Effective Insertion Of DNA Molecules Into Cells For Gene Therapies

For years, researchers have attempted to harness the full potential of gene therapy, a technique that inserts genes into a patient’s cells to treat aggressive diseases such as cancer. But getting engineered DNA molecules into cells is not an easy task.

J. Mark Meacham, assistant professor of mechanical engineering & materials science at Washington University in St. Louis, leads a team of researchers that has developed a method enabling effective insertion of large molecules — such as DNA, RNA and proteins into cells and propels them into the cell nucleus. By combining a technique known as Acoustic Shear Poration (ASP) with electrophoresis, the approach uses ultrasound waves and focused mechanical force to create nanoscale holes, or pores, in the cell membrane that are big enough for large macromolecules or nanoparticles to pass into the cell’s interior.

Operation of the acoustic shear poration (ASP) device in Meacham’s lab

The researchers wrote that so far, ASP has achieved greater than 75 percent delivery efficiency of macromolecules. DNA insertion, or transfection, which is of most interest in gene therapy, is significantly more challenging. Yet the combined application of mechanical and electrical forces pioneered by Meacham and colleagues yields roughly 100 percent improvement in transfection versus pure mechanoporation. Results of the research are published in Scientific Reports.

Source: https://engineering.wustl.edu/

How To Detect Cancer With a Urine Test

Researchers centered at Nagoya University (Japan) develop a nanowire device able to detect microscopic levels of urinary markers potentially implicated in cancerCells communicate with each other through a number of different mechanisms. Some of these mechanisms are well-known: in animals, for example, predatory threats can drive the release of norepinephrine, a hormone that travels through the bloodstream and triggers heart and muscle cells to initiate a “fight-or-flight” response. A far less familiar mode of cellular transport is the extracellular vesicle (EV). EVs can be thought of as small “chunks” of a cell that are able to pinch off and circulate throughout the body to deliver messenger cargo to other cells. These messengers have become increasingly recognized as crucial mediators of cell-to-cell communication.

In a new study reported in Science Advances, researchers centered at Nagoya University have developed a novel medical device that can efficiently capture these EVs, and potentially use them to screen for cancer.


EVs are potentially useful as clinical markers. The composition of the molecules contained in an EV may provide a diagnostic signature for certain diseases,” lead author Takao Yasui explains. “The ongoing challenge for physicians in any field is to find a non-invasive diagnostic tool that allows them to monitor their patients on a regular basis–for example, a simple urine test.”

Among the many molecules EVs have been found to harbor are microRNAs, which are short pieces of ribonucleic acid that play diverse roles in normal cellular biology. Critically, the presence of certain microRNAs in urine might serve as a red flag for serious conditions such as bladder and prostate cancer. While this important cargo could therefore theoretically aid physicians in cancer diagnoses, there are still many technological hurdles that need to be overcome. One such hurdle: finding a feasible method to capture EVs in sufficient quantities to analyze them in a routine clinical setting.

The content of EVs in urine is extremely low, at less than 0.01% of the total fluid volume. This is a major barrier to their diagnostic utility,” Yasui notes. “Our solution was to embed zinc oxide nanowires into a specialized polymer to create a material that we believed would be highly efficient at capturing these vesicles. Our findings suggest that the device is indeed quite efficient. We obtained a collection rate of over 99%, surpassing ultracentrifugation as well as other methods that are currently being used in the field.

Source: http://en.nagoya-u.ac.jp/

Swiss Army Knife NanoVaccine To Fight Tumors

Scientists are using their increasing knowledge of the complex interaction between cancer and the immune system to engineer increasingly potent anti-cancer vaccines.
Now researchers at the National Institute ofBiomedical Imaging and Bioengineering (NIBIB) have developed a synergistic nanovaccine packing DNA and RNA sequences that modulate the immune response, along with anti-tumor antigens, into one smallnanoparticle. The nanovaccine produced an immune response that specifically killed tumor tissue, while simultaneously inhibiting tumor-induced immune suppression. Together this blocked lung tumor growth in a mouse model of metastatic colon cancer.

Large particles (left) containing the DNA and RNA components are coated with electronically charged molecules that shrink the particle. The tumor-specific neoantigen is then complexed with the surface to complete construction of the nanovaccine.
Upper left: electron micrograph of large particle


The molecular dance between cancer and the immune system is a complex one and scientists continue to identify the specific molecular pathways that rev up or tamp down the immune system. Biomedical engineers are using this knowledge to create nanoparticles that can carry different molecular agents that target these pathways. The goal is to simultaneously stimulate the immune system to specifically attack the tumor while also inhibiting the suppression of the immune system, which often occurs in cancer patients. The aim is to press on the gas pedal of the immune system while also releasing the emergency brake.

A key hurdle is to design a system to reproducibly and efficiently create a nanoparticle loaded with multiple agents that synergize to mount an enhanced immune attack on the tumor. Engineers at the NIBIB report the development and testing of such a nanovaccine in the journal Nature Communications.

Source: https://www.nibib.nih.gov/

Editing Genes In Human Embryos

Two new CRISPR tools overcome the scariest parts of gene editing.The ability to edit RNA and individual DNA base pairs will make gene editing much more precise. Several years ago, scientists discovered a technique known as CRISPR/Cas9, which allowed them to edit DNA more efficiently than ever before.
Since then, CRISPR science has exploded; it’s become one of the most exciting and fast-moving areas of research, transforming everything from medicine to agriculture and energy. In 2017 alone, more than 14,000 CRISPR studies were published.

But here’s the thing: CRISPR, while a major leap forward in gene editing, can still be a blunt instrument. There have been problems with CRISPR modifying unintended gene targets and making worrisome, and permanent, edits to an organism’s genome. These changes could be passed down through generations, which has raised the stakes of CRISPR experiments — and the twin specters of “designer babies” and genetic performance enhancers — particularly when it comes to editing genes in human embryos.
So while CRISPR science is advancing quickly, scientists are still very much in the throes of tweaking and refining their toolkit. And on Wednesday, researchers at the Broad Institute of MIT and Harvard launched a coordinated blitz with two big reports that move CRISPR in that safer and more precise direction.
In a paper published in Science, researchers described an entirely new CRISPR-based gene editing tool that targets RNA, DNA’s sister, allowing for transient changes to genetic material. In Nature, scientists described how a more refined type of CRISPR gene editing can alter a single bit of DNA without cutting it — increasing the tool’s precision and efficiency.

The first paper, out Wednesday in Science, describes a new gene editing system. This one, from researchers at MIT and Harvard, focuses on tweaking human RNA instead of DNA.

Our cells contain chromosomes made up of chemical strands called DNA, which carry genetic information. Those genes have recipes for proteins that lead to a bunch of different traits. But to carry out the instructions in any one recipe, DNA needs another type of genetic material called RNA to get involved.

RNA is ephemeral: It acts like a middleman, or a messenger. For a gene to become a protein, that gene has to be transcribed into RNA in the cell, and the RNA is then read to make the protein. If the DNA is permanent — the family recipe book passed down through generations — the RNA is like your aunt’s scribbled-out recipe on a Post-It note, turning up only when it’s needed and disappearing again.

With the CRISPR/Cas9 system, researchers are focused on editing DNA. (For more on how that system works, read this Vox explainer.) But the new Science paper describes a novel gene editing tool called REPAIR that’s focused on using a different enzyme, Cas13, to edit that transient genetic material, the RNA, in cells. REPAIR can target specific RNA letters, or nucleosides, that are involved in single-base changes that regularly cause disease in humans.

This is hugely appealing for one big reason: With CRISPR/Cas9, the changes to the genome, or the cell’s recipe book, are permanent. You can’t undo them. With REPAIR, since researchers can target single bits of ephemeral RNA, the changes they make are transient, even reversible. So this system could fix genetic mutations without actually touching the genome (like throwing away your aunt’s Post-It note recipe without adding it to the family recipe book).

Source: https://www.vox.com/

Faulty DNA Linked To Fatal Heart Condition Removed From Embryo

Scientists have modified human embryos to remove genetic mutations that cause heart failure in otherwise healthy young people in a landmark demonstration of the controversial procedure. It is the first time that human embryos have had their genomes edited outside China, where researchers have performed a handful of small studies to see whether the approach could prevent inherited diseases from being passed on from one generation to the next.

While none of the research so far has created babies from modified embryos, a move that would be illegal in many countries, the work represents a milestone in scientists’ efforts to master the technique and brings the prospect of human clinical trials one step closer. The work focused on an inherited form of heart disease, but scientists believe the same approach could work for other conditions caused by single gene mutations, such as cystic fibrosis and certain kinds of breast cancer.

This embryo gene correction method, if proven safe, can potentially be used to prevent transmission of genetic disease to future generations,” said Paula Amato, a fertility specialist involved in the US-Korean study at Oregon Health and Science University.

The scientists used a powerful gene editing tool called Crispr-Cas9 to fix mutations in embryos made with the sperm of a man who inherited a heart condition known as hypertrophic cardiomyopathy, or HCM. The disease, which leads to a thickening of the heart’s muscular wall, affects one in 500 people and is a common cause of sudden cardiac arrest in young people. Humans have two copies of every gene, but some diseases are caused by a mutation in only one of the copies. For the study, the scientists recruited a man who carried a single mutant copy of a gene called MYBPC3 which causes HCM.

Source: https://www.theguardian.com/

How To Capture Quickly Cancer Markers

A nanoscale product of human cells that was once considered junk is now known to play an important role in intercellular communication and in many disease processes, including cancer metastasis. Researchers at Penn State have developed nanoprobes to rapidly isolate these rare markers, called extracellular vesicles (EVs), for potential development of precision cancer diagnoses and personalized anticancer treatments.

Lipid nanoprobes

Most cells generate and secrete extracellular vesicles,” says Siyang Zheng, associate professor of biomedical engineering and electrical engineering. “But they are difficult for us to study. They are sub-micrometer particles, so we really need an electron microscope to see them. There are many technical challenges in the isolation of nanoscale EVs that we are trying to overcome for point-of-care cancer diagnostics.”

At one time, researchers believed that EVs were little more than garbage bags that were tossed out by cells. More recently, they have come to understand that these tiny fat-enclosed sacks — lipids — contain double-stranded DNA, RNA and proteins that are responsible for communicating between cells and can carry markers for their origin cells, including tumor cells. In the case of cancer, at least one function for EVs is to prepare distant tissue for metastasis.

The team’s initial challenge was to develop a method to isolate and purify EVs in blood samples that contain multiple other components. The use of liquid biopsy, or blood testing, for cancer diagnosis is a recent development that offers benefits over traditional biopsy, which requires removing a tumor or sticking a needle into a tumor to extract cancer cells. For lung cancer or brain cancers, such invasive techniques are difficult, expensive and can be painful.

Noninvasive techniques such as liquid biopsy are preferable for not only detection and discovery, but also for monitoring treatment,” explains Chandra Belani, professor of medicine and deputy director of the Cancer Institute,Penn State College of Medicine, and clinical collaborator on the study.

We invented a system of two micro/nano materials,” adds Zheng. “One is a labeling probe with two lipid tails that spontaneously insert into the lipid surface of the extracellular vesicle. At the other end of the probe we have a biotin molecule that will be recognized by an avidin molecule we have attached to a magnetic bead.”

Source: http://news.psu.edu/

Osteoarthritis: NanoParticles Stop Destruction Of Cartilage

Osteoarthritis is a debilitating condition that affects at least 27 million people in the United States, and at least 12 percent of osteoarthritis cases stem from earlier injuries. Over-the-counter painkillers, such as anti-inflammatory drugs, help reduce pain but do not stop unrelenting cartilage destruction. Consequently, pain related to the condition only gets worse. Now, researchers at Washington University School of Medicine in St. Louis have shown in mice that they can inject nanoparticles into an injured joint and suppress inflammation immediately following an injury, reducing the destruction of cartilage.

osteoarthritisResearchers at Washington University School of Medicine in St. Louis have found that injecting nanoparticles into an injured joint can inhibit the inflammation that contributes to the cartilage damage seen in osteoarthritis. Shown in green is an inflammatory protein in cartilage cells. After nanoparticles are injected, the inflammation is greatly reduced


I see a lot of patients with osteoarthritis, and there’s really no treatment,” said senior author Christine Pham, MD, an associate professor of medicine. “We try to treat their symptoms, but even when we inject steroids into an arthritic joint, the drug only remains for up to a few hours, and then it’s cleared. These nanoparticles remain.

Frequently, an osteoarthritis patient has suffered an earlier injury — a torn meniscus or ACL injury in the knee, a fall, car accident or other trauma. The body naturally responds to such injuries in the joints with robust inflammation. Patients typically take drugs such as acetaminophen and ibuprofen, and as pain gets worse, injections of steroids also can provide pain relief, but their effects are short-lived.

In this study, the nanoparticles were injected shortly after an injury, and within 24 hours, the nanoparticles were at work taming inflammation in the joint. But unlike steroid injections that are quickly cleared, the particles remained in cartilage cells in the joints for weeks.

The nanoparticles used in the study are more than 10 times smaller than a red blood cell, which helps them penetrate deeply into tissues. The particles carry a peptide derived from a natural protein called melittin that has been modified to enable it to bind to a molecule called small interfering RNA (siRNA). The melittin delivers siRNA to the damaged joint, interfering with inflammation in cells.

Source: https://source.wustl.edu/

Vaccine That Is Programmable In One Week

MIT engineers have developed a new type of easily customizable vaccine that can be manufactured in one week, allowing it to be rapidly deployed in response to disease outbreaks. So far, they have designed vaccines against Ebola, H1N1 influenza, and Toxoplasma gondii (a relative of the parasite that causes malaria), which were 100 percent effective in tests in mice. The vaccine consists of strands of genetic material known as messenger RNA, which can be designed to code for any viral, bacterial, or parasitic protein. These molecules are then packaged into a molecule that delivers the RNA into cells, where it is translated into proteins that provoke an immune response from the host.

In addition to targeting infectious diseases, the researchers are using this approach to create cancer vaccines that would teach the immune system to recognize and destroy tumors.

MIT-Program-Vaccines_0 (1)

This nanoformulation approach allows us to make vaccines against new diseases in only seven days, allowing the potential to deal with sudden outbreaks or make rapid modifications and improvements,” says Daniel Anderson, an associate professor in MIT’s Department of Chemical Engineering and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES).

Anderson is the senior author of a paper describing the new vaccines in the Proceedings of the National Academy of Sciences. The project was led by Jasdave Chahal, a postdoc at MIT’s Whitehead Institute for Biomedical Research, and Omar Khan, a postdoc at the Koch Institute; both are the first authors of the paper.

Source: http://news.mit.edu/

How To Map RNA Molecules In The Brain

Cells contain thousands of messenger RNA molecules, which carry copies of DNA’s genetic instructions to the rest of the cell. MIT engineers have now developed a way to visualize these molecules in higher resolution than previously possible in intact tissues, allowing researchers to precisely map the location of RNA throughout cells. Key to the new technique is expanding the tissue before imaging it. By making the sample physically larger, it can be imaged with very high resolution using ordinary microscopes commonly found in research labs.

MIT RNA-Imaging

Now we can image RNA with great spatial precision, thanks to the expansion process, and we also can do it more easily in large intact tissues,” says Ed Boyden, an associate professor of biological engineering and brain and cognitive sciences at MIT, a member of MIT’s Media Lab and McGovern Institute for Brain Research, and the senior author of a paper describing the technique in the July 4 issue of Nature Methods.

Studying the distribution of RNA inside cells could help scientists learn more about how cells control their gene expression and could also allow them to investigate diseases thought to be caused by failure of RNA to move to the correct location.

Source: http://news.mit.edu/

Blood Test Can Diagnose Pancreatic Cancer

Indiana University cancer researchers have found that a simple blood test might help diagnose pancreatic cancer, one of the most deadly forms of the disease.

In research published today in the American Journal of Gastroenterology, Murray Korc, M.D., Professor of Cancer Research at the Indiana University School of Medicine and a researcher at the Indiana University Melvin and Bren Simon Cancer Center, and colleagues found that several microRNAs – small RNA molecules — circulate at high levels in the blood of pancreatic cancer patients.

blood cells

This is a new finding that extends previous knowledge in this field,” Dr. Korc said. “The key new feature here is that there is a panel of microRNAs that can be measured accurately in the plasma component of blood to determine if a patient has pancreatic cancer.”

Specifically, the IU research team found that an increased expression of miRNA-10b, -155, and -106b in plasma appears highly accurate in diagnosing pancreatic ductal adenocarcinoma. Pancreatic ductal adenocarcinoma is by far the most common type of pancreatic malignancy.

Source: http://news.medicine.iu.edu/

How To Heal Diabetic Skin Wounds

A new high-tech but simple ointment applied to the skin may one day help diabetic patients heal stubborn and painful ulcers on their feet, Northwestern University researchers report.

Scientist and dermatologist Amy S. Paller and chemist Chad A. Mirkin are the first to develop a topical gene regulation technology that speeds the healing of ulcers in diabetic animals. They combined spherical nucleic acids (SNAs, which are nanoscale globular forms of RNA) with a common commercial moisturizer to create a way to topically knock down a gene known to interfere with wound healing.

Type 2 diabetes and its enormous associated costs are on the rise in the United States. More than one-fifth of the 27 million type 2 diabetics in the country have chronic, non-healing skin wounds, and many undergo amputation. The Northwestern discovery offers a possible solution to this serious problem.
Finding a new way to effectively heal these resistant diabetic wounds is very exciting,” said Dr. Paller, director of Northwestern’s Skin Disease Research Center. “But, in addition, this study further proved that SNAs — in nothing but common moisturizer — can penetrate the skin barrier, a challenge that other therapies have been unable to conquer.

Source: http://www.northwestern.edu/

Cell Reprogramming

In 1953 Watson and Crick first published the discovery of the double helix structure of the DNA. They were able to visualize the DNA structure by means of X-Ray diffraction. Techniques, such as electron microscopy, allowed scientists to identify nucleosomes, the first and most basic level of chromosome organisation. Until now it was known that our DNA is packaged by regular repeating units of those nucleosomes throughout the genome giving rise to chromatin. However, due to the lack of suitable techniques and instruments, the chromatin organisation inside a cell nucleus could not be observed in a non-invasive way with the sufficient resolution. Now, for the first time, a group of scientists at the Center for Genomic Regulation CRG and ICFO in Barcelona (Spain), have been able to visualise and even count the smallest units which, packaged together, form our genome. This study was possible thanks to the use of super-resolution microscopy, a new cutting-edge optical techniquethat received the Nobel Prize in Chemistry in 2014. In combination with innovative quantitative approaches and numerical simulations, they were also able to define the genome architecture at the nano-scale. Most importantly, they found that the nucleosomes are assembled in irregular groups across the chromatin and nucleosome-free-DNA regions separate these groups.
Genome Sequencing

By using the STORM technique, a new super-resolution microscopy method, we have been able to view and even count nucleosomes across the chromatin fibers and determine their organisation. STORM overcomes the diffraction limit that normally restricts the spatial resolution of conventional microscopes and enables us to precisely define the chromatin fibre structure”, states Prof. Melike Lakadamyali, group leader at ICFO.This enabling technique allowed the researchers to go deeper and, by comparing stem cells to Differentiated cells (specialised cells that have already acquired their role), they observed key differences in the chromatin fibre architectures of both cells.

We found that stem cells have a different chromatin structure than somatic (specialised) cells. At the same time, this difference correlates with the level of pluripotency. The more pluripotent a cell is, the less dense is its packaging. It gives us new clues to understand the stem cells functioning and their genomic structure, which will be helpful for example, in studying cell reprogramming”, explains Pia Cosma, group leader and ICREA research professor at the CRG.
Source: http://www.crg.eu/en/