delivery vehicles by employing cutting-edge artificial intelligence (AI) approaches.
Our approach achieves the highest functional diversity of any capsid library thus far. It unlocks vast areas of functional but previously unreachable sequence space, with many potential applications for generating improved viral vectors, like AAVs with much reduced immunogenicity and much improved target tissue selectivity, and also for highly efficient gene therapies.
— Eric Kelsic, Co-founder and CEO, Dyno Therapeutics
Using multiple design strategies, the team first generated smaller data sets on which they could train several machine learning models. These were collections of AAV capsids with variable numbers of mutations introduced in a 28 amino acid segment of the AAV2 VP3 protein that forms part of the capsid and exposes it to neutralizing antibodies. A high-throughput method enabling the synthesis of mutated capsid sequences and in vitro experiments for testing which ones efficiency produced viable stable capsids, provided a highly effective test bed for their overall approach. The results from this first experimental study then were used by the team as training data for three alternative machine learning models that generated much larger numbers of diverse capsid variants to be tested with a final validation experiment.
A central bottleneck in the creation of diverse AAV capsids and variants that can evade neutralization is the production of capsids that remain stable: most of the variants will fail to assemble into functional capsids or package their AAV genomes. “The deep neural network models that we deployed with our Google collaborators accurately predicted capsid viability across extremely diverse variants. Reaching this level of diversity in the capsid segment is an important milestone that we can build on to find immune-evading capsids for gene therapy,” said co-first author Sam Sinai, Ph.D., a former graduate student of Church who joined Kelsic’s team at the Wyss Institute and is a co-founder leading the machine learning team at Dyno Therapeutics. “And we can take similar approaches to create AAV capsids with much improved tissue selectivity.”
In 2019, a former Wyss team including Kelsic, Sinai, and their mentor Church published a related approach in Science in which they mutated one by one each of the 735 amino acids within the entire AAV2 capsid in different ways. What they called a “wide” search resulted in a large AAV library that identified changes affecting AAV2’s viability and its “homing” potential to specific organs in mice, as well as a previously unknown accessory protein that binds to cell membranes and which was hidden within the capsid-encoding DNA sequence. In their previous study, the researchers used a simple experimental model to optimize the tissue targeting ability of the virus.
“This new study involving machine learning models developed with Google Research nicely complements our earlier work in that it focuses on a small, but very important, region of the AAV capsid with an unprecedented resolution,” said co-corresponding author Church. “It shows that neural networks combined with the high-throughput synthetic testing developed in our lab is changing the way we design gene delivery vehicles and protein drugs.” Church is the lead of the Wyss Institute’s Synthetic Biology Platform where the project was started, and Professor of Genetics at Harvard Medical School and of Health Sciences and Technology at Harvard and MIT.
[Our work with Google Research] shows that neural networks combined with the high-throughput synthetic testing developed in our lab is changing the way we design gene delivery vehicles and protein drugs.
— George Church
“This work gives a glimpse into the future as artificial intelligence approaches, such as machine learning, are opening up vast new design spaces that enable the development of entirely new drugs and drug delivery approaches for combating innumerable challenges to human health. It also highlights the Wyss Institute’s commitment to computational problem-solving in areas where new therapies are desperately needed,” said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at SEAS.
Other authors on the study were co-first authors Drew H. Bryant and Ali Bashir, as well as Patrick F. Riley at Google Research; Nina K. Jain and Pierce J. Ogden at the Wyss Institute and HMS; and co-corresponding author Lucy J. Colwell at Google Research and the University of Cambridge, UK. It was funded by Harvard’s Wyss Institute for Biologically Inspired Engineering.
Featured image: In their machine learning-based capsid diversification strategy, the team focused on a 28 amino acid peptide within a segment of the AAV2 VP3 capsid protein that exposes the AAV capsid to neutralizing antibodies produced by individuals and thus can be the cause of an immune response against the virus. More purple colored portions of this peptide are buried deeper in the capsid, while yellow parts are exposed on the virus’ surface. Credit: Wyss Institute at Harvard University (original by Drew Bryant)
The Wyss Institute’s eRapid electrochemical sensor technology now enables sensitive, specific, and multiplexed detection of blood biomarkers at low cost with potential for many clinical applications
Many life-threatening medical conditions, such as sepsis, which is triggered by blood-borne pathogens, cannot be detected accurately and quickly enough to initiate the right course of treatment. In patients who suffer infection by an unknown pathogen that then progresses to overt sepsis, every additional hour that an effective antibiotic cannot be administered significantly increases the mortality rate, so time is of the utmost essence.
The challenge with rapidly diagnosing sepsis stems from the fact that measuring only one biomarker often does not allow a clear-cut diagnosis. Engineers have struggled for decades to simultaneously quantify multiple biomarkers in whole blood with high specificity and sensitivity for point-of-care (POC) diagnostic applications, as this would avoid time-consuming and costly blood processing steps in which informative biomarker molecules could potentially be lost.
Now, a multi-disciplinary team at Harvard’s Wyss Institute for Biologically Inspired Engineering and the University of Bath, UK, led by Wyss Founding Director Donald Ingber, M.D., Ph.D., and Wyss Senior Staff Scientist Pawan Jolly, Ph.D., has further developed the Institute’s eRapid technology as an affinity-based, low-cost electrochemical diagnostic sensor platform for the multiplexed detection of clinically relevant biomarkers in whole blood. The device uses a novel graphene nanocomposite-based surface coating and was demonstrated to accurately detect three different sepsis biomarkers simultaneously. The findings are reported in Advanced Functional Materials.
“In this study, we have taken an important step towards deploying our electrochemical sensor platform in clinical settings for fast and sensitive detection of multiple analytes in human whole blood. As the nanocomposite coating we developed here is inexpensive, it has the potential to revolutionize point-of-care diagnostics not only to test for sepsis biomarkers, but a much broader range of biomarkers that can be multiplexed in sets to report on the states of many diseases and conditions,” said Ingber, who also is a lead of the Wyss Institute’s Bioinspired Therapeutics and Diagnostics Platform, and the Judah Folkman Professor of Vascular Biology at Harvard Medical School and Boston Children’s Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and Applied Sciences.
Ingber, Jolly, and their Wyss team are currently also developing eRapid electrochemical sensors with the newly engineered graphene-based nanocomposite coating as a critical component of a point-of-care diagnostic for COVID-19, traumatic brain injury, myocardial infarction, and many other disorders.
By developing their electrochemical sepsis-sensing technology, Ingber’s team built on earlier work published in Nature Nanotechnology, in which they had solved the problem of “biofouling” of electro-chemical sensing elements with their eRapid technology. In theory, electrochemical biosensors would be preferred for many clinical applications because of their ability to quantify the content of biological samples by directly converting the binding event of a biomarker to an electronic signal, their low power consumption and cost, and easy integration with diagnostic readers. However, especially when using whole blood, many blood components nonspecifically bind to the surface coatings of the sensors’ electrodes and lead to their degradation, as well as electric noise in the form of false signals.
The team’s eRapid technology uses a novel antifouling nanocomposite coating for electrodes to which binding reagents are attached that capture biomarker molecules from small quantities of blood and other complex biological fluids. Upon chemically detecting any one of these biomarker molecules with high sensitivity and selectivity, the eRapid platform generates an electrical signal at the electrodes that correlates in strength with the levels of target molecules that are detected. The initial nanocomposite coating allowed excellent conversion of chemical to electrical signals, and relied on tiny electrically conductive gold nanowires that were embedded in a matrix of a crosslinked protein known as bovine serum albumin. However, the high costs of the gold materials had been the major barrier to commercializing eRapid for clinical applications.
“In our advanced eRapid version, we replaced the coating’s gold nanowires with graphene oxide nanoflakes that also have anti-fouling and electrochemical properties, but they are much less expensive and allow even more sensitive measurements. In fact, the costs of fabricating the nanocomposite were reduced to a fraction of its original cost, which together with the sensing technology’s speed, efficiency, and versatility should enable the eRapid platform to have immediate commercial impact,” said Jolly.
After optimizing and characterizing their nanocomposite coating in binding assays for the inflammatory cytokine interleukin 6, the team applied it to the diagnosis of sepsis. Essentially, by attaching an antibody molecule to the coating that binds procalcitonin (PCT), and adding a second PCT-specific antibody to the complex that is linked to an enzyme, a precipitate is formed from a chemical substrate and deposited on the coating. This changes the current of electrons reaching the electrode, and helps register the PCT binding event as an electronic signal.
“We demonstrated that this electrochemical sensor element can detect PCT with high accuracy in whole blood, and validated it by quantifying PCT levels in 21 clinical samples, directly comparing it with a conventional ELISA assay – with excellent correlation,” said first-author Uroš Zupančič, who was a visiting scholar in Ingber’s group from the University of Bath. Zupančič is a Ph.D. candidate mentored by the study’s co-authors Despina Moschou, Ph.D., a Lecturer at the University of Bath, and Pedro Estrela, Ph.D., Associate professor and the head of the Centre for Biosensors, Bioelectronics and Biodevices at the University.
The team then extended their approach to simultaneously detecting multiple sepsis biomarkers by also designing sensor elements for C-reactive protein, another sepsis biomarker, and pathogen-associated molecular patterns (PAMPs). The PAMP sensor element in particular leverages the Wyss Institute’s broad-spectrum pathogen capture technology that uses a genetically engineered protein called FcMBL, which binds more than 100 different pathogens of all classes, as well as molecules on their surfaces that are released into blood when pathogens are killed (PAMPs) and act to trigger the sepsis cascade.
Video: This animation features an eRapid technology on-a-chip and shows how engineered and spatially separated binding systems capture specific biomarkers without interfering with each other to allow the multiplexed analysis of sepsis in whole blood. Credit: Wyss Institute at Harvard University
“Assembling three dedicated electrochemical sensor elements for biomarkers that can be present in blood at vastly different concentrations on a single chip posed a significant challenge. However, the three elements in the final sensor exhibited specific responses within the clinically significant range without interfering with each other, and they did so with a turnaround time of 51 minutes, which meets the clinical need of sepsis diagnosis within the first hour,” said Zumpančič.
To make the current eRapid technology even more effective and useful for clinical sample analysis, the team integrated it with a microfluidic system that takes out the human element involved in handling the sensor in the laboratory, and enhances the number of biomarker binding events at its surface. This allows biomarker analysis with the system to be automated, and enabled the researchers to decrease the turnaround time for measuring PCT to seven minutes.
The study was funded by the Defense Advanced Research Projects Agency (DARPA) under contract W911NF-16-C-0050, the Wyss Institute for Biologically Inspired Engineering at Harvard University, the Rosetrees Trust under project M681, and the U.S. Army Research Office.
Featured image: Wyss Institute researchers have developed eRapid technology as an affinity-based, low-cost electrochemical diagnostic sensor platform for the multiplexed detection of clinically relevant sepsis biomarkers in whole blood. Credit: Wyss Institute at Harvard University
Reference: Sabaté del Río, J., Henry, O.Y.F., Jolly, P. et al. An antifouling coating that enables affinity-based electrochemical biosensing in complex biological fluids. Nat. Nanotechnol. 14, 1143–1149 (2019). https://doi.org/10.1038/s41565-019-0566-z