Cellular healthcare is an approach that goes beyond clinical laboratory testing to identify the location of specific cancer cells and aid in treatment decisions
Advances in synthetic biology and genetic engineering are leading to development of bacterial biosensors that could eventually aid pathologists and clinical laboratories in diagnosis of many types of cancers.
One recent example comes from researchers at the University of California San Diego (UCSD) who worked with colleagues in Australia to engineer bacteria that work as “capture agents” and bind to tumorous material.
The KRAS gene is associated with colorectal cancer. The researchers named their development the Cellular Assay for Targeted CRISPR-discriminated Horizontal gene transfer (CATCH).
CATCH successfully detected cancer in the colons of mice. The researchers believe it could be used to diagnose cancers, as well as infections and other diseases, in humans as well, according to a UCSD news release.
“If bacteria can take up DNA, and cancer is defined genetically by a change in its DNA, then, theoretically, bacteria could be engineered to detect cancer,” gastroenterologist Daniel Worthley, PhD, a cancer researcher at Colonoscopy Clinic in Brisbane, Australia, told MedicalResearch.com. This research could eventually provide clinical laboratories and anatomic pathologists with new tools to use in diagnosing certain types of cancer. (Photo copyright: Colonoscopy Clinic.)
Tapping Bacteria’s Natural Competence
In their Science paper, the researchers acknowledged other synthetic biology achievements in cellular biosensors aimed at human disease. But they noted that more can be done by leveraging the “natural competence” skill of bacteria.
“Biosensors have not yet been engineered to detect specific extracellular DNA sequences and mutations. Here, we engineered naturally competent Acinetobacter baylyi (A. baylyi) to detect donor DNA from the genomes of colorectal cancer cells, organoids, and tumors,” they wrote.
“Many bacteria can take up DNA from their environment, a skill known as natural competence,” said Rob Cooper, PhD, co-first author of the study and a scientist at US San Diego’s Synthetic Biology Institute, in the news release. A. baylyi is a type of bacteria renowned for success in doing just that, the NCI article pointed out.
This enabled them to explore “free-floating DNA sequences on a genomic level.”
Those sequences were compared to “known cancer DNA sequences.”
A. baylyi (genetically modified) was tested on its ability to detect “mutated and healthy KRAS DNA.”
Only bacteria that had “taken up mutated copies of KRAS … would survive treatment with a specific drug.”
“It was incredible when I saw the bacteria that had taken up the tumor DNA under the microscope. The mice with tumors grew green bacterial colonies that had acquired the ability to be grown on antibiotic plates,” said Josephine Wright, PhD, Senior Research Fellow, Gut Cancer Group, South Australian Health and Medical Research Institute (SAHMRI), in the news release.
Detecting DNA from Cancer Cells In Vitro and in Mice
Findings in vitro and in mice include the following:
The engineered bacteria enabled detection of DNA with KRAS G12D from colorectal cancer cells made in the lab, NCI reported.
When mice were injected with colorectal cancer cells, the researchers’ technology found tumor DNA, Engadget reported.
The study adds to existing knowledge of horizontal gene transfer from bacteria to bacteria, according to UCSD.
“We observed horizontal gene transfer from the tumor to the sensor bacteria in our mouse model of colorectal cancer. This cellular assay for targeted, CRISPR-discriminated horizontal gene transfer (CATCH) enables the biodetection of specific cell-free DNA,” the authors wrote in Science.
“Colorectal cancer seemed a logical proof of concept as the colorectal lumen is full of microbes and, in the setting of cancer, full of tumor DNA,” gastroenterologist Daniel Worthley, PhD, a cancer researcher at Colonoscopy Clinic in Brisbane, Australia, told MedicalResearch.com.
Finding More Cancers and Treatment
More research is needed before CATCH is used in clinical settings. The scientists are reportedly planning on adapting CATCH to multiple bacteria that can locate other cancers and infections.
“The most exciting aspect of cellular healthcare … is not in the mere detection of disease. A laboratory can do that,” wrote Worthley in The Conversation. “But what a laboratory cannot do is pair the detection of disease (a diagnosis) with the cells actually responding to the disease [and] with appropriate treatment.
“This means biosensors can be programmed so that a disease signal—in this case, a specific sequence of cell-free DNA—could trigger a specific biological therapy, directly at the spot where the disease is detected in real time,” he added.
Clinical laboratory scientists, pathologists, and microbiologists may want to stay abreast of how the team adapts CATCH, and how bacterial biosensors in general continue to develop to aid diagnosis of diseases and improve ways to target treatment.
CRISPR-Act 3.0 could significantly increase crop yields and plant diversity worldwide and help fight against global hunger and climate change
Clinical laboratory professionals and pathologists who read Dark Daily are highly aware of CRISPR gene editing technology. We’ve covered the topic in multiple ebriefings over many years. But how many know there’s a version of CRISPR specifically designed for editing and activating plant genes?
Scientists at the University of Maryland (UMD) developed a new version of CRISPRa (CRISPR Activation) for plants which they claim has four to six times the activation capacity of currently available CRISPRa systems and can activate up to seven genes at once. They call their new and improved CRISPRa technology “CRISPR-Act 3.0.”
According to a paper published in the journal Nature Plants, titled, “CRISPR-Act3.0 for Highly Efficient Multiplexed Gene Activation in Plants,” the UMD researchers developed “a highly robust CRISPRa system working in rice, Arabidopsis (rockcress), and tomato, CRISPR-Act 3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenes Cas9 (dSpCas9).
“While my lab has produced systems for simultaneous gene editing [multiplexed editing] before, editing is mostly about generating loss of function to improve the crop,” said Yiping Qi, PhD (above), one of the authors of the UMD study, in a new release. “But if you think about it,” he added, “that strategy is finite, because there aren’t endless genes that you can turn off and actually still gain something valuable. Logically, it is a very limited way to engineer and breed better traits, whereas the plant may have already evolved to have different pathways, defense mechanisms, and traits that just need a boost.” (Photo copyright: University of Maryland.)
CRISPR-Act 3.0 Increases Function of Multiple Genes Simultaneously
The UMD researchers successfully applied CRISPR-Act 3.0 technology to activate many types of genes in plants, including the ability to expedite the breeding process via faster flowering. They hope that activating genes in plants to improve functionality will result in better plants and crops.
“Through activation, you can really uplift pathways or enhance existing capacity, even achieve a novel function. Instead of shutting things down, you can take advantage of the functionality already there in the genome and enhance what you know is useful,” said Yiping Qi, PhD, associate professor, Department of Plant Science and Landscape Architecture at the University of Maryland, in a UMD new release.
The scientists also noted that there may be other advantages to this type of multiplexed activation of genes.
“Having a much more streamlined process for multiplexed activation can provide significant breakthroughs. For example, we look forward to using this technology to screen the genome more effectively and efficiently for genes that can help in the fight against climate change and global hunger,” Qi added. “We can design, tailor, and track gene activation with this new system on a larger scale to screen for genes of importance, and that will be very enabling for discovery and translational science in plants.”
The researchers hope this technology can have a major impact on the efficiency of crop and food production.
“This type of technology helps increase crop yield and sustainably feed a growing population in a changing world,” Qi said. “I am very pleased to continue to expand the impacts of CRISPR technologies.”
Feeding the World’s Hungry with CRISPR
CRISPR is a robust tool used for editing genomes that typically operates as “molecular scissors” to cut DNA. CRISPR-Act 3.0, however, uses deactivated CRISPR-Cas9 which can only bind and not cut. This allows the system to work on the activation of proteins for designated genes of interest by binding to certain segments of DNA. The UMD researchers believe there is significant potential for expanding the multiplexed activation further, which could alter and improve genome engineering.
“People always talk about how individuals have potential if you can nurture and promote their natural talents,” Qi said in the UMD news release. “This technology is exciting to me because we are promoting the same thing in plants—how can you promote their potential to help plants do more with their natural capabilities? That is what multiplexed gene activation can do, and it gives us so many new opportunities for crop breeding and enhancement.”
CRISPR is being developed and enhanced in many research settings, and knowledge of how to best use the gene editing technology is rapidly advancing. Though more research on CRISPR-Act 3.0 is needed to ensure its reliability, it’s exciting to consider the potential of gene activation for massively increasing crop yield worldwide.
Not to mention how new CRISPR technologies continue to drive innovations in clinical laboratory diagnostics and precision medicine treatments.
VCU scientists used the technique to measure mutations associated with acute myeloid leukemia, potentially offering an attractive alternative to DNA sequencing
More accurate but less-costly cancer diagnostics are the Holy Grail of cancer research. Now, research scientists at Virginia Commonwealth University (VCU) say they have developed a clinical laboratory diagnostic technique that could be far cheaper and more capable than standard DNA sequencing in diagnosing some diseases. Their method combines digital polymerase chain reaction (dPCR) technology with high-speed atomic force microscopy (HS-AFM) to generate nanoscale-resolution images of DNA.
The technique allows the researchers to measure polymorphisms—variations in gene lengths—that are associated with many cancers and neurological diseases. The VCU scientists say the new technique costs less than $1 to scan each dPCR reaction.
“We chose to focus on FLT3 mutations because they are difficult to [diagnose], and the standard assay is limited in capability,” said physicist Jason Reed, PhD, Assistant Professor in the Virginia Commonwealth University Department of Physics, in a VCU press release.
Reed is an expert in nanotechnology as it relates to biology and medicine. He led a team that included other researchers in VCU’s physics department as well as physicians from VCU Massey Cancer Center and the Department of Internal Medicine at VCU School of Medicine.
“The technology needed to detect DNA sequence rearrangements is expensive and limited in availability, yet medicine increasingly relies on the information it provides to accurately diagnose and treat cancers and many other diseases,” said Jason Reed, PhD (above center, with Andrey Mikheikin, PhD, on left and Sean Koebley, PhD, on right), in a press release from Virginia Commonwealth University (VCU). “We’ve developed a system that combines a routine laboratory process with an inexpensive yet powerful atomic microscope that provides many benefits over standard DNA sequencing for this application, at a fraction of the cost.” (Photo copyright: Virginia Commonwealth University.)
Validating the Clinical Laboratory Test
The physicists worked with two VCU physicians—hematologist/oncologist Amir Toor, MD, and hematopathologist Alden Chesney, MD—to compare the imaging technique to the LeukoStrat CDx FLT3 Mutation Assay, which they described as the “current gold standard test” for diagnosing FLT3 gene mutations.
The researchers said their technique matched the results of the LeukoStrat test in diagnosing the mutations. But unlike that test, the new technique also can measure variant allele frequency (VAL). This “can show whether the mutation is inherited and allows the detection of mutations that could potentially be missed by the current test,” states the VCU press release.
“We plan to continue developing and testing this technology in other diseases involving DNA structural mutations,” Reed said. “We hope it can be a powerful and cost-effective tool for doctors around the world treating cancer and other devastating diseases driven by DNA mutations.”
“In our approach we first used digital PCR, in which a mixed sample is diluted to less than one target molecule per aliquot and the aliquots are amplified to yield homogeneous populations of amplicons,” he said. “Then, we deposited each population onto an atomically-flat partitioned surface.”
The VCU researchers “scanned each partition with high-speed atomic force microscopy, in which an extremely sharp tip is rastered across the surface, returning a 3D map of the surface with nanoscale resolution,” he said. “We wrote code that traced the length of each imaged DNA molecule, and the distribution of lengths was used to determine whether the aliquot was a wild type [unmutated] or variant.”
In Diagnostics World, Reed said the method “doesn’t really have any more complexity than a PCR assay itself. It can easily be done by most lab technicians.”
Earlier Research
A VCU press release from 2017 noted that Reed’s research team had developed technology that uses optical lasers (similar to those in a DVD player) to accelerate the scanning. The researchers previously published a study about the technique in Nature Communications, and a patent is currently pending.
“DNA sequencing is a powerful tool, but it is still quite expensive and has several technological and functional limitations that make it difficult to map large areas of the genome efficiently and accurately,” Reed said in the 2017 VCU press release. “Our approach bridges the gap between DNA sequencing and other physical mapping techniques that lack resolution. It can be used as a stand-alone method or it can complement DNA sequencing by reducing complexity and error when piecing together the small bits of genome analyzed during the sequencing process.”
Using CRISPR technology, the team also developed what they described as a “chemical barcoding solution,” placing markers on DNA molecules to identify genetic mutations.
New DNA Clinical Laboratory Testing?
Cancer diagnostics are constantly evolving and improving. It is not clear how long it will be before VCU’s new technique will reach clinical laboratories that perform DNA testing, if at all. But VCU’s new technique is intriguing, and should it prove viable for clinical diagnostic use it could revolutionize cancer diagnosis. It is a development worth watching.
‘Prime editing’ is what researchers are calling the proof-of-concept research that promises improved diagnostics and more effective treatments for patients with genetic defects
Known as Prime Editing, the scientists developed this technique as a more accurate way to edit Deoxyribonucleic acid (DNA). In a paper published in Nature, the authors claim prime editing has the potential to correct up to 89% of disease-causing genetic variations. They also claim prime editing is more powerful, precise, and flexible than CRISPR.
The research paper describes prime editing as a “versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit.”
And a Harvard Gazette article states, “Prime editing differs from previous genome-editing systems in that it uses RNA to direct the insertion of new DNA sequences in human cells.”
Assuming further research and clinical studies confirm the
viability of this technology, clinical laboratories would have a new diagnostic
service line that could become a significant proportion of a lab’s specimen
volume and test mix.
In that e-briefing we wrote that Liu “has led a team of scientists in the development of a gene-editing protein delivery system that uses cationic lipids and works on animal and human cells. The new delivery method is as effective as protein delivery via DNA and has significantly higher specificity. If developed, this technology could open the door to routine use of genome analysis, worked up by the clinical laboratory, as one element in therapeutic decision-making.”
Now, Liu has taken that development even further.
“A major aspiration in the molecular life sciences is the ability to precisely make any change to the genome in any location. We think prime editing brings us closer to that goal,” David Liu, PhD (above), Director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, told The Harvard Gazette. “We’re not aware of another editing technology in mammalian cells that offers this level of versatility and precision with so few byproducts.” (Photo copyright: Broad Institute.)
Cell Division Not Necessary
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is considered the most advanced gene editing technology available. However, it has one drawback not found in Prime Editing—CRISPR relies on a cell’s ability to divide to generate desired alterations in DNA—prime editing does not.
This means prime editing could be used to repair genetic mutations in cells that do not always divide, such as cells in the human nervous system. Another advantage of prime editing is that it does not cut both strands of the DNA double helix. This lowers the risk of making unintended, potentially dangerous changes to a patient’s DNA.
The researchers claim prime editing can eradicate long lengths of disease-causing DNA and insert curative DNA to repair dangerous mutations. These feats, they say, can be accomplished without triggering genome responses introduced by other forms of CRISPR that may be potentially harmful.
“Prime editors are more like word processors capable of
searching for targeted DNA sequences and precisely replacing them with edited
DNA strands,” Liu told NPR.
The scientists involved in the study have used prime editing to perform over 175 edits in human cells. In the test lab, they have succeeded in repairing genetic mutations that cause both Sickle Cell Anemia (SCA) and Tay-Sachs disease, NPR reported.
“Prime editing is really a step—and potentially a significant step—towards this long-term aspiration of the field in which we are trying to be able to make just about any kind of DNA change that anyone wants at just about any site in the human genome,” Liu told News Medical.
Additional Research Required, but Results are Promising
Prime editing is very new and warrants further
investigation. The researchers plan to continue their work on the technology by
performing additional testing and exploring delivery mechanisms that could lead
to human therapeutic applications.
“Prime editing should be tested and optimized in as many cell types as researchers are interested in editing. Our initial study showed prime editing in four human cancer cell lines, as well as in post-mitotic primary mouse cortical neurons,” Liu told STAT. “The efficiency of prime editing varied quite a bit across these cell types, so illuminating the cell-type and cell-state determinants of prime editing outcomes is one focus of our current efforts.”
Although further research and clinical studies are needed to
confirm the viability of prime editing, clinical laboratories could benefit
from this technology. It’s worth watching.
With $191 million in startup capital, the genomics startup will draw on existing genetic databases to create personalized medicine therapies for chronic diseases
Why do some people get sick while others do not? That’s what genetic researchers at Maze Therapeutics want to find out. They have developed a new approach to using tools such as CRISPR gene editing to identify and manipulate proteins in genetic code that may be the key to providing personalized protection against specific diseases.
If viable, the results of Maze’s research could mean the development of specific drugs designed to mimic genetic code in a way that is uniquely therapeutic to specific patients. This also would create the need for clinical laboratories to sequence and analyze patients’ DNA to determine whether a patient would be a candidate for any new therapies that come from this line of research.
Based in San Francisco, Maze Therapeutics (Maze) is studying modifier genes—genes that affect the phenotype or physical properties of other genes—and attempting to create drugs that replicate them, reported MIT Technology Review. Maze believes that genetic modifiers could afford a “natural form of protection” against disease.
“If you have a disease-causing gene, and I have the disease-causing gene, why is it that you may be healthy and I may be sick? Are there other genes that come into play that provide a protective effect? Is there a drugging strategy to recover normal phenotype and recover from the illness?” Maze Chief Executive Officer Jason Coloma, PhD, asked in an interview with FierceBiotech.
In 2019, Maze received $191 million in financing from Third Rock Ventures, ARCH Venture Partners, and others, to find ways to translate their findings into personalized medicines, according to a news release. And with the availability of international public genetic databases and CRISPR gene editing, now may be good timing.
“This was the perfect time to get into this space with the tools that were being developed and the amount of data that has been accumulated on the human genetic side,” Charles Homcy, MD, Third Rock Ventures Partner and Maze Scientific Founder, told Forbes, which noted that Maze is tapping existing population-wide genetic databases and large-scale studies, including the United Kingdom’s Biobank and Finland’s Finngen.
To help find genetic modifier drug targets, Maze is accessing CRISPR gene editing capabilities. Jonathan Weissman, PhD, Maze Scientific Founder and Professor of Cellular Molecular Pharmacology at University of California, San Francisco (UCSF), told MIT Technology Review: “You take a cell with a disease-causing gene and then see if you can turn it back to normal. We can do 100,000 experiments at once because each cell is its own experiment.”
“At Maze, we are focused on expanding our understanding of the natural disease protection provided by genetic modifiers through an integrated approach that combines studying natural human genetic variation across the globe and conducting large-scale experiments of gene perturbations,” Charles Homcy, MD (above), Founder and interim CEO of Maze and a partner at Third Rock Ventures, said in a news release. “Through our integrated approach, we believe we will create novel medicines based around those modifiers to treat a number of diseases.” (Photo copyright: Forbes.)
Using CRISPR to Identify the Cause of Disease
One drug research program reportedly progressing at Maze involves developing gene therapy for the neurogenerative disease amyotrophic lateral sclerosis (ALS). The program borrows from previous research conducted by Aaron Gitler, PhD, Professor of Genetics at Stanford University and Maze co-founder, which used CRISPR to find genetic modifiers of ALS. The scientists found that when they removed the protein coding gene TMX2 (Thioredoxin Related Transmembrane Protein 2), the toxicity of proteins building the disease was reduced, reported Chemical and Engineering News.
“We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells,” Gitler and colleagues wrote in Nature Genetics. “Together, our results demonstrate the promise of using CRISPR-Cas9 screens in defining the mechanisms of neurodegenerative diseases.”
“We have the flexibility to think differently. We like to
think of ourselves as part of this new breed of biotech companies,” Coloma told
FierceBiotech.
It’s an exciting time. Clinical laboratories can look
forward to new precision medicine diagnostic tests to detect disease and
monitor the effects of patient therapies. And the research initiatives by Maze
and other genetic companies represent a new approach in the use of genetic code
to create specific drug therapies targeted at specific diseases that work best
for specific patients.
The companion diagnostics that may come from this research would
be a boon to anatomic pathology.
