Journal Club

This blog originated as a press release from the Max Planck Institute for Medical Research in Heidelberg. Thanks to them for allowing us to repost it here.

Scientists create synthetic exosomes with natural functionalities and present their therapeutic application.

Scientists from the Max Planck Institute for Medical Research in Heidelberg and colleagues at the DWI Leibniz Institute for Interactive Materials in Aachen have engineered synthetic exosomes that regulate cellular signaling during wound closure. The synthetic structures are built to resemble naturally occurring extracellular vesicles (EV) that play a fundamental role in communication between cells during various processes in our bodies. The scientists uncovered key mechanisms in the regulation of wound healing and the formation of new blood vessels. They designed and built programmable fully-synthetic EVs from scratch rather than isolating natural EVs from cells. Inspired by the roles of the natural counterparts, the scientists demonstrate for the first time that fully synthetic exosomes with therapeutic functions can be constructed.

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This blog originated as a press release from the Technical University of Denmark (DTU). Thanks to them for allowing us to repost it here.

DTU Health Tech researchers have developed a method for detection of SARS-CoV-2 RNA that can be adapted to detect other diseases.

Current SARS-CoV-2 RNA detection methods recommended by the World Health Organization profoundly rely on the roles of biological enzymes. High cost, stringent transportation and storage conditions, as well as a global supply shortage of enzymes, limit large-scale testing. The result is that most countries have to prioritize testing on vulnerable cases, which creates delay in diagnostics and identification of positive cases, which again can hamper pandemic mitigation and suppression.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is still the gold standard for whole genome detection and has been playing a key role in controlling the COVID-19 pandemic. However, the sample-to-result time for qRT-PCR is several hours, and the method requires a complex thermocycler instrument to raise and lower the temperature of the reaction in discrete steps.

Simpler and less expensive

Non-enzymatic isothermal amplification methods, being simpler and faster, have shown promising potential to substitute for qRT-PCR. Although these methods perform very well when the target gene is short, they are yet to function efficiently for detection of whole viral genomes (long DNA or RNA targets).

During the COVID-19 pandemic, the Euro area alone experienced a 3.8% drop in GDP within the first quarter of 2020 (Eurostat 2020). Thus, developing a lower-cost methodology for pathogen detection would be highly beneficial for both patients and the healthcare systems aiming to battle future pandemics.

Associate Professor Yi Sun and Postdoc researcher Mohsen Mohammadniaei at DTU Health Tech have invented a one-pot assay, which they have named NISDA (Non-enzymatic Isothermal Strand Displacement and Amplification assay). The assay is for rapid detection of SARS-CoV-2 RNA without the need for the RNA reverse transcription step of the qRT-PCR methodology. Being one-pot enables a single step detection routine. The user only needs to add the sample into a single tube, place it in the instrument, and wait for 30 minutes to obtain the result.

The assay works at constant temperature, requires no enzymes, and is based on the toehold-mediated strand displacement (TMSD) approach. TMSD is an enzyme-free molecular tool from which one strand of DNA or RNA (output) is displaced by another strand (input) to form a more stable duplex structure.

High accuracy and sensitivity

The NISDA assay was able to detect a very low concentration of RNA (10 copies/µL) in only 30 minutes. In collaboration with Hvidovre Hospital and Bispebjerg Hospital, the research team clinically validated the NISDA assay, acheiving 100% specificity as well as 96.77% and 100% sensitivity when setting up in the laboratory and hospital, respectively.

Associate Professor Yi Sun elaborates, “We exploited the TMSD approach and designed three DNA probes. One probe exchanged the whole genome to a short DNA strand and the other two probes utilized the exchanged short DNA for triggering a fluorescence signal amplification cascade reaction. The beauty of NISDA assay is its simplicity. We removed the usage of enzymes to reduce the assay cost and enhance its robustness at room temperature.”

In the assay workflow, the extracted RNA from throat swab samples is added to the reaction mixture and incubated at 42°C for 30 minutes. The next step is fluorescence measurement, and samples with significantly higher fluorescence signals than that of the control samples are considered positive.

Schematic of NISDA assayThe NISDA assay comprises a single tube containing three DNA probes. After the addition of the extracted RNA from swab samples and incubation at 42°C for 30 min, positive samples show higher fluorescent signals than negative samples.

Towards a multiple disease diagnostics tool

“Being directly involved in improving people’s health is the ultimate dream of a biomedical researcher and we believe that the NISDA assay has given us this wonderful chance to attain that ambition”, Postdoctoral Researcher Mohsen Mohammadniaei says.

“The next step is to further design the NISDA assay for detecting different pathogens and develop a point-of-care diagnostic device for multiple disease diagnostics. Another advantage of the NISDA assay is its ability to be designed for short RNA targets such as cancer biomarker microRNA. We are currently exploring different schemes for the commercialization of the NISDA assay and we are certain that the NISDA assay will become widely-known in the near future”, Associate Professor Yi Sun finishes.

Reference

Mohammadniaei M et al., A non-enzymatic, isothermal strand displacement and amplification assay for rapid detection of SARS-CoV-2 RNA. (2021) Nat Comm 12: 5089. doi: 10.1038/s41467-021-25387-9 PMID: 34429424.

This blog originated as a press release from the Broad Institute of MIT and Harvard. Thanks to MIT News for allowing us to repost it here.

Made of components found in the human body, the programmable system is a step toward safer, targeted delivery of gene editing and other molecular therapeutics.

Molecular therapies graphicA new system to deliver molecular therapies to cells, called SEND, can be programmed to encapsulate and deliver different RNA cargoes, potentially provoking less of an immune response than other delivery approaches.
Credit: Courtesy of the researchers

Researchers from MIT, the McGovern Institute for Brain Research at MIT, the Howard Hughes Medical Institute, and the Broad Institute of MIT and Harvard have developed a new way to deliver molecular therapies to cells. The system, called SEND, can be programmed to encapsulate and deliver different RNA cargoes. SEND harnesses natural proteins in the body that form virus-like particles and bind RNA, and it may provoke less of an immune response than other delivery approaches.

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This blog originated on the NIH Intramural Research Program’s “I Am Intramural” blog. Thanks to them for allowing us to repost it here.

First-Trimester Blood Analysis Could Enable Earlier, More Effective Intervention

Imagine a world in which pregnant women routinely travel to places of healing and meet with wise sages who examine a bit of their blood to divine when their babies will be born. While this may sound like something out of Greek mythology, it may soon become a reality, as researchers from the NIH Intramural Research Program (IRP) have developed a test that was able to use blood samples taken early in pregnancy to identify women who would later deliver their babies prematurely.

Mother and baby By analyzing blood samples taken in the first trimester of pregnancy, IRP researchers were able to accurately identify most of the women who later went on to deliver their babies prematurely.

Women typically expect to deliver their babies after carrying them for roughly 40 weeks, a period of time known as gestation. In reality, only four percent of mothers deliver on their ‘due date.’ Roughly one in ten babies is born ‘premature’ or ‘preterm,’ meaning they are delivered before 37 weeks of gestation, and most of these births are ‘spontaneous,’ occuring with no prior warning. The earlier in pregnancy a baby is born, the higher the baby’s odds of experiencing short- and long-term health effects, including behavioral problems, learning disabilities, breathing difficulties, and infections. What’s more, because African American women deliver preterm much more often than other groups of women, premature birth is a significant contributor to racial health disparities.

If doctors were able to predict preterm births, they could intervene early to delay delivery. Right now, the best predictors for premature births are a history of preterm delivery and a shorter-than-normal cervix, the tube of tissue connecting the vagina and uterus. However, the former factor cannot be applied to first-time mothers, and most premature births occur in women who have neither of those risk factors. Measuring cervical length also requires significant resources and is only done 16 to 24 weeks into pregnancy.

Dr. Roberto Romero Senior author Dr. Roberto Romero

“Cervical length is the most powerful predictor of spontaneous preterm birth, but the patient must be seen at a healthcare facility and we need equipment and expertise,” says IRP senior investigator Roberto Romero, M.D., D.Med.Sci., the new study’s senior author. “It would be great if we could have a simple blood test that could predict spontaneous preterm delivery.”

The IRP team’s study used blood samples collected from women who had been pregnant for 6 to 13 weeks, significantly earlier than cervical length can be used to predict preterm birth. First, the researchers analyzed samples from nine women who went on to deliver preterm and 70 women who delivered at term to measure the concentrations in their blood of 45 different microRNA molecules — short strands of genetic material that help control the behavior of genes. This analysis showed that 12 specific microRNAs were much more abundant in blood from women who delivered prematurely.

Next, using blood collected from 78 other women during the same early period of pregnancy, Dr. Romero’s team measured the levels of those 12 microRNAs to attempt to retroactively identify which of them ultimately delivered prematurely. Overall, their analysis method correctly classified nearly 90 percent of the women who went on to deliver preterm, with a low rate of false negatives. Moreover, when the IRP researchers separately analyzed samples from women whose blood was taken before 10 weeks of gestation and those whose blood was taken after that time point, they found that they were able to identify 80 percent of the women who went on to deliver prematurely in the former group and all of the women who delivered preterm in the latter group.

Diagram of how micoRNAs affect protein production by genes Diagram of how micoRNAs affect protein production by genes

MicroRNAs regulate the activity of genes by reducing the amount of protein they produce.

“I never want to claim anything is 100 percent, but the point is if we have a blood test based on microRNAs, we can predict spontaneous preterm delivery,” Dr. Romero says. “The idea that there is a blood test that can help us assess a person’s risk for preterm delivery is not a dream.”

The researchers also consulted an online database to identify biological pathways that those 12 microRNAs are involved in. These include processes that help transport biological molecules around the cell, control the activity of estrogen-related genes, and activate enzymes involved in cell death. This information provides clues as to the biological triggers of some cases of preterm birth, knowledge that will help scientists develop therapies that prevent it.

Meanwhile, doctors already have some methods to reduce the risk of preterm birth, and these approaches are more effective the earlier in pregnancy they are implemented. If future studies in larger and more diverse populations confirm the results of Dr. Romero’s research, clinicians could identify patients who may need those treatments using a simple blood test.

“We are interested not just in prediction but also in prevention,” Dr. Romero says. “There are substantial advantages if we can use blood markers during pregnancy for this purpose, and the earlier we can do that the better because then there is a window in which we can intervene.”

Reference
Winger EE, Reed JL, Ji X, Gomez-Lopez N, Pacora P, Romero R. MicroRNAs isolated from peripheral blood in the first trimester predict spontaneous preterm birth. (2020) PLoS ONE 15:e0236805. doi: 10.1371/journal.pone.0236805. PMID: 32790689.

This blog originated as a press release from The Ohio State University. Thanks to them for allowing us to repost it here.

Inserting genetic material into the body to treat diseases caused by gene mutations can work, scientists say – but getting those materials to the right place safely is tricky.

Scientists from The Ohio State University have reported in the journal Science Advances that the lipid-based nanoparticles they engineered, carrying two sets of protein-making instructions, showed in animal studies that they have the potential to function as therapies for two genetic disorders.

In one experiment, the payload-containing nanoparticles prompted the production of the missing clotting protein in mice that are models for hemophilia. In another test, the nanoparticles’ cargo reduced the activation level of a gene that, when overactive, interferes with clearance of cholesterol from the bloodstream.

Dr. Yizhou Dong Dr. Yizhou Dong

Each nanoparticle contained an applicable messenger RNA molecule that translates genetic information into functional proteins.

“We demonstrated two applications for lipid-like nanomaterials that effectively deliver their cargo, appropriately biodegrade, and are well-tolerated,” said Yizhou Dong, senior author of the study and associate professor of pharmaceutics and pharmacology at The Ohio State University.

“With this work, we have lowered potential side effects and toxicity and have broadened the therapeutic window. This gives us confidence to pursue studies in larger animal models and future clinical trials.”

This work builds upon a collection of lipid-like spherical compounds that Dong and colleagues had previously developed to deliver messenger RNA. This line of particles was designed to target disorders involving genes that are expressed in the liver.

The team experimented with various structural changes to those particles, effectively adding “tails” of different types of molecules to them, before landing on the structure that made the materials the most stable. The tiny compounds have a big job to do: embarking on a journey through the bloodstream, carrying molecules to the target location, releasing the ideal concentration of messenger RNA cargo at precisely the right time, and safely degrading.

The tests in mice suggested these particles could do just that.

The researchers injected nanoparticles containing messenger RNA holding the instructions to produce a protein called human factor VIII into the bloodstream of normal mice and mouse models for hemophilia. A deficiency of this protein, which enables blood to clot, causes the bleeding disorder. Within 12 hours, the deficient mice produced enough human factor VIII to reach 90 percent of normal activity. A check of the organs of both protein-deficient mice and normal mice showed that the treatment caused no organ damage.

“It can be helpful to think of this as a protein-replacement therapy,” Dong said.

In the second experiment, nanomaterials were loaded with two types of instructions: messenger RNA carrying the genetic code for a DNA base editor, and a guide RNA to make sure the edits occurred in a specific gene in the liver called PCSK9. Dozens of mutations that increase this gene’s activity are known to cause high cholesterol by reducing clearance of cholesterol from the bloodstream.

Analyses showed that the treatment resulted in the intended mutation of about 60 percent of the target base pairs in the PCSK9 gene, and determined that only a low dose was needed to produce high editing effect.

Dong credited academic and industry partners for helping advance this work. Co-corresponding authors include Denise Sabatino of Children’s Hospital of Philadelphia and Delai Chen from Boston-based Beam Therapeutics, who provided expertise in hemophilia and DNA base editing, respectively.

Dong and first author Xinfu Zhang are inventors on patent applications filed by Ohio State related to the lipid-like nanoparticles. This technology has been licensed for further clinical development.

This work was supported by the National Institute of General Medical Sciences, the National Heart, Lung and Blood Institute, and a startup fund from Ohio State’s College of Pharmacy.

Additional co-authors are Giang N. Nguyen of Children’s Hospital of Philadelphia; Weiyu Zhao, Chengxiang Zhang, Chunxi Zeng, Jingyue Yan, Shi Du, Xucheng Hou, Wenqing Li, Justin Jiang, Binbin Deng and David McComb of Ohio State; and Robert Dorkin, Aalok Shah, Luis Barrera, Francine Gregoire and Manmohan Singh of Beam Therapeutics.

Reference
Zhang X et al. Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing. (2020) Sci Adv 6:eabc2315. doi: 10.1126/sciadv.abc2315. PMID: 32937374.

This blog originated as a press release from The Ohio State University. Thanks to them for allowing us to repost it here.

Dr. Raphael Pollock has earned the reputation as one of the world’s best surgical oncologists for patients facing one of the toughest cancers to treat, sarcoma. Frequently these tumors start out in the very deepest recesses of the retroperitoneum, the part of the abdomen where the kidneys, pancreas and inferior vena cava are located.

Dr. Raphael Pollock Dr. Raphael Pollock

Director of The Ohio State University Comprehensive Cancer Center, Pollock’s 30 years of experience in the operating room naturally led him to ponder the steps before surgery, specifically if there were better ways to diagnose or detect sarcomas. In early 2019, he tapped into Ohio State’s scientific breadth and depth to investigate a new diagnostic method based upon research he conducted while at MD Anderson Cancer Center in Houston. He reached out to Shaurya Prakash, associate professor of mechanical and aerospace engineering and an expert in microfluidics.

Currently, there are two predominant options to acquire a diagnostic biopsy of a tumor deep in the abdomen: invasive surgery under general anesthesia; or a method utilizing computed tomography (CT) scans to guide a long needle through the skin to acquire tissue from the mass. Both are expensive and take time to schedule.

“I’ve been interested for a while in the role of exosomes in the spread of cancers,” Pollock said. Exosomes are extracellular vesicles containing constituents—protein, DNA, and RNA—of the cells that secrete them. They can affect function and behavior of other cells with which they interact. Until recently, they were regarded as merely cell waste products without much clinical research relevance.

Dr. Shaurya Prakash Dr. Shaurya Prakash

“We learned that there are a number of things inside the exosomes that interact potentially with cells in the tumor microenvironment,” he added. “Then they circulate in the bloodstream and land in other parts of the body.”

So Pollock asked Prakash if there could be an efficient way of extracting these exosomes from a peripheral blood sample to obtain the contents that might be used to diagnose a tumor deep within the body. The microfluidics expert was intrigued.

“I learned that often by the time sarcomas are diagnosed, the disease state is very advanced,” Prakash said. “The value of isolating these circulating biomarkers is earlier detection. Prognosis is better with earlier detection and diagnosis.”

In the past, Pollock had employed ultracentrifugation to isolate exosomes from blood, but it was arduous and expensive. He and Prakash reviewed the literature and realized there might be several different engineering concepts that could be leveraged to improve the process.

Size-based filtration was first, since exosome size is quite specific. Prakash’s previous water treatment research was useful in developing a microfluidic filtration system. Their second area of focus was targeting a surface marker or protein with monoclonal antibodies to attach, secure and extract the exosomes.

Microfluidic device prototype Microfluidic device prototype

The duo’s prototype microfluidic device integrates size-based separation followed by immunoaffinity-based capture of extracellular vesicles in one process. They also are exploring the use of electrical charge to enhance the exosome filtering.

Prakash and Pollock have submitted two manuscripts—one of which was published recently in the Journal of Microelectromechanical Systems—demonstrating their device is more effective than ultracentrifugation in terms of time, yield, and purity.

The collaboration is just the latest example of an emerging partnership between the College of Engineering and The Ohio State University Comprehensive Cancer Center – James Cancer Hospital and Solove Research Institute.

“These circulating biomarkers are a very small fraction of the overall constituency of blood,” explained Prakash. “The real engineering challenge is extracting that proverbial needle from a haystack. And how do you sort that out and get the right needle.”

Exploded view of microfluidic channels Exploded view of microfluidic channels separated by a nanocapillary array membrane. The dotted line represents the cross-section that was utilized for SEM characterization of the device, as seen inset. Only a portion of captured exosomes may have been connected to the tumor, so capturing as many as possible within a blood sample is critical.

Beyond the manuscripts, the research team is gearing up to submit a proposal. Coincidentally, the National Cancer Institute is now seeking proposals that focus on developing new methodologies to extract exosomes to investigate whether the cargo inside may be applicable as biomarkers for cancer.

“We’re now in a position to really drill down into the engineering concepts of the device,” Pollock said.

He added that while this type of device could be applied to many types of cancers, it is especially advantageous for sarcoma diagnosis.

“After a long operation to remove a confirmed tumor, it is very difficult in scans to differentiate tumor recurrence from post-surgical scarring,” he explained. “But if you can detect something a tumor releases in the bloodstream, that provides you with a higher index of suspicion of what you may be seeing on a scan.

“Instead of relying on repeat scans over months to determine size increase or decrease, we can potentially identify recurrence at a very early point when the total volume of recurrence is small and more amenable to treatment. We’re very excited about the potential.”

Looking ahead, Prakash and Pollock want to build toward a systematic clinical trial. While there is nothing in the prototype device that cannot be used in a clinical trial, Prakash said some optimization would be required.

“It’s been a total partnership,” Pollock said. “None of this would have happened without the mutual interest and opportunities to communicate about possibilities.”

The research team included mechanical and aerospace engineering PhD student Prashanth Mohana Sundaram, and Lucia Casadei, Gonzalo Lopez, Danielle Braggio and Gita Balakirsky from the Comprehensive Cancer Center.

This blog originated as a press release from Cedars-Sinai. Thanks to them for allowing us to repost it here.

Analysis of particles shed by tumors points to new, less invasive way to diagnose malignancies

A recent study sheds light on proteins in particles called extracellular vesicles, which are released by tumor cells into the bloodstream and promote the spread of cancer. The findings suggest how a blood test involving these vesicles might be used to diagnose cancer in the future, avoiding the need for invasive surgical biopsies.

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This blog originated as a press release from the University of Sussex. Thanks to them for allowing us to repost it here.

Scientists at the University of Sussex have identified a potential pattern within blood which signals the presence of motor neuron disease; a discovery which could significantly improve diagnosis.

Currently, it can take up to a year for a patient to be diagnosed with amyotrophic lateral sclerosis (ALS), more commonly known as motor neuron disease (MND).

But after comparing blood samples from patients with ALS, those with other motor-related neurological diseases, and healthy patients, researchers were able to identify specific biomarkers which act as a diagnostic signature for the disease.

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This blog originated as a press release from ISGlobal, the Barcelona Institute for Global Health. Thanks to ISGlobal for permission to post it here.

A new study shows that extracellular vesicles from the malaria parasite Plasmodium vivax promote parasite adhesion to spleen cells

Extracellular vesicles (EVs) play a role in the pathogenesis of malaria vivax, according to a study led by researchers from the Barcelona Institute for Global Health (ISGlobal), an institution supported by the ”la Caixa” Foundation, and the Germans Trias i Pujol Research Institute (IGTP). The findings, published in Nature Communications, indicate that EVs from P. vivax patients communicate with spleen fibroblasts promoting the adhesion of parasite-infected red blood cells. These data provide important insights into the pathology of vivax malaria. The study was carried out at the Can Ruti Campus, with the participation of the IGTP Genomics platform, the Nephrology service of the Germans Trias i Pujos Hospital, and researchers from the Irsicaixa AIDS Research Institute.

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This blog originated as a press release from UCSD News.

Researchers at the University of California San Diego discovered that high blood levels of RNA produced by the PHGDH gene could serve as a biomarker for early detection of Alzheimer’s disease. The work could lead to the development of a blood test to identify individuals who will develop the disease years before they show symptoms.

The team published their findings in Current Biology.

The PHGDH gene produces RNA and proteins that are critical for brain development and function in infants, children, and adolescents. As people get older, the gene typically ramps down its production of these RNAs and proteins. The new study, led by Sheng Zhong, a professor of bioengineering at the UC San Diego Jacobs School of Engineering in collaboration with Dr. Edward Koo, a professor of neuroscience at the UC San Diego School of Medicine, suggests that overproduction of extracellular RNA (exRNA) by the PHGDH gene in the elderly could provide an early warning sign of Alzheimer’s disease.

“Several known changes associated with Alzheimer’s disease usually show up around the time of clinical diagnosis, which is a little too late. We had a hunch that there is a molecular predictor that would show up years before, and that’s what motivated this study,” Zhong said.

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