July 2021 ERCC webinar by Steven Jay

ERCC webinar July 1st: Steven Jay from the University of Maryland, College Park

This blog originated as a press release from the Singopore-MIT Alliance for Research and Technology (SMART). Thanks to them for allowing us to repost it here.

Four times faster than conventional PCR methods, a new approach called RADICA is highly specific, sensitive, and resistant to inhibitors.

● RApid DIgital Crispr Approach (RADICA) is a molecular rapid testing methodology that allows absolute quantification of viral nucleic acids in 40-60 minutes.

● RADICA is four times faster and significantly less expensive than conventional polymerase chain reaction (PCR) methods as it does not require costly equipment for precise temperature control and cycling.

● The method has been tested on SARS-CoV-2 synthetic DNA and RNA, Epstein–Barr virus in human B cells and serum, and can be easily adapted to detect other kinds of viruses.


Researchers from Critical Analytics for Manufacturing Personalized-Medicine (CAMP), an Interdisciplinary Research Group (IRG) at the Singapore-MIT Alliance for Research and Technology (SMART), MIT’s research enterprise in Singapore, have developed a new method for rapid and accurate detection of viral nucleic acids – a breakthrough that can be easily adapted to detect different DNA/RNA targets in viruses like the coronavirus.

The pandemic has highlighted the importance of rapid diagnostics and improved methods to detect viruses, especially as the world seeks to be prepared for future pandemics or the next dangerous pathogen. Particularly, the biomanufacturing industry, with the unique challenges of using cells as cell therapy products, is looking for innovations in rapid methods to detect virus contamination as part of their quality control processes and in release testing. While the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered a gold standard for viral detection, there are limitations and they can often produce variable results.

A more accurate version is the digital PCR method that allows absolute quantification – meaning it reveals the copy number of viruses in a sample – can allow for setting clear thresholds of virus contamination, and is not susceptible to potential fluctuations of reference gene required by standard qPCR methods. However, digital PCR demands a relatively long reaction time of around four hours. Another drawback of all current PCR-based methods is that they need expensive equipment for precise temperature control and cycling.

The new methodological development by CAMP – the RApid DIgital Crispr Approach (RADICA) – allows absolute quantification of viral nucleic acids in 40-60 minutes in an isothermal manner in a water bath, a widely available and inexpensive piece of laboratory equipment. The team’s research is explained in a paper titled “Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids” published recently in the journal Biomaterials.

Schematic of RADICA methodSchematic illustration of RADICA. After DNA/RNA extraction, samples can be used for the detection and quantification of various targets. RPA-Cas12a reaction in each positive partition exponentially amplifies target DNA, triggering more Cas12a activation and increasing the fluorescence readout. In this way, the reaction time of RADICA is reduced 4 times (from 4h to 1h compared to dPCR). Credit: Singapore-MIT Alliance for Research and Technology (SMART)

The RADICA method has been tested on SARS-CoV-2 synthetic DNA/RNA as well as the Epstein-Barr virus in cultured B cells and patient serum. The researchers say the method can be adapted to detect other kinds of viruses, and in other types of samples such as saliva and cell culture media. RADICA is also able to distinguish the virus from their close relatives.

“This is the first reported method of detecting nucleic acids to utilise the sensitivity of isothermal amplification and specificity of CRISPR based detection in a digital format – allowing rapid and specific amplification of DNA without the time consuming and costly need for thermal cycling,” says Dr Xiaolin Wu, Postdoctoral Associate at SMART CAMP. “RADICA offers four times faster absolute quantification compared to conventional digital PCR methods.”

The team uses extracted DNA/RNA of the sample and divides a 15 µL reaction into thousands of independent partitions. In each partition, the DNA/RNA is amplified and identified by Cas12a protein, an enzyme that can turn the target signal into a fluorescent signal. This allows absolute quantification to be achieved by counting the number of partitions that have the target DNA/RNA and are lit up.

“The last year has shown us the importance of detecting viruses quickly and accurately, and RADICA can help fill existing gaps in this area,” says National University of Singapore Professor Hanry Yu, co-corresponding author and Co-Lead Principal Investigator at SMART CAMP. “Cell therapy products have a very short shelf life and patients are usually in need of treatment urgently. Current sterility tests need around 14 days, which is too slow for clinical needs but RADICA shortens the process into hours.”

Professor Tim Lu, who is co-corresponding author, CAMP Principal Investigator and Associate Professor of Biological Engineering and Electrical Engineering and Computer Science at Massachusetts Institute of Technology, said the team’s method is faster, cheaper and more efficient than what is used today, and its digital format makes it more tolerant to contamination or inhibitors that may be present in biological samples – often the case with cell therapy products. Professor Lu adds that on top of detecting the presence of a target virus, RADICA also identifies how many viruses there are in the sample which can help doctors and researchers in deciding the course of treatment, as well as production and inventory management of cell therapy products.

While the researchers at CAMP developed RADICA for monitoring cell therapy manufacturing processes and biosafety release testing of cell therapy products, Dr Wu says the method can also be used to detect DNA/RNA targets of different viruses and adapted to devices commonly found in hospitals and service laboratories – providing a potential new way to tackle pandemics.

The research is carried out by SMART and supported by the National Research Foundation (NRF) Singapore under its Campus for Research Excellence And Technological Enterprise (CREATE) programme.

Reference
Wu X. et al., Digital CRISPR-based method for the rapid detection and absolute quantification of nucleic acids. (2021) Biomaterials 274: 120876. doi: 10.1016/j.biomaterials.2021.120876 PMID: 34034027.

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.