Tag Archives: #disease

A New Technique For Correcting Disease-causing Mutations (Medicine)

Novel method, developed by McGovern Institute researchers, may lead to safer, more efficient gene therapies.

Gene editing, or purposefully changing a gene’s DNA sequence, is a powerful tool for studying how mutations cause disease, and for making changes in an individual’s DNA for therapeutic purposes. A novel method of gene editing that can be used for both purposes has now been developed by a team led by Guoping Feng, the James W. (1963) and Patricia T. Poitras Professor in Brain and Cognitive Sciences at MIT.

“This technical advance can accelerate the production of disease models in animals and, critically, opens up a brand-new methodology for correcting disease-causing mutations,” says Feng, who is also a member of the Broad Institute of Harvard and MIT and the associate director of the McGovern Institute for Brain Research at MIT. The new findings were published online May 26 in the journal Cell.

Genetic models of disease

A major goal of the Feng lab is to precisely define what goes wrong in neurodevelopmental and neuropsychiatric disorders by engineering animal models that carry the gene mutations that cause these disorders in humans. New models can be generated by injecting embryos with gene editing tools, along with a piece of DNA carrying the desired mutation.

In one such method, the gene editing tool CRISPR is programmed to cut a targeted gene, thereby activating natural DNA mechanisms that “repair” the broken gene with the injected template DNA. The engineered cells are then used to generate offspring capable of passing the genetic change on to further generations, creating a stable genetic line in which the disease, and therapies, are tested.

Although CRISPR has accelerated the process of generating such disease models, the process can still take months or years. Reasons for the inefficiency are that many treated cells do not undergo the desired DNA sequence change at all, and the change only occurs on one of the two gene copies (for most genes, each cell contains two versions, one from the father and one from the mother).

In an effort to increase the efficiency of the gene editing process, the Feng lab team initially hypothesized that adding a DNA repair protein called RAD51 to a standard mixture of CRISPR gene editing tools would increase the chances that a cell (in this case a fertilized mouse egg, or one-cell embryo) would undergo the desired genetic change.

As a test case, they measured the rate at which they were able to insert (“knock-in”) a mutation in the gene Chd2 that is associated with autism. The overall proportion of embryos that were correctly edited remained unchanged, but to their surprise, a significantly higher percentage carried the desired gene edit on both chromosomes. Tests with a different gene yielded the same unexpected outcome.

“Editing of both chromosomes simultaneously is normally very uncommon,” explains postdoc Jonathan Wilde. “The high rate of editing seen with RAD51 was really striking, and what started as a simple attempt to make mutant Chd2 mice quickly turned into a much bigger project focused on RAD51 and its applications in genome editing,” says Wilde, who co-authored the Cell paper with research scientist Tomomi Aida.

A molecular copy machine

The Feng lab team next set out to understand the mechanism by which RAD51 enhances gene editing. They hypothesized that RAD51 engages a process called interhomolog repair (IHR), whereby a DNA break on one chromosome is repaired using the second copy of the chromosome (from the other parent) as the template.

To test this, they injected mouse embryos with RAD51 and CRISPR but left out the template DNA. They programmed CRISPR to cut only the gene sequence on one of the chromosomes, and then tested whether it was repaired to match the sequence on the uncut chromosome. For this experiment, they had to use mice in which the sequences on the maternal and paternal chromosomes were different.

They found that control embryos injected with CRISPR alone rarely showed IHR repair. However, addition of RAD51 significantly increased the number of embryos in which the CRISPR-targeted gene was edited to match the uncut chromosome.

“Previous studies of IHR found that it is incredibly inefficient in most cells,” says Wilde. “Our finding that it occurs much more readily in embryonic cells and can be enhanced by RAD51 suggest that a deeper understanding of what makes the embryo permissive to this type of DNA repair could help us design safer and more efficient gene therapies.”

A new way to correct disease-causing mutations          

Standard gene therapy strategies that rely on injecting a corrective piece of DNA to serve as a template for repairing the mutation engage a process called homology-directed repair (HDR).

“HDR-based strategies still suffer from low efficiency and carry the risk of unwanted integration of donor DNA throughout the genome,” explains Feng. “IHR has the potential to overcome these problems because it relies upon natural cellular pathways and the patient’s own normal chromosome for correction of the deleterious mutation.”

Feng’s team went on to identify additional DNA repair-associated proteins that can stimulate IHR, including several that not only promote high levels of IHR, but also repress errors in the DNA repair process. Additional experiments that allowed the team to examine the genomic features of IHR events gave deeper insight into the mechanism of IHR and suggested ways that the technique can be used to make gene therapies safer.

“While there is still a great deal to learn about this new application of IHR, our findings are the foundation for a new gene therapy approach that could help solve some of the big problems with current approaches,” says Aida.

This study was supported by the Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, the Poitras Center for Psychiatric Disorders Research at MIT, an NIH/NIMH Conte Center Grant, and the NIH Office of the Director.

Featured image: Staining for RAD51 (bright cyan-colored dot) in a fertilized one-cell mouse embryo shows repair of a CRISPR-induced DNA break. Credits: Image courtesy of the researchers.

Paper: “Efficient Homozygous Gene Conversion in Embryos via RAD51-Enhanced Interhomolog Repair”

Provided by MIT

New Method Targets Disease-causing Proteins for Destruction (Medicine)

Scientists at the University of Wisconsin–Madison have developed a way to use a cell’s own recycling machinery to destroy disease-causing proteins, a technology that could produce entirely new kinds of drugs.

Some cancers, for instance, are associated with abnormal proteins or an excess of normally harmless proteins. By eliminating them, researchers believe they can treat the underlying cause of disease and restore a healthy balance in cells.

The new technique builds on an earlier strategy by researchers and pharmaceutical companies to remove proteins residing inside of a cell, and expands on this system to include proteins outside or on the surface of liver cells.

“In the past, to develop a new drug, we often needed to find a molecule that bound to the protein of interest and also changed the function of the protein. But there are many potential proteins associated with diseases whose function you cannot block easily,” says Weiping Tang, a professor of pharmacy and chemistry at the University of Wisconsin–Madison who led the new research. “With the targeted protein degradation strategy, we can go after many of those proteins.”

Tang, with colleagues in the UW–Madison School of Pharmacy, demonstrated the new method in proof-of-concept experiments on lab-grown liver cells. They were able to neutralize multiple extracellular proteins, including EGFR, a cancer-associated protein. The scientists published their findings March 4 in the journal ACS Central Science.

The technology works much like a city’s trash-collection system. Antibodies attach tags for destruction to specific proteins, marking them as unwanted. A shuttle protein on the liver cell, serving as a kind of garbage truck, recognizes these markers, engulfs the protein, and ferries it to the protein-digesting compartment of the cell, which breaks down the protein into reusable parts.

Many researchers over the past several years, including the Tang group, have developed tools to selectively target for destruction certain cellular proteins found on the inside of cells, named PROTACs. Multiple PROTAC-based drugs are now in clinical trials to treat several cancers.

The Tang lab is now expanding the scope of targets to additional proteins found outside of or on the surface of the liver cell. They have turned to the lysosome, a compartment of the cell that digests and destroys all kinds of materials, including what the cell engulfs from outside. Since the liver is the primary organ that breaks down proteins in the body, it is an ideal tissue to selectively degrade undesired proteins.

“I believe there’s a great future ahead of us, but we need to invest our resources and time to investigate the protein degradation strategy further”

— says Tang

To gain access to the lysosome, the researchers relied on a lysosome shuttle named ASGPR. The shuttle is primarily found on the surface of liver cells. It recognizes certain sugars on proteins and delivers the proteins to the lysosome for digestion.

To help encourage ASGPR to recognize and help destroy disease-causing proteins, researchers in earlier studies learned they could attach three specific sugars to the proteins. But first they had to figure out how to go about attaching them to the proteins they wanted to eliminate.

Tang’s group focused on antibodies, which are ideal candidates since they recognize and bind to specific proteins. The team attached the sugary tag that would activate ASGPR to an antibody that would track down the specific proteins they hoped to destroy. In this way, the scientists could shuttle the protein to the lysosome of liver cells with this highly targeted trash-collection system.

They tested their technology against several proteins, including EGFR, which tumors produce in excess. By attaching the sugary tag to an EGFR antibody, the scientists were able to deplete a significant amount of the protein that otherwise accumulated outside of cancerous liver cells grown in the lab.

Tang’s lab is now working to refine the ASGPR method by making it more effective, and to expand the strategy to destroy proteins on the surface of other types of cells. They are also interested in collaborating with other researchers to help them test the removal of a wider array of disease-associated proteins.

“I believe there’s a great future ahead of us, but we need to invest our resources and time to investigate the protein degradation strategy further,” says Tang.

Featured image: Weiping Tang, a professor of pharmacy and chemistry at the University of Wisconsin–Madison. PHOTO BY SALLY GRIFFITH-OH

Reference: Yaxian Zhou, Peng Teng, Nathan T. Montgomery, Xiaolei Li, and Weiping Tang, “Development of Triantennary N-Acetylgalactosamine Conjugates as Degraders for Extracellular Proteins”, ACS Cent. Sci. 2021.

Provided by Wisconsin-Madison

Researchers Identify Disease-related Gene Changes in Kidney Tissue (Medicine)

Researchers from Indiana University have identified key genetic changes in the interstitial kidney tissue of people with diabetes, a discovery that signifies the potential for a revolutionary new genetic approach to the treatment of kidney disease. They will contribute their findings to the Kidney Precision Medicine Project’s (KPMP) “cell atlas,” a set of maps used to classify and locate different cell types and structures within the kidney. 

They shared their groundbreaking findings in a study published on February 10, 2021, in Science Advances.

In the study, researchers investigated the kidney tissue of healthy people and people with diabetes using a technique called “regional transcriptomics.” This technique involves a rapid stain of kidney tissue, and then using a laser to cut out microscopic regions of interest. 

They found that important genes change when a scar forms on the interstitium, said Daria Barwinska, PhD, the lead author of the study and an Assistant Scientist in the Department of Medicine at Indiana University School of Medicine.

“The interstitium is the ‘glue’ that holds the kidney together. It is one of the least characterized parts of the kidney, but scars in the interstitium caused by diseases such as diabetes can contribute to kidney disease,” said Barwinska. 

Acute kidney injury (AKI) and chronic kidney disease (CKD) affect millions of people in the United States and globally. However, no effective therapies exist for AKI, and only a few are available for CKD. The KPMP, a multi-site project focused on understanding and finding new treatments to AKI and CKD, is seeking to bring treatment for these conditions “into the molecular era,” according to Michael Eadon, MD. 

IU is one of KPMP’s many “tissue interrogation sites” across the country. Collectively, these sites are working together bring cutting-edge technologies to aid in the interrogation of human kidney biopsies.

“Many diseases can look the same under the microscope, but they have very different causes,” said Eadon, who is the study’s corresponding author and an Assistant Professor of Medicine in the Department of Medicine at IU School of Medicine. “We’re seeking to understanding how different genes contribute to very common kidney diseases.”

This study could usher in the era of new and better treatments for millions of people with AKI and CKD. 

“A personalized medicine approach that understands how different diseases affect a patient’s genes will aid in finding potential treatments for kidney disease,” said Barwinska. “This approach can meet any single patient’s needs.”

Contributors to this study were funded by the Indiana University Grand Challenge Precision Health Initiative.

Reference: Daria Barwinska, Tarek M. El-Achkar, Ricardo Melo Ferreira, Farooq Syed, Ying-Hua Cheng, Seth Winfree, Michael J. Ferkowicz, Takashi Hato, Kimberly S. Collins, Kenneth W. Dunn, Katherine J. Kelly, Timothy A. Sutton, Brad H. Rovin, Samir V. Parikh, Carrie L. Phillips, Pierre C. Dagher, Michael T. Eadon, for the Kidney Precision Medicine Project, “Molecular characterization of the human kidney interstitium in health and disease”, Science Advances  10 Feb 2021: Vol. 7, no. 7, eabd3359 DOI: 10.1126/sciadv.abd3359

Provided by Indiana University School of Medicine

Winner-takes-all Synthetic Gene Circuit Process Opens New Pathways to Disease Treatment (Biology)

Division of synthetic gene circuit workloads will make therapy more effective.

A new process for inserting synthetic gene circuits into host cells, developed by a team of bioengineers at Arizona State University, has broad implications for improving the effectiveness of a range of disease therapies.

Synthetic biology is an interdisciplinary research field that uses engineering principles to create biological components that don’t exist in the natural world. These synthetic components mimic naturally evolved organisms, but are customized to fight disease, including cancer.

A paper recently published in BioRxiv, “Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions,” outlines how gene circuits can be reconfigured so that they do not overwhelm the host cells.

“We connect circuits together like a Lego chain and insert them into a host cell,” explained lead author Xiaojun Tian, an assistant professor in the School of Biological and Health Systems Engineering at ASU. “The circuits in the chain are designed to perform different functions, but they must compete with each other for the cell’s limited resources.”

Competition for resources has been a challenge in the synthetic biology field since its inception 20 years ago. “We would find circumstances where one gene circuit in a chain would consume 90 percent of a host cell’s available resources, leaving only 10 percent for the remaining circuit.”

Tian’s team devised a way to insert individual gene circuits into multiple host cells that work collectively. Each cell performs a specific function, eliminating the undesired competition for resources of any host cell. “Instead of dividing resources, each cell can perform 100 percent of its assigned workload,” said Tian. “The host cells perform as a connected unit without depleting any one cell’s resources – and each gene circuit becomes a winner.”

The technology has broad implications for cancer treatment, with future applications for other diseases on the horizon. Ninety percent of cancer deaths are due to metastasis – the spread of cancer cells to other sites in the body. However, treatment resistance is still a major problem in cancer therapeutics.

“There are many different kinds of cells in a cancer mass,” said Tian. “Some cells are responsive to chemotherapy and others are not, causing treatment resistance.

New multitasking synthetic gene circuitry configuration can be constructed to prevent the cells from metastasizing in the first place, while simultaneously making them more receptive to treatment.”

Tian explains that multicellular synthetic circuits will be a much more effective way to treat cancer.

The research team also includes Rong Zhang, Hanah Goetz, Juan Melendez-Alvarez, Jiao Li, and Xiao Wang from Arizona State University and Tian Ding from Zhejiang University in Hangzhou, Zhejiang, China. The research was funded by the National Science Foundation and the ASU Dean’s Fellowship.

Featured image: Tian’s research reveals a novel winner-takes-all resource competition between synthetic gene circuits within one host cell. © Graphic created by Xiaojun Tian/ASU.

Reference: Rong Zhang, Hanah Goetz, Juan Melendez-Alvarez, Jiao Li, Tian Ding, Xiao Wang, Xiao-Jun Tian, “Winner-Takes-All Resource Competition Redirects Cascading Cell Fate Transitions”, bioRxiv 2020.05.23.103259; doi: https://doi.org/10.1101/2020.05.23.103259

Provided by Arizona State University

First Patient Dosed in Clinical Trial of Gene Therapy For Tay-Sachs and Sandhoff Diseases (Medicine)

Gene therapy program based on research at UMass Medical School

Sio Gene Therapies, a company focused on developing gene therapies to radically transform the lives of patients with neurodegenerative diseases, announced that the first patient with infantile Tay-Sachs disease has been dosed in a Phase I/II trial evaluating AXO-AAV-GM2, an investigational gene therapy for the treatment of GM2 gangliosidosis, which causes Tay-Sachs and Sandhoff diseases.

Sio, formerly known as Axovant, licensed exclusive worldwide rights from UMass Medical School for the development and commercialization of gene therapy programs for GM1 gangliosidosis and GM2 gangliosidosis, including Tay-Sachs and Sandhoff diseases.

“The families of children with Sandhoff and Tay-Sachs diseases show incredible bravery in choosing to participate in investigational studies of novel therapeutics like AXO-AAV-GM2. We share their hope that this treatment can halt or reverse the otherwise inexorable course of these tragic diseases,” said Terence R. Flotte, MD, the Celia and Isaac Haidak Professor, executive deputy chancellor, provost and dean of the School of Medicine and principal investigator of the trial.

The study is an open-label, two-stage, clinical trial designed to evaluate safety and dose-escalation and safety and efficacy of surgical delivery of AXO-AAV-GM2 directly to the brain and spinal cord of pediatric participants with both infantile and juvenile GM2 gangliosidosis.

AXO-AAV-GM2 has been granted Orphan Drug and Rare Pediatric Disease Designation by the FDA and is the first investigational gene therapy to enter clinical trials for GM2 gangliosidosis. In 2019, clinical evidence from two patients under an investigator-initiated study found that treatment with AXO-AAV-GM2 was generally well tolerated and associated with improved bioactivity outcomes.

Research into the causes and potential therapies for lysosomal storage diseases such as Tay-Sachs and Sandhoff diseases and GM1 gangliosidosis at UMass Medical School by Miguel Sena-Esteves, PhD, associate professor of neurology and a principal scientist of the research program at UMMS; and Heather Gray-Edwards, PhD, DVM, assistant professor of radiology at UMass Medical School and a part of the development team for the research discoveries; in conjunction with Douglas Martin, PhD, professor of anatomy, physiology and pharmacology at the Auburn University College of Veterinary Medicine, has led to significant advances in the field, including development of the gene therapy vector used to deliver functioning copies of the defective genes that cause disease.

GM2 gangliosidosis is a set of rare, monogenic neurodegenerative lysosomal storage disorders caused by mutations in the genes that encode the enzyme β-Hexosaminidase A. It can be categorized into two distinct diseases, Tay-Sachs disease and Sandhoff disease. Children affected by GM2 gangliosidosis suffer from a progressively debilitating disease course and reduced life expectancy.

“We are proud to bring the first potentially disease-modifying treatment for GM2 gangliosidosis to the clinic, which is a milestone both for Sio, for patients and for the field of gene therapy,” said Gavin Corcoran, MD, chief R&D officer of Sio. “By restoring lysosomal enzyme activity where it is essential, AXO-AAV-GM2 has the potential to change the course of this disease and help affected children attain and retain important neurodevelopmental milestones. The prior investigator-initiated study of AXO-AAV-GM2 provided important proof-of-concept data and we look forward to the results of the first stage of our study as we strive to develop a treatment for children suffering from this rapidly progressive and fatal disease.”

Sio aims to advance the program through strategic partnerships with leading research organizations. The company has a partnership with Viralgen, an AskBio subsidiary, to support AAV-based vector manufacturing of clinical trial material for the registrational study. Additionally, through an existing genetic testing collaboration with Invitae, ongoing partnership with GM2 gangliosidosis patient groups, and collaboration with leading academic researchers at UMass Medical School and Massachusetts General Hospital, Sio has begun patient identification and site startup activities for the ongoing clinical study.

Featured image: Miguel Sena-Esteves, PhD, and Heather Gray-Edwards, PhD, DVM © UMass Med

This news is confirmed by us from UMass Medical School Communications

Provided by University of Massachusetts Medical school

Potential Preventative Treatment Demonstrated for Crohn’s Disease (Gastroenterology / Medicine)

A potential preventive treatment for Crohn’s disease, a form of inflammatory bowel disease, has been demonstrated in a mouse model and using immune-reactive T cells from patients with Crohn’s disease.


This research, led by University of Alabama at Birmingham researcher Charles O. Elson, M.D., professor of medicine, focused on a subset of T cells known as T memory, or Tm cells. The UAB researchers used a triple-punch treatment to remove Tm cells and increase the number of T regulatory, or Treg, cells. Both of these results were able to prevent colitis in a T cell transfer mouse model, and they had similar inhibitory effects on immune-reactive CD4-positive T cells isolated from Crohn’s disease patient blood samples. 

These results, Elson says, support a potential immunotherapy to prevent or ameliorate inflammatory bowel disease.

Some background is needed to understand how and why the triple-punch treatment, which was reported in the journal Science Immunology, works. 

Inflammatory bowel diseases result from an over-activation of the immune response against gut microbes in genetically susceptible hosts. One specific microbial antigen causing this over-reaction by short-lived T effector cells is flagellin, the protein-subunit of bacterial flagella, the long tail-like structures that twirl like a propeller to make some bacterial motile. 

One group of immuno-dominant flagellins are those from the Lachnospiraceae family, including CBir1; more than half of Crohn’s disease patients have elevated serological reactivity to CBir1 and related flagellins. 

Unlike the short-lived T effector cells that act like soldiers to help fight infections, T memory cells serve as sentinels that remember a previous encounter with flagellins. They are long-lived and quiescent, with a low level of metabolism. If reactivated by a fresh encounter with flagellin antigens, they undergo a profound metabolic transition and quickly expand into large numbers of pathogenic T effector cells. 

This metabolic switch is controlled by a signaling protein, mTOR, located in the Tm cell.  

Thus, activation of mTOR is necessary for T cell expansion, making it an inescapable metabolic checkpoint to create activated Tm cells. It is also the checkpoint for T naïve cells that are encountering flagellin for the first time.

So, Elson and colleagues hypothesized that activation of CD4-positive Tm or T naïve cells by flagellin antigens, while at the same time shutting down the metabolic checkpoint through the use of mTOR inhibition, would result in the death or an absence of the normal immune response to an antigen, which is called anergy. These effects comprise two parts of the triple-punch treatment, with the third being induction of Treg cells. 

The activation was prompted by a synthetic peptide that had multiple repeats of one CBir1 epitope. Such a peptide can selectively stimulate memory cells without activating an innate immune response. 

To shut down the metabolic checkpoint, the UAB researchers used two existing drugs, rapamycin and metformin. Rapamycin directly inhibits mTOR, and metformin adds to that inhibition by activating a kinase called AMPK that negatively regulates mTOR activity. 

Elson calls this treatment cell activation with concomitant metabolic checkpoint inhibition, or CAMCI.

Parenteral application of CAMCI in mice successfully targeted microbiota flagellin-specific CD4-positive T cells, leading to significant antigen-specific CD4-positive T cell death, impaired development and impaired reactivation of CD4-positive memory responses, and substantial induction of a CD4-positive Treg cell response. It prevented colitis in the mouse model and had similar inhibitory effects on microbiota-flagellin-specific CD4-positive T cells isolated from patients with Crohn’s disease.

For a potential future treatment of patients with Crohn’s disease, only targeting a single flagellin is unlikely to have much effect, Elson says. “Instead, we anticipate the future use of a synthetic multi-epitope peptide containing multiple CD4-positive T cell flagellin epitopes to target many microbiota-flagellin-reactive CD4-positive Tm cells,” Elson said. “Depending on the serologic or CD4-positive T cell response to certain microbiota antigens, this CAMCI approach could be tailored to individuals with different combinations of epitopes as a personalized immunotherapy.”

Elson says he envisions this CAMCI approach as an intermittent pulse therapy to maintain remission in patients with Crohn’s disease. “And with autoantigen epitopes better studied in the future,” he said, “this approach could be expanded to treat other inflammatory or autoimmune diseases such as Type 1 diabetes or multiple sclerosis.” 

In developed countries, three of every 1,000 people have inflammatory bowel disease. Its major forms, Crohn’s disease and ulcerative colitis, have substantial morbidity and large medical care costs, and no current therapy alters the natural history of these diseases. 

Co-authors with Elson, in the study “CD4-positive T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis,” are Qing Zhao, Lennard W. Duck, Katie L. Alexander and Peter J. Mannon, UAB Department of Medicine; Fengyuan Huang, UAB Department of Genetics, Informatics Institute; and Craig L. Maynard, UAB Department of Pathology.

Support came from a Litwin IBD Pioneers grant from the Crohn’s and Colitis Foundation, and National Institutes of Health grants DK071176, DK118386, AI27667, AR048311 and RR-20136.

At UAB, Elson holds the Basil I. Hirschowitz Endowed Chair in Gastroenterology.

Reference: Qing Zhao, Lennard W. Duck, Fengyuan Huang, Katie L. Alexander, Craig L. Maynard, Peter J. Mannon, Charles O. Elson, “CD4+ T cell activation and concomitant mTOR metabolic inhibition can ablate microbiota-specific memory cells and prevent colitis”, Science Immunology 11 Dec 2020: Vol. 5, Issue 54, eabc6373 DOI: 10.1126/sciimmunol.abc6373 https://immunology.sciencemag.org/content/5/54/eabc6373

Provided by University of Alabama at Birmingham

Pacify The Protein and Win Over A Disease (Medicine)

Will it be enough to pacify the activity of certain proteins in order to hold back the development of many dangerous diseases including Alzheimer’s disease? An article on a breaking through discovery has just been published in PNAS, a prestigious magazine of American Academy of Sciences. Its first author is Karolina Mikulska-Rumińska, Dr., a biophysicist from NCU.

Dr Karolina Mikulska-Ruminska from the Department of Biophysics in the Institute of Physics, Nicolaus Copernicus University © Andrzej Romanski/Nicolaus Copernicus University

The interests of Karolina Mikulska-Rumińska, Dr., from the Department of Biophysics in the Institute of Physics, NCU focus on widely understood biological structures, especially proteins. In her work, she applies calculation methods in computer simulations. During her postdoctoral internship in the group of Ivet Bahar, Prof. at the University of Pittsburgh, she already began her cooperation with medical doctors from Los Angeles and the researchers from the University of Pittsburgh.

The latest effects of her scientific inquiries can be read about in the article “Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation”, which has just been published in PNAS. Doctor Mikulska-Rumińska and Jargalsaikhan Dagvadorj from Cedars-Sinai Medical Center in Los Angeles are the first authors of the publication. The article refers to the mechanisms of the activities of the immune system under inflammatory conditions.

One of the main objectives of our research was to define what role is played by the protein – prointerleukin-1 alpha (pro-IL-1α). It is known that the mature form of this pro-inflammatory cytokine (IL-1α) is responsible for the formation of inflammatory states, fever and sepsis. It was a huge discovery to find out that the same protein in an inactive form of precursor (pro-IL-1α) plays such a key role in regulating the response of the immune system – explains Mikulska-Rumińska, Dr.

Dangerous inflammation

Interleukins (IL) are a group of cytokines, that is proteins, which take part in inflammatory processes of the immune system. The proteins are numbered – a few dozen have been classified. Dr. Mikulska-Rumińska got her interest in the first group: IL-1. This is a collective term which refers to the cytokines of key importance in inflammation processes. They are secreted in response to various types of antigens, for instance of viral, bacterial, or fungal origin. Out of 10 different strains, one of the most significant is, already mentioned, IL-1α, which most often occurs as a membrane cell and it interacts with the neighboring cells and IL-1β. It is these cytokines that have become the focus of the examination of the Polish researcher and American scientists.

At this point, it is necessary to mention the inflammasome NLRP3. This is a structure within a cell which consists of many specialized proteins.  In a healthy cell it remains inactive. It becomes activated when the organism is endangered with, for instance, the presence of microorganisms or cells which appeared due to the damage of tissues, and metabolic disorders – explains Dr. Mikulska-Rumińska. Active inflammasomes NLRP3 transform the interleukin 1 beta (IL-1β) and interleukine18 (IL-18) into their active forms, due to which an inflammatory state is generated in the organism.

– Obviously, there is quite a numerous group of inflammations which are necessary for the organisms, for instance to eliminate bacteria or viruses. A chronic state, however, leads to many dangerous diseases. The best example of this is so called “cytokine storm”, which is hyperreaction of the immune system, which is a serious problem in the most serious cases of COVID-19, which medical doctor are fighting against at the moment – explains Dr. Mikulska-Rumińska. – Hyperactivity of NLRP3 triggers off numerous pathological conditions, including atherosclerosis, arthritis, inflammatory bowel disease or type 2 diabetes.

A lot of scientists also find connection between chronic inflammatory state and Alzheimer’s disease, which is progressive neurodegenerating disease that leads to gradual atrophy of cognitive and memory functions. Although the pathophysiological mechanism of the disease has not yet been entirely discovered, researchers believe it is related to the activation of NLRP3 inflammasome.

Our research reveals the mechanism due to which cells modulate the activation of NLRP3 inflammasome. This is very crucial as it is connected with the possible therapeutic application, for example while treating the above-mentioned diseases – says Dr. Mikulska-Rumińska.

– Shortly speaking, we have found that the cells which lack pro-IL-1α are characterized by a decreased activity of NLRP3 inflammasome and caspase-1 (an element of the inflammasome which activates cytokines). This leads to a lesser release of IL-1β and decreased damage of mitochondria. The role of pro-IL-1α is therefore extremely important – it regulates the activation of the inflammasome, and thus is a key protein to initiate an inflammatory state – explains Dr. Mikulska-Rumińska. 

Competing proteins

The scientists have also become interested in one more issue. Dr. Mikulska-Rumińska has discovered that the fragment IL-1α (so called signal peptide) is extremely similar to another protein – LC3b, which plays a key role in purifying the organism from damaged or redundant cells. The sequences of both of them are almost identical.

Interaction of signal peptid  pro-IL1α (protein fragment) with cardiolipin (CL) in the lipid membrane © Nicolaus Copernicus University in Torun

– We have shown that pro-IL-1α interacts in the mitochondrial membrane with cardiolipin. Cardiolipin is an important phospholipid which normally binds with another protein – LC3b and, at the same time, sends a signal “eat me” to eliminate calls – explains Dr. Mikulska-Rumińska.

Simulations of the molecular dynamics for such a set have shown what structural elements are the key elements for interactions with cardiolipin. What is more, it has turned out that the same structural elements are present in the protein LC3b.

– These proteins may compete with each other, which may trigger off serious consequences – explains Dr. Mikulska-Rumińska. – When pro-IL-1α in a, for instance, damaged cell connects with cardiolipin, LC3b is not able to perform its job, which is to communicate to the organism that it should be “thrown out”. This happens because cardiolipin gets intercepted by pro-IL-1α and can no longer interact with LC3b.

This discovery may also contribute to developing new strategies of treatment of inflammatory states responsible for the development of numerous diseases.

Provided by Nicolas Copernicus University in Torun

New Drug Molecules Hold Promise For Treating Rare Inherited Terminal Childhood Disease (Medicine)

Scientists at the University of Exeter have identified a way to “rescue” cells that have genetically mutated, paving the way to a possible new treatment for rare terminal childhood illness such as mitochondrial disease.

Mitochondria. Credit: Wikipedia commons

The research, funded by the United Mitochondrial Disease Foundation in the U.S., was led by Professors Matt Whiteman and Tim Etheridge. In the study, published in the Journal of Inherited Metabolic Disease, the team used novel drugs being developed at the University of Exeter, which “metabolically reprogramme” mitochondria—the cellular energy production centers in cells, by providing them with an alternative fuel source to generate metabolic energy in the form of minute quantities of hydrogen sulfide.

The team used microscopic worms (C. elegans) with specific genetic mutations affecting energy production, that match mutations that cause human diseases such as Leigh Syndrome. The team found that administering the new compounds to these animals successfully normalized or improved energy production needed to keep them healthy and active.

Professor Tim Etheridge, of the University of Exeter, one of the study authors, said: “Worms are a very powerful genetic tool to study human health and disease and offer an ideal platform to quickly identify new potential therapeutics. The worms used in this study had genetic defects in how their mitochondria regulate cellular energy production to model different human mitochondrial diseases. The novel compounds we are developing at the University of Exeter are able to bypass some of these defects and keep the worms, and their mitochondria healthy. We know this because we saw improvements in physical activity and improvements in muscle and mitochondrial integrity. The animals also lived for longer after treatment but more importantly, they remained active for longer, because of metabolic reprogramming.”

The team had previously shown that the compounds had potent therapeutic effects in mammalian models with defective mitochondria. In those studies, the animals’ mitochondria became defective as result of a disease process. In the latest study however, the defective mitochondria were the direct cause of the disease, as in human mitochondrial disease and were still successfully treated with the Exeter compounds. The fact that the compounds could reverse some of these inherited defects in energy metabolism strongly suggest that their effect will translate to humans, and the team is confident this can be tested in the near future.

Lead author Professor Matt Whiteman, of the University of Exeter, said: “Mitochondrial diseases, and their related conditions, are areas of huge and desperate unmet clinical need. Our study is an important first step and a lot of work still needs to be done. For the first time, we have demonstrated that our new molecules have successfully metabolically reprogrammed, or rescued, cells in animals with genetic defects in their mitochondria. We’re currently testing newer and more potent molecules able to do the same task, through slightly different approaches, and we’re looking for commercial partners to help our efforts to progress our molecules through to clinical testing.”

The paper is titled “The mitochondria-targeted hydrogen sulfide donor AP39 improves health and mitochondrial function in a C. elegans primary mitochondrial disease model.”

Reference: Bridget C. Fox et al. The Mitochondria‐targeted Hydrogen Sulfide Donor Ap39 Improves Health and Mitochondrial Function in A C. Elegans Primary Mitochondrial Disease Model, Journal of Inherited Metabolic Disease

Provided by University of Exeter

Immune Cell That Drives Breast Cancer Could be Effective Target in Novel Immunotherapies (Medicine)

Breast cancer is the most common cancer in women worldwide, but many immunotherapies have had limited success in treating aggressive forms of the disease.

New research findings from Paula Bos, Ph.D., identified a type of immune cells that acts as a major driver of breast cancer growth by preventing the accumulation of a specific protein that induces anti-tumor responses. Credit: VCU Massey Cancer Center

“A deeper understanding of the immunobiology of breast cancer is critical to the success in harnessing immunotherapeutic approaches to improve breast cancer survival,” said Paula Bos, Ph.D., member of the Cancer Biology research program at VCU Massey Cancer Center and assistant professor in the Department of Pathology at the VCU School of Medicine.

New research findings from Bos, published in Cell Reports, identified a type of immune cells that acts as a major driver of breast cancer growth by preventing the accumulation of a specific protein that induces anti-tumor responses. This new knowledge could be utilized for the development of novel immunotherapeutic approaches to treat the disease.

Regulatory T cells (Treg cells) are a special class of immune cells that possess a unique ability to suppress the function of other immune cells. This function serves to protect the organism from overreacting to certain molecules created within the body; however, in many cases it subdues the immune system’s ability to attack cancer cells. Therefore, Treg cells are often abundant in solid tumors, particularly breast cancers, and are commonly associated with worse outcomes.

In previous research, Bos demonstrated that targeting Treg cells in breast cancer models significantly reduced tumor growth and metastasis; however, it remained unclear on a molecular level why this tumor reduction was happening.

There is a specific protein called interferon gamma (IFN-g) that has powerful anti-tumor properties, including the activation of macrophages, which are cells that can initiate inflammation and prevent cancer growth.

Bos’ latest study suggests that Treg cells suppress IFN-g production by CD4 T lymphocytes (a type of white blood cells), further instigating disease progression. After analyzing breast cancer models in which Treg cells had been targeted and destroyed, Bos discovered an increased presence of IFN-g and functional reprogramming of macrophages into tumor-fighting cells.

“Additionally, we demonstrated better overall survival in human cancers with similar genetic patterns to those observed in mice with breast cancer whose Treg cells were eliminated,” Bos said.

This research is the first of its kind to study the mechanistic function of Treg cells in breast cancer.

Bos said these findings validate the potential for adoptive transfer therapeutics using macrophages programmed with the IFN-g protein to effectively treat breast cancer. Adoptive transfer refers to the process of transferring external cells into a patient to improve immune function or response.

“Our work raises the possibility that white blood cells can be extracted from cancer patients, reprogrammed outside of their body through brief exposure to the IFN-g protein and re-infused back into the patient, contributing to the generation of anti-tumor responses,” Bos said.

Bos is currently studying the function of Treg cells in metastatic cancer and plans to design follow-up studies testing the utilization of IFN-g as an adoptive transfer therapeutic agent in cancer mouse models.

References: Nicholas M. Clark et al, Regulatory T Cells Support Breast Cancer Progression by Opposing IFN-γ-Dependent Functional Reprogramming of Myeloid Cells, Cell Reports (2020). DOI: 10.1016/j.celrep.2020.108482

Provided by Virginia Commonwealth University