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A comprehensive guide to PCR and some of the most popular variations of this essential molecular biology technique.

Polymerase chain reaction (PCR) is an essential molecular biology technique used to amplify specific segments of DNA. PCR was first developed by Kary Mullis who won the Nobel Prize for Chemistry in 1993 for his revolutionary method.¹ PCR is essential for a wide range of applications such as genetic mapping, bacterial & viral detection, cloning, sequencing, and forensics.

Although there is a vast array of specialized PCR methods, the general workflow and components for the reaction remain the same. PCR relies on fluctuations in temperature achieved by a thermal cycler and Taq polymerase, a special polymerase that is functional even at elevated temperatures. The necessary components for any PCR reaction are a DNA template strand, oligonucleotide primers, deoxynucleotide triphosphates (dNTPS), buffer, and Taq polymerase.² A general PCR amplification cycle follows the same principal steps:

Denature DNA - The template DNA is heated above 94 °C to separate the double stranded DNA (Figure 1).

Anneal Primers - The temperature is then lowered to between 40-60 °C, enabling the primers to anneal to the separated DNA strands at a specific sequence (Figure 1).

Extend Primers - Lastly, the temperature is increased to an optimal 70-74 °C, enabling Taq polymerase to elongate the new strand from the primer by inserting nucleotides complementary to the template DNA in the 5' to 3' direction (Figure 1).

Figure 1. Overview of the key steps during PCR and the corresponding temperatures they take place at.

The PCR cycle will repeat anywhere from 20-40 times to yield millions of copies of the desired DNA strand. Highly specific primers are critical during the annealing step to obtain sufficient yields of your desired PCR product and to avoid amplification of off-target sequences. Read our complete guide on How to Design Primers for more information on how to design effective PCR primers for different kinds of PCR.

When PCR was first developed, PCR products could only be quantified after the reaction was complete by using endpoint analysis techniques, like gel electrophoresis. However, an alternate method known as quantitative Real Time PCR (or simply qPCR) was developed to quantify your PCR product as the reaction is running. During qPCR, fluorescent reporter molecules (e.g. DNA binding dyes, fluorescently labeled PCR primers or probes) are incorporated into the DNA strand to quantify the PCR product in real time using fluorescence.³ The resulting qPCR curve can be used to monitor amplification in real time ( Figure 2). Special thermal cyclers can detect fluorescence and perform amplification simultaneously to accurately detect and quantify fluorescence with each amplification cycle. The benefits of qPCR over end-point PCR include the ability to accurately quantify initial template DNA input as well as increased sensitivity. To save time, researchers commonly use qPCR premix kits that provide many of the reaction components in just one tube, which streamlines the preparation of the reaction.

Figure 2. An example of a qPCR Curve. As an increasing number of PCR cycles are performed, the amount of PCR product increases exponentially, resulting in increasing fluorescence until the fluorescence eventually plateaus as the reaction reagents are depleted. The Cq (quantification cycle) is the number of cycles at which the fluorescence level is above a pre-defined threshold, set to determine amplification from background fluorescence. The Cq value can be used to quantify the starting DNA input and final amplicon output if standards are run in parallel to generate a standard curve.

For several applications, such as RNA virus detection or gene expression studies, it is necessary to amplify RNA segments instead of DNA. RNA can be amplified using PCR, but it first must be converted to cDNA to create a suitable template for Taq polymerase. A reverse transcriptase is employed to convert RNA to cDNA, hence the name RT-PCR (reverse transcription PCR). There are two approaches to RT-PCR: one-step and two-step RT-PCR. During one-step RT-PCR, the reverse transcription and PCR amplification step occur in the same tube while during two-step RT-PCR the reverse transcription and amplification steps are done separately. One-step RT-PCR is more efficient and well suited for high-throughput sample processing. However, if the PCR parameters need to be fine-tuned, two-step RT-PCR is the preferred method. Choosing the proper RT-PCR method will depend on your desired throughput and application. There are commercially available RT premix kits that include all the necessary reagents in a ready-to-use master mix for simplifying the cDNA synthesis step.

Droplet digital PCR is a variation of PCR that utilizes a water-oil emersion to separate amplified PCR products. The partitioning of accumulating amplicons works like wells on a 96-well plate but instead of 96-wells the sample is separated into 20,000 droplets, enabling the precise quantification of DNA molecules without a reference standard.⁴ The molecule-level sample processing enables decreased sample input requirements compared to other PCR methods and incredibly sensitive detection of slight differences in amplicon count between samples.

At room temperature, PCR primers can anneal to regions of the template DNA that are not perfectly complementary. If Taq polymerase elongates these off-target primers, undesired PCR products will be generated. To mitigate amplification of off-target DNA segments, researchers can employ a "hot start" DNA polymerase that has been impaired with chemical modifiers to block the polymerase's enzymatic activity at room temperature. When thermal cycling begins and the temperature reaches 94 °C the covalent bonds adhering the modifier to the DNA polymerase break, restoring the functionality of the polymerase. It is also possible to perform hot start PCR manually by leaving out a component of the PCR reaction until the reaction is heated to 60 °C.⁵

Multiplex PCR makes it possible to amplify multiple targets at once by employing two or more pairs of PCR primers. Multiplex PCR is an efficient technique that enables the comparison of multiple amplicons simultaneously. However, optimizing annealing conditions and ensuring high primer specificity is especially important for successful multiplex PCR. Some of the challenges to overcome when doing multiplex PCR include non-specific amplification and reduced efficiency if there is any competition between the primers. If considering doing a multiplex reaction, ensure your primers are highly unique and that your amplicons are different sizes so they can be distinguished on a gel.

Nested PCR uses two pairs of PCR primers for improved yield and specificity of amplicons, making this technique effective for amplifying low-input samples or samples that require increased specificity.⁶ During the first round of PCR, the DNA strand is denatured and then the first pair of primers anneal to outer primer sites that flank the amplicon of interest ( Figure 3). The product of this initial round of PCR is then amplified again using a second pair of primers that bind to primer sites that are "nested" within the beginning and end of the DNA sequence of interest, hence the term nested PCR ( Figure 3.). The two rounds of amplification halt the production of non-specific products because in the off chance an off-target segment is amplified in the first round of PCR, it is very unlikely that the non-specific region will be recognized and amplified by the nested primer set during the second round of PCR.

PCR amplification is a fundamental molecular biology tool that is essential to many downstream applications such as cloning and NGS. However, no matter the application, the best way to set yourself up for PCR success is to start with high-quality template nucleic acid. Zymo Research's Quick-DNA andQuick-RNA kits are robust extraction solutions that ensure eluted nucleic acid is high yield, ultra-pure and suitable for PCR amplification. In addition, if you are working with samples high in polyphenolics, like plants, soil, or skin tissue, theOneStep PCR Inhibitor Removal Kits are ideal for the complete removal of inhibitors for highly efficient PCR amplification. If you aren't getting the purity or concentration you need from your existing extraction method, the DNA Clean & Concentrator Kits can be used to clean-up template DNA to be used for PCR or to completely remove salts, polymerases, and endonucleases from your PCR product, leaving you with DNA that is suitable for even the most sensitive downstream applications. If your workflow involves extracting a PCR product from agarose gel, the Zymoclean Gel DNA Recovery Kits ensure your recovered DNA is suitable for highly sensitive downstream applications including sequencing, ligation, and endonuclease digestion. Finally, the ZymoPURE Plasmid Purification Kits are ideal for producing plasmid that is pure enough for PCR reactions or transfections. Discover which DNA and RNA purification kit best suits your research goals today.

Citations

1. National Human Genome Research Institute. (2020). Polymerase Chain Reaction (PCR) fact sheet. Retrieved December 5, 2024, from https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet

2. Lorenz T. C. (2012). Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies [Video]. Journal of Visualized Experiments.https://doi.org/10.3791/3998

3. Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M. W., Shipley, G. L., Vandesompele, J., & Wittwer, C. T. (2009). The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical chemistry, 55(4), 611-622. https://doi.org/10.1373/clinchem.2008.112797

4. Quan, P. L., Sauzade, M., & Brouzes, E. (2018). dPCR: A Technology Review. Sensors, 18(4), 1271. https://doi.org/10.3390/s18041271

5. Green, M. R., & Sambrook, J. (2018). Hot Start Polymerase Chain Reaction (PCR). Cold Spring Harbor protocols, 2018(5), 10.1101/pdb.prot095125. https://doi.org/10.1101/pdb.prot095125

6. Green, M. R., & Sambrook, J. (2019). Nested Polymerase Chain Reaction (PCR). Cold Spring Harbor protocols, 2019(2), 10.1101/pdb.prot095182. https://doi.org/10.1101/pdb.prot095182

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A comprehensive guide to PCR and some of the most popular variations of this essential molecular biology technique including qPCR, RT-PCR, & Hot Start PCR.

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https://www.fiercebiotech.com/research/microbiome-research-project-ids-good-bacteria-combat-obesity

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By Bernat Olle, PureTech Ventures
The last few years have seen an explosion of academic work in the field of the human microbiome and the National Institutes of Health has made a big bet that this field can transform medicine. To make a long story short, scientists are finding that the bugs that colonize your gut, airways, skin, etc., do a whole lot of good things without which human life would be pretty miserable (see for example the collection of articles in the most recent issue of Science Translational Medicine, or the flood of articles in and ), including protecting us from opportunistic infections, keeping allergies and autoimmune diseases at bay or supplying key nutrients. The tricks that our microbes use to make all this goodness happen and the chaos that results from messing with them hold critical clues for developing a whole new class of therapies. Future microbiome modulators could be (among others) orally administered commensal microbes, or natural or synthetic molecules that mimic their effects. For full disclosure, at PureTech Ventures we strongly believe in this field, too, and have started new company initiatives to mine this space including this company.

Other promising therapeutic modalities that captured the public's imagination at the time they emerged, such as recombinant DNA (rDNA) and RNAi, have taken a long time to materialize (or have not been translated into products yet, in the case of RNAi). Given all the attention the microbiome field is getting now, and the lessons of the past, striking a note of caution when considering translation of microbiome therapies to the clinic seems wise.
Not your average new therapeutic modality
However, there are several reasons why drugs that modulate the microbiome could be translated to the clinic significantly faster than other new therapeutic modalities such as rDNA and RNAi:

No fundamental technological barrier: Notwithstanding the technical complexity of the microbiome field, which requires integration of many technologies from genome sequencing, new bioinformatics tools, unique animal models, and metabolite profiling tools (more on this later), there isn't one fundamental technological barrier that needs to be resolved to enable clinical translation. That was not the case for rDNA and RNAi therapeutics. Manufacturing stood in the way of translating rDNA drugs to the clinic. Delivery stands in the way of RNAi drugs. Initially, protein products were only available in miniscule quantities or were very costly to produce in large-scale. MAbs were not feasible until hybridoma technologies came around. Early pioneers of the rDNA industry had to figure out ways to express protein products in recombinants hosts, at high yields, with correct folding, with the right glycosylation patterns, at large scales, and with the right purity. That took time. Similarly, delivering RNAi therapies systemically to where they are needed in the body remains stubbornly complicated, despite some progress in using lipids and nanoparticles to address targets in the liver.

In contrast, the technology needed to manufacture products based on live microbes already exists. With some tweaks, essentially the same cell banking, fermentation and analytical methods already in use to manufacture biologics can be applied to manufacture live organisms. Purifying a microbe from a fermentation broth is trivial in comparison to purifying a protein. And over millions of years of evolution, our microbes have evolved intelligent ways of delivering themselves into our gut via the oral route and without the need for fancy delivery systems. Humans are born sterile, and we get colonized by microbes that use a number of strategies to reach their natural niche in the human body, such as forming spores.

Established proof of principle that the microbiome can be modulated and that this is of value to patients: OK, so there is exciting data coming out of microbiome research projects--but are there any simple applications of this therapeutic modality that have worked in the clinic? RNAi, stem cells, and genomics companies did not have a satisfactory answer to this question. In contrast, transplantation of the microbiota in humans ("fecal transplantation" ... yep, eating somebody else's poop) has been performed for decades with very promising results. At present, it is only used to treat refractory patients with selected conditions such as C. difficile infections or IBD. Whether the procedure is repulsive is beside the point. It works, and it establishes proof of principle that manipulating the microbiome (1) can be done and (2) is of clinical value.

You can't change your "human" genome, but you can change your microbiome: In fact, it happens all the time. It changes depending on what you eat, your hygiene, your age, or what drugs you take (particularly antibiotics). This provides much more flexibility for intervening. Identification of microbiome anomalies points to alterable fates.

Safety: Humans have lifelong exposure to massive amounts of commensal microbes (we have on the order of a hundred trillion microbes in our gut). So you'd think we have evolved to tolerate a wide range of doses of certain live organisms. Yes, rDNA drugs too were expected to be safer than small molecules because they are more "natural" but they proved to be immunogenic sometimes. That has not been a problem with probiotics used as supplements and food ingredients, which have an extremely safe track record of human use.
It can't possibly be that simple
Sure, it probably will not be simple. Some notes of caution often heard these days merit attention:

"Mapping out the biological pathways is a scientific process that takes a long time." True, no matter what therapeutic modality is being used. But has this been the rate limiting step for RNAi therapies to make it to the clinic? And while mapping what role our microbiome plays in a highly complex disease like cancer will take time, there is low hanging fruit to go after in other therapeutic areas in infectious disease and inflammation, just as certain hormones and growth factors were a logical starting point for rDNA therapies.

"It is not always clear if microbiome changes are cause or effect, or what caused the microbiome changes in the first place." While often it is not known whether microbiome alterations were caused by hygiene, diet, antibiotic use, genotype, immune responses, enteric infections, or combinations of the above, this has not stopped clinicians from intervening and shifting the microbiome of patients back to a "healthy state" (See fecal transplantation comments above). Surely, some of the correlative connections coming up in the literature will prove to have no role in driving disease (they may still lead to very useful biomarkers). But there already is very compelling evidence that in other cases alterations of the microbiome are a driver in pathology, regardless of whether they were the initial disease trigger. Numerous experiments have demonstrated that transplanting the microbiota of diseased animals into healthy hosts transfers disease phenotypes and influences onset and/or progression of obesity and chronic inflammatory diseases such as allergies and autoimmune diseases. It is also well accepted among clinicians, and supported by animal experiments, that microbial communities promote pathology in IBD.

"We don't know how much variation there is in the human microbiome across individuals and within individuals." Actually we are starting to get a good sense of this variation thanks to $140 million from the NIH and a growing suite of open source tools (bottom line: yes, there is variation in species but their functions, e.g. metabolic pathways, vary little across individuals). In certain instances, this variation will uncover subtypes of individuals within a complex disease spectrum, improving the odds of success in clinical trials.

"Probiotics in foods and supplements have shown little to substantiate their health claims and don't even colonize the gut." Again, true in most cases. In making probiotics, just as in making antibodies, you get what you select for. And food companies have focused on selecting strains based on their technological properties (manufacturability, ability to formulate in food matrixes, acid tolerance, shelf live, etc.), not based on their functions in human biology, which is a theme for a whole separate blog entry, coming up soon.
Dr. Bernat Olle is a principal at PureTech Ventures. He has been a member of the founding teams of Follica, Vedanta Biosciences and Enlight Biosciences. He serves as the chief operating officer and a member of the board of directors of Vedanta Biosciences. He completed his doctoral work at the Chemical Engineering Department at MIT, where he co-developed a novel method to increase oxygen transfer in bioreactors by using colloidal nanoparticles.
Editor's note: This piece originally appeared June 15 on Giving Life to Science: The Official Blog of PureTech Ventures.

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Industry Voices: Microbiome Therapies - Coming to a Clinic Near You

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By Bernat Olle, PureTech Ventures The last few years have seen an explosion of academic work in the field of the human microbiome and the National Institutes of Health has made a big bet that this field can transform medicine.

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Y Combinator-backed uBiome launched the world's first sequencing-based microbiome screening test, which will provide patients and their physicians with more information about the bacteria in their gut.

The test, dubbed SmartGut, uses 16S rRNA gene sequencing and a custom bioinformatics pipeline to identify a number of microbes that are linked to specific infections, health risks and gut conditions, uBiome said in a statement. Patients collect a stool sample at home and mail it directly to a lab. The test identifies both pathogenic and commensal bacteria and is easier and more convenient to use than other tests on the market, according to the statement.

"The SmartGut test is a transformation of the way we think about clinical microbiology. It leverages the power of DNA sequencing to test for various microorganisms simultaneously and puts the power of microbial genomics into the hands of clinicians," said Zachary Apte, uBiome's chief technology officer, in the statement. "uBiome and our citizen science partners have assembled the largest database of human microbiomes ever, allowing us to define reference ranges from healthy individuals for every single target."

uBiome also announced the closing of a "significant round of Series B investment," which was led by 8VC. The company has raised a total of $22 million. Founded in 2012, uBiome is also backed by Y Combinator and Andreessen Horowitz, among other investors.

"We learn more about health every year, but unnecessarily negative health outcomes are common because the system doesn't encourage people to take preventative action," said Joe Lonsdale, 8VC founding partner, in the statement. "By making gut health testing faster and easier, uBiome will unquestionably have a massive impact."

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uBiome launches gut microbiome sequencing test, reels in Series B

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Y Combinator-backed uBiome launched the world's first sequencing-based microbiome screening test, which will provide patients and their physicians with more information about the bacteria in their gut. The test, dubbed SmartGut, uses 16S rRNA gene sequencing and a custom bioinformatics pipeline to identify a number of microbes that are linked to specific infections, health risks and gut conditions, uBiome said in a statement.

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Vedanta, NYU aim for microbiome-based cancer immunotherapies

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Vedanta Biosciences has partnered with the NYU Langone Medical Center to develop microbiome-derived immunotherapies specifically for use paired with checkpoint inhibitors. The idea is to use the microbiome to help improve the efficacy of this major developing class of oncology drugs as well as, potentially, for standalone use to treat cancer.

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CAMBRIDGE, MA, February 5, 2015 - In the largest longitudinal study of the microbiome to date, researchers from the Broad Institute of MIT and Harvard, Massachusetts General Hospital (MGH), and the DIABIMMUNE Study Group have identified a connection between changes in gut microbiota and the onset of type 1 diabetes (T1D). The study, which followed infants who were genetically predisposed to the condition, found that onset for those who developed the disease was preceded by a drop in microbial diversity - including a disproportional decrease in the number of species known to promote health in the gut. These findings, published by Cell, Host & Microbe, could help pave the way for microbial-based diagnostic and therapeutic options for those with T1D.

The human microbiome, which consists of the trillions of microorganisms (bacteria, viruses, and other assorted "bugs") that reside in our bodies, has become an area of growing interest to the medical community as researchers have begun to probe the role it plays in human health and disease. While most bugs in our microbiome are harmless, and even beneficial, changes in the microbiome (and in the interactions microbial species share with their human hosts) have been linked to various disease states, including diabetes and Inflammatory Bowel Disease (IBD).

To explore the possible connection between changes in the microbiome and type 1 diabetes, a team led by Ramnik Xavier, an Institute Member of the Broad and Chief of Gastroenterology at MGH, followed 33 infants (out of a much larger cohort of Finnish and Estonian children) who were genetically predisposed to T1D. From birth to age 3, the team regularly analyzed the subjects' stool samples, collecting data on the composition of their gut microbiome.

In the handful that developed T1D during this period, the team observed a 25% drop in community diversity (in other words, in the number of distinct species present in the microbiome) one year prior to the onset of the disease. They also noted that this population shift included a decrease in bacteria known to help regulate health in the gut, along with an increase in potentially harmful bacteria that are known to promote inflammation. The findings are further evidence of a previously identified link between inflammation of the gut and type 1 diabetes.

"We know from previous human studies that changes in gut bacterial composition correlate with the early development of type 1 diabetes, and that the interactions between bacterial networks may be a contributing factor in why some people at risk for the disease develop type 1 diabetes and others don't," said Jessica Dunne, Director of Discovery Research at JDRF, which funded the study. "This is the first study to show how specific changes in the microbiome are affecting the progression to symptomatic T1D."

Previous studies have shown that transferring microbiota from mice that were predisposed to autoimmune diabetes (the mouse equivalent of T1D) to mice that were not predisposed increased the prevalence of autoimmune diabetes in mice that were otherwise unlikely to develop the disease. Studies in humans have also shown an association between T1D and the bacterial composition of the gut. However, those studies were retrospective, meaning they were conducted after the patients developed the disease, making causality difficult to prove.

"This study is unique because we have taken a cohort of children at high risk of developing type 1 diabetes and then followed what changes in the microbiome tip the balance toward progression to the disease," Xavier said.

Aleksandar Kostic, a postdoctoral fellow in Xavier's lab and first author of the study, agreed, calling the study "a compelling piece of evidence pointing toward a direct role of the microbiome in type 1 diabetes."

Since the study also followed infants who did not ultimately develop type 1 diabetes, the researchers were also able to gain insights into the normal development of the microbiome during infancy. They found that, while the species of bacteria present in the gut microbiome vary greatly between individuals, the composition of the microbiome is generally stable within the individual over time.

Moreover, using metabolomic analysis (looking at the metabolites - the tiny molecules produced during metabolism - in subject stool samples), the researchers were also able to see that, while bacterial species varied between individuals, the biological functions served by the various species in the microbiome remained consistent over time, and from person to person.

"Whether the bacterial community is very small, as it is in early infancy, or if it's larger as it is later in life, the community is always serving the same major functions regardless of its composition. No matter which species are present, they encode the same major metabolic pathways, indicating that they're doing the same jobs," Kostic said.

By revealing patterns in the development of the microbiome in healthy individuals, and in those progressing toward T1D onset, the findings may ultimately have diagnostic or therapeutic implications. In terms of diagnostics, understanding how the microbiome shifts prior to the onset of disease could ultimately help clinicians spot early microbial features of T1D.

As for therapeutics, Xavier, who is also the Kurt Isselbacher Chair in Medicine at Harvard Medical School and Co-Director of the Center for Microbiome Informatics and Therapeutics at MIT, says that knowing which species are absent and which are flourishing in the gastrointestinal tract of children with T1D may help make it possible to slow progression of the disease after onset by revealing ways to manipulate the microbiome and, in-turn, microbiome-induced immunoregulation.

The next step, he says, is to broaden the sample pool to determine what factors in the environment and in the microbiome might be making Finns - who are at exceptionally high risk of T1D - more predisposed to the disease than other populations. That includes revisiting the hygiene hypothesis, which holds that a lack of childhood exposure to microbiota and other potentially infectious agents may hinder the development of the immune system and increase susceptibility to immunological disorders.

The researchers are also examining the metagenomic data gathered in the study to determine what biological pathways the microbiota are acting upon - or what metabolites they may be producing - that could be contributing to disease.

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Researchers find link between microbiome, type 1 diabetes

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CAMBRIDGE, MA, February 5, 2015 - In the largest longitudinal study of the microbiome to date, researchers from the Broad Institute of MIT and Harvard, Massachusetts General Hospital (MGH), and the...

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Novartis has stepped in to back Paris-based Seventure Partners' biotech fund focused exclusively on the emerging field of the microbiome as well as nutrition, bringing its fund to $176 million. Over the last two years Seventure has been playing angel to the microbiome field, backing Enterome, Maat pharma, Eligo bioscience, Targedys, Micropharma and others. Novartis ($NVS) is joining a group that includes Danone, Tereos, Lesaffre, Bel, Tornier, the Institut National de Recherche Agronomique, the Danish Technology Institute, and Chalmers University of Technology in Gothenburg. "Seventure Partners believes that large worldwide pharmaceutical groups are increasingly interested in the microbiome field in anticipation of finding new ways to conduct research and developing drugs with accompanying diagnostics; especially for infectious and gastrointestinal diseases, and other indications, as well as immuno-oncology approaches," said Isabelle de Cremoux, CEO and Managing Partner of Seventure. Release

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Novartis backs $176M microbiome biotech fund

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Novartis has stepped in to back Paris-based Seventure Partners' biotech fund focused exclusively on the emerging field of the microbiome as well as nutrition, bringing its fund to $176 million. Over the last two years Seventure has been playing angel to the microbiome field, backing Enterome, Maat pharma, Eligo bioscience, Targedys, Micropharma and others.

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The U.S. government has launched a new public-private microbiome research fund as it looks to build up the development of bacteria-based meds and other new tech.

Announced from the White House this morning, its Science and Technology Policy group--in collaboration with Federal agencies and private-sector stakeholders--is launching the National Microbiome Initiative (NMI) to "foster the integrated study of microbiomes across different ecosystems."

Microbiomes are the communities of microorganisms that live on or in people, plants, soil, oceans, and the atmosphere. They are vital in maintaining healthy function of these diverse ecosystems, influencing human health, climate change, food security, and other factors.

But dysfunctional microbiomes are associated with issues including human chronic diseases such as obesity, diabetes, and asthma. Along with a slew of biotechs, the new deal is set to help better understand how these microbiomes work, and how they can be exploited to create new medicines.

In a statement, the White House said: "The NMI aims to advance understanding of microbiome behavior and enable protection and restoration of healthy microbiome function. In a year-long fact-finding process, scientists from Federal agencies, academia, and the private sector converged on three recommended areas of focus for microbiome science, which are now the goals of the NMI."

These include: Supporting interdisciplinary research; developing platform technologies; and expanding the microbiome workforce.

The NMI will launch with a combined Federal agency investment of more than $121 million in its first year for funding in microbiome studies across several areas, including human health and agriculture.

This includes $10 million from the Department of Energy, double that from the NIH, and $16 million from the National Science Foundation.

On the charity and private side, the Bill and Melinda Gates Foundation will invest $100 million over 4 years to investigate and develop tools to study human and agricultural microbiomes, while the JDRF will stump up $10 million over 5 years to address microbiome research related to type I diabetes.

Meanwhile, Cambridge, MA-based Evelo Biosciences is dedicating $1 million to award up to 10 academic research grants for joint studies exploring the human microbiome and cancer. This new funding will support collaborative, multidisciplinary approaches aimed at illuminating how the human microbiome influences cancer susceptibility and progression. Evelo will work with researchers to identify and develop new microbiome-based cancer therapeutics.

As part of today's announcement, Novartis ($NVS) has also said it has teamed up with the University of California, San Francisco and the Broad Institute to mine microbiome data. The four-year, $8.1 million collaboration will use bioinformatics, synthetic biology and other tools to predict the chemical structures of compounds that can be made by microbiomes.

A number of microbiome startups are already populating the biotech field, including France's Enterome, which has been making progress while Cambridge, MA-based Seres Therapeutics attracted investors to a 2015 IPO that raised $134 million. Seres' lead drug targets Clostridium difficile infections by altering patients' bacterial makeup.

One of the pioneers in the space, Second Genome, is currently pursuing a Phase I program for its lead therapy, SGM-1019, as it explores an inflammatory bowel disease pathway leading to a potential new therapy for ulcerative colitis. Just last month it raised $42.6 million in a Series B round led by the venture arms of Roche ($RHHBY) and Pfizer ($PFE).

Big Pharma has been getting in on the act as well. Last year, J&J ($JNJ) beefed up its R&D efforts in the microbiome space as its subsidiary Janssen Biotech inked a deal to collaborate with Boston-based Vedanta on a new bacterial treatment for inflammatory bowel disease. And it handed over an undisclosed upfront payment and promises of up to $241 million in milestones if their partnership can blaze a path to the market eventually.

And not to be left out, Janssen's Human Microbiome Institute is also involved in the initiative, and said it will develop a network of entrepreneurs and scientists in the microbiome research field. The JHMI will in addition make "strategic investments" in order to build a portfolio of microbiome therapies and diagnostics, the U.S. drugmaker said.

- check out the release
- see more from FierceBiotechIT on the Novartis deal

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White House launches $121M-plus microbiome research initiative

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The U.S. government has launched a new public-private microbiome research fund as it looks to build up the development of bacteria-based meds and other new tech.

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Making drug-resistant bacteria susceptible to antibiotics

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Scientists at the New York University School of Medicine have uncovered an innate defense mechanism that protects bacteria from normally lethal doses of antibiotics. A drug inhibiting an enzyme called cystathionine gamma-lyase enhanced the effect of antibiotics in mice.

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Penn team uses CRISPR to edit out genetic disease before and after birth in mice

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University of Pennsylvania researchers delivered a base editor to prenatal and postnatal mouse models of Hurler syndrome, a genetic, lysosomal storage disease that causes organ swelling, muscle abnormalities, heart disease and a range of other problems. They say the results offer proof-of-concept fo…

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Gene therapy delivered to the brain shows promise in children with rare neurodegenerative disease

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Scientists from Ohio State University developed a novel method for delivering gene therapy to specific regions of the brain and tested it in children with a rare neurodegenerative disorder called AADC deficiency. Based on positive results from the trial, they’re now eyeing the technology in more wid…

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Glaucoma can cause blindness and has no cure, as current therapies only slow vision loss by decreasing elevated eye pressure. Mount Sinai researchers have developed a one-time gene therapy that they hope will prevent vision loss induced by glaucoma and other conditions that cause retinal degeneration.

The researchers found that by reactivating an enzyme called CaMKII in retinal ganglion cells, which are responsible for sending images to the brain to process visual cues, they were able to prevent vision loss in mouse models of glaucoma. The results of the new study, published Thursday in the online version of the journal Cell, have led to commercial interest that could one day translate into new treatments.

The team started by testing CaMKII (calcium/calmodulin-dependent protein kinase II pathway) in a wide range of eye-disease models. They discovered that it is critical to the survival of retinal ganglion cells, making it a "desirable therapeutic target" for preserving vision, said senior author Bo Chen, Ph.D., director of the Ocular Stem Cell Program at Mount Sinai's Icahn School of Medicine, in a statement.

The researchers then used an adeno-associated viral vector to introduce an over-active version of CaMKII into the retinal ganglion cells of the mouse models just before they became damaged.

In the gene therapy-treated mice, 77% of the retinal ganglion cells survived 12 months after they were damaged, compared to 8% in the control group. The same results occurred six months after optic nerve crush, which leads to slower degeneration, versus 7% in the control mice.

"The fact that manipulation of CaMKII would involve a one-time transfer of a single-gene adds to its vast potential to treat serious retinal conditions in humans," Chen said. "The next step is testing this in larger animal models, which may pave the way for starting clinical trials."
RELATED: Astellas gene therapy repairs damage in neurodegenerative disease models
CaMKII has been studied in gene therapy research for other indications, as well. In July 2019, researchers at Boston Children's Hospital said they created a gene therapy that targeted irregularities in CaMKII to treat cardiac diseases marked by irregular heartbeats.

In March, scientists at the University of Cambridge said they developed a gene therapy expressing two therapeutic proteins that showed promise in mouse models of glaucoma and of misfolded tau protein, which is linked to Alzheimer's and dementia. A modified version of that therapy is now being developed by Astellas.

There have been some disappointments in bringing forward gene therapies for eye diseases, however. Biogen said in June that its candidate for choroideremia, a rare genetic retinal disease that causes progressive vision loss and ultimately blindness, had failed a late-stage clinical trial.

Roche also struggled to advance a gene therapy for choroideremia. Ten days after Biogen's blunder, Roche said it was walking away from a deal with 4D Molecular Therapeutics on a sponsored trial. The small biotech said it will move forward with developing the therapy after it regains the full rights in September.

The Mount Sinai team is now filing patent applications for its gene therapy and is in active discussions with multiple companies to move the treatment into the clinic, Mount Sinai said.

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New gene therapy approach shows signs of preventing vision loss from glaucoma

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Mount Sinai researchers said a study of a gene therapy in mice was able to prevent vision loss and blindness induced by serious retinal injury and diseases such as glaucoma. The approach, which they're working to commercialize, involves reactivating the enzyme CaMKII in retinal ganglion cells.

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Fruit compound reverses brain damage in mouse models of Parkinson’s

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The compound farnesol, found in berries and other fruits, could inspire new Parkinson’s disease treatments, Johns Hopkins researchers said. In mouse studies, the team found that it prevented the loss of dopamine neurons and reversed symptoms of the disease.

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Several years ago, researchers at Massachusetts General Hospital (MGH) figured out a way to convert skin cells into muscle cells that were self-renewing and seemed promising for treating injuries and degenerative diseases like muscular dystrophy. But they weren't quite sure how the conversion was happening.

Now they know-and they believe their insights could yield recipes for generating patient-matched muscle cells to treat a range of disorders.

The MGH researchers call their muscle cells myogenic progenitor cells (iMPCs). In a new study, published in the journal Genes & Development, they explain how adding three chemicals to skin cells causes them to transform into iMPCs.

A gene called MyoD can convert skin cells into muscle cells, but mature muscle cells cannot divide to create new muscle. In previous experiments, the researchers hit on three chemicals that could make skin cells revert to a stem-cell-like state instead, which would allow them to transform into self-renewing muscle cells.

The key, the MGH researchers discovered in the new study, is that the chemicals remove marks called methyl groups that are added to DNA in a process known as DNA methylation.

"DNA methylation typically maintains the identity of specialized cells, and we showed that its removal is key for acquiring a muscle stem cell identity," said lead author Masaki Yagi, Ph.D., a research fellow at MGH, in a statement.
RELATED: Zebrafish reveal regenerative protein that could inspire new treatments for muscle-wasting diseases and aging
It's the latest study focused on finding new ways to regenerate muscle. Earlier this year, an Australian team reported that a protein called NAMPT could regenerate muscle in zebrafish and mouse models by amplifying a natural healing process in the body that occurs when macrophages migrate to injury sites.

Other research groups have focused on the role of the MyoD protein in healing. A Sanford Burnham Prebys team, for example, discovered that in aging people, old muscle stem cells can trigger a DNA damage response that blocks MyoD and prevents muscle cells from forming.

The MGH scientists believe their findings could be useful beyond muscle regeneration. The three-chemical cocktail could be used to generate stem cells for a variety of tissue types, senior author Konrad Hochedlinger, Ph.D., a principal investigator at the Center for Regenerative Medicine at MGH and a professor of medicine at Harvard Medical School, said.

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MGH team shows how injured muscles might be repaired with patients' own skin cells

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Several years ago, researchers at Massachusetts General Hospital (MGH) figured out a way to convert skin cells into muscle cells that were self-renewing and seemed promising for treating injuries and degenerative diseases like muscular dystrophy. But they weren't quite sure how the conversion was happening.

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Oncolytic viruses have long held promise for cancer treatment, but their first generation hasn't quite delivered. Armed with new tools and technology-including 3D printing-Humane Genomics is part of a wave of companies working on oncolytic viruses 2.0.

The only FDA-approved oncolytic virus is Amgen's Imlygic, which scored a nod in 2015 for the treatment of melanoma. Based on a herpes virus, the treatment is injected into tumors, where it replicates and produces GM-CSF, a protein that stimulates the immune response.

However, Imlygic doesn't work in metastatic disease, and cancer-killing viruses as a whole face other barriers. A major hurdle is delivery: The viruses must be injected directly into tumors, because the immune system may catch and destroy them before they reach cancer cells if they are given intravenously. Another challenge is efficacy. The treatments don't seem to work well on their own, and multiple companies are pursuing combinations, such as with checkpoint inhibitors and other immunotherapies.
RELATED: Candel reels in downsized $72M IPO to get cancer-killing viruses through the clinic
New York-based Humane Genomics is building a platform that can design cancer-killing viruses from scratch as well as produce them and test them more quickly and cheaply than other methods.

"We can 3D print or synthesize DNA based on a computer file," said Humane Genomics CEO Peter Weijmarshausen. "We take a file that contains A, G, C and T in the right order and feed it into a machine that produces DNA fragments," he added, referring to the nucleotide bases or letters that are the building blocks of DNA.

Humane Genomics aims to develop viruses that not only target cancer cells while sparing healthy ones but that can also carry drugs to boost the virus's effects or dial up the immune response against the tumor.

Weijmarshausen founded Humane Genomics with Andrew Hessel and Chad Moles, the company's chief science officer, who focused on synthetic virology during his graduate study in biotechnology. The company picked up $125,000 in seed funding from Y Combinator this year and stands to raise more after pitching its work to investors at the startup accelerator's Demo Day next week.
RELATED: To turn off disease-causing genes, Talus Bio wants to knock proteins off of DNA
"It turns out to make a virus that is really good at identifying cancer cells and killing them requires quite a bit of engineering," Weijmarshausen said. "It's not that we didn't know how to do it, but until quite recently, science lacked the tools to do it well."

Synthetic biology, the science of redesigning organisms such as viruses for useful purposes, is just one part of the equation. The rising availability and decreasing costs of genome sequencing is the other.

Sequencing has helped drug developers understand how cancer cells are different from healthy cells and led to the creation of drugs that zero in on cancer-driving genes.

"The same is true for viruses," Weijmarshausen said. With sequencing, scientists have learned how viruses found in nature work and can use that understanding to build synthetic viruses to fight cancer.
RELATED: MIT team employs synthetic biology to create 'circuits' that could detect signs of disease
"We can make a new virus in about a week, and it costs a few hundred dollars," Weijmarshausen said. In contrast, using CRISPR-based gene editing to modify a naturally occurring virus could take many more weeks and thousands of dollars, he added.

"As a result, we can take some wild gambles ... This platform is completely synthetic. We are not making edits, but writing viruses from scratch, which allows us to test concepts that are going to be difficult otherwise," Weijmarshausen said.

Instead of modifying natural viruses like herpes virus or vesicular stomatitis virus to fight cancer-and putting up with potential trade-offs or side effects-Human Genomics is cherry-picking the most desirable qualities found in natural viruses and using them to build synthetic viruses.

Oncolytic viruses (red dots) at work in a lab dish (Human Genomics)

The company is developing its first programs for the treatment of bone cancer, liver cancer, small-cell lung cancer and a form of brain cancer called glioblastoma. It's starting with these cancers because they're on the rarer side and they don't have great treatments, Weijmarshausen said.

In the near term, Humane Genomics will work on experiments to show its viruses work in mice. It is also on the hunt for partners among institutions that treat patients with the cancer types it's interested in.

Further down the line, Weijmarshausen sees synthetic oncolytic viruses as personalized treatments to be deployed alongside routine blood testing for the earliest signs of cancer.

"Based on that detection in the blood, we'll already know how to engineer a virus that can take care of that kind of cancer. We make it, or have it on the shelf, and you get a shot," he said. "You may have a bit of a flu for a few days, but without you noticing you had one form of cancer or another, the virus will have killed it."

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Humane Genomics is 3D-printing oncolytic viruses to treat rare cancers

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Humane Genomics is building a platform that can design cancer-killing viruses from scratch and test them more quickly than other methods. It is working on viruses that not only target cancer cells while sparing healthy ones but that can also carry drugs to boost the virus's effects or dial up the immune response against the tumor.

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Gene therapy biotechs are ready with an arsenal of clinical trials to test the potentially groundbreaking treatments in humans. But an array of complicated safety concerns remain to be addressed-including identifying the correct animal models to use in preclinical research to ensure human testing is safe and determining how to deliver the medicines safely.

That's the focus of a two-day marathon advisory committee meeting hosted by the FDA this week.

Two gene therapies were examined in an FDA presentation: Novartis' Zolgensma for spinal muscular atrophy and Spark Therapeutics' Luxturna for inherited retinal disease. Both use an adeno-associated virus, or AAV, to deliver a therapeutic gene to patients.

More than 40 therapies using these vectors were submitted to the FDA in 2020 for early consultation ahead of human testing and more than 10 were in consideration for human testing that year. The number of these submissions has been rising.
RELATED: Novartis back with bid for 'multibillion-dollar' Zolgensma expansion in older SMA patients as FDA lifts clinical hold
But despite the promise in these gene therapies, the FDA has flagged safety concerns ranging from liver toxicity to kidney injury to a loss of neurons. These issues have long been noted as gene therapies moved toward market and patients died during earlier clinical trials.

The committee is trying to answer complex questions about the merits and limitations of animal studies to determine the risk of liver toxicity in humans. Which animals should be used in preclinical testing to best model what might happen in humans? How can patients be screened based on their risk of experiencing adverse liver events? What strategies should be used with gene therapy treatment itself to mitigate the risks? Should there be a limit on vector dosing?

About one-third of the 500 patients who have received Zolgensma since its approval have experienced a liver-related adverse event, including one case of serious liver failure, the FDA documents said. The therapy has a black box warning of the possibility and patients should be treated with systemic corticosteroids.

The Swiss Big Pharma was the only company to formally present at the advisory committee meeting. The presentation Friday will include details of adverse events that emerged last year during a post-marketing trial for Zolgensma. But even before the committee heard from the company, plenty of discussion was had with the approved gene therapy as a case study on the first day.
RELATED: Roche bulks up in gene therapy and hemophilia with $4.3B Spark buyout
Nine SMA patients out of the 1,400 who have received a dose of Zolgensma to date have experienced thrombotic microangiopathy, a rare disorder that can cause low platelet counts, organ damage and other serious issues, and in some cases death.

One patient who has received the SMA gene therapy died from sepsis, while the rest recovered or were listed as improving. The events led to a revision of Zolgensma's label, according to the Novartis presentation.

Novartis also noted that the company is not alone in seeing some of these adverse events during clinical development. The presentation highlighted a Duchenne muscular dystrophy gene therapy developed by an unnamed company that saw four cases of TMA out of nine patients dosed.

Gene therapies tend to treat rare, life-threatening conditions so a patient's benefit-risk profile needs to be considered when prescribing these medicines, according to Novartis. Patients should be screened for TMA early, given the risk is known, and treatment for the condition should be implemented immediately. The risk should also be added to the drug label for future AAV gene therapies.

More than 1,000 children have now received Zolgensma since the therapy was approved in 2019, according to advisory committee member Barry Byrne, M.D., Ph.D. He serves as associate chair of pediatrics and director of the Powell Gene Therapy Center at the University of Florida. This presents an opportunity to establish a long-term follow-up study in patients who did not have underlying liver disease before being exposed to a gene therapy.
RELATED: After cutting dose because of deaths, Astellas pauses gene therapy trial as new liver issue arises
Pfizer gene therapy toxicologist Jing Yuan, Ph.D. argued during a public comment session held Thursday that existing clinical monitoring for liver disease and potential tumors that could be linked to gene therapy treatment "is a reasonable approach and should be adequate during clinical development."

These long-term follow-up recommendations "should continue to be adequate," Yuan said.

Are standards needed?

During the discussion on the first day, various experts recommended different ways to tackle the safety concerns.

"I heard general agreement that animal studies are, in general, problematic with their relationship to humans. Sometimes they show us what is possible and other times, they are not sufficiently representative of the human situation," said the committee's chair, Lisa Butterfield, Ph.D., an expert in tumor immunology and VP of the PICI Research Center at the University of California, San Francisco.

Charles Vite, Ph.D., suggested that dogs may be the best preclinical models for future trials, given the average 10-year life span of the animals and their similarity to humans. Vite is a professor of neurology in the Department of Clinical Sciences at the University of Pennsylvania.
RELATED: FDA lifts clinical hold on uniQure's gene therapy amid cancer concerns; shares jump
"I think it's possible to get at these questions because the science is so much more advanced in understanding physiology in dogs than in pigs," Vite said.

A more formal risk assessment for patients is needed for future trials, according to Theo Heller, M.D. Heller is section chief of translational hepatology in the National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health.

"We have strong suggestion that pre-existing hepatic conditions may predict the risk of serious liver injury in multiple human models which would make this more important to do carefully," Heller said.

Committee member Tabassum Ahsan, Ph.D. argued that it's too early to create general recommendations for preclinical studies. Ahsan serves as the head of analytical development and characterization for therapeutic discovery at MD Anderson Cancer Center.
RELATED: Novartis touts latest Zolgensma data as SMA competition against Biogen, Roche heats up
"The technology is still evolving, but we do need to start to drive towards standards, otherwise, we're really not going to be able to generate the database of information that we need to make informed decisions as we move forward," Ahsan said.

Butterfield noted that "there seems to be a strong call for longer assessment" of patients following dosing with a gene therapy.
Editor's note: This story was updated at 12:20 p.m. ET on Sept. 3, 2021, to clarify the number of patients who have received Zolgensma in clinical trials and since FDA approval.

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FDA committee takes on complex gene therapy safety questions with Novartis' Zolgensma providing lessons learned

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Gene therapy biotechs are ready with an arsenal of clinical trials to test the potentially groundbreaking treatments in humans. But an array of complicated safety concerns remain to be addressed-including the correct animal models to use in preclinical research to ensure human testing is safe and how to deliver the medicines safely.

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Researchers at the University of California, San Diego (UCSD) have grown miniature human lungs in a lab dish that they say come the closest to mimicking the real thing-complete with all the myriad cell types found in the body.

The small organoids of human tissue could help rapidly iterate the testing of new treatments for respiratory and infectious diseases including COVID-19 by allowing scientists to explore their preliminary effects without needing a full study.

"This human disease model will now allow us to test drug efficacy and toxicity and reject ineffective compounds early in the process, at 'Phase 0,' before human clinical trials begin," Pradipta Ghosh, M.D., professor at the UCSD School of Medicine, said in a statement.

In a paper published in the journal eLife, Ghosh and her colleagues described how they built three lines of organoids out of adult stem cells, taken from the surgically removed organ of a patient with lung cancer-and showed that infecting those models with the coronavirus could replicate real-world cases. The mini-lungs also helped reveal how the organ's different types of cells work when faced with an infection.

The researchers were able to produce the cells that make up both the lungs' upper and lower airways as well as the specialized cells that line the alveoli and help exchange oxygen and carbon dioxide. They found that the upper airway cells help the virus establish an infection, while the lower cells aid in the body's immune response.

Both types can also feed into the dangerously hyperactive immune response known as a cytokine storm, which has been seen in some cases of COVID-19.
RELATED: GlaxoSmithKline taps TARA's 'beating-heart-on-a-chip' to simulate cardiac drug responses
A separate team of computational researchers evaluated the organoids' gene expression patterns and compared them to the records of patients who died of the disease. They found the mini-lungs behaved similarly to real-world infections better than other models, such as those including only one type of cell or derived from other methods.

"Our lung organoids are now ready to use to explore the uncharted territory of COVID-19, including post-COVID complications such as lung fibrosis," said Soumita Das, founding co-director of UCSD's HUMANOID Center of Research Excellence, which focuses on cell organoid models and drug discovery.

"We have already begun to test drugs for their ability to control viral infection-from entry to replication to spread-the runaway immune response that is so often fatal, and lung fibrosis," Das said.

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Researchers replicate COVID-19 infections with lab-grown 'mini-lungs'

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Researchers at the University of California, San Diego (UCSD) have grown miniature human lungs in a lab dish that they say come the closest to mimicking the real thing-complete with all the myriad cell types found in the body.

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Why grab just a pair of scissors when you could get more functionality with a Swiss knife?

A team of researchers at Stanford University think they've built such a tool to apply to CRISPR gene editing, which could help advance the platform and make it easier to deliver into cells. the results were reported Friday in the journal Molecular Cell.

CRISPRs, or clustered regularly interspaced short palindromic repeats, are part of the bacterial immune system that defend against invading viruses. They are made up of repeating sequences of genetic code that are interrupted by pieces of genetic code from previous invaders, which allows the cell to detect and destroy returning invaders. The CRISPR-Cas9 system is a form of gene engineering that targets specific parts of the genetic code to edit DNA at specific locations. This enables researchers to modify the genes in living cells.

The gene editing field recently reached a major landmark when Intellia Therapeutics presented first-in-human data in June. But the technology as it exists now can only be applied to a small portion of the thousands of discovered CRISPRs because the Cas9 proteins in the system are too big. The Stanford researchers have now engineered a tinier version of the CRISPR-Cas system, aptly named CasMINI, which could unlock more applications and get into more mammalian cells.
RELATED: With first-in-human trial results, Intellia shows the world that gene editing has arrived
As it stands today, less than 1% of discovered CRISPRs can work in human cells because they are too big to fit, which limits their use in gene-editing technologies, said senior study author Stanley Qi, an assistant professor at Stanford University, in a statement. CasMINI could help build better tumor-killing lymphocytes, reprogram stem cells or treat more genetic diseases in the eye, muscle or liver.

The large sizes of CRISPR-Cas systems can restrict delivery into cells and negatively impact their potential for clinical viability. Adeno-associated virus, a vector applied in AAV gene therapies that helps deliver the medicine, has a limited amount of space, meaning it can't fit many Cas fusion proteins.

Stanford's new miniature tool works with Cas12f, which could be the answer for fitting into those tight spaces, given the protein is less than half the size of common CRISPR systems that use Cas9 and Cas12a. Prior to the mini system, researchers were unsure whether Cas12f could be used in mammalian cells, but after some tinkering, the Stanford team was able to produce a class of Cas12f variants.

"If people sometimes think of Cas9 as molecular scissors, here we created a Swiss knife containing multiple functions. It is not a big one, but a miniature one that is highly portable for easy use," Qi said.

The Stanford team claims to be the first to turn a non-working CRISPR into a working one through RNA and protein engineering. The engineered CasMINI is 529 amino acids, compared to Cas9's 1,368 amino acids, which could fit better into available delivery mechanisms such as AAV packaging, lipid nanoparticles or other RNA-delivery modalities, the researchers said.
RELATED: Regenerative medicine nears banner year with $14.1B cash infusion, regulatory milestones and a well-stocked pipeline
CasMINI is in its infancy and needs more work to be optimized for base and gene editing. The research team plans to test the system for in vivo gene therapies and has filed for provisional patents through the university.

"It is on our wish list that it will become a therapy to treat genetic diseases, to cure cancer, and to reverse organ degeneration," Qi said.

Qi has founded and holds stakes in Epicrispr Biotechnologies, a CRISPR biotech in stealth, and Refuge Biotechnologies, a cell therapy startup working with MD Anderson Cancer Center on trials of solid tumor drugs.

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A mini-CRISPR system could be 'Swiss knife' in gene editing

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Researchers at Stanford University say they have created a mini version of the CRISPR-Cas system that could be easier to deliver into cells for gene therapies. The mini gene-editing system comes after CRISPR pioneer Intellia revealed first-in-human gene editing data in June.

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iECURE is emerging from stealth with $50 million and a mission to go after the "holy grail" of gene editing with an in vivo approach that is mutation agnostic.

The biotech will use the funds for a slate of gene insertion programs for liver disorders out of the lab of gene therapy pioneer Jim Wilson, M.D., Ph.D. at the University of Pennsylvania.

Versant Ventures, which has backed gene-editing companies CRISPR Therapeutics and Graphite Bio, is launching iECURE alongside OrbiMed Advisors. The series A gives the biotech access to up to 13 programs out of Wilson's Gene Therapy Program at Penn.

Wilson's lab will conduct the preclinical, toxicology and manufacturing support and then iECURE will sign the regulatory filings and move the assets through the clinic and onto the market, CEO Joseph Truitt told Fierce Biotech.

"It's a much more mature company than you would typically start off with if you were just licensing some technology from an academic center," Truitt said. By getting access to Wilson's team, iECURE doesn't have to recruit dozens of employees, the CEO said.
RELATED: FDA committee takes on complex gene therapy safety questions with Novartis' Zolgensma providing lessons learned
Wilson's lab has already shown long-term evidence of genome editing in non-human primates, which is important because translating into species larger than mice is often difficult, said Tom Woiwode, Ph.D., iECURE chairman and Versant managing director, in a joint interview with Truitt and Wilson.

Wilson has thought of the liver as a great target for gene therapy since the late-1980's, when he was in the lab as a post-doc, because "so many diseases have their root in the liver [and] it's accessible to biologics." Wilson is also director of the Orphan Disease Center and a professor in medicine and pediatrics at Penn's School of Medicine.

iECURE and Wilson's lab are approaching the "holy grail of genome editing" by using an in vivo approach that inserts genes inside organs, inside the human body, Woiwode said. In another adventurous analogy, Woiwode said the company is also going after the "untapped frontier of gene editing" by using a mutation agnostic approach. This is critical given many genetic diseases have multiple, "sometimes hundreds," of individual mutations. That many targets would not lend well to creating therapies at scale, the chairman added.

The series A will help iECURE bankroll the first three indications in the program of up to 13, Truitt said. More indications down the pipeline are still under evaluation. The financing is slated to take iECURE through the early part of 2023, the CEO said.

The first program goes after familial hypercholesterolemia, or high cholesterol caused by abnormalities in the gene PCSK9. The treatment reduced PCSK9 protein levels by 85% and LDL cholesterol by 56% in nonhuman primates for three years, Wilson's lab reported in Molecular Therapy in February.
RELATED: Moderna donates ultra-rare disease therapy to nonprofit founded by late Takeda R&D chief
The lab has been working with Precision Biosciences on that asset, which is ready to enter the clinic. As part of iECURE's deal with Wilson's lab, iECURE will conduct the phase 1 study and then return the program to Precision, Truitt said.

The next two indications in the pipeline are the rare metabolic disorders phenylketonuria and ornithine transcarbamylase deficiency, Truitt said.

The gene-editing technology used by iECURE and Wilson's lab is delivered via adeno-associated virus, or AAV. Toxicity and safety issues have arisen with AAV gene therapies, which resulted in an FDA advisory committee meeting last week to discuss and make recommendations on animal and human testing with these therapies.

Wilson took part in two panels during the two-day meeting and said he doesn't see the results of that meeting having an impact on iECURE.

"In terms of the work with IECURE, there really isn't anything that I heard at the committee that impacts on our preclinical plan, our clinical plan or our discussions with health authorities," Wilson said.

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iECURE emerges with $50M to search for 'holy grail' and 'untapped frontier' of gene editing

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iECURE is emerging from stealth with $50 million and a mission to go after the "holy grail" of gene editing with an in vivo approach that is mutation agnostic. The biotech will use the funds for a slate of gene insertion programs for liver disorders out of the lab of gene therapy pioneer Jim Wilson, M.D., Ph.D.

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Recent clinical holds of several studies and an FDA advisory committee meeting has put the safety of gene therapy under the spotlight. To increase the efficacy of gene therapy and reduce the risk of side effects like liver toxicity, scientists at the Broad Institute of MIT and Harvard and Harvard University have developed a new group of adeno-associated viruses (AAVs) as delivery vehicles.

The vectors, dubbed MyoAAV, were more efficient at targeting muscle tissue with lower accumulation in the liver when compared to traditional vectors currently used in clinical trials, according to a new study published in Cell.

The top variants from the MyoAAV family showed strong delivery of therapeutic genes across human cells in test tubes, mice and monkeys. Thanks to improved efficiency, the vectors could help gene therapy achieve efficacy in genetic muscle diseases at about 100 to 250 times lower doses than traditional viral vectors can, potentially reducing the risk of liver damage and other serious side effects, the researchers suggested.

Targeting muscle with more tissue-specific vectors

AAV vectors are made from the outer protein shells of AAVs, called capsids. To make the MyoAAVs, the Broad team developed a platform called DELIVER. The system allows investigators to screen millions of capsid variants in different animal models and select those that can effectively transfer genetic material to host cells of interest, Sharif Tabebordbar, Ph.D., the study's lead author, explained in an email interview with Fierce Biotech Research.

Tabebordbar and colleagues started with AAV9, a commonly used gene therapy vector, and added snippets of amino acids to the naturally occurring capsids. They injected the capsids into mice and monkeys and sequenced the animals' muscle tissue to identify capsids that had successfully transduced the gene.
RELATED: After patients die, FDA clamps hold on Astellas gene therapy trial
A group of top capsid variants that were highly expressed in muscles shared a unique surface structure. The team picked one, MyoAAV 1A, for further testing.

In mice, injections of MyoAAV 1A resulted in transgene expression of a fluorescent protein that was 10 to 29 times higher in skeletal muscles than it was in mice that got AAV9, the team reported. What's more, a lower amount of MyoAAV 1A landed in the liver than was the case with AAV9, and expression of the transgene was lower in the liver of the MyoAAV 1A-injected animals compared to AAV9 recipients, Tabebordbar noted.

In a mouse model of Duchenne muscular dystrophy, MyoAAV 1A carrying a CRISPR-Cas9 gene-editing restored the desired dystrophin protein in muscle tissue at a greater level than what was possible with the traditional AAV9 approach, the researchers found. The mice treated with the novel vector showed higher levels of strength, as well.

The team also tested MyoAAV 1A in a mouse model of X-linked myotubular myopathy (XLMTM), a rare neuromuscular disorder that causes muscle weakness. In mice that lacked the Mtm1 gene, all of the animals that got AAV9 encoding the human version of the gene had to be euthanized between 11 weeks to 21 weeks because of a lack of efficacy. By contrast, all of the mice injected with the same dose of MyoAAV 1A-based gene therapy survived and showed similar activity levels as normal mice did, the researchers reported.

Notably, the dose used for the study was significantly lower than doses used in previously published animal studies of XLMTM gene therapy. It was also 100 times lower than the dose Astellas is using in its clinical trial of AAV8-based AT132, an XLMTM gene therapy the Japanese pharma got from its $3 billion acquisition of Audentes Therapeutics, the researchers noted.

Avoiding liver toxicity with a lower dose

The FDA placed a clinical hold in mid-2020 on AT132's trial after three patients who had received a now-discontinued high dose of the gene therapy died of liver failure. Astellas paused the study again last week after a patient on the low dose of the therapy showed abnormal liver function.

In 2018, scientists at the University of Pennsylvania Perelman School of Medicine warned that a high-dose AAV delivery of a functional SMN gene designed to treat spinal muscular atrophy led to serious adverse events in animals, including the death of a monkey after acute liver failure and shock.

"Liver toxicity after AAV-mediated muscle gene therapy is dose-dependent and it has been observed in clinical trials [at doses] currently required for achieving therapeutic efficacy with naturally-occurring capsids," Tabebordbar noted.
RELATED: FDA committee takes on complex gene therapy safety questions with Novartis' Zolgensma providing lessons learned
Liver toxicity, one of the most common side effects linked to gene therapies, was a key topic of a two-day FDA advisory committee meeting last week focused on gene therapy safety. During the conference, experts in the field suggested researchers should conduct more robust screening for preexisting liver conditions in patients. While the advisors stopped short of placing an upper limit on the gene therapy doses, the liver problems appeared to get worse as the dose increases, they noted.

That's where the Broad team believes MyoAAV could help. "MyoAAV class of capsids provide equivalent therapeutic efficacy at dramatically lower doses, and on top of that are detargeted from the liver compared to wild type capsids," Tabebordbar said. "These two characteristics give the MyoAAV family of capsids an advantage to overcome liver toxicity issues."

After further analyzing the mechanism of MyoAAV 1A, the researchers found that its transduction is dependent on cell surface structures known as integrins. With that knowledge, the team further modified amino acids on MyoAAV 1A and used DELIVER to screen second-generation variants.

This process gave rise to a second generation, MyoAAV 2A. Compared with an AAV9-based gene therapy, a low dose of gene therapy with MyoAAV 2A led to greater and more widespread expression of microdystrophin in multiple muscle groups in a mouse model of DMD, the researchers reported. MyoAAV 2A delivered 12 to 46 times higher numbers of AAV genomes per nucleus in different mouse muscle tissues but 2.5 times lower levels in the liver compared with AAV9.

The scientists also developed several MyoAAV variants for monkeys, which they said outperformed the variants identified in mice.

Broader gene therapy use beyond muscle disease

Now the Broad team is looking for industry partners to move the MyoAAV class of capsids toward the clinic, Tabebordbar said.

"These vectors have the potential to advance muscle-directed therapeutic gene delivery for a large number of musculoskeletal disorders," the researchers wrote in the study. More broadly, DELIVER could help evolve capsid variants for targeting any cell type of interest by selecting variants that express their transgene in the desired tissue or cell type, Tabebordbar added.

The lower dosage requirements could also reduce manufacturing costs for gene therapy, the team noted. Currently, Novartis' AAV9-vectored SMA gene therapy Zolgensma, at a list price of $2.125 million, is the world's most costly drug.

"We anticipate that adoption of DELIVER to additional tissue and organ systems will have a far-reaching impact in accelerating the development and translation of gene therapy and other genomic medicine approaches for a variety of human diseases," the researchers wrote.

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As gene therapy safety faces scrutiny, Broad Institute proposes a safer and more efficient delivery tool

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A team led by scientists at the Broad Institute has developed adeno-associated viruses as gene therapy vectors. The vehicles can target muscle tissue, and therapies based them are effective in animals at low doses, potentially reducing the risk of liver damage, they said.

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How do faulty genes drive cancer? There’s a surprising immune connection, study finds

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A study out of Howard Hughes Medical Institute found that more than 100 mutated cancer-suppressor genes don’t act directly on cancer cells to cause them to grow, but rather block the immune system from recognizing and destroying tumors. They believe further research into those immune pathways could…

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Unlike many tissues in the human body, the heart lacks the ability to regenerate, rendering it unable to self-replenish heart muscle cells known as cardiomyocytes after injury. Scientists have been exploring various ways to help the heart replace damaged cells and restore its function, but those efforts have yet to yield marketable therapies.

Now, a research team led by scientists at the Max Planck Institute for Heart and Lung Research is proposing a promising new method to give the heart regenerative capabilities. The approach involves transiently returning adult cardiomyocytes to a fetal-like state by selectively expressing four genes that are essential for cell renewal: Oct4, Sox2, Klf4 and c-Myc, collectively known as OSKM.

In mouse models of heart attack, heart-specific expression of OSKM for a short period of time either before or immediately after an induced heart incident helped regenerate cardiomyocytes, reduced scar size and improved cardiac function, the team reported in a study published in Science.

Previous studies have shown that OSKM over-expression in mice can induce the so-called "partial reprogramming" process and reverse mature cells to an embryonic state. So the Max Planck-led team applied that approach to heart cells.

For their studies, the researchers used the antibiotic doxycycline to achieve heart-specific OSKM expression in mice. The team then treated them with a six-day doxycycline regimen either six days before inducing heart attacks in the animals or one or six days afterward.
RELATED: Gene therapy repairs cardiac tissue after heart attack in animal models
A heart attack leaves a fibrotic scar. In the Max Planck-led study, all doxycycline-treated rodents showed a significant decrease in scar size compared with untreated controls. However, only pretreated mice and those that got doxycycline immediately after an attack showed improved cardiac function. Mice that received the treatment six days afterward didn't show significant improvements, the team reported.

The six-day treatment duration was important. In healthy mice, six days of continuous doxycycline administration didn't affect normal left ventricular functioning, the researchers found. But extended expression of OSKM for another six days led to significant expression of smooth muscle actin-a sign of scarring-as well as reduction of cardiac function.

Furthermore, early signs of abnormal heart function as a result of OSKM over-expression can be reversed after Day 6 by stopping doxycycline, but the problem became irreversible in healthy mice that got the drug for 12 days. And long-term treatment seemed to force cardiomyocytes into a stem cell-like state, enabling them to grow out of control and form tumors, the team found.

"This is an important reminder that the OSKM genes are potent oncogenic factors that can erase cellular identity in the process of reprogramming, and tight control of their timing and amount is necessary to achieve a therapeutic effect," two researchers at the Stanford University School of Medicine noted in an accompanying editorial.
RELATED: Healing the heart by returning it to its infancy
Giving heart cells regenerative abilities has become a popular research path in cardiology. Scientists at the Texas Heart Institute recently used short hairpin RNAs to suppress the SAV gene in the Hippo signaling pathway in heart cells. The gene therapy showed promising results in improving heart function in pig models of heart attack.

Last year, a team at the UT Southwestern Medical Center found that inhibiting a protein called calcineurin-which regulates two other proteins, Meis1 and Hoxb13-could prolong the time cardiomyocytes divide after their formation.

The Max Planck-led researchers believe their study showed that transient reprogramming of adult cardiomyocytes with OSKM expression could restore heart functions after a heart attack, especially if the treatment is started early. It might also be applied to other organs that fail to regenerate by themselves, they suggested.

A gene therapy based on the OSKM mechanism may be challenging to develop for people, because it requires precision in targeting and dosage control, the two Stanford scientists noted in their piece. "Identification of molecular thresholds that promote dedifferentiation but avoid a point of no return will be critical to engineering a safe therapy," they wrote.

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Repairing heart damage by transiently turning back the clock

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A research team led by scientists at the Max Planck Institute for Heart and Lung Research has proposed a promising new method to give the heart regenerative capabilities. It involves transiently returning adult cardiomyocytes to a fetal-like state by selectively expressing four genes.

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There are many regions of the body that are difficult to reach with drug treatments, most notably the brain, which is surrounded by the famously impenetrable blood-brain barrier. Now, researchers led by the City of Hope have developed a novel drug-delivery system that they say can carry an anti-HIV drug cargo into the brain and other remote tissues in mice-an advance they believe could ultimately be applied to a range of diseases.

The City of Hope team, in conjunction with Menzies Health Institute Queensland at Griffith University, packaged a protein-based anti-HIV drug into "nanosized" cell fragments called exosomes. When the researchers treated mouse models of HIV with the drug, it tamped down HIV levels in the brain as well as in the bone marrow and spleen, they reported in Nature Communications.

One of the main challenges in treating HIV is that the virus can lay dormant in the body for long periods in "reservoirs" that can later become active. HIV reservoirs in the brain are among the hardest to target because of the blood-brain barrier.

The City of Hope researchers set out to find a way to "block and lock" the expression of HIV, they said in a statement, effectively preventing the virus from reactivating. They created the drug by fusing a particular zinc finger protein to a type of enzyme called a DNA methyl transferase, resulting in a "ZPAMt HIV protein repressor." They then packaged it into the exosome nanoparticles.

The researchers reported that 10 weeks after treating the mice, they found that the suppression of HIV in brain, bone marrow and spleen was greater in ZPAMt exosome-treated animals than it was in controls.
RELATED: How 'duoCAR-T' cells could clear HIV and prevent resurgence of virus reservoirs
Although antiretroviral treatments have revolutionized the treatment of HIV, the drugs haven't proven effective at targeting virus reservoirs. Several research teams are investigating alternatives. Engineered cell therapies that target HIV reservoirs, for example, are being developed by scientists at the Albert Einstein College of Medicine and the University of Pittsburgh, the University of California, Los Angeles and the University of North Carolina.

The City of Hope and Menzies researchers see potential for their ZPAMt exosomes well beyond HIV. Engineered exosomes could carry several different types of cargo, they said, including drugs that treat infectious diseases or gene therapies that could cripple pathogens. And, because they can penetrate the brain, they could ultimately prove useful in treating Alzheimer's, Parkinson's and other neurological disorders, they said.

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Novel 'nanosized' drug carriers tamp down HIV in the brain and other remote tissues in mice

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Researchers led by the City of Hope have developed a novel drug-delivery system that can carry an anti-HIV drug cargo into the brain and other remote tissues in mice, locking the virus into a dormant state. They believe the advance could ultimately be applied to a range of diseases.

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The human gut houses a nervous system that operates largely independently of the body's central nervous system-a "second brain" that's of particular interest to a team of researchers at Michigan State University. They believe their latest findings about this second brain, known in scientific circles as the enteric nervous system, could inspire new treatments for common intestinal diseases such as irritable bowel syndrome.

The MSU team studied the cells of the enteric nervous system, including glia, which are non-neuronal cells that don't produce electrical signals. The researchers discovered that glial cells actively modify signaling networks in the gut to maintain normal functions like motility, they explained in Proceedings of the National Academy of Science.

Neurology researchers have long assumed that because glia are not electrically active, they likely play a supporting role to neurons, but figuring out what exactly that role is has been challenging. The MSU researchers found that enteric glia modify neuronal activity via two pathways called purinergic signaling and cholinergic signaling.

To better understand the interplay between glia and the two pathways, the researchers created a mouse model that allowed them to selectively activate glia and then study the effects of the manipulation on cells in the animals' colons.

"Thinking of this second brain as a computer, the glia are the chips working in the periphery," said co-author Brian Gulbransen, an MSU Foundation professor of physiology, in a statement. "They're an active part of the signaling network, but not like neurons. The glia are modulating or modifying the signal.
RELATED: Kintai's obesity drug that works via the gut microbiome moves closer to the clinic
The MSU research comes on the heels of another study from the same team in which they described how enteric glial cells indirectly activate an inflammatory process in irritable bowel syndrome.

Research aimed at manipulating the enteric nervous system is picking up steam. Kintai Therapeutics, which is backed by Flagship Pioneering, is developing small-molecule drugs that manipulate enteric signaling. It reported positive preclinical data on an obesity candidate last year. Other startups targeting the enteric nervous system include Neurogastrx and Enterin.

The growing understanding of glial cells in the gut could inform efforts to develop drugs that target the cells and change their function, Gulbransen said. That could lead to new treatments not just for IBD but also for other motility disorders and for rare conditions in which parts of the gut stop working for no obvious reason, he added.

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New insights into the 'second brain' in the gut could inspire treatments for intestinal diseases, MSU team reports

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Michigan State University researchers discovered that glial cells actively modify electrical signals in the gut to maintain normal functions like motility. They believe the findings could inspire new treatments for common conditions like inflammatory bowel disease and other motility disorders.

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Investor excitement over the development of products that can be injected directly into patients to correct inherited diseases, or "in vivo gene editing," has largely been fueled over the past few months by Intellia Therapeutics and its experimental therapy NTLA-2001 to address the rare liver disease transthyretin (ATTR) amyloidosis. But Intellia's huge run-its stock is up 70% year to date-has overshadowed some of the players in the field, including LogicBio Therapeutics, which has seen its shares lose half their value this year.

Investors may still be underestimating the potential of in vivo gene editing, however. That was the conclusion reached by analysts at Jefferies after they examined new animal data that Intellia and LogicBio presented at the European Society of Gene and Cell Therapy (ESGCT) conference last week.

Intellia rolled out data from a nonhuman primate study in the disease alpha-1 antitrypsin deficiency (AATD). The liver normally makes alpha-1 antitrypsin protein, which protects the lungs, but in patients with AATD, the protein is misshaped, raising the risk of both lung and liver disease. Intellia had shown in a previous primate study that it could insert a healthy form of the gene SERPINA1, which produced normal levels human alpha-1 antitrypsin for 52 weeks.

At the ESGCT event, Intellia presented updated data demonstrating a second step in the process, which involved knocking out the abnormal SERPINA1 gene after inserting the healthy version.

"This insertion followed by knockout led to the continued production of normal human levels of functioning A1AT protein-substantially higher than what has been seen with other treatment approaches-as well as reduction of the disease-associated protein," Intellia said in a statement. The ability to complete both steps would be vital for treating diseases that manifest in both the lung and the liver, like AATD, the company added.

The primate data "showed impressive control of [Intellia's] tech" for inserting one gene and then knocking out another, Jefferies analysts said in a Monday report. They have a price target on the company's stock of $195. It's currently trading around $132.

NTLA-2001, which is partnered with Regeneron, is in early-stage clinical trials, as is NTLA-2002, an in vivo therapy Intellia is developing to treat hereditary angioedema. The company has not yet disclosed a timeline for moving its AATD therapy into clinical trials.
RELATED: In vivo gene editing grabs the spotlight after Intellia triumph, but challenges loom
LogicBio is taking a different approach to correcting hereditary liver diseases. Its platform technology, GeneRide, delivers functioning copies of defective genes into cells' nuclei, setting off natural DNA-repair mechanisms that insert the functional gene into the chromosome. At ESGCT, LogicBio presented data on the use of GeneRide in mouse models of two inherited liver diseases: hereditary tyrosinemia type 1 (HT1) and Wilson disease.

In the HT1 models, GeneRide-treated hepatocytes replaced diseases hepatocytes in the liver within four weeks of treatment, LogicBio announced. The animals no longer required chronic drug treatments for the disease and their liver function returned to normal, the company said.

In the models of Wilson disease, which causes a toxic copper buildup, hepatocytes corrected with GeneRide spread through the liver, improving organ function and reducing the amount of copper excreted by the animals, the company said.

LogicBio had previously reported similar results in mouse models of methylmalonic acidemia (MMA), and a few days before the ESGCT event, it described data from a phase 1/2 trial of GeneRide in children with MMA, a disorder in which the body cannot break down some fats and proteins. The therapy inserts a corrected copy of the MMUT gene with the goal of restoring a protein to correct the condition.

The company announced that biomarker levels in two pediatric patients indicated successful insertion of the gene, and the FDA cleared the company to continue enrolling children as young as six months and to try a higher dose of the treatment in some patients. LogicBio has promised to release additional data from that trial by the end of the year.

LogicBio hasn't had quite as easy a ride with investors as Intellia has, however. Last year, the FDA slapped a hold on the MMA trial due to safety-monitoring concerns, prompting the company to revise the protocol.

Still, Jefferies analysts saw reason for optimism in the mouse data presented at ESGCT. "Data in these unique mouse models provides some [additional] support" for LogicBio's mechanism of action, they said in the new report. They're so optimistic, in fact, that they predicted LogicBio's shares would jump from the current price of about $4 a share to $18.

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Intellia, LogicBio fuel hope for in vivo gene editing with new animal data in rare diseases: report

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Jefferies analysts examined new animal data that in vivo gene editing players Intellia and LogicBio presented at the European Society of Gene and Cell Therapy (ESGCT) conference and concluded that investors are still underestimating the potential of the emerging technology, which is designed to be injected directly into patients to correct inherited diseases.

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Gene-editing technologies such as CRISPR have shown promise as both research tools and therapies for a range of diseases. But existing tools don't always alter the genome as intended, and they can cause unwanted and potentially dangerous mutations.

To better understand the mechanisms behind gene editing, scientists at Princeton University and the Massachusetts Institute of Technology developed a screening method called Repair-seq, which allows researchers to identify genetic elements and associated cellular processes that occur during gene editing.

In two studies published in the journal Cell, the researchers used Repair-seq to generate insights they say could help in the design of more accurate and efficient gene-editing systems. They mapped out the contribution of several pathways in the process by which cells repair double-stranded DNA breaks induced by CRISPR/Cas9 or Cas12a. They also worked with gene-editing pioneer David Liu at the Broad Institute of MIT and Harvard to pinpoint a cellular process that impedes another gene-editing method called "prime editing," which doesn't cause double-stranded breaks.

Repair-seq pairs CRISPR-based screens with site-specific deep genetic sequencing to profile mutations at targeted DNA lesions, according to the team. In one Cell study, the team used Repair-seq to disable 476 genes involved in the repair of double-stranded breaks and examine various outcomes from editing with the Cas9 and Cas12a enzymes. The experiments provided a high-resolution atlas that links repair outcomes with specific genetic factors.
RELATED: Prime Medicine uncloaks with $315M to push 'search and replace' gene-editing treatments
"Systematic exploration of this atlas revealed that repair outcomes with superficially similar sequence architectures can show marked differences in genetic dependencies," the researchers wrote in the study. For example, the researchers showed that the DNA1 and MCM10 genes modulate how fragments of genomic sequences repair double-stranded breaks.

In a separate Cell study published last week, the team worked with Liu to study prime editing using a Repair-seq screen. Liu's team first described prime editing in 2019. The platform involves a guide RNA called pegRNA that sends Cas9 to its target, where the enzyme cuts one strand of DNA. It uses an RNA template to install the desired sequence at the site of the nicked DNA, which then serves as the template to remake the other strand, completing the edit.

The team turned off each of 476 DNA repair genes and examined the outcomes of prime editing. They found that the DNA mismatch repair process-which corrects base errors during DNA replication-interferes with prime editing. Based on that discovery, the researchers designed new prime editing systems that expressed a protein called MLH1dn to temporarily mute mismatch repair.

Liu and colleagues combined the novel prime editing tool with engineered pegRNAs, as described in a recent Nature Biotechnology study, and achieved markedly enhanced editing efficiency, they said.
RELATED: In vivo gene editing grabs the spotlight after Intellia triumph, but challenges loom
Liu previously worked with the same team to deploy Repair-Seq toward identifying factors that affect base editing, which tweaks one nucleotide base rather than changing a long piece of DNA. The results were shared in a Nature Biotechnology study published in June. Liu was a co-founder of Beam Therapeutics, which is working on base-editing therapies for a range of disorders including blood diseases and cancers.

The new research is stocked with gene-editing pioneers, in fact. The co-corresponding author of one of the Cell studies is Cecilia Cotta-Ramusino, who helped get another CRISPR startup co-founded by Liu, Editas Medicine, off the ground. She joined "gene writer" startup Tessera Therapeutics in 2019 as head of platform development.

Liu is now directing his attention to moving prime editing forward. He co-founded Prime Medicine, which decloaked in July with $315 million. The biotech is now incorporating the new findings on pegRNA and prime editing into its platform, it said Tuesday.

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Princeton and MIT gene-editing pioneers uncover clues for improving CRISPR and other tools

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Scientists at Princeton and MIT, including gene-editing pioneer David Liu, used a new screening method called Repair-seq to identify pathways that affect different outcomes in CRISPR/Cas9, prime editing and base editing. The findings could help design more accurate and efficient gene-editing systems, they say.

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A cadre of big and small biotechs and pharmas, plus nonprofit organizations, have signed on to a new U.S. initiative to go after gene therapies for rare diseases.

Called the Bespoke Gene Therapy Consortium, or BGTC, the initiative is headed up by the FDA and the National Institutes of Health (NIH) and is designed to accelerate development of gene therapies. The agencies are putting up $76 million over five years.

On the list of companies are Biogen, Johnson & Johnson's Janssen pharmaceutical unit, Pfizer, the Novartis Institutes for BioMedical Research, Regenxbio, Spark Therapeutics, Takeda, Taysha Gene Therapies, Thermo Fisher Scientific and Ultragenyx. The Alliance for Regenerative Medicine, the American Society of Gene and Cell Therapy and other groups are also involved.

While the diseases are rare, the collective group could make an impact on conditions that affect 30 million Americans. Just two rare diseases out of the 7,000 recognized have approved gene therapies: Novartis' Zolgensma in spinal muscular atrophy and Spark's Luxturna for RPE65 mutation-associated retinal dystrophy.

The BGTC, which is part of the NIH Accelerating Medicines Partnership program, will optimize and streamline the development process for gene therapies for rare diseases to help fill the unmet need.
RELATED: FDA committee takes on complex gene therapy safety questions with Novartis' Zolgensma providing lessons learned
"Most rare diseases are caused by a defect in a single gene that could potentially be targeted with a customized or 'bespoke' therapy that corrects or replaces the defective gene," said NIH Director Francis Collins, M.D., Ph.D. "There are now significant opportunities to improve the complex development process for gene therapies that would accelerate scientific progress and, most importantly, provide benefit to patients by increasing the number of effective gene therapies."

Developing gene therapies is a highly complex process that takes significant time and resources. Companies tend to tackle one disease at a time, and the right tools can be hard to come by. There's also no standardization across the field, the FDA release said.

"Rare diseases affect 25 to 30 million Americans, but because any given rare disorder affects so few patients, companies often are reluctant or unable to invest the years of research and millions of dollars necessary to develop, test and bring individualized gene therapy treatments for a single disease to market," said Joni Rutter, Ph.D., acting director of NIH's National Center for Advancing Translational Sciences.

If the companies can come together and standardize a therapeutic development model with a common gene therapy delivery technique-also known as a vector-the process could be more efficient. A main goal of the BGTC will be to better understand the basic biology of the adeno-associated virus vector that delivers gene therapies and develop a standard test to aid manufacturing and enhance therapeutic benefits.
RELATED: The next generation of gene therapy for rare diseases forges ahead as developers weather hurdles
"The BGTC aims to make it easier, faster and less expensive to pursue bespoke gene therapies in order to incentivize more companies to invest in this space and bring treatments to patients," Rutter added.

The initiative will support four to six clinical trials focused on rare, single-gene diseases or commercial programs that are further along in development that could be scaled up quickly. Streamlining the regulatory process will also be part of the BGTC.

There's plenty of opportunity for companies that do get a rare disease gene therapy across the finish line. Novartis just reported $375 million in sales for Zolgensma for the third quarter amid steady sales in the U.S. and as the therapy became more available across Europe.

Biogen, Novartis and many of the companies on the list all have pipelines full of gene therapies that could meet the rare disease criteria. But the path has been bumpy, with many failures and safety concerns sidelining promising medicines. An FDA advisory committee recently reviewed safety of these therapies and questioned the best animal models to use in early research to ensure human safety once testing advances.

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Pfizer, Novartis sign on to accelerate gene therapies for rare diseases with US agencies

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A cadre of big and small biotechs and pharmas, plus nonprofit organizations, have signed on to a new U.S. initiative to go after gene therapies for rare diseases. Called the Bespoke Gene Therapy Consortium, or BGTC, the initiative is headed up by the FDA and the National Institutes of Health (NIH) and is designed to accelerate development of gene therapies.

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CAR-T therapies, which arm a patient's own immune cells with cancer-fighting proteins, are currently used to treat some patients with blood cancers. But the technology has been hard to apply to other cancers, partly because of a lack of tumor-specific antigens for the T cells to target. Now, a research team at the University of Pennsylvania's Children's Hospital of Philadelphia (CHOP) has developed a new type of CAR-T cell designed to fight solid tumors by targeting proteins inside cancer cells.

These "peptide-centric" CAR-T cells hunt down fragments of cancer-related proteins that are made accessible to the immune system by antigen-presenting MHC proteins. Such a CAR-T therapy selectively eliminated patient-derived neuroblastoma tumors in mice, the researchers reported in a new Nature study.

The discovery could open up a much broader range of therapeutic targets for cancer immunotherapy, the team said. Working with collaborators at Australia-based antibody discovery biotech Myrio Therapeutics, the CHOP scientists aim to move the CAR-T product into clinical trials in late 2022 or early 2023, said CHOP's John Maris, M.D., senior author of the study and professor of pediatrics at Penn's Perelman School of Medicine, in a statement.

In solid tumors, most of the proteins responsible for driving tumor growth and survival reside in the nuclei of cancer cells, rather than on the cell surface, where they would readily be accessible to immune cells, including CAR-T cells. Tumor cells also reveal some fragments of intracellular proteins through MHC, which is known for its ability to present foreign objects to the immune system. However, low rates of mutations and MHC expression have made solid tumors difficult to target with immunotherapies.

Nevertheless, the CHOP team believed that some tumor-specific peptides from key oncoproteins could be targeted with synthetic peptide-centric (PC) CARs. The team focused its research on neuroblastoma, an aggressive form of pediatric cancer that has few mutations and is instead driven by abnormal gene-expression networks.
RELATED: Enhancing cancer-fighting T cells by manipulating their microenvironment
By surveying a large genomic database of neuroblastoma samples, the researchers identified 7,608 peptides accessible to T cells. They narrowed the pool down to peptides that were unique to neuroblastoma, played essential roles in tumor growth and had strong binding ability with MHC.

An unmutated peptide derived from the PHOX2B gene emerged as the best hit. PHOX2B is neuroblastoma dependency gene, and its expression is routinely used in diagnosing neuroblastoma.

The team then engaged Myrio to select PHOX2B-directed PC-CAR constructs that could specifically bind to the related peptide-MHC complex.

In mice engrafted with patient-derived neuroblastoma tumors, treatment with the CAR-T cells led to complete tumor responses, the team reported. What's more, the PC-CARs cells were able to target the same PHOX2B peptide on different MHC types.

The latest study from CHOP reflects a growing interest among immuno-oncology researchers in expanding CAR-T therapies to solid tumors. Penn, which collaborated with Novartis to develop the CAR-T treatment Kymriah, has an ongoing collaboration with Tmunity Therapeutics focused on CAR-T programs for solid tumors. These include a CHOP partnership with the Philadelphia biotech around a glypican 2 CAR-T therapy for neuroblastoma.
RELATED: Tmunity expands Penn pact, picking up another CAR-T program, rights to manufacturing tech
In a Nature Cancer study published a year ago, a Penn team combined a PAK4 inhibitor, which blocks excessive growth of tumor-feeding blood vessels, with EGFR-targeted CAR-T cells and showed encouraging early results in mouse models of glioblastoma.

The new study expands the pool of potential targets for cancer immunotherapy, the researchers said.

"We are excited about this work because it allows us to now go after essential cancer drivers that have been considered 'undruggable' in the past," Maris said in the statement. "We think that PC-CARs have the potential to vastly expand the pool of immunotherapies and significantly widen the population of eligible patients."

In addition to moving a neuroblastoma CAR-T into the clinic, Myrio and CHOP plan to use the same approach to target other pediatric cancers, Myrio CEO Pete Smith said in a statement.

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university of pennsylvania school of medicine

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Turning CAR-T tech against solid tumors by targeting protein fragments in cancer cells

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A University of Pennsylvania team has developed a new type of engineered immune cell called "peptide-centric" CAR-T that's designed to shrink solid tumors by targeting proteins inside cancer cells. CAR-T cells developed with the technology eradicated neuroblastoma in mouse models and could move into human trials in 2022 or 2023.

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Levodopa, the commonly prescribed dopamine-restoring drug for Parkinson's disease, loses its effectiveness over time. Researchers at Northwestern University say they've found a potential method for reviving the drug's benefits: gene therapy.

The researchers restored the ability of neurons to convert levodopa into dopamine in mice with a gene therapy that targets the substantia nigra region of the brain. By effectively recreating a healthy environment in the brain, the therapy eliminated abnormal brain activity that causes movement difficulties in Parkinson's patients, the team reported in Nature.

The new findings also provided insights into why dopamine-releasing neurons wither away in Parkinson's. By studying the genetic features of the neurons in Parkinson's models, the Northwestern researchers showed that damage to the mitochondria, the power suppliers inside of dopamine-producing neurons, triggers events that lead to Parkinson's.

"Whether mitochondrial damage was a cause or consequence of the disease has long been debated. Now that this issue is resolved, we can focus our attention on developing therapies to preserve their function and slow the loss of these neurons," said James Surmeier, Ph.D., chair of neuroscience at Northwestern's Feinberg School of Medicine, in a statement.

The insights could be used to develop tests that identify Parkinson's in people five to 10 years before it manifests, Surmeier suggested.
RELATED: Neurocrine exits $165M Parkinson's pact with Voyager after FDA hold
Efforts to develop gene and cell therapies for Parkinson's are underway, with mixed results so far. Bayer has started two early-stage trials: a gene therapy being developed by its subsidiary AskBio and a stem cell treatment from its unit BlueRock Therapeutics.

Voyager Therapeutics has suffered several setbacks in its efforts to develop a gene therapy for Parkinson's. Sanofi ended its deal with Voyager in October 2017, AbbVie nixed its pact in August 2020 and Neurocrine Biosciences axed its tie-up in February of this year after a clinical hold was placed on a phase 2 trial last December. Pfizer inked a $630 million pact with Voyager last month to use its capsids in neurologic and cardiovascular gene therapies, though the specific disease targets were not disclosed.

Other researchers are also looking for innovative ways to spruce up dopamine-producing neurons. A team at the University of San Diego, California developed a gene therapy technique that turned astrocyte cells into dopamine-producing neurons, for example.

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Parkinson's gene therapy restores responses to dopamine-boosting drug in mouse models

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Northwestern University researchers found that a gene therapy targeting the region of the brain that houses dopamine-releasing neurons could boost the effectiveness of the widely used Parkinson's drug levodopa in mice. The team also found a a path to diagnosing the disease early.

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Treating drug-resistant prostate cancer by exploiting tumor-suppressing RNA

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Researchers at the Washington University School of Medicine discovered a long noncoding RNA that regulates the androgen receptor in prostate cancer. Dialing up its expression in mouse models of the disease caused tumors to shrink, they reported.

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One popular idea for regenerating injured spinal cords is to implant artificial scaffolds bearing healing proteins near the injury site, but the idea has not panned out so far. Now, researchers at Northwestern University say they've found a way to make such a technique work, with two peptides that are altered so they amp up nerve regeneration.

The Northwestern team developed an injectable liquid containing the modified peptides, which form nanofibers similar to the spinal cord's extracellular matrix. In mouse models of spinal cord injury, a single injection of the therapy promoted the regrowth of axons and restored walking ability, they reported in Science.

The key to the therapy is "dancing molecules," explained lead author Samuel Stupp, Ph.D., a professor at Northwestern. His team mutated two peptide sequences: one that reduced scarring and the other that promoted the formation of blood vessels. That controlled the motion of the molecules in the nanofiber matrix in ways that promoted regeneration, Stupp said.

"By making the molecules move, 'dance' or even leap temporarily out of these structures, known as supramolecular polymers, they are able to connect more effectively with receptors," Stupp said in a statement.
RELATED: CRISPR screen points to spinal repair pathway in zebrafish
The moving molecules in the Northwestern study triggered signals critical to spinal cord repair, the Northwestern team observed. Promoting the regrowth of axons, which are the long tails at the end of neurons, improved communication between the brain and body. The therapy also reduced scarring, and the regrowth of blood vessels fed vital nutrients to cells needed to repair critical tissues.

After 12 weeks, the injected materials degraded into nutrients that continued to feed regenerative cells, the Northwestern team reported. There were no obvious side effects, they said.

This is one of several innovative approaches being investigated to prevent paralysis after spinal cord injury. Earlier this year, a University of Edinburgh team used the gene-editing technology CRISPR in zebrafish to pinpoint the gene TGFB1 as essential in promoting spinal cord repair.

UT Southwestern and Indiana University researchers genetically engineered cells to overproduce the healing protein Sox2, which improved mobility in mouse models of spinal cord injury. And a Temple University team found that boosting levels of the protein Lin28 in injured spinal cords prompted axon regrowth in mice.

The Northwestern researchers are planning to apply to the FDA to start human trials of their therapy, they said. They also believe their technique could be applied to other disorders marked by the loss of tissues in the central nervous system, including Parkinson's and Alzheimer's diseases, they added.

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Injectable 'dancing molecules' repair spinal cord injury, restore mobility in paralyzed mice

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A Northwestern team developed an injectable liquid containing modified peptides, which form nanofibers similar to the spinal cord's extracellular matrix. In mouse models of spinal cord injury, the therapy promoted the regrowth of axons and restored walking ability, they said.

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Fine-tuning targeted therapy for multiple myeloma with ‘multi-omics’ data

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Mount Sinai scientists analyzed biological datasets from multiple myeloma patients and generated a computational model that allowed them to identify new subtypes of the disease. Armed with the results, they identified potential targeted treatments for patients who fall into those subtypes.

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Alcohol accounts for about 3 million deaths worldwide annually, and there are only four approved drugs to treat alcohol use disorder. Psilocybin, the active ingredient in magic mushrooms, has drawn interest as a potential remedy for the disorder, and now a team of German researchers is backing up the idea with intriguing preclinical data.

Researchers at the University of Heidelberg treated rat models of alcohol addiction with psilocybin and found that the psychedelic was effective at staving off cravings, they reported in the journal Science Advances.

The promising effects of psilocybin stem from the compound's ability to restore expression of a specific glutamate receptor known as mGluR2, the researchers explained. Reduced expression of the receptor causes both alcohol cravings and impairments in "executive functions," such as self-control and decision-making, they demonstrated in the study.

The team started by exposing the rats to alcohol vapor that made them intoxicated at similar levels seen in people with clinical alcohol addiction. Long-lasting behavioral changes and molecular changes in the brain occurred as the rats became dependent on alcohol over seven weeks, the researchers reported.
RELATED: Psilocybin clears biggest depression test yet, but Compass stock falls amid chatter about adverse events, durability
The researchers gave the rats one of two different doses of psilocybin to see whether the magic mushroom ingredient was effective at reducing relapses. They found that both doses were effective compared to a control treatment, the team said in a statement.

Little is understood about the molecular mechanisms that contribute to the crumbling of executive function in alcohol use disorder, the researchers said. That led them to perform the tests, which found that lower levels of mGluR2 expression in a certain region of the cortex trigger alcohol-seeking behavior and impaired cognitive flexibility in the animal model.

The team then set out to see whether psilocybin could restore the receptor's expression and tamp down the desire to seek out alcohol while also restoring flexibility in cognition. In addition to reducing cravings, the treatment lessened alcohol-seeking behavior in the rat models and reduced the risk of relapse, they reported.

The Heidelberg team's research comes amid a wave of investor interest in psychedelics as treatments for depression, addiction and other conditions. Alcohol use disorder is the focus of Journey Colab's lead drug candidate, which is derived from a psychedelic compound found in some cacti. The startup snagged $12 million in September to move the drug into clinical trials next year.

As for psilocybin, it's being tested in several diseases, including depression. Compass Pathways reported earlier this month that its psilocybin candidate showed improved symptoms in people with treatment-resistant depression in a phase 2b trial.

Hurdles remain, though, including a federal law that has hampered research in the field, ethics concerns, intellectual property challenges and a stigma surrounding psychedelics. What's more, Compass' trial raised concerns about suicidal ideation in some patients and the durability of the treatment.

The German team mapped out a strategy for testing psilocybin in people with alcohol dependency. As part of their study, they used positron emission tomography to identify a biomarker they believe could be used to identify people who might benefit from interventions targeting the mGluR2 receptor.

They suggested starting by enrolling patients identified with the biomarker into a trial of psilocybin and using MRI scans to track improvements in "functional connectivity" in the brain after a single dose, they wrote. If such improvements are observed, a longer controlled trial could be designed to test whether psilocybin reduces alcohol cravings and prevents relapses, they proposed.

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Psychedelic psilocybin cuts cravings and boosts key brain functions in rat models of alcohol use disorder

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Researchers at the University of Heidelberg treated rat models of alcohol addiction with the active ingredient in magic mushrooms, psilocybin, and found that the psychedelic restored expression of a glutamate receptor called mGluR2. That reduced cravings and improved executive functioning, they found.

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Medulloblastoma is a common brain tumor in children that becomes particularly tough to treat once it spreads. In an effort to find an immunotherapy that might offer hope to these young patients, scientists at Children's National Hospital in Washington, D.C., used a specific type of DNA analysis to devise a novel therapy that's showing promise in preclinical studies.

The team identified peptides that are unique to medulloblastoma tumors and then engineered T cells so they could recognize and target those proteins. The T cells eliminated medulloblastoma cells in test tubes, they reported in Nature Communications.

The researchers started by sequencing DNA from patient samples, analyzing all of the proteins in the tumors that affect cancer biology-a process called "low-input proteogenomics," they explained in a statement from Children's National. Then the researchers developed T cells that targeted specific proteins, or neoantigens, in each sample that were unique to the cancer and not present in healthy cells.
RELATED: Startup targets glioblastoma tumors with CAR-T therapy
Tumor cells typically take on mutations when they copy their DNA, and those mutated genes create abnormal proteins that are unique to each patient's cancer. For their process to work correctly, the Children's National researchers had to develop a technology to filter out normal peptides.

"Targeting antigens that are completely specific to the tumor, and expressed nowhere else in the body, will potentially increase the strength of tumor antigen-specific T cell products while decreasing the toxicity," said first author Samuel Rivero-Hinojosa, Ph.D., staff scientist at Children's National, in the statement.

Personalized CAR-T therapies have proven effective in blood cancers but have not yet been successfully translated to the treatment of solid tumors. Several techniques for adapting the technology to brain tumors are under investigation.

Canadian scientists reported last year they developed a CAR-T to target CD133, also called prominin-1, a protein on neural stem cells in some brain tumors. The treatment is being developed by startup Empirica Therapeutics.

In April of this year, University of California, San Francisco scientists described an approach they developed to arm T cells to target solid tumors, including glioblastoma, via molecular circuits that prime cancer cells for killing by T cells.

The Children's National Hospital team hopes to design a clinical trial during which they will take T cells from medulloblastoma patients, train them to recognize unique tumor neoantigens and then reinfuse them back into the patients, the hospital said.

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Personalized T cells could offer an immunotherapy option to children with brain tumors

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Scientists at Children's National Hospital identified peptides that are unique to medulloblastoma tumors and then engineered T cells so they could recognize and target those proteins. They believe the therapy could offer a personalized immuno-oncology option for children diagnosed with this common tumor type.

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CAR-T cell therapy, which modifies a patient's own T cells to recognize specific antigens on tumors, has proven effective in treating blood cancers. But the engineered immune cells have precious little time to proliferate in the body, which often limits their anti-tumor efficacy. Now, scientists at the Curie Institute in France have identified a potential mechanism that could be targeted to improve T-cell therapies.

By using genomewide CRISPR-Cas9 screen in mice, the Curie Institute team pinpointed the protein SOCS1 as an important "brake" that inhibits the expansion and survival of CD4+ T cells, according to results published in Science Immunology.

Crippling SOCS1 in human CD19-targeted CAR-T cells markedly improved the persistence of CD4+ CAR-T cells and the function of CD8 CAR-T cells in a mouse model. Thus SOCS1 inhibition could boost the efficacy of T-cell therapies against cancer, the team suggested.

When cancer relapses or progresses after patients initially respond well to CAR-T treatments, the culprit is thought to be poor persistence of the engineered cells. One of the key components of CAR-T cell therapy, CD8+ T cells, can continuously self-proliferate after stimulation. But another critical element, CD4+ T cells, require repeated antigen stimulation to continue growing.
RELATED: Enhancing CAR-T therapy by leveraging its communication with the immune system
To understand the inhibitory mechanism of CD4+ T cells, the Curie Institute team used CRISPR screening to search for genes that, when inactivated, would restore the proliferation of antigen-experienced CD4+ T cells.

The gene encoding for SOSC1 emerged as a hit. In two different TCR CD4+ T-cell models, inactivating the gene helped the T cells expand, while the the proliferation of untreated cells quickly stopped, the researchers reported.

Further analysis showed that SOCS1 moderates the immune system, blocking several signaling pathways and crippling CD4+ T cell proliferation.

The researchers challenged mice with a type of bladder tumor and treated them with anti-tumor CD4+ T cells. Treatment with T cells that had SOCS1 inactivated rejected the tumors, as the T cells proliferated. The researchers also found that the SOCS1-deleted T cells infiltrated tumors more efficiently than control cells by nearly tenfold.

SOCS1 also played a role in regulating CD8+ T cells, the study found. In mice with melanoma, the scientists observed a significant and durable rejection of tumors in animals that received SOCS1-deleted, tumor-specific CD8+T cells as compared with control T cells. In SOCS1-inactivated T cells that entered tumors, the researchers found higher numbers of cytotoxic molecules and effector T cells, likely explaining the strong anti-tumor effect, they said.

The French team validated their findings with human CD4+ and CD8+ CAR-T cells, reporting that in mouse models of lymphoblastic leukemia, treatment with SOCS1-inactivated CAR-T cells produced better tumor control, and the number of CAR-T cells accumulating in bone marrow was twofold higher than it was with unmodified CAR-T cells.

The promise that engineered T-cell therapy holds against various cancers has prompted scientists across the globe to explore ways to enhance its efficacy. A team at the University of Pennsylvania recently proposed inhibiting BET proteins to prevent T-cell exhaustion in CAR-T cell treatment of chronic lymphocytic leukemia, for example.

Scientists at France's Pasteur Institute found that the interaction between interferon gamma by CAR-T cells and interleukin 12 by host cells is critical to the cancer-killing activity of CAR-T cells. And a team at the Fred Hutchinson Cancer Research Center has designed new CARs with fragments involved in T-cell signaling to increase their ability to recognize tumor antigens.

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Improving CAR-T cell therapies for cancer by targeting a protein 'brake'

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CAR-T cells to treat cancer have precious little time to proliferate in the body, which often limits their anti-tumor efficacy. A team at the Curie Institute pinpointed the protein SOCS1 as an inhibitor of CAR-T expansion and survival and suggested it could be targeted to improve T-cell therapies.

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Scientists have known about rare and common genetic variants that influence cardiovascular disease, but few gene mutations have been found to impact more than one risk for coronary artery disease. That gap in knowledge has limited the development of new treatments targeted to individual risk factors.

Now, scientists at the University of Maryland say they have found a genetic variant in the Old Order Amish population in Pennsylvania that could inform new medical approaches to decreasing or preventing the world's leading cause of morbidity and death. The population was found to have increased levels of heart protection, ameliorating their risk for heart disease, the researchers explained in new findings published in the journal Science.

Using exome sequencing, the researchers found that people in the Old Order Amish community have a "missense" variant, or a single base pair substitution, in the gene B4GALT1. That correlates with a lower risk of cardiovascular disease. The gene codes for a protein that transfers the sugar molecule galactose across the cell membrane.

The University of Maryland team delivered the gene mutation to mouse models of cardiovascular disease using CRISPR-Cas9 technology. Animals with the variant experienced a 38% drop in blood low-density lipoprotein cholesterol, or LDL-C, and also saw a decline in the amount of the blood-clotting protein fibrinogen.
RELATED: Fending off heart attacks by getting at their genetic roots
The University of Maryland has studied the Old Order Amish since 1993, with more than 7,000 Amish adults participating in at least one study on genetic determinants of cardiometabolic health as part of a research program at the institution.

The group comprises about 40,000 in people in the Lancaster County, Pennsylvania, region, and most of the individuals can trace their ancestry back 15 generations to the 750 or so founders of the community.

While the group is relatively small-and variants such as the one found in B4GALT1 are typically rare or absent in the broader population-the new findings could still point to therapeutic targets that would pave the way for treatments relevant to most people, the scientists suggested.

Other academics and biopharmas are looking at genetic approaches to heart disease. A team at the University of Virginia, for example, found that several naturally occurring gene variants influence the buildup of fatty plaques in blood vessels, which can lead to heart attacks or strokes. The team also found that one gene, MIA3, plays a central role in forming protective caps to stabilize plaque lesions in blood vessels.

Researchers across Harvard University and Imperial College London have built an atlas of the 500,000 cells in the heart that keep humans on the go. The cross-institutional team found that each of the six regions of the heart has its own subsets of cells, which could shed light on variable responses to treatments for cardiac diseases.

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Genetic variant linked to Amish community could pave way for new heart disease therapies

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Researchers at the University of Maryland say a genetic variant linked to the Old Order Amish community in Pennsylvania reduce levels of LDL cholesterol and a blood-clotting protein. The findings could lead to new paths for preventing or treating cardiovascular disease.

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Most therapies for vascular diseases aim to treat risk factors like high blood pressure and cholesterol rather than repairing the damaged blood vessels. Now, a team of researchers at the University of Chicago has shown that a targeted nanomedicine may be able to address vascular lesions.

The researchers designed a peptide that targets vascular cell adhesion molecule 1, which populates inflamed endothelial cells at high levels, and tested it in mouse models of atherosclerosis, a vascular disease in which the walls of blood vessels form plaques that clog the arteries. The vascular lesions shrank, they reported in the Proceedings of the National Academy of Sciences.

Now, the scientists who led the study planning to form a company to prepare the drug for clinical trials, said Matthew Tirrell, Ph.D., dean of the Pritzker School of Molecular Engineering, in an interview. "We can deliver an inflammation-reducing therapeutic oligonucleotide to sites of inflammation in the vasculature and we showed how this retards the progression of atherosclerotic plaques" in animals, Tirrell said.

Tirrell said the results are what the team anticipated, but they didn't predict how effective the treatment could be.

"If you deliver the same oligonucleotide systemically, without packaging it in our nanoparticle, it also retards the development of atherosclerosis, but our way of doing it is ... twice as effective. It really showed a clear benefit in delivering the therapeutic right to the site of inflammation in the vascular endothelium," Tirrell said.

The researchers will likely test the treatment in other conditions first, because vascular disease can be hard to diagnose. One potential route would be to test the nanoparticles during angioplasty or stent insertions, which cause inflammation and disturbed blood flow. The team's nanoparticles are able to assuage blood-flow issues in mouse models, Tirrell said. Another potential use for the drug is in arteriovenous fistulas, or AVFs, which connect arteries to veins and are critical for vascular access in kidney dialysis.
RELATED: How a discovery in 'junk' DNA could protect against heart disease
Other researchers working on new approaches to atherosclerosis include a team at Brigham and Women's Hospital that found a long, noncoding RNA molecule played a role in the cellular aging of blood vessels. And a team at the University of Oxford discovered Plexin D1, part of a key cell-surface receptor group, that helps promote plaque formation, which leads to atherosclerosis.

The University of Chicago researchers believe the nanoparticle constitutes a platform technology that could show promise in reducing inflammation in other endothelial tissues. The team also has preliminary data on acute respiratory distress in COVID-19 and other respiratory diseases, Tirrell said.

The next step for the team is to design large-animal studies to prove out the treatment's effectiveness in various complications of vascular disorders.

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proceedings of the national academy of sciences

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Nanomedicine shrinks vascular lesions in mice and could pave path to new treatments

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University of Chicago researchers say their novel nanomedicine shrank vascular lesions in mice and stopped the progression of plaque buildup that leads to vascular diseases. The team is working to secure funding for a larger animal study and form a company to test the treatment in people.

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Super-fit mice reveal anti-inflammatory protein that could boost search for Alzheimer’s drugs

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A Stanford team discovered that a protein called clusterin is prevalent in exercising mice and prompts anti-inflammatory, brain-boosting effects. They concluded that a drug that reduces inflammation in the brain by mimicking the binding of clusterin to endothelial cells could prove effective in Alzh…

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Explore more searches like transgenic animals

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Use of Transgenic Animals in Biotechnology: Prospects and Problems

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During the past two decades, there have been numerous attempts at using animals
in order to produce recombinant human proteins and monoclonal antibodies.
However, it is only recently that the first two therapeutic agents isolated
from the milk of transgenic ...

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uniQure - Gene Therapy - Manufacturing Facility Video

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Overview

You're the newest member of the Quality Assurance department at CATT - the Center for the Advancement of Toast Technology. Your mission is to audit the company and determine if its SOPs, notebooks, equipment, personnel, reagent labels, and test animals are GLP compliant.

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You're the newest member of the Quality Assurance department at CATT - the Center for the Advancement of Toast Technology. Your mission is to audit the company and determine if its SOPs, notebooks, equipment, personnel, reagent labels, and test animals are GLP compliant.

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Overview

In this interactive exercise, you will learn the importance of batch documents by reviewing the core values that define them, exploring their importance to the manufacturing process, and identifying and correcting the most common types of errors encountered in batch documentation.

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In this interactive exercise, you will learn the importance of batch documents by reviewing the core values that define them, exploring their importance to the manufacturing process, and identifying and correcting the most common types of errors encountered in batch documentation.

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Gibco Cell Culture Basics | Thermo Fisher Scientific - US

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The Cell Culture Basics handbook from Gibco and videos can help your lab attain reproducible results every day. Cell culture is critical to your success, so make sure everyone in your lab uses the best procedures and products to consistently get the outcomes you expect.

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BLAST for beginners
This tutorial is designed as a quick introduction to the BLAST family of sequence analysis programs.

These slides show a progression of steps in using blastn, beginning at the home page for the National Center for Biotechnology Information (NCBI) ( www.ncbi.nlm.nih.gov) and ending at PubMed, a tool for searching scientific literature. In this series, you will see how to submit a nucleotide sequence, compare it to other sequences using blastn, and interpret some of the results.

Before you begin, here are some helpful hints:

Click the green arrows to move to the next slide.

In most slides, the green arrows point to the button or link that you should select in an actual BLAST search.

To repeat the information, reload the page, or return to a previous slide by using the "Back" button on your browser.

In most cases, the links shown on the following slides are not active; only the green arrows have active links.

It's helpful to have two or more web browser windows open at the same time so that you can view the tutorial in one window and the live site in the other.

This series is not meant as a comprehensive description of the BLAST programs, merely a quick guide to help you get started. For more information about BLAST, see https://blast.ncbi.nlm.nih.gov

Click on the green arrow to begin

Funding for this project was provided by the National Science Foundation's Course, Curriculum, / and Laboratory Improvement Program under grants DUE-0088153 and DUE 0127599

Privacy | Using Molecule World Images | Contact

2019 Digital World Biology® ©Digital World Biology LLC. All rights reserved.

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BLAST for beginners This tutorial is designed as a quick introduction to the BLAST family of sequence analysis programs. These slides show a progression of steps in using blastn, beginning at the home page for the National Center for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov) and ending at PubMed, a tool for searching scientific literature.

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Take a Walk on the Lab side: a Virtual Field Trip to Amgen

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Are you interested in seeing the inside of a lab but don't know anyone who could get you inside one? Amgen's Virtual Lab can give you a fascinating look into what goes on in a production laboratory.

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Best video on 10 Principles of GMP | Good Manufacturing Practices

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FDA ensures the quality of drug products by carefully monitoring drug manufacturers' compliance with its Current Good Manufacturing Practice (CGMP) regulations. The CGMP regulations for drugs contain minimum requirements for the methods, facilities, and controls used in manufacturing, processing, and packing of a drug product. The regulations make sure that a product is safe for use, and that it has the ingredients and strength it claims to have.

The approval process for new and generic drug marketing applications includes a review of the manufacturer's compliance with the CGMPs. FDA assessors and investigators determine whether the firm has the necessary facilities, equipment, and ability to manufacture the drug it intends to market.

Code of Federal Regulations (CFR). FDA's portion of the CFR is in Title 21, which interprets the Federal Food, Drug and Cosmetic Act and related statutes, including the Public Health Service Act. The pharmaceutical or drug quality-related regulations appear in several parts of Title 21, including sections in parts 1-99, 200-299, 300-499, 600-799, and 800-1299.

The regulations enable a common understanding of the regulatory process by describing the requirements to be followed by drug manufacturers, applicants, and FDA.

21 CFR Part 314 For FDA approval to market a new drug.

21 CFR Part 210. Current Good Manufacturing Practice in Manufacturing Processing, packing, or Holding of Drugs.

21 CFR Part 211. Current Good Manufacturing Practice for Finished Pharmaceuticals.

21 CFR Part 212. Current Good Manufacturing Practice for Positron Emission Tomography Drugs.

21 CFR Part 600. Biological Products: General.

The following pages provide more information with respect to regulations for particular product types and manufacturing considerations:

Contact for Further Information:
CDER-OPQ-Inquiries@fda.hhs.gov

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Current Good Manufacturing Practice (CGMP) Regulations

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Regulations help to ensure quality drug products. FDA monitors drug manufacturers' compliance with Current Good Manufacturing Practice (CGMP) regulations. This web page provides links to resources to help drug manufacturers comply with the CGMP regulations; including guidance documents, federal regulations, MaPPs, and questions and answers.

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E-Mail: lseidman@madisoncollege.edu This page explains how to use the internet to obtain the current Good Manufacturing Practices (cGMP) regulations. It describes how the regulations are organized and how they can be searched and retrieved.
Address: Madison College
1701 Wright St. Madison, WI 53704
Revised November 8, 2012
The Code of Federal Regulations (CFR) is a codification of the general and permanent rules of the federal government [1]. The CFR contains the complete and official text of the regulations that are enforced by federal agencies.
Footnotes:
The CFR is organized as follows:

The CFR is divided into 50 titles that represent broad areas subject to Federal regulations.

Each title is divided into chapters that are assigned to various agencies issuing regulations pertaining to that broad subject area.

Each chapter is divided into parts covering specific regulatory areas.

Large parts may be subdivided into subparts.

Each part or subpart is then divided into sections -- the basic unit of the CFR.

Sometimes sections are subdivided further into paragraphs or subsections. Citations pertaining to specific information in the CFR will usually be provided at the section level.

An example of a typical CFR citation is 21 CFR 211.67(a). Here is what this citation actually means:

The number 21 is the CFR title [2]. The broad subject area is Food and Drugs.[3]

The number 211 is the part. Part 211 is entitled "Current Good Manufacturing Practice for Finished Pharmaceuticals".

The number .67 refers to a particular section. This section is about Equipment Cleaning and Maintenance.

The (a) is the first paragraph or subsection within the section.

There are several ways to access the portions of the CFR relating to the manufacture of pharmaceuticals using the web [4]:

1. The FDA web site includes the CFR dealing with cGMP https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfCFR/CFRSearch.cfm.

2. The FDA website https://www.fda.gov/industry includes along with the federal regulations, notes, proposed changes in the regulations and other pertinent information concerning cGMP.

3. You can find any part of the Code of Federal Regulations at https://www.govinfo.gov/app/collection/cfr/

4. A good site for information about FDA regulations is a commercial site hosted by The GMP Institute. This site includes the following useful items:

You can purchase inexpensive booklets from the GMP Institute that contain the text of the GMP regulations.

You can also go to their web site to link to the cGMP regulation 21 CFR Parts 210 and 211 for the drug industry.

They also have links to the regulations for the medical device industry (21 CFR Part 820), the food industry (21 CFR Part 110) and the blood products industry (21 CFR Part 606).

This site also has an inclusive list of from the FDA.

The as published in the September 29, 1978 issue of the Federal Register, can also be found on their web site. The Preamble is an interesting document that contains comments from the public relating to the GMP regulations and the official response to those comments.

1 This explanation is based largely on information from the National Archives web site, https://www.archives.gov/federal-register/cfr. This government web site explains what the Federal Register is and provides access to government documents.

2 Each of the titles of the CFR is assigned to a specific agency. The Government agencies that have regulatory control can be determined by the relevant CFR Titles, Subchapters or Chapters.

3 Title 21 contains three Chapters that relate to Food and Drugs. Often the Chapter and Subchapter are not cited, but the citation in the example here is found under Chapter 1. The agency that is responsible for compliance with Chapter 1 is The Food and Drug Administration(FDA). The other Chapters are controlled by other federal agencies. Chapter 1 has seven subchapters and 1299 Parts. Subchapter C covers regulations for General Drugs. Other Subchapters within this Chapter cover regulations for Food, Food additives, and other products regulated by FDA.

4 Federal regulations are first published in the Federal Register (FR) by the executive departments and agencies of the Federal Government. The Federal Register is a legal newspaper published every business day by the National Archives and Records Administration (NARA). It contains Federal agency regulations; proposed rules and notices; and Executive orders, proclamations and other Presidential documents. The Federal Register informs citizens of their rights and obligations and provides access to a wide range of Federal benefits and opportunities for funding. The CFR is kept up-to-date by the daily Federal Register. For the most up-to-date information, these two publications must be used together to determine the latest version of any given rule. When a Federal agency publishes a regulation in the Federal Register, that regulation usually is an amendment to the existing CFR in the form of a change, an addition, or a removal.

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Access the Code of Federal GMP Regulations

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Authors: Noreen Warren and Lisa A. Seidman, Ph.D. E-Mail: lseidman@madisoncollege.eduAddress: Madison College1701 Wright St. Madison, WI 53704 Revised November 8, 2012 This page explains how to use the internet to obtain the current Good Manufacturing Practices (cGMP) regulations. It describes how the regulations are organized and how they can be searched and retrieved.

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THE PARTS OF A STANDARD OPERATING PROCEDURE (SOP) Text Authors: Noreen Warren and Lisa A. Seidman, Ph.D. SOP authors: Michael Barazia (Identification Methods for Copper Sulfate) and Jay Bucher, Senior Metrologist, Promega Corporation (Balance and Scale Calibration Procedure) E-Mail: lseidman@madisoncollege.edu Madison Area Technical College, Madison, WI 53704 Revision November 8, 2012 This page describes Standard Operating Procedures (SOPs) and their component sections and provides examples of a SOP for students to review.

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The differences between Good Manufacturing Practices (GMPs) and Good Laboratory Practices (GLPs) aren't always obvious. Both share similar terminology and at times, somewhat similar applications.

But for those who apply these practices, or collaborate with those who do, the differences are important. Confusing one for the other can lead to serious compliance problems and rework.

This guide offers a quick and simple explanation of the differences, starting with the most pronounced.

Laboratory testing

This is one of the most dangerous areas to get the two confused. While GMPs and GLPs serve similar purposes in other applications, they're quite different in the lab.

The simplest way to understand the differences between the two is where they lie in the larger product development process. GLPs are focused squarely on preclinical development. More specifically, nonclinical laboratory safety studies that support or are intended to support the research or marketing applications of the product. These include biocompatibility, toxicology, pharmacology, etc.

On top of this, GLPs only apply to studies performed in test systems like live organisms, plants, or microorganisms-not chemistry-based or microbiological quality testing. More simply, GLPs are based in study. Their purpose is to give FDA the data it needs to scrutinize and audit the scientific validity of research studies.

GMPs, on the other hand, are concerned with process. They're intended to demonstrate to the FDA whether or not batches of regulated products are manufactured according to pre-defined criteria.

Lot release or lot conformance testing for products on the market are almost always required to be done under GMP while testing of safety and efficacy should be done under GLP in most cases.

Validation

Unfortunately, the differences between GLPs and GMPs are not so apparent during validation. This continues to be an area of debate within the industry. "It depends" is the basic idea you'll come away with from most interpretations.

The best advice is to carefully consider which set of practices are more appropriate for a particular product and ensure that all equipment and testing instrumentation is calibrated, qualified, and maintained according to SOPs.

Want to learn more about laboratory developed testing and regulatory challenges that may be coming? Grab our free white paper: Laboratory Developed Tests: Issues & Anticipated Regulations

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GMP vs. GLP in Laboratory Testing & Validation

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The differences between Good Manufacturing Practices (GMPs) and Good Laboratory Practices (GLPs) aren't always obvious. Both share similar terminology and at times, somewhat similar applications. But for those who apply these practices, or collaborate with those who do, the differences are important. Confusing one for the other can lead to serious compliance problems and rework.

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What is the Difference between GLP, GCP, and GMP Laboratory Testing Regulations? | Eurofins CRL

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What is the Difference between GLP and GMP Laboratory Testing Regulations? | Microchem Laboratory

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People are often confused by differences between Good Laboratory Practice (GLP) regulations and Good Manufacturing Practice (GMP) regulations as they relate to laboratory testing. This is understandable, since both GLPs and GMPs cover lab testing but are very different. In addition, scientists and q…

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Transgenic Purple Tomato. A purple tomato is genetically modified to contain a cancer-fighting compound. A gene for the compound was transferred into normal red

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Biotechnology has been used to create transgenic crops. Transgenic crops are genetically modified with new genes that code for traits useful to humans. 

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Tips and Considerations for Sensitive PCR Assays

Primer design is a critical step in the design of a good real time PCR experiment. There are many elements to consider when designing primers that can help minimize troubleshooting down the line. Here are important recommendations on creating specific and effective primers for several types of PCR assays.

Polymerase chain reaction (PCR) is a biochemical process that uses repeated cycles of heating and cooling to amplify a genetic region of interest, called the amplicon. In each cycle the double stranded DNA is first denatured and then cooled, allowing short oligonucleotide primers to anneal to the 3' end of each DNA strand of the amplicon. These primers are then used by the DNA polymerase to build new complementary strands, which will result in the doubling of the number of DNA molecules of the targeted DNA sequence for each PCR cycle. While this system is incredibly useful to amplify and precisely quantify a gene of interest, there are several obstacles that can lower primer efficiencies and jeopardize your experiment. Here are the top recommendations for how to design primers for PCR and real time quantitative PCR (qPCR), reverse transcription quantitative PCR (RT-qPCR), bisulfite PCR, and methylation specific PCR (MSP).

Tips for primer design:

Keep the melting temperatures (Tm) of each primer pair within 2°C of one another. The Tm can be approximately calculated by the formula Tm = (A+T) x 2 + (G+C) x 4, however, more precise, and elaborate Tm calculation tools are available online. Having a similar Tm between primers ensures that the forward and reverse primers will be bound to their complementary DNA strands at the same time, reducing the chance that the primer with the highest Tm will bind to nonspecific DNA sequences.

Use an annealing temperature (Ta) 3-5°C below the melting temperature. The annealing temperature is the temperature at which the primers will bind to a new template strand. This needs to be lower than the Tm so that the primers can efficiently bind to the target, but not too low that the primers bind to nonspecific targets.

Keep primers between 18-22 base pairs long. This primer length is long enough to ensure binding specificity while also short enough to keep the Tm within an appropriate range.

Design primers with a GC content of 35-65%. A GC content between 35% and 65% without long stretches (> 4 bases) of the same nucleotide will ensure enough sequence complexity for optimal primer specificity.

Minimize G/C repeats, especially at the 3' end of the primer. Cytosine and guanine have stronger binding affinity than adenine and thymine and repeats of more than 4 G or C can bind to many places in the genome with high affinity. If these repeats are at the 3' end of the primer, DNA polymerase can extend amplicons in off-target locations, which can ultimately decrease the PCR efficiency.

Limit amplicon length to 140 base pairs. Amplicons between 70-140 base pairs are generally long enough to allow the design of two efficient primers and a probe (if a TaqMan-based assay is desired) for qPCR assays. Longer amplicons are possible, although it may be necessary to adjust the thermal cycler protocol to allow for complete elongation of the new strand. Additionally, if the sample contains heavily fragmented DNA, it is best to keep amplicons smaller to increase the likelihood of having intact target sequences for primer binding.

Tips for TaqMan® probe design:

Keep the melting temperatures of the probe 4 to 8°C higher than the primers. For TaqMan-based qPCR assays, probes are designed to anneal to the target DNA before the primers start to elongate the strands. This will keep the signal as high as possible without compromising specificity.

Keep the probe length between 20-25 base pairs. Probes that are slightly longer than primers will remain bound to the target throughout elongation and will likely be more specific for the targeted DNA region.

Do not overlap probe and primer binding sites. Probes and primers with overlapping binding sites cannot be used in the same reaction.

Avoid guanine at the 5' end of the probe. Guanine has a quenching effect on the fluorophores attached to TaqMan probes which can dampen the signal in your reaction.

Considerations for both primers and probes:

Do not design primers or probes containing common SNPs. Check genome data to ensure that primer and probe binding sites do not contain common single nucleotide polymorphisms (SNPs). SNPs can interfere with primer or probe binding by introducing sequence variations in a subset of test samples. You can use UCSC's Genome Browser tool to check for SNPs.

Check oligonucleotide sequences for hairpins, self-dimers, and hetero dimers. Avoid using oligonucleotides that have strong interactions with themselves (hairpins or self-dimers) or with other oligonucleotides in the reaction mixture (hetero-dimers), especially if the interaction occurs at the 3' end of the primer.

Check the oligonucleotide sequences against the host genome. Primers should be specific to the target sequence of interest. To ensure specificity to the right genomic location, you can use NCBI's BLAST tool to cross reference the primer sequence against the entire genome.

ZymoTaq DNA Polymerase (Catalog No. E2001) makes it easy to perform hot-start qPCR while reducing primer dimers and nonspecific product formation. For more information, view our collection of ZymoTaq Polymerase products.

The above tips are also applicable to a slightly different form of PCR called quantitative reverse transcription PCR (RT-qPCR). RT-qPCR allows you to perform PCR starting from RNA instead of DNA. RNA is first converted into cDNA (reverse transcription, or RT), and then amplified with PCR as described in the previous section. During maturation, RNA is subjected to splicing, which removes introns from pre-mRNA sequences. For this reason, mRNA sequences differ from genomic DNA sequences. Thanks to the splicing it is possible to design a PCR assay specific for the RNA transcript.

Design primers over an exon-exon junction to quantify mRNA instead of gDNA. If possible, design at least one primer over an exon-exon junction to increase the reaction specificity for mRNA over gDNA (this will be useful in case genomic DNA has not been completely removed from the RNA sample). Most of the 5' end of the primer sequence should be located on one exon leaving only 3 to 4 bases at the 3' end in the next exon.

Zymo Research provides high-quality reagents for efficient cDNA synthesis and robust qPCR amplification that can simplify and improve your RT-qPCR workflow. Using the ZymoScript RT PreMix Kit (Catalog No. R3012), high yield, full-length cDNA synthesis can be achieved in less than 15 minutes. Alternatively, fast reverse transcription and highly sensitive qPCR amplification can be completed in a single reaction using the ZymoScript One-Step RT-qPCR Kit (Catalog No. R3014). The ZymoScript One-Step RT-qPCR Kit is compatible with both SYBR Green and TaqMan probe-based assays to allow ultimate flexibility. Both kits use blue tracking dye to simplify the reaction set-up and save time. RT-qPCR design and set-up has never been more convenient and powerful. For more information on the new ZymoScript product line, please reach out to our scientists at tech@zymoresearch.com or call (949) 679-1190 ext. 3.

Bisulfite PCR is a multi-step process that enables users to determine the methylation status of CpG sites within a PCR amplicon. The first step in bisulfite PCR is bisulfite conversion of the DNA. Bisulfite conversion is a process that uses sodium bisulfite to convert all unmethylated cytosines in DNA to uracil. In contrast, methylated cytosines are left unaltered by the sodium bisulfite treatment. After the bisulfite conversion step, a PCR reaction is run to determine the methylation status of CpG sites located between the two primers. During this PCR, the converted uracil bases are read and amplified as thymine, while the methylated cytosines continue to be amplified as cytosine. Thus, cytosines that were methylated in the original DNA will be the only cytosines left in the DNA sequence after amplification. Based on this principle it is possible, by sequencing the bisulfite PCR product, to identify the methylation status of the cytosines in the original DNA sample. While this method is useful to learn the methylation status of DNA, the conversion of unmethylated cytosines to uracil causes the two DNA strands to lose complementarity and strongly decreases the complexity of the DNA sequence. This makes the design of primers specific for the DNA region of interest more challenging.

Tips for bisulfite primer design:

Design primers that are between 26-30 base pairs long. This will increase the chance to design a specific primer with an adequate Tm.

Keep amplicons between 70-300 base pairs. Since bisulfite treatments tend to fragment the DNA, the amplification of long amplicons is more challenging than short amplicons. For optimal results keep the amplicon length between 70 to 300 bp.

Avoid CpG sites in primer sequences. If it is unavoidable to include a CpG site in a primer sequence, locate it at the 5' end of the primer and add a degenerate base (Y) complementary to the cytosine position in the opposite strand.

Allow the reaction to run for 35 to 40 cycles. Bisulfite conversion is a harsh process that leads to DNA fragmentation and damage. Design PCR programs with at least 35 thermal cycles to compensate for DNA fragmentation.

Use a hot start polymerase for bisulfite PCR to increase specificity. Nonspecific amplification is relatively common with bisulfite-converted DNA due to it being AT-rich. Using a hot-start polymerase and optimized primer design and protocol will help reduce nonspecific products.

Keep annealing temperatures between 55-60°C to increase specificity.

For more on bisulfite conversion, see our Bisulfite Beginner Guide. For fast and complete bisuflite conversion of DNA and RNA for methylation analyses, see our range of bisulfite conversion kits, and ZymoTaq polymerase collections.

Methylation Specific PCR (MSP) relies on amplification to assess the methylation status at specific CpG sites after bisulfite conversion. Success with this system depends on the differential amplification of the template using methylated (M) and non-methylated (U) primer sets. While most of the considerations for MSP primer design are identical for those for bisulfite PCR, CpG sites within the primers are treated differently. Bisulfite PCR examines the methylation status of all the CpG sites present in a specific region, while MSP assesses the methylation at a single CpG site complementary to the 3' end of the primer sequence.

Create M and U primer sets with CpG sites at the 3' end. Depending on the location of the CpG sites on the original sense or anti-sense DNA strands, the two primer sets will contain different bases in the CpG position. Methylated primers will contain either C or G in CpG locations and non-methylated primers will contain either T or A. The primer set (M or U) that amplifies the target-sequence will inform about the methylation status of that CpG site.

For more information on MSP, see the MSP section of our Bisulfite Beginner Guide.

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How to Design Primers for PCR Experiments

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Primer design is a critical step in the design of a good real time PCR experiment. There are many elements to consider when designing primers that can help minimize troubleshooting down the line. Here are important recommendations on creating specific and effective primers for several types of PCR assays.

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Nanoengineers from UC San Diego have developed nanosponges that soak up a dangerous pore-forming toxin produced by MRSA, and have shown that their nanosponge vaccine was safe and more effective than toxoid vaccines made from heat-treated staph toxin.
Nanosponges that soak up a dangerous pore-forming toxin produced by MRSA (methicillin-resistant Staphylococcus aureus) could serve as a safe and effective vaccine against this toxin. This "nanosponge vaccine" enabled the immune systems of mice to block the adverse effects of the alpha-haemolysin toxin from MRSA-both within the bloodstream and on the skin. Nanoengineers from the University of California, San Diego described the safety and efficacy of this nanosponge vaccine in the December 1 issue of Nature Nanotechnology.

The nanosponges at the foundation of the experimental "toxoid vaccine" platform are bio-compatible particles made of a polymer core wrapped in a red-blood-cell membrane. Each nanosponge's red-blood-cell membrane seizes and detains the Staphylococcus aureus (staph) toxin alpha-haemolysin without compromising the toxin's structural integrity through heating or chemical processing. These toxin-studded nanosponges served as vaccines capable of triggering neutralizing antibodies and fighting off otherwise lethal doses of the toxin in mice.

Toxoid vaccines protect against a toxin or set of toxins, rather than the organism that produces the toxin(s). As the problem of antibiotic resistance worsens, toxoid vaccines offer a promising approach to fight infections without reliance on antibiotics.

"With our toxoid vaccine, we don't have to worry about antibiotic resistance. We directly target the alpha-haemolysin toxin," said Liangfang Zhang, a nanoengineering professor at UC San Diego Jacobs School of Engineering and the senior author on the paper. Targeting the alpha-haemolysin toxin directly has another perk. "These toxins create a toxic environment that serves as a defense mechanism which makes it harder for the immune system to fight Staph bacteria," explained Zhang.

Beyond MRSA and other staph infections, the nanosponge vaccine approach could be used to create vaccines that protect against a wide range of toxins, including those produced by E. coli and H. pylori.

This work from Zhang's Nanomaterials and Nanomedicine Laboratory at the UC San Diego included nanoengineering post-doctoral researcher Che-Ming "Jack" Hu, nanoengineering graduate student Ronnie Fang, and bioengineering graduate student Brian Luk.

The researchers found that their nanosponge vaccine was safe and more effective than toxoid vaccines made from heat-treated staph toxin. After one injection, just 10 percent of staph-infected mice treated with the heated version survived, compared to 50 percent for those who received the nanosponge vaccine. With two more booster shots, survival rates with the nanosponge vaccine were up to 100 percent, compared to 90 percent with the heat-treated toxin.

"The nanosponge vaccine was also able to completely prevent the toxin's damages in the skin, where MRSA infections frequently take place," said Zhang, who is also affiliated with the Moores Cancer Center at UC San Diego.
Fighting Pore-Forming Toxins
This work is a twist on a project the UC San Diego nanoengineers presented earlier this year: a nanosponge that can sop up a variety of pore-forming toxins-from bacterial proteins to snake venom-in the body.

Pore-forming toxins work by punching holes in a cell's membrane and letting the cell essentially leak to death. But when toxins attack the red blood cell membrane draped over the nanoparticle, "nothing will happen. It just locks the toxin there," Zhang explained.

The nanoengineers wondered what would happen if they loaded one of their nanosponges with staph toxin in this way, and presented the whole package to an essential part of the immune system called dendritic cells. Could the loaded particles trigger an immune response and work as a toxoid vaccine?

Staph toxin is so powerful that it kills immune cells in its unaltered form. Most vaccine candidates, therefore, use a heat or chemically processed version of the toxin that unravels some of its proteins and makes it a little weaker. But this process also makes the immune response to the toxin a little weaker.

"The more you heat it, the safer the toxin is, but the more you heat it, the more you damage the structure of the protein," Zhang explained. "And this structure is what the immune cell recognizes, and builds its antibodies against."

The nanosponge toxoid vaccine gets around this problem by detaining-but not changing-the staph toxin. Like a dangerous but handcuffed prisoner, the staph toxin can be led to the dendritic cells of the immune system without causing any harm.

Before this, "there was no way you could deliver a native toxin to the immune cells without damaging the cells," Zhang said. "But this technology allows us to do this."

Each vaccine particle is approximately 85 nanometers in diameter; for comparison, about 1000 of them would fit across the width of a single human hair. They are cleared from the body after injection in about two weeks, the researchers found.

Staphylococcus aureus

Staph bacteria are one of the most common causes of skin infections, and can cause blood poisoning and surgical infections as well as pneumonia. According to the Centers for Disease Control and Prevention, about 80,000 Americans suffer from invasive MRSA infections each year, and over 11,000 of those individuals die. At the moment, there are no vaccines approved to protect humans against the toxins associated with staph infections, including those caused by MRSA strains.

The idea for a staph vaccine came about when the researchers considered the success of their nanosponge. If the particle was so good at collecting toxins, they wondered, what were the potential uses of a particle full of toxin? "To be honest, we never thought about the vaccine use from the beginning," Zhang noted. "But when we do research, we always want to look at a problem in reverse."

In a way, the toxoid vaccine hearkens back to their first use for the particles, as a cancer drug delivery device, Zhang noted.

The particles "work so beautifully," Zhang said, that it might be possible to detain several toxins at once on them, creating "one vaccine against many types of pore-forming toxins," from staph to snake venom.

The research was funded by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (award no. R01DK095168) and by the National Science Foundation (grant DMR-1216461).

"Nanoparticle-detained toxins for safe and effective vaccination," by Che-Ming J. Hu, Ronnie H. Fang and Liangfang Zhang in the Department of NanoEngineering at the University of California, San Diego; and Brian T. Luk in the Department of Bioengineering at the University of California, San Diego.

Publication: Che-Ming J. Hu, et al., "Nanoparticle-detained toxins for safe and effective vaccination," Nature Nanotechnology, 2013; doi:10.1038/nnano.2013.254

Image: UC San Diego Department of NanoEngineering

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Nanoengineers Develop 'Nanosponge Vaccine' to Fight MRSA Toxins

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Nanoengineers from UC San Diego have developed nanosponges that soak up a dangerous pore-forming toxin produced by MRSA, and have shown that their nanosponge vaccine was safe and more effective than toxoid vaccines made from heat-treated staph toxin. Nanosponges that soak up a dangerous pore-form

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Animation of Nanosponges and SARS COV-2 Coronavirus

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Powerful COVID-19 Research Tool: Scientists Create Noninfectious Versions of SARS-CoV-2

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Powerful COVID-19 Research Tool: Scientists Create Noninfectious Versions of SARS-CoV-2

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https://scitechdaily.com/powerful-covid-19-research-tool-scientists-create-noninfectious-versions-of-sars-cov-2/?utm_source=TrendMD&utm_medium=cpc&utm_campaign=SciTechDaily_TrendMD_0

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Using a new delivery system, researchers from MIT have developed nanoparticles that can deliver the CRISPR genome-editing system and specifically modify genes, eliminating the need to use viruses for delivery. With the new technique, the researchers were able to cut out certain genes in about 80 percent of liver cells, the best success rate ever achieved with CRISPR in adult animals.

"What's really exciting here is that we've shown you can make a nanoparticle that can be used to permanently and specifically edit the DNA in the liver of an adult animal," 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).

One of the genes targeted in this study, known as Pcsk9, regulates cholesterol levels. Mutations in the human version of the gene are associated with a rare disorder called dominant familial hypercholesterolemia, and the FDA recently approved two antibody drugs that inhibit Pcsk9. However these antibodies need to be taken regularly, and for the rest of the patient's life, to provide therapy. The new nanoparticles permanently edit the gene following a single treatment, and the technique also offers promise for treating other liver disorders, according to the MIT team.

Anderson is the senior author of the study, which appears in the November 13 issue of Nature Biotechnology. The paper's lead author is Koch Institute research scientist Hao Yin. Other authors include David H. Koch Institute Professor Robert Langer of MIT, professors Victor Koteliansky and Timofei Zatsepin of the Skolkovo Institute of Science and Technology, and Professor Wen Xue of the University of Massachusetts Medical School.
Targeting disease
Many scientists are trying to develop safe and efficient ways to deliver the components needed for CRISPR, which consists of a DNA-cutting enzyme called Cas9 and a short RNA that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut.

In most cases, researchers rely on viruses to carry the gene for Cas9, as well as the RNA guide strand. In 2014, Anderson, Yin, and their colleagues developed a nonviral delivery system in the first-ever demonstration of curing a disease (the liver disorder tyrosinemia) with CRISPR in an adult animal. However, this type of delivery requires a high-pressure injection, a method that can also cause some damage to the liver.

Later, the researchers showed they could deliver the components without the high-pressure injection by packaging messenger RNA (mRNA) encoding Cas9 into a nanoparticle instead of a virus. Using this approach, in which the guide RNA was still delivered by a virus, the researchers were able to edit the target gene in about 6 percent of hepatocytes, which is enough to treat tyrosinemia.

While that delivery technique holds promise, in some situations it would be better to have a completely nonviral delivery system, Anderson says. One consideration is that once a particular virus is used, the patient will develop antibodies to it, so it couldn't be used again. Also, some patients have pre-existing antibodies to the viruses being tested as CRISPR delivery vehicles.

In the new Nature Biotechnology paper, the researchers came up with a system that delivers both Cas9 and the RNA guide using nanoparticles, with no need for viruses. To deliver the guide RNAs, they first had to chemically modify the RNA to protect it from enzymes in the body that would normally break it down before it could reach its destination.

The researchers analyzed the structure of the complex formed by Cas9 and the RNA guide, or sgRNA, to figure out which sections of the guide RNA strand could be chemically modified without interfering with the binding of the two molecules. Based on this analysis, they created and tested many possible combinations of modifications.

"We used the structure of the Cas9 and sgRNA complex as a guide and did tests to figure out we can modify as much as 70 percent of the guide RNA," Yin says. "We could heavily modify it and not affect the binding of sgRNA and Cas9, and this enhanced modification really enhances activity."
Reprogramming the liver
The researchers packaged these modified RNA guides (which they call enhanced sgRNA) into lipid nanoparticles, which they had previously used to deliver other types of RNA to the liver, and injected them into mice along with nanoparticles containing mRNA that encodes Cas9.

They experimented with knocking out a few different genes expressed by hepatocytes, but focused most of their attention on the cholesterol-regulating Pcsk9 gene. The researchers were able to eliminate this gene in more than 80 percent of liver cells, and the Pcsk9 protein was undetectable in these mice. They also found a 35 percent drop in the total cholesterol levels of the treated mice.

The researchers are now working on identifying other liver diseases that might benefit from this approach, and advancing these approaches toward use in patients.

"I think having a fully synthetic nanoparticle that can specifically turn genes off could be a powerful tool not just for Pcsk9 but for other diseases as well," Anderson says. "The liver is a really important organ and also is a source of disease for many people. If you can reprogram the DNA of your liver while you're still using it, we think there are many diseases that could be addressed."

"We are very excited to see this new application of nanotechnology open new avenues for gene editing," Langer adds.

The research was funded by the National Institutes of Health (NIH), the Russian Scientific Fund, the Skoltech Center, and the Koch Institute Support (core) Grant from the National Cancer Institute.

Publication: Hao Yin, et al., "Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing," Nature Biotechnology, 2017; doi:10.1038/nbt.4005

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Researchers Use CRISPR-Carrying Nanoparticles To Edit Genomes

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Using a new delivery system, researchers from MIT have developed nanoparticles that can deliver the CRISPR genome-editing system and specifically modify genes, eliminating the need to use viruses for delivery. With the new technique, the researchers were able to cut out certain genes in about 80 percent of liver cells, the best success rate ever achieved with CRISPR in adult animals.

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New Genetic Systems Created by Biologists to Neutralize Gene Drives

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Two active genetics strategies help address concerns about gene-drive releases into the wild. In the past decade, researchers have engineered an array of new tools that control the balance of genetic inheritance. Based on CRISPR technology, such gene drives are poised to move from the laboratory in

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Neurodegenerative diseases present unique challenges that must be addressed in novel ways. Decades of research in neurology and the neuroscience fields have shown that many neurodegenerative diseases share a common underlying mechanism: a decline in the cell’s ability to produce energy. Although thi…

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Safety

Documentation

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Counting Cells/Trypan Blue Staining

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DNA Maxi Prep

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Western Blot

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The Promise of Nanomedicine | Joy Wolfram | TEDxJacksonville

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