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Archive for the ‘Genetic Engineering’ Category

Genetic modification is ready to serve humanity The Miscellany News – Miscellany News

Sunday, October 18th, 2020

On Oct. 7, 2020, Dr. Emmanuelle Charpentier and Dr. Jennifer A. Doudna were awarded the Nobel Prize in Chemistry for their work in the field of gene editing. On top of breaking barriers as the first two women jointly awarded the chemistry prize, Charpentier and Doudnas recognition is a huge step forward for the controversial field of genetic engineering.

Humans have been practicing a form of genetic engineering ever since we started cultivating plants and livestock. Grafting two plants together dates back centuries in both the East and the West, and selective breeding was a staple technique used by even the earliest farmers. These techniques arent using advanced technology to target and change certain genes, but nevertheless the point of these exercises was to eliminate or diminish unwanted characteristics and promote the characteristics that the farmer found most useful. Wild cabbage was bred to create broccoli, brussel sprouts and domesticated cabbage. Cattle were bred to increase their edible volume. This was all uncontroversial, but it was all gene editing.

Today the techniques have changed, but the underlying mission has stayed the same: improve quality of life. Public opinion has shifted, however. Currently, more than half of adults in the U.S. believe that using genetically modified organisms (GMOs) as a food source is worse for your health than using non-modified foods. Of those, 88 percent believe that GMO foods will lead to health problems for the general populace. There is no such thing as non-modified food, but there is a stigma against food modified in a lab.

Part of this bias may be due to the way direct modification was introduced in the 1950s. In order to increase variation in plants so that selective breeding could be done more efficiently, scientists bombarded plants with radiation. This process, known as mutation breeding, was part of an effort to discover a peaceful use for the nuclear knowledge that was proliferating in the aftermath of World War II. Radiation was poorly understood by the general public in the mid-20th century. The possibilities of mutation due to radiation caused imagination to run rampant over reality: 1954s Them! stars giant insects caused by nuclear testing in the area.

The 1957 film Beginning of the End has grasshoppers eat mutated plants and then grow to enormous sizes. Even some of the most famous pop culture characters that exist today were formulated along these lines. In 1961 the Fantastic Four were given their powers by cosmic radiation. Spider-Man has had eight movies over the last 20 years, and he was famously bitten by a radioactive spider. These examples dont insinuate that people really believed that radiation could produce superheroes and skyscraper-sized insects, but they do reflect a general fear of the unknown that the gene modification of radiation could produce.

Radiation is no longer the bugaboo of the modern day, but fear of radiation has been displaced by fear of targeted gene editing, like the Crispr-Cas9 technique pioneered by Charpentier and Doudna. Some of this fear may be well founded: Theres no definite way to know that a gene edited plant or animal wont act similar to an invasive species. Presumably freed from some ailment or deficit that was limiting its growth, it is possible that a plant may grow at a pace that is higher than wanted by its creators. Nature is a delicate balance, and intervening must be done in a reasonable way that weighs the potential costs and benefits.

Mosquito reduction or elimination may not seem to be a worthwhile risk for something with unknown side effects, but that initial intuition would be wrong. Malaria, a disease transmitted mainly through mosquito bites, kills around 400,000 people per year. Zika and West Nile virus, while less deadly, are also transmitted into the human populace via mosquito. No other creature kills humans at the rate of mosquitoes. Despite the environmental damage that may be wreaked by the adjustment of the other flora and fauna to a lack of mosquitoes, gene editing to reduce mosquito population is a clear path to saving hundreds of thousands of lives every year.

With this sort of benefit in mind, the United States Environmental Protection Agency and Florida state government recently came to an agreement that will release over 750 million genetically modified mosquitoes into Florida. This is no small action and could potentially disrupt the entire food web of Florida, and possibly beyond.

The plan in Florida is to introduce a strain of Aedes Aegypti mosquitoes, a spreader of the Zika virus, that are genetically engineered so that their female offspring die off. Mosquitoes bite to extract human blood, and in this exchange mosquitoes can transfer any diseases they are carrying. Mosquitoes only bite so that they can extract iron and proteins in human blood and transfer it to the fertilized eggs that will be the next generation of that mosquitos bloodline. As such, the only mosquitoes that bite, and thus have the chance to transfer diseases, are adult females. The firm Oxitec produced a modified mosquito whose female offspring cant grow out of the larval stage. No adult females means no blood sucking, which means no disease transmission and no new mosquito larvae being produced.

A similar plan was executed in Brazil, where the Aedes Aegypti mosquito population was cut by 89 to 96 percent. With such a large reduction in mosquito population, the benefits move beyond that of just public health. Thousands of tracts of land would become more usable and see an increase in value if mosquitoes died out. Even day-to-day activities like gardening or talking walks could become much more pleasant in the absence of mosquitoes.

2020 has already shown the effects of disease and failures of public health. COVID-19 has killed over a million people; over the last 10 years, malaria has killed over four million. We have to live with COVID-19 for the foreseeable future, but gene editing has given us a tool to end malaria. Genetically modified mosquitoes should not end in Florida or with Aedes Aegypti: they should be of all species, placed all over the globe. For months the world has lived under a new biological terror. Its time we release a new biological salvation.

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Dyslexia shows the inborn nature of visual imagining and cognition – Genetic Literacy Project

Sunday, October 18th, 2020

Reading is a learned skill; no one is born reading. But learning to read relies on inborn human capacities for language and speech. And dyslexia is ageneticcondition that compromises thesebrain networks.

Yet laypeople are convinced that dyslexia results from troubleswith vision. And these errors matter. A parent who holds these views might fail to recognize her childs difficulties with rhymes and pig Latin (both require phonemic awareness) as warning signs. So why are we so wrong about dyslexia? Why do we mistake dyslexia for word blindness?

At first blush, these misconceptions seem rather innocent; laypeople, by definition, arent reading experts, so perhaps they just dont know better. But aspiringteachers, with ample educational training, make similar mistakes. Moreover, the pattern of mistakes suggests a deeper problem.

While these biases are unconscious, they demonstrably veer off reasoning in numerous areas, from our irrational fascination with the brain to ourfear of artificial intelligence; our troubles with dyslexia, then, are but one of its many victims. To counter these errors, information alone wont sufficea real change requires that we take a hard look within.

Reading, then, rests on decoding in more ways than one. For children to successfully decode printed words, we must all improve our decoding of the human mind.

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AGC Biologics Shifts Leadership Structure at United States and Copenhagen Sites to Support the Continued Development and Growth of the Regions -…

Sunday, October 18th, 2020

SEATTLE, Oct. 15, 2020 /PRNewswire/ -- AGC Biologics, a leading global biopharmaceutical contract development and manufacturing organization (CDMO), has announced a leadership update at the United States and Copenhagen facilities. The changes are being made to strengthen the strategic development and executive oversight of the rapid growing facilities in the US and Copenhagen, and are effective at the date of release, October 15, 2020.

Jeffrey D. Mowery will join the Global Executive Team in the role of Senior Vice President of US Operations, based at company headquarters in Seattle, Washington. Andrea C. Porchia will become the General Manager and Site Head for the Copenhagen Operation.

In his new position, Mr. Mowery will oversee the new Boulder, Colorado facility rollout and ensure that progress is maintained at the expanding Seattle site. Mr. Mowery draws on more than two decades of industry expertise in small molecule, biologic and cell and gene therapy production and technology transfer expertise to deliver quality in his work at AGC Biologics.

"In his most recent role as General Manager of the Copenhagen, Denmark facility, J.D. Mowery achieved a period of strong growth, even with today's challenges from the COVID pandemic. We believe the US sites, and ultimately our customers, will benefit from his leadership skills, results oriented approach and broad operational expertise in the same way that Copenhagen has," said Kasper Moller, CTO of AGC Biologics. He continued, "as part of this transition, Andrea C. Porchia has been promoted to General Manager of the Copenhagen site where her broad and deep biologics experience, and ability to effectively navigate all aspects of biomanufacturing and development will be an indispensable asset for the Copenhagen Site and to our valued customers."

Through more than seven years at AGC Biologics, Ms. Porchia has taken on increasing responsibilities, both at the Copenhagen site and globally as Project Director, Business Development Representative, Global Head of Project Management and now General Manager. She leverages more than two decades of research and process expertise to enhance business operations with a critical focus on project management and customer service.

To learn more about the AGC Biologics global network of facilities, please visit:

About AGC Biologics

AGC Biologics is a leading global biopharmaceutical contract development and manufacturing organization (CDMO) committed to delivering a high standard of service to solve complex customer challenges. The company is driven by innovation and continuously invests in technologies to complement decades of proven expertise in drug development and manufacturing, including working through FDA, PDMA and EMA approvals. A range of customizable bioprocessing services includes development and manufacturing of mammalian and microbial-based therapeutic proteins, protein expression, plasmid DNA (pDNA) support, antibody drug development and conjugation, viral vector production, genetic engineering of cells, cell line development with a proprietary CHEF1 Expression System, cell banking and storage.

AGC Biologics employs more than 1,400 professionals worldwide who are dedicated to supporting customers at all phases of development through to commercialization, with critical expertise in process development, formulation, and analytical testing. The global service network boasts locations in the United States at Seattle, Washington and Boulder, Colorado; across Europe in Copenhagen, Denmark; Heidelberg, Germany; Milan and Bresso, Italy; and in Asia at Chiba, Japan.

Learn more at, or find us on LinkedIn at and Twitter @agcbiologics.

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AGC Biologics Shifts Leadership Structure at United States and Copenhagen Sites to Support the Continued Development and Growth of the Regions -...


Scientists Found a New Way to Control the Brain With LightNo Surgery Required – Singularity Hub

Sunday, October 18th, 2020

If I had to place money on a neurotech that will win the Nobel Prize, its optogenetics.

The technology uses light of different frequencies to control the brain. Its a brilliant mind-meld of basic neurobiology and engineering that hijacks the mechanism behind how neurons naturally activateor are silencedin the brain.

Thanks to optogenetics, in just ten years weve been able to artificially incept memories in mice, decipher brain signals that lead to pain, untangle the neural code for addiction, reverse depression, restore rudimentary sight in blinded mice, and overwrite terrible memories with happy ones. Optogenetics is akin to a universal programming language for the brain.

But its got two serious downfalls: it requires gene therapy, and it needs brain surgery to implant optical fibers into the brain.

This week, the original mind behind optogenetics is back with an update that cuts the cord. Dr. Karl Deisseroths team at Stanford University, in collaboration with the University of Minnesota, unveiled an upgraded version of optogenetics that controls behavior without the need for surgery. Rather, the system shines light through the skulls of mice, and it penetrates deep into the brain. With light pulses, the team was able to change how likely a mouse was to have seizures, or reprogram its brain so it preferred social company.

To be clear: were far off from scientists controlling your brain with flashlights. The key to optogenetics is genetic engineeringwithout it, neurons (including yours) dont naturally respond to light.

However, looking ahead, the study is a sure-footed step towards transforming a powerful research technology into a clinical therapy that could potentially help people with neurological problems, such as depression or epilepsy. We are still far from that visionbut the study suggests its science fiction potentially within reach.

To understand optogenetics, we need to dig a little deeper into how brains work.

Essentially, neurons operate on electricity with an additional dash of chemistry. A brain cell is like a living storage container with doorscalled ion channelsthat separate its internal environment from the outside. When a neuron receives input and that input is sufficiently strong, the cells open their doors. This process generates an electrical current, which then gallops down a neurons output brancha biological highway of sorts. At the terminal, the electrical data transforms into dozens of chemical ships, which float across a gap between neurons to deliver the message to its neighbors. This is how neurons in a network communicate, and how that network in turn produces memories, emotions, and behaviors.

Optogenetics hijacks this process.

Using viruses, scientists can add a gene for opsins, a special family of proteins from algae, into living neurons. Opsins are specialized doors that open under certain frequencies of light pulses, something mammalian brain cells cant do. Adding opsins into mouse neurons (or ours) essentially gives them the superpower to respond to light. In classic optogenetics, scientists implant optical fibers near opsin-dotted neurons to deliver the light stimulation. Computer-programmed light pulses can then target these newly light-sensitive neurons in a particular region of the brain and control their activity like puppets on a string.

It gets cooler. Using genetic engineering, scientists can also fine-tune which populations of neurons get that extra powerfor example, only those that encode a recent memory, or those involved in depression or epilepsy. This makes it possible to play with those neural circuits using light, while the rest of the brain hums along.

This selectivity is partially why optogenetics is so powerful. But its not all ponies and rainbows. As you can imagine, mice dont particularly enjoy being tethered by optical fibers sprouting from their brains. Humans dont either, hence the hiccup in adopting the tool for clinical use. Since its introduction, a main goal for next-generation optogenetics has been to cut the cord.

In the new study, the Deisseroth team started with a main goal: lets ditch the need for surgical implants altogether. Immediately, this presents a tough problem. It means that bioengineered neurons, inside a brain, need to have a sensitive and powerful enough opsin door that responds to lighteven when light pulses are diffused by the skull and brain tissue. Its like a game of telephone where one person yells a message from ten blocks away, through multiple walls and city noise, yet you still have to be able to decipher it and pass it on.

Luckily, the team already had a candidate, one so good its a ChRmine (bad joke cringe). Developed last year, ChRmine stands out in its shockingly fast reaction times to light and its ability to generate a large electrical current in neuronsabout a 100-fold improvement over any of its predecessors. Because its so sensitive, it means that even a spark of light, at its preferred wavelength, can cause it to open its doors and in turn control neural activity. Whats more, ChRmine rapidly shuts down after it opens, meaning that it doesnt overstimulate neurons but rather follows their natural activation trajectory.

As a first test, the team used viruses to add ChRmine to an area deep inside the brainthe ventral tegmental area (VTA), which is critical to how we process reward and addiction, and is also implicated in depression. As of now, the only way to reach the area in a clinical setting is with an implanted electrode. With ChRmine, however, the team found that a light source, placed right outside the mices scalp, was able to reliably spark neural activity in the region.

Randomly activating neurons with light, while impressive, may not be all that useful. The next test is whether its possible to control a mouses behavior using light from outside the brain. Here, the team added ChRmine to dopamine neurons in a mouse, which in this case provides a feeling of pleasure. Compared to their peers, the light-enhanced mice were far more eager to press a lever to deliver light to their scalpsmeaning that the light is stimulating the neurons enough for the mice to feel pleasure and work for it.

As a more complicated test, the team then used light to control a population of brain cells, called serotonergic cells, in the base of the brain, called the brainstem. These cells are known to influence social behaviorthat is, how much an individual enjoys social interaction. It gets slightly disturbing: mice with ChRmine-enhanced cells, specifically in the brainstem, preferred spending time in their test chambers social zone versus their siblings who didnt have ChRmine. In other words, without any open-brain surgery and just a few light beams, the team was able to change a socially ambivalent mouse into a friendship-craving social butterfly.

If youre thinking creepy, youre not alone. The study suggests that with an injection of a virus carrying the ChRmine geneeither through the eye socket or through veinsits potentially possible to control something as integral to a personality as sociability with nothing but light.

To stress my point: this is only possible in mice for now. Our brains are far larger, which means light scattering through the skull and penetrating sufficiently deep becomes far more complicated. And again, our brain cells dont normally respond to light. Youd have to volunteer for what amounts to gene therapywhich comes with its own slew of problemsbefore this could potentially work. So keep those tin-foil hats off; scientists cant yet change an introvert (like me) into an extrovert with lasers.

But for unraveling the inner workings of the brain, its an amazing leap into the future. So far, efforts at cutting the optical cord for optogenetics have come with the knee-capped ability to go deep into the brain, limiting control to only surface brain regions such as the cortex. Other methods overheat sensitive brain tissue and culminate in damage. Yet others act as 1990s DOS systems, with significant delay between a command (activate!) and the neurons response.

This brain-control OS, though not yet perfect, resolves those problems. Unlike Neuralink and other neural implants, the study suggests its possible to control the brain without surgery or implants. All you need is light.

Image Credit: othebo from Pixabay

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Scientists Found a New Way to Control the Brain With LightNo Surgery Required - Singularity Hub


No evidence that coronavirus genetic sequences were fabricated, contrary to preprint by Li-Meng Yan and colleagues – Health Feedback

Sunday, October 18th, 2020


Fabricated genetic sequences were used to support the hypothesis that the virus arose naturally


Inadequate support: The preprint by Yan et al. offers no evidence to support their claim that the genetic sequences of other coronavirus strains were fabricated to support the hypothesis that SARS-CoV-2 arose naturally.Incorrect: The fact that multiple coronavirus strains share highly similar or identical genetic or protein sequences is not evidence that those viruses were fabricated. Shared genetic or protein sequences is common among viruses that belong to the same family and indicates their evolutionary relatedness.


There is no evidence supporting the claim by Yan et al. that genetic sequences of several coronaviruses were fabricated to support the hypothesis that SARS-CoV-2 arose naturally. The presence of highly similar or identical gene and protein sequences are common among organisms that are evolutionarily related to each other. Therefore, it is expected that members of the coronavirus family share similar or identical genetic or protein features. Scientific evidence supports the hypothesis that the virus arose naturally in wildlife before it crossed over to humans.

REVIEW Uncertainty surrounding the origin of the novel coronavirus has provided fertile ground for breeding conspiracy theories, some of which Health Feedback previously found to be inaccurate and unsubstantiated (see here and here). The recent claim by virologist Li-Meng Yan that the SARS-CoV-2 virus is manmade is the latest in a long series of conspiracy theories stretching back to the beginning of the coronavirus pandemic.

On 14 September 2020, Yan and her colleagues published a preprint on the online repository Zenodo claiming that the SARS-CoV-2 virus is a product of genetic engineering. A preprint is a research paper that has not been peer-reviewed by other scientists yet. Experts who examined the preprint found it was highly flawed and provided no supporting evidence for their claims, as detailed in this Health Feedback review.

Yan et al. published a second preprint on 8 October 2020 claiming that the virus is an unrestricted bioweapon and alleging that the genetic sequences of ten other coronaviruses are fabricated and do not exist in nature. Contrary to this claim, these ten coronaviruses, including RaTG13which is the closest known relative to SARS-CoV-2 and has about 96% genome sequence identity to SARS-CoV-2[1]and some pangolin coronaviruses, were analyzed by other scientists and found to support the natural origin hypothesis for SARS-CoV-2[2-7]. The second preprint from Yan et al. received more than 130,000 views on Zenodo since it was published, and was promoted by outlets known for publishing misinformation, such as Zero Hedge and National Pulse.

The alleged motivation for fabricating genetic sequences is related to one of the primary claims by Yan et al., specifically that the bat coronaviruses ZC45 and ZXC21 provided the genetic backbone for SARS-CoV-2. In support of this claim, Yan et al. point to the 100% identity in the envelope (E) protein sequence that exists between these three viruses. The E protein is a small protein on the surface of the membrane that encloses the viral genome and is important for producing virus particles that can efficiently infect cells[8].

Firstly, the claim that the bat coronaviruses ZC45 and ZXC21 provided the genetic backbone to artificially create SARS-CoV-2 was presented in the first preprint by Yan et al. This claim was debunked by scientists, who pointed out that the genetic sequences of ZC45 and ZXC21 are very different to that of SARS-CoV-2. In fact, the virus ZC45 is only 89% related to SARS-CoV-2, said Stanley Perlman, a professor at the University of Iowa who studies coronaviruses, in this article:

Perlman said it would be nearly impossible to make the reverse genetics system needed to manipulate the virus and changing its sequence to arrive at SARS-CoV-2 would be virtually impossible since it would not be known how to manipulate the virus.

Kristian Andersen, a professor at Scripps Research who studies the evolution of viruses including SARS-CoV-2, also pointed out the incongruency of the claim on Twitter: This simply cant be true there are more than 3,500 nucleotide differences between SARS-CoV-2 and these viruses.

Marvin Reitz, a virologist at the University of Maryland, put it more bluntly in his review of the first preprint: [I]t still would require more than 3,000 nucleotide substitutions [for ZC45] to become SARS-CoV-2. This is not even slightly credible; it beggars reason.

A response by scientists at the Johns Hopkins University Center for Health Security also provides a detailed rebuttal of the claims made by Yan et al. in their first preprint. It also highlights the implausible use of ZC45 and ZXC21 as the genetic backbone for SARS-CoV-2.

In short, ZC45 and ZXC21 are very different from SARS-CoV-2 in terms of genome identity. Altering a backbone from either of the two to transform it into the genome of SARS-CoV-2 would require a feat of genetic engineering that is extremely difficult, if not impossible, to accomplish with current technology.

Based on their spurious initial assumption that ZC45 and ZXC21 provided the genetic backbone for SARS-CoV-2, Yan et al. claim that the genetic sequences of RaTG13 and the other coronaviruses were fabricated to obscure the link between SARS-CoV-2 and ZC45/ZXC21, and that RaTG13 and the other coronaviruses do not exist. To support this claim, they point to the observation that all these viruses also have an E protein sequence that is 100% identical to that of ZC45 and ZXC21.

The argument by Yan et al. that the genetic sequences of some coronaviruses were fabricated to support the hypothesis that SARS-CoV-2 arose naturally does not hold up to scrutiny. In a Business Insider interview, Emma Hodcroft, a postdoctoral fellow at the University of Basel and co-developer of the Nextstrain project that studies the evolution of pathogens, including SARS-CoV-2, pointed out that most of the samples that Yans group says are fake predate the start of the pandemic. Hodcroft also explained:

This accusation implies there were years of coordination and fake sequence generation, Hodcroft said, adding: This is an incredible claim, and would require a significant evidence burden to back it up, which is missing from the paper.

Virologists have also analyzed the genome sequence of RaTG13 and found it to be authentic and supported by good-quality data.

Although some coronaviruses share certain identical genetic sequences with SARS-CoV-2, this is not evidence that the other coronaviruses were fabricated. Instead, similar or identical genetic and protein sequences of coronaviruses are evidence of their evolutionary relatedness, which is expected since these viruses all belong to the coronavirus family. Specifically, the E protein sequence of SARS-CoV-2, RaTG13, and the other coronaviruses analyzed in the preprint by Yan et al. are indeed identical to that of ZC45 and ZXC21, but this in itself does not indicate that the RaTG13 and the other coronaviruses were fabricated to mimic the E protein sequence of ZC45 and ZXC21.

Lastly, one feature of concern in both preprints by Yan and her co-authors is the listing of their affiliations as the Rule of Law Society and the Rule of Law Foundation. These two organizations have no prior experience in conducting biological research and are linked to Stephen Bannon and Wengui Guo, both of whom have published COVID-19 misinformation in the past.

Overall, the claims in the second preprint by Yan and her colleagues are as ill-founded as the claims made in their first preprint. Evidence supporting claims that the virus was engineered is lacking. In contrast, scientific analyses support the hypothesis that SARS-CoV-2 arose naturally in wildlife before crossing over to humans during a zoonotic infection (transmission of pathogens from animals/insects to humans). There are numerous examples of emerging zoonotic pathogens causing disease outbreaks throughout human history and across the world[9].

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No evidence that coronavirus genetic sequences were fabricated, contrary to preprint by Li-Meng Yan and colleagues - Health Feedback


Nobel Prize in Chemistry 2020 Winners from a Patent Perspective – Lexology

Sunday, October 18th, 2020

Further to my recent article about the 2020 Nobel Prize announcements, the winners of the 2020 Chemistry Nobel Prize were announced as Emmanuelle Charpentier and Jennifer Doudna on 7 October 2020 "for the development of a method for genome editing". This, of course, refers to the revolutionary genetic scissors technology, CRISPR/Cas9. For this article, Ive teamed up with my colleague Jamie Atkins, whose specialisms include prosecution of CRISPR-related patent applications at the EPO, to get into the details of the winning technology.

In this article, we explore:

Diversity in the Chemistry Nobel Prize;

The winning technology;

What is CRISPR and how does it work?;

How can CRISPR be used?;

CRISPR Patents; and

The future could CRISPR be used to combat COVID -19?

Diversity in the Chemistry Nobel Prize

Before looking at the patent side of things, it is worth noting that this is the first Nobel Prize awarded to two women. Charpentier commented shortly after the prize announcement that:

My wish is that this will provide a positive message to the young girls who would like to follow the path of science, and to show them that women in science can also have an impact through the research that they are performing This is not just for women, but we see a clear lack of interest in following a scientific path, which is very worrying.

While there is still a significant gender gap in the Laureates of the Chemistry Nobel Prize, it is encouraging that there are now an additional two female winners to add to the previous five: Marie Curie (1911), Irne Joliot-Curie (1935), Dorothy Crowfoot Hodgkin (1964), Ada Yonath (2009) and Frances H. Arnold (2018). We hope this figure continues to increase each year, along with wider recognition of other under-represented groups, for example in terms of BAME and LGBTQ+ representation.

The winning technology

At face value, CRISPR/Cas9 (CRISPR) seems to be more biological than chemical, but this only serves to highlight the breadth of chemistry as a field. As described in Nobels will, the Nobel Prize in Chemistry is to be awarded to the person who shall have made the most important chemical discovery or improvement, and this requirement is surely met by CRISPR.

What is CRISPR and how does it work?

The CRISPR/Cas9 editing tool developed by the Nobel Prize winners is based on the discovery of a naturally occurring system used by bacteria to defend against viral infection. When a virus is detected, the bacteria produce short RNA sequences that guide a DNA cutting enzyme (Cas9) to viral DNA matching the RNA sequence. Cas9 cuts the viral DNA, thereby disabling the virus. Doudna and Charpentier made several important discoveries leading to a better understanding of this bacterial system, developed a simplified version of the system and crucially showed that it could be programmed to target almost any DNA sequence of interest, as reported in the seminal Jinek et al. 2012 paper:

''Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNAprogrammable genome editing.

The basic components of the CRISPR/Cas9 system are the DNA cutting enzyme (Cas9) and the guide RNA based on the target DNA sequence, which directs the nuclease to the desired cutting location. Once the DNA in a cell is cut, the cell tries to fix the break using its own repair mechanisms. Due to the error-prone nature of such mechanisms, this fix can actually disable a gene. Alternatively, by supplying a template sequence together with the CRISPR machinery, the cells DNA repair mechanisms can be exploited to replace a section of DNA with the template sequence of choice.

Building on this foundational work of the Nobel Laureates, many complementary genome editing tools based on the CRISPR principle have been developed. For example, in a technique known as base editing, a deactivated Cas9 is fused to a cytidine deaminase enzyme, which allows targeted conversion of the cytidine base (C) to thymine (T) without cleaving the DNA (Komor et al, 2016). Another example is prime editing, in which deactivated Cas9 is fused to reverse transcriptase. This is coupled with a guide RNA that specifies the target site and encodes a desired replacement sequence, allowing new genetic information to be written into a specified DNA target site (Anzalone et al, 2019).

How can CRISPR be used?

There are innumerable exciting possibilities that stem from the ability to edit the genome of any living cell in a targeted manner using these basic principles. For example, CRISPR is already revolutionising genetic research by providing a quicker and easier way for researchers to knock out specific genes in order to investigate the function of those genes and their role in cellular pathways. There also important applications in agriculture, where it is being used to speed up the generation of improved crop varieties and could play an important role in food security.

Another key application is in the filed of diagnostics (more on that below), but perhaps one of the most exciting and lucrative CRISPR applications is in the field of medicine. Doudna is a co-founder of Intellia and Charpentier is a co-founder of CRISPR Therapeutics, both of which are developing CRISPR-based therapies, some of which are already in early stage clinical trials, e.g. for the treatment of sickle cell anaemia. Many current trials involve editing the genome of cells extracted from the body (e.g. hematopoietic stem cells) before reinserting the modified cells back into the patient. An alternative approach also being explored is delivering the CRISPR machinery directly into the body, for example to disable faulty disease-causing genes. This year saw the first delivery of CRISPR machinery to a patient in an attempt to treat an inherited form of blindness called Leber congenital amaurosis 10 (LCA10).

CRISPR patents

Any new technology with such a great potential for commercial application is an ideal candidate for patent protection. The European Patent Office (EPO) has published 32 European patent applications naming Doudna as an inventor and 7 European patent applications naming Charpentier as an inventor. Patent applications are published 18 months after their effective filing date, so there may be many more unpublished patent applications that have already been filed naming these Laureates as inventors.

CRISPR patents have also been at the centre of attention in both Europe and the US over recent years. In Europe, weve seen the high-profile CRISPR priority appeal, in which one of the Broad Institutes fundamental CRISPR patents (EP2771468, claiming an earliest priority date of 12 December 2012) was revoked for lack of novelty over some of the seminal CRISPR papers. These papers became prior art because the patent was found not to be entitled to its claimed priority dates (see our articles here, here and here from earlier this year for the details). Doudna and Charpentiers patents have also come under attack in Europe; their 2013 patent EP2800811 was opposed by seven parties, and maintained in amended form in May 2020.

In the US, high profile interference proceedings between University of California and others and the Broad Institute and others (Broad) before the US Patent Trial and Appeal Board culminated in a decision in favour of Broad, which was upheld in 2018 by the Court of Appeals for the Federal Circuit. Further such proceedings are currently in progress.

Stay tuned for a more in-depth discussion of the ongoing challenges relating to CRISPR patents.

The future could CRISPR be used to combat COVID -19?

Shortly after the Nobel Prize was announced, Charpentier was asked whether CRISPR could be used to make a vaccine for COVID-19. She indicated this was unlikely in a direct way, but that it could be useful indirectly by allowing researchers to understand the virus in ways that help them develop a vaccine (e.g. understanding what is important for the virus to replicate). The full Q&A with Charpentier is available here.

It has in fact already been deployed in a fast and accurate diagnostic test for Covid-19. This test, referred to as SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), harnesses the targeting function of the guide RNAs of the CRISPR system to bind to coronavirus sequences, and the cutting function of Cas12 (a nuclease related to Cas9) to cleave a reporter molecule, to confirm detection of the virus. Fittingly, Doudnas own lab recently announced its own CRISPR-based diagnostic test that can detect SARS-CoV-2 in just 5 minutes. This high processing speed is achieved by avoiding the need to amplify the viral genome (as required by earlier assay formats). Instead, the new test uses combinations of CRISPR RNA which target different parts of the virus RNA and activate multiple Cas nucleases (Cas13a) per piece of viral RNA, boosting the fluorescent signal generated when a reporter molecule is cut. Moreover, the researchers showed that the fluorescence could be measured with a mobile phone camera, demonstrating the simplicity and portability of the assay.

As mentioned above, clinical trials involving CRISPR-based approaches are already underway, and we look forward to seeing more success stories in the coming years. While these are no doubt exciting times, it is clear that extreme caution must be exercised to fully understand and mitigate the risk of CRISPR acting off-target. There are also ethical debates to be had about how far to take gene editing. Should scientists be permitted to introduce heritable changes into the genome even if this can be done safely and efficiently?

One thing is for sure, the work conducted by Doudna and Charpentier has revolutionised the field of genetic engineering, and for that work these inspirational inventors should be celebrated.

Nobel Prize in Chemistry 2020 Winners from a Patent Perspective - Lexology


Viewpoint: Greenpeace-funded study backfires, undermining case to treat gene-edited crops as GMOs – Genetic Literacy Project

Sunday, October 18th, 2020

The anti-GMO movement dominated the public discourse about crop biotechnology for decades. Led by committed activists who knew how to manipulate the media, they effectively steamrolled a scientific community that wasnt ready for the PR war that Greenpeace and other NGOs launched against frankenfoods in the mid-1990s. Thirty years later, were beginning to see that dynamic shift as plant breeding technology improves and experts successfully defend it against activist attacks.

In August, John Fagan, organic food champion, biologist and Raja of World Peace in the Maharishi organization, led a Greenpeace-funded study claiming that gene-edited crops developed with new breeding techniques (NBTs) like CRISPR can be detected.

This may seem unimpressive to most people, but the result is a big deal to anti-GMO activists like Fagan. Gene-edited crops may be essentially identical to conventionally bred plants; the only difference is that gene editing dramatically speeds up the breeding process, saving time, money and getting enhanced seeds into farmers fields much more quickly than was previously possibleall without inserting foreign DNA into the crops genome. This is the primary distinction between gene editing and transgenesis (GMO in the vernacular).

These facts aside, European law treats gene-edited and GMO crops the same. Commercial cultivation of both is effectively banned in the EU (farmers have access to only one transgenic corn variety), though political pressure is building to reform Europes strict regulations. Anti-biotech groups have been on a campaign to block these reforms since July 2018, just after the European Court of Justice first ruled on crop gene editing. Fagans paper was the latest contribution to this effort. If gene-edited and conventional crops can be distinguished, the argument goes, then the former should be regulated as GMOs.

But theres a problema big one in fact: Fagans study actually demonstrated that its not possible to detect most gene-edited plants, therefore destroying the EUs justification for regulating them as GMOs. Its the perfect example of what we fondly call an own goal. And it illustrates how anti-science groups flog disinformation with the help of gullible journalists who stenograph their questionable claims for wide distribution.

Given the onslaught of disinformation we face in a post-COVID world, Fagans paper offers us the perfect opportunity to review the activist playbook and immunize ourselves against bad science and its harmful consequences.

As gene editing becomes an increasingly effective tool for improving agricultural production and reducing its environmental impacts, many countries (the US, Canada, Brazil, Argentina among dozens more) have split from the European Union (EU) on NBTs, exempting them from the expensive and exhausting regulations that govern GMO crops.

This is not only scientifically sound, its pragmatic. There is no way to detect gene edits in most cases. These changes look just like natural mutations found in wild plants, or the genetic changes induced by old-fashioned and EU-approved practices like bathing seeds in mutagens or irradiating them, or changes that occur in plants produced via tissue culture. The tests at our disposal cannotI repeat, cannotdistinguish mutations caused by any of these techniques.

This creates a problem for Food Purity Rajas and other opponents of biotechnology. If these gene-edited crops gain public acceptance and dont count as GMO, the organic industry will be at a competitive disadvantage. How could they justify a premium price for Non-GMO Project-certified corn flakes in those circumstances? Naturally, they have to challenge the efficacy and safety of gene editing to prevent such an outcome. Fear is their go-to currency in this effort, as John Fagan explained almost six years ago in a mailing list for anti-GMO campaigners, run by Claire Robinson of the activist website GM Watch. Its part of a long-term plan to make people fear all engineered food.

Fagan recently gave credit for Americas rejection of GMOs to transcendental meditation (TM), which caused a sharp increase in coherence in U.S. collective consciousness, when a large permanent group of TM practitioners was assembled in Iowa, USA. I havent seen the evidence for that, though Id be happy to take a look at the research if anyone can locate it. What is more likely, and supported by data, is that a long-term misinformation campaign made up of bad science and shock marketing scared parents everywhere into buying organic fruit snacks to avoid scary GMOs.

Down to the last detail, this tried and true activism strategy was deployed to influence the current discourse around gene editing. Besides his enthusiasm for TM, John Fagan also has training in molecular biology and operates a non-profit lab with the necessary testing capacity (incidentally, he also started a company that certifies products for the Non-GMO Project). Greenpeace, meanwhile, has an effective, stunt-based PR machine that can churn out multimedia presentations and widely read press releases, which sympathetic NGOs can dutifully amplify. But things didnt turn out as intended this time.

Its impossible to know which came first: the idea for a campaign to attack gene-edited crops, which needed supporting science, or a study in need of the PR muscle Greenpeace could leverage. Someday an intrepid investigative journalist might be able to work this out. But in any case, the outcome on September 7, 2020 was the release of a paper in the peer-reviewed journal Foods, which claimed to reveal a test that could uniquely and specifically detect the first commercial gene-edited crop, a variety of herbicide-resistant canola developed by the seed company Cibus. With a press release, media blitz, and slickly produced website, Greenpeace and other funders launched the #NowhereToHide campaign to promote Fagans paper and encourage EU regulators to treat this herbicide-tolerant canola as a GMO.

Similar to the guy who claimed he invented email, Fagans team implied they developed a new test to identify this crop. Thats not the case; the qPCR (polymerase chain reaction) method used in the study is well established for canola. Fagans novelty claim is therefore quite erroneous, as one scientist noted on Twitter. Nobody disputes that you can find point mutations, changes to a single DNA base pair, with qPCR. The key is that its impossible to determine if a variation is naturally occurring or purposefully induced. Many experts have pointed this out in response to the paper. The test would likewise detect herbicide-resistant plants that have been known to scientists and regulators since in 2002, from wild populations with that same mutation. Etienne Bucher, a plant geneticist based in Switzerland, tried to help Greenpeace grasp this:

But heres the kicker: this canola is not gene edited. It is a somaclonal mutation that was found in the screenings for an herbicide-tolerant variety, one of those changes that occurs in tissue culture. So what Fagans team has, in fact, definitively proved: they cannot detect edited canola this way. Additionally, it appears this rapeseed could be classified as non-GMO in Europe, since it was developed with one of the grandfathered techniques not subject to the onerous EU GMO approval process.

Own. Goal. Reminds me of the time anti-vaxxers commissioned research that confirmed vaccines do not cause autism.

Let the goal-post moving commence!

After the scientific community made quick work of Fagans study, Greenpeace and activists like Claire Robinson at GMWatch began furiously backpedaling. Maharishi TM trainer and geneticist Michael Antoniou told the anti-GMO website that the method they have developed reliably detects a single DNA base unit change, regardless of how it came about. But he went on to assert that EU regulators should still rely on this test to detect the canola variety. This makes absolutely no sense, as the German Central Committee for Biosafety experts observed [automated Google translation]:

The publication by Chhalliyil et al does not add any new knowledge to the current state of science and technology. Rather, it proves that it is not possible to distinguish genome-edited plants from plants with spontaneously occurring mutations. Without prior knowledge of the manufacturing process [my emphasis], no statement can be made as to whether or not it is a GMO within the meaning of the ECJ [European Court of Justice] ruling.

GM Watch agreed, though it fell back on a legal argument to excuse the studys weakness:

What the test cannot do is detect the technique by which a mutation was brought about but under EU law it doesnt need to. The way that the law deals with proof of origin for all GMOs products of gene editing included is to require the developer to declare that their product is a GMO and provide a test method and reference material.

In other words, if Cibus tells regulators its canola is gene edited, then regulators can determine if the canola is gene edited.

The media blitz around Fagans study was designed to promote organic food, but ironically enough, all this talk about identifying the source of mutations dredged up a potentially serious problem for the organic industry. In 2014, the USDAs National Organic Standards Board investigated what kinds of genetic modifications led to many of the key organic crops, only to realize how difficult it would be to classify breeding and laboratory mutations:

Exploring this issue has brought to the attention of the subcommittee that engineered genetic manipulation of plant breeding materials has already occurred in many of the crop varieties that are currently being used in organic farming. A partial list:

Many of these techniques that were used in initial crosses that have now passed down through many generations may not be traceable any longer

The board realized that many of the crops in organic production right now were the result of laboratory processes (genetic engineering, one could say) that are undetectable with molecular testing. Why is this significant? Well, a cynical scientist could use Fagans test to detect mutations in organic tangerines just as well as Cibus canola, demonstrating the inanity of labeling the tangerine non-GMO and the canola genetically engineered.

Bungled though it was, this parallel science PR stunt helpfully illustrated how disinformation can sow confusion and lead to nonsensical policy. For example, Greenpeace celebrated when an Austrian health minister declared that Fagans test should be used to enforce the EUs GMO rules. Well-known anti-crop biotech German politicians also eagerly embraced the results of the study. If people with so much influence over food safety rules in their countries can be fooled, you can see why junk science poses the risk it does.

Still, Greenpeace clearly lost this round. The activist-media juggernaut went down in flames before it could do too much damageand GM Watch spent most of September explaining away Fagans study in the face of intense expert scrutiny. Expect the anti-science crusaders to fall back on these same tactics in the future, because its all they know how to do. But look forward to the fact that there are now scientists, battle hardened by years in the social media trenches, ready to blow air horns the next time an NGO launches a scheme like this.

Mary Mangan holds a PhD in cell, molecular, and developmental biology from the University of Rochester. She co-founded OpenHelix, a company that provides awareness and training on open source genomics software tools. Follow her on Twitter @mem_somerville

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Viewpoint: Greenpeace-funded study backfires, undermining case to treat gene-edited crops as GMOs - Genetic Literacy Project


We’ve learned much in this crisis including what we don’t know, by John McGauley – The Keene Sentinel

Sunday, October 18th, 2020

In the ninth month of the COVID scourge which covered the land, the people lamented their fate and looked to the heavens for relief and succor.

That is, I imagine, how the Bible might recount the pandemic weve been in since February, if the Good Book were being written today.

Around here, at least, weve been spared the worst from this thing. But if you recall when it all started, and for several months hence, the hospital geared up for scores of deathly ill patients and local doctors and nurses were told to expect reporting for emergency duty. A mobile refrigerated morgue was brought in. They even retrofitted parts of Keene State College to handle the overflow. Main Street downtown was bereft of traffic; for a couple of months, it was always 6 a.m. Sunday morning down there. Planes, trains and buses stopped; there were no pedestrians. There was a mad rush for PPE (we all learned what that acronym meant), and ventilators.

No one knows for sure where this pandemic is headed, and those up here in the northern climes are wondering if it will get worse as the winter winds drive us all inside until April and May. What will our Thanksgiving and Christmas holidays look like this year?

Assessing the past nine months, Ive had a number of observations, and you may have had similar thoughts.

For starters, I think that society came unglued. And, I think I know why. We collided with something that all of our sophisticated science and technology couldnt handle, and we freaked out. Our new Apple phones, PET scans, 5G networks, genetic engineering all the kings horses and all the kings men were helpless in the face of one of the worlds oldest adversaries: pestilence, which is one of the Four Horsemen of the Apocalypse in the Book of Revelation, to use another biblical reference. While astronauts aboard the International Space Station circle the earth, ensconced in the most amazing science, they look down upon an earth stymied and suffering from a primordial disease.

Secondly, we learned that government has severe limitations. Like our belief in technology, we have come to think in the past century that the state and feds can come up with solutions and come to our rescue. Thats just not true, and never was. You can blame whoever you want, but governments response was and is severely flawed. Its not a failure of the Republicans or Democrats or career bureaucrats at the NIH or CDC its just the fact that government cannot solve our problems when the you-know-what hits the fan. Never could. If youve ever been through the aftermath of a severe hurricane or a strong earthquake, you know that there comes a time when youre just on your own. When a crisis hits, you learn immediately that the emperor lives very far away.

Thirdly, most people behaved, but enough didnt that it showed the sour side of humanity. Panic buying without cause, denying others of necessities. A refusal to wear masks in public. We can sometimes be a stupid bunch.

Fourth, our economy is very fragile, despite being the richest country in the world. That showed itself right away. Our weakest link proved to be our distribution network for goods and services. One of the correct responses by the federal government was the rapid carpet-bombing of cash to companies and individuals. Well have to pay that big bill later, but it saved us from a collapsed system.

Fifth, we have, in fact, dodged the bullet. Despite the number of hospitalizations and deaths, COVID-19 could have been worse, with a much higher mortality rate. If we didnt already possess a sophisticated medical system, the death rate might have been off the charts.

Sixth, instant communication and 24/7 media coverage is half good, half terrible. It disseminates at the speed of light erroneous theories, rumors, dubious statistics, malevolent gossip, wild statements from wrong-headed people parading as scientists, shoddy studies and surveys, and inflammatory rhetoric from politicians and bureaucrats. Cant change that; information and misinformation spread at the same rate, and people believe weird things. Well see that when a vaccine is finally developed, and millions refuse to take it.

Lastly, if there is a silver lining to all this, its that well be prepared for the next disaster, or disease, that comes our way. That is, if we remember what we did right and what we did wrong this time. Unfortunately, humans have a propensity to reinvent the wheel over and over.

John McGauley, an author and local radio talk-show host, writes from Keene. He can be contacted at

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We've learned much in this crisis including what we don't know, by John McGauley - The Keene Sentinel


Was the MERS virus a model for the creation of COVID-19? – WION

Sunday, October 18th, 2020

First reported in 2012 in Saudi Arabia, Middle East Respiratory Syndrome (MERS) is a respiratory illness caused by a coronavirus with symptoms similar to the COVID-19 coronavirus, namely, fever, cough and shortness of breath with a range from none, to mild, to severe.

As of January 2020, about 2,500 cases of MERS have been reported worldwide. Human-to-human transmission typically requires close contact with an infected person, the spread being uncommon outside of hospitals.

In contrast to COVID-19, the death rate from MERS is about 35%.

MERS is believed to have originated in bats, was transmitted to camels as an intermediate host, then infecting humans, who had contact with the infected animals.

Although the COVID-19 virus has structural similarities to bat coronaviruses, its precise origin has yet to be identified.

The most distinguishing and unique structural feature of the COVID-19 virus is the furin polybasic cleavage site, a sequence of amino acids that interacts with human cell enzymes, which cut or cleave parts of the viral structure, thus contributing to the life cycle of the virus.

In the case of COVID-19, that sequence of amino acids is usually identified as proline-arginine-arginine-alanine or, in scientific notation, PRRA, which precedes an arginine-serine cleavage point, R-S.

It is unknown from where the PRRA sequence originated because it does not exist in any of the bat coronaviruses identified as close relatives of the COVID-19 virus.

A model for such a structure, however, does exist in the MERS coronavirus, which has a proline-arginine-serine-valine or PRSV sequence preceding the R-S cleavage point and having the following alignment:



Both sequences begin with proline (P), both are polybasic having more than one arginine (R) and both have a non-polar amino acid in the fourth position, alanine (A) and valine (V), respectively, prior to the cleavage point, R-S.

It is important to note that COVID-19 and MERS are from two completely different families of coronaviruses, so one could not have evolved from the other.

According to the scientific article Structures and dynamics of the novel S1/S2 protease cleavage site loop of the SARS-CoV-2 spike glycoprotein," the presence of proline (P) is highly unusual.

Unlike other amino acids, proline produces structural rigidity in proteins and is found in only 5 out of 132 identified furin cleavage site sequences.

Likewise, alanine (A) located just prior to the R-S cleavage point exists in only 5 out of 132 furin cleavage site sequences.

In an early June scientific article, A novel bat coronavirus closely related to SARS-CoV-2 contains natural insertions at the S1/S2 cleavage site of the spike protein, the authors claimed to have identified a bat coronavirus, called RmYN02, that appears to have a precursor of the COVID-19 furin polybasic cleavage site.

RmYN02 has a proline-alanine-alanine (PAA) insertion roughly in a similar position to the COVID-19 virus, but PAA is chemically neutral, lacks any basic amino acids and has no R-S point to be cleaved.

RmYN02's PAA sequence, therefore, cannot be considered a precursor of the COVID-19 furin polybasic cleavage site.

So, the question remains, if no yet identified close relative of COVID-19 has a similar furin polybasic cleavage site, from where did such a unique structural feature with amino acids in unusual positions arise?

Furin polybasic cleavage sites are known to increase viral infectivity and pathogenicity. Genetic engineering techniques for inserting such cleavage sites have existed for at least fifteen years.

At present, no natural evolutionary pathway has been identified to explain the presence of COVID-19s furin polybasic cleavage site.

Those who may have manufactured the COVID-19 virus, could have been trying to mimic the cleavage site found in MERS.

Furthermore, the high rate of human-to-human transmission found for COVID-19, may have resulted from "pre-adapting" the virus for human infection by serial infection or passaging of the virus using animal models genetically-engineered to express the human coronavirus receptor.

There is now a preponderance of evidence that the COVID-19 virus was the product of laboratory experimentation rather than a natural infectious "jump" from bats to humans.

China still has a lot of explaining to do.

(Lawrence Sellin, Ph.D. is a retired U.S. Army Reserve colonel, who previously worked at the U.S. Army Medical Research Institute of Infectious Diseases and conducted basic and clinical research in the pharmaceutical industry. His email address is

(Disclaimer: The opinions expressed above are the personal views of the author and do not reflect the views of ZMCL.)

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Was the MERS virus a model for the creation of COVID-19? - WION


CAR T-cell Therapies for the Treatment of Patients with Acute Lymphoblastic Leukemia – OncoZine

Sunday, October 18th, 2020

Acute Lymphoblastic Leukemia (AML), also called acute myeloblastic leukemia, acute myelogenous leukemia, acute myeloid leukemia, or acute nonlymphocytic leukemia, is an aggressive, fast-growing, heterogenous group of blood cancers that arise as a result of clonal expansion of myeloid hematopoietic precursors in the bone marrow. Not only are circulating leukemia (blast) cells seen in the peripheral blood, but granulocytopenia, anemia, and thrombocytopenia are also common as proliferating leukemia cells interfere with normal hematopoiesis.

Approximately 40-45% of younger and 10-20% of older adults diagnosed with AML are cured with current standard chemotherapy. However, the outlook for patients with relapsed and/or refractory disease is gloomy. Relapse following conventional chemotherapy remains is a major cause of death.

The process of manufacturing chimeric antigen receptor (CAR) T-cell therapies. [1] T-cells (represented by objects labeled as t) are removed from the patients blood. [2] Then in a lab setting the gene that encodes for the specific antigen receptors is incorporated into the T-cells. [3] Thus producing the CAR receptors (labeled as c) on the surface of the cells. [4] The newly modified T-cells are then further harvested and grown in the lab. [5]. After a certain time period, the engineered T-cells are infused back into the patient. This file is licensed by Reyasingh56 under the Creative Commons Attribution-Share Alike 4.0 International license.Today, the only curative treatment option for patients with AML is allogeneic hematopoietic stem cell transplantation or allo-HSCT, which through its graft-vs.-leukemia effects has the ability to eliminate residual leukemia cells. But it is an ption for only a minority. And despite a long history of success, relapse following allo-HSCT is still a major challenge and is associated with poor prognosis.

In recent years, rresearchers learned a lot about the genomic and epigenomic landscapes of AML. This understanding has paved the way for rational drug development as new drugable targets, resulting in treatments including the antibody-drug conjugate (ADC) gemtuzumab ozogamycin (Mylotarg; Pfizer/Wyeth-Ayerst Laboratories).

CAR T-cell TherapiesChimeric antigen receptor (CAR) T-cells therapies, using a patients own genetically modified T-cells to find and kill cancer, are one of the most exciting recent developments in cancer research and treatment.

Traditional CAR T-cell therapies are an autologous, highly personalised, approach in which T-cells are collected from the patient by leukopheresis and engineered in the laboratory to express a receptor directed at a cancer antigen such as CD19. The cells are then infused back into the patient after administration of a lymphodepletion regimen, most commonly a combination of fludarabine and cyclophosphamide. Durable remissions have been observed in pediatric patients with B-ALL and adults with NHL.

CD19-targeted CAR T-cell therapies, have, over the last decade, yielded remarkable clinical success in certain types of B-cell malignancies, and researchers have made substantial efforts aimed at translating this success to myeloid malignancies.

While complete ablation of CD19-expressing B cells, both cancerous and healthy, is clinically tolerated, the primary challenge limiting the use of CAR T-cells in myeloid malignancies is the absence of a dispensable antigen, as myeloid antigens are often co-expressed on normal hematopoietic stem/progenitor cells (HSPCs), depletion of which would lead to intolerable myeloablation.

A different approachBecause autologous CAR T-cell therapies are patient-specific, each treatment can only be used for that one patient. Furthermore, because CAR T-cells are derived from a single disease-specific antibody, they are, by design, only recognized by one specific antigen. As a consequence, only a small subset of patients with any given cancer may be suited for the treatment.

This specificity means that following leukopheresis, a lot of work needs to be done to create this hyper personalised treatment option, resulting in 3 5 weeks of manufacturing time.

The manufacturing process of CAR T-cell therapies, from a single academic center to a large-scale multi-site manufacturing center further creates challenges. Scaling out production means developing processes consistent across many collection, manufacturing, and treatment sites. This complexity results in a the realitively high cost of currently available CAR T-cell therapies.

To solve some of the concerns with currently available CAR T-cell therapies, researchers are investigating the option to develop allogenic, off-the-shelf Universal CAR T-cell (UCARTs) treatments that can be mass manufactured and be used for multiple patients.

Allogeneic CAR T-cell therapy are generally created from T-cells from healthy donors, not patients. Similar to the autologous approach, donor-derived cells are shipped to a manufacturing facility to be genetically engineered to express the antibody or CAR, however, in contrast to autologous CAR T-cells, allogeneic CAR T-cells are also engineered with an additional technology used to limit the potential for a graft versus host reaction when administered to patients different from the donor.

One unique benefit ofn this approach is that because these therapies hey are premade and available for infusion, there is no requirement to leukopheresis or a need to wait for the CAR T-cells to be manufactured. This strategy also will benefit patients who are cytopenic (which is not an uncommon scenario for leukemia patients) and from whom autologous T-cell collection is not possible.

PioneersAmong the pioneers of developing allogeneic CAR-T therapies are companies including Celyad Oncology, Cellectis, Allogene Therapeutics, and researchers at University of California, Los Angeles (UCLA) in colaboration with Kite/Gilead.

Researchers at UCLA were, for example, able to turn pluripotent stem cells into T-cells through structures called artificial thymic organoids. These organoids mimic the thymus, the organ where T-cells are made from blood stem cells in the body.

Celyad OncologyBelgium-based Celyad Oncology is advancing a number of both autologous and allogeneic CAR T-cell therapies, including proprietary, non-gene edited allogeneic CAR T-cell candidates underpinned by the companys shRNA technology platform. The shRNA platform coupled with Celyads all-in-one vector approach provides flexibility, versatility, and efficiency to the design of novel, off-the-shelf CAR T-cell candidates through a single step engineering process.

In July 2020, the company announced the start of Phase I trials with CYAD-211, Celyads first-in-class short hairpin RNA (shRNA)-based allogeneic CAR T candidate and second non-gene edited off-the-shelf program. CYAD-211 targets B-cell maturation antigen (BCMA) for the treatment of relapsed/refractory multiple myeloma and is engineered to co-express a BCMA-targeting chimeric antigen receptor and a single shRNA, which interferes with the expression of the CD3 component of the T-cell receptor (TCR) complex.

During the 2020 American Society of Clinical Oncology (ASCO) Virtual Scientific Program in May 2020, the company presented updates from its allogeneic programs, including additional data from the alloSHRINK study, an open-label, dose-escalation Phase I trial assessing the safety and clinical activity of three consecutive administrations of CYAD-101, an investigational, non-gene edited, allogeneic CAR T-cell candidate engineered to co-express a chimeric antigen receptor based on NKG2D (a receptor expressed on natural killer (NK) cells that binds to eight stress-induced ligands and the novel inhibitory peptide TIM TCR Inhibitory Molecule), for the treatment of metastatic colorectal cancer (mCRC).

The expression of TIM reduces signalling of the TCR complex, which is responsible for graft-versus host disease.every two weeks administered concurrently with FOLFOX (combination of 5-fluorouracil, leucovorin and oxaliplatin) in patients with refractory metastatic colorectal cancer (mCRC).

The safety and clinical activity data from the alloSHRINK trial in patients with mCRC demonstrated CYAD-101s differentiated profile as an allogeneic CAR T-cell candidate. Furthermore, the absence of clinical evidence of graft-versus-host-disease (GvHD) for CYAD-101 confirms the potential of non-gene edited approaches for the development of allogeneic CAR-T candidates.

Interim data from the alloSHRINK trial showed encouraging anti-tumor activity, with two patients achieving a confirmed partial response (cPR) according to RECIST 1.1 criteria, including one patient with a KRAS-mutation, the most common oncogenic alteration found in all human cancers. In addition, nine patients achieved stable disease (SD), with seven patients demonstrating disease stabilization lasting more than or equal to three months of duration.

Based on these results, clinical trials were broadened to include evaluating CYAD-101 following FOLFIRI (combination of 5-fluorouracil, leucovorin and irinotecan) preconditioning chemotherapy in refractory mCRC patients, at the recommended dose of one billion cells per infusion as an expansion cohort of the alloSHRINK trial. Enrollment in the expansion cohort of the trial is expected to begin during the fourth quarter of 2020.

CellectisCellectis is developping a universal CAR T-cell (UCART) platform in an attempy to create off-the-shelf CAR T-cell therapies. The companys pipeline includes UCART123, a CAR T-cell therapy designed to targets CD123+ leukemic cells in acute myeloid leukemia (AML). The investigational agent is being studied in two open-label Phase I trials: AML123 studying the therapys safety and efficacy in an estimated 156 AML patients, and ABC123 studying the therapys safety and activity in an estimated 72 patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN).

UCART22Another investigational agent in clinical trials is UCART22 which is designed to treat both CD22+ B-cell acute lymphoblastic leukemia (B-ALL) and CD22+ B-cell non-Hodgkin lymphoma (NHL). Cellectis reported that UCART22 is included in an open-label, dose-escalating Phase I trial to study its safety and activity in relapsed or refractory CD22+ B-ALL patients.

UCART22 harbors a surface expression of an anti-CD22 CAR (CD22 scFv-41BB-CD3z) and the RQR8 ligand, a safety feature rendering the T-cells sensitive to the antibody rituximab. Further, to reduce the potential for alloreactivity, the cell surface expression of the T-cell receptor is abrogated through the inactivation of the TCR constant (TRAC) gene using Cellectis TALEN gene-editing technology.[1]

Preclinical data supporting the development of UCART22 was presented by Marina Konopleva, M.D., Ph.D. and her vteam during the 2017 annual meeting of the American Society of Hematology (ASH) meeting. [1]

Cellectis is also developing UCARTCS1 which is developed to treat CS1-expressing hematologic malignancies, such as multiple myeloma (MM). UCARTCLL1 is in preclinical development for treating CLL1-expressing hematologic malignancies, such as AML.

Cellectis and Allogene Therapeutics, another biotech company involved in the developmen t of CAR T-cell therapies, are developing ALLO-501, another CAR T-cell therapy which targets CD19 and is being developed for the the treatment of patients with relapsed or refractory NHL. Allogene Therapeutics is also developing ALLO-715, an investigational CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) for treating relapsed or refractory multiple myeloma and ALLO-819, which targets CD135 (also called FLT3), for treating relapsed or refractory AML.

Allogene, in collaboration with both Cellectis, Pfizer (which has a 25% stake in Allogene) and Servier have numerous active open-label, single-arm Phase I trials for an off-the-shelf allogeneic CAR-T therapy UCART19* in patients with relapsed or refractory CD19+ B-ALL. Participating patients receive lymphodepletion with fludarabine and cyclophosphamide with alemtuzumab, followed by UCART19 infusion. Adults patients with R/R B-ALL are eligible.

The PALL aims to evaluate the safety and feasibility of UCART19 to induce molecular remission in pediatric patients with relapsed or refractory CD19-positive B-cell acute lymphoblastic leukemia (B-ALL) in 18 pediatric patients.

The CALM trial is a dose-escalating study evaluating the therapys safety and tolerability in 40 adult patients; and a long-term safety and efficacy follow-up study in 200 patients with advanced lymphoid malignancies.

Allogene reported preliminary proof-of-concept results during the annual meeting of the American Society of Hematology (ASH) in December 2018.

Data from the first 21 patients from both the PALL (n=7) and CALM (n=14) Phase I studies were pooled. The median age of the participating patients was 22 years (range, 0.8-62 years) and the median number of prior therapies was 4 (range, 1-6). Sixty-two percent of the patients (13/21) had a prior allogeneic stem cell transplant.

Of the 17 patients who received treatment with UCART19 and who received lymphodepletion with fludarabine, cyclophosphamide and alemtuzumab, an anti-CD52 monoclonal antibody, 14 patients (82%) achieved CR/CRi, and 59% of them (10/17) achieved MRD-negative remission.

In stark contrast, the four patients who only received UCART19 and fludarabine and cyclophosphamide without alemtuzumab did not see a response and minimal UCART19 expansion.

Based on these results, researchers noted that apparent importance of an anti-CD52 antibody for the efficacy of allogeneic CAR-T therapies. In addition, safety data also looked promising. The trial results did not include grade 3 or 4 neurotoxicity and only 2 cases of grade 1 graft-versus-host disease (10%), 3 cases of grade 3 or 4 cytokine release syndrome which were considered manageable (14%), 5 cases of grade 3 or 4 viral infections (24%), and 6 cases of grade 4 prolonged cytopenia (29%).

Precision BiosciencesPrecision Biosciences is developing PBCAR0191, an off-the-shelf investigational allogeneic CAR T-cell candidate targeting CD19. The drug candidate is being investigated in a Phase I/IIa multicenter, nonrandomized, open-label, parallel assignment, dose-escalation, and dose-expansion study for the treatment of patients with relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) or R/R B-cell precursor acute lymphoblastic leukemia (B-ALL).

The NHL cohort includes patients with mantle cell lymphoma (MCL), an aggressive subtype of NHL, for which Precision has received both Orphan Drug and Fast Track Designations from the U.S. Food and Drug Administration (FDA).

A clinical trial with PBCAR0191 Precision Biosciences is exploring some novel lymphodepletion strategies in addition to fludarabine and cyclophosphamide. Patients with R/R ALL, R/R CLL, R/R Richter transformation, and R/R NHL are eligible. Patients with MRD+ B-ALL are eligible as well. This trial is enrolling patients.

In late September 2020, Precision BioSciences, a clinical stage biotechnology amd Servier, announced the companies have added two additional hematological cancer targets beyond CD19 and two solid tumor targets to its CAR T-cell development and commercial license agreement.

PBCAR20APBCAR20A is an investigational allogeneic anti-CD20 CAR T-cell therapy being developed by Precision Biosciences for the treartment of patients with relapsed/refractory (R/R) non-Hodgkin lymphoma (NHL) and patients with R/R chronic lymphocytic leukemia (CLL) or R/R small lymphocytic lymphoma (SLL). The NHL cohort will include patients with mantle cell lymphoma (MCL), an aggressive subtype of NHL, for which Precision BioSciences has received orphan drug designation from the United States Food and Drug Administration (FDA).

PBCAR20A is being evaluated in a Phase I/IIa multicenter, nonrandomized, open-label, dose-escalation and dose-expansion clinical trial in adult NHL and CLL/SLL patients. The trial will be conducted at multiple U.S. sites.

PBCAR269APrecision Biosciences is, in collaboration with Springworks Therapeutics, also developing PBCAR269A, an allogeneic BCMA-targeted CAR T-cell therapy candidate being evaluated for the safety and preliminary clinical activity in a Phase I/IIa multicenter, nonrandomized, open-label, parallel assignment, single-dose, dose-escalation, and dose-expansion study of adults with relapsed or refractory multiple myeloma. In this trial, the starting dose of PBCAR269A is 6 x 105 CAR T cells/kg body weight with subsequent cohorts receiving escalating doses to a maximum dose of 6 x 106 CAR T cells/kg body weight.

PBCAR269A is Precision Biosciencess third CAR T-cell candidate to advance to the clinic and is part of a pipeline of cell-phenotype optimized allogeneic CAR T-cell therapies derived from healthy donors and then modified via a simultaneous TCR knock-out and CAR T-cell knock-in step with the =companys proprietary ARCUS genome editing technology.

The FDA recently granted Fast Track Designation to PBCAR269A for the treatment of relapsed or refractory multiple myeloma for which the FDA previously granted Orphan Drug Designation.

TCR2 TherapeuticsTCR2 Therapeutics is developing a proprietary TRuC (TCR Fusion Construct) T-cells designed to harness the natural T cell receptor complex to recognize and kill cancer cells using the full power of T-cell signaling pathways independent of the human leukocyte antigen (HLA).

While succesful in hematological malignancies, CAR T-cells therapies have generally struggled to show efficacy against solid tumors. Researchers at TCR2 Therapeutics believe this is is caused by the fact that CAR T-cell therapies only utilize a single TCR subunit, and, as a result, do not benefit from all of the activation and regulatory elements of the natural TCR complex. By engineering TCR T-cells, which are designed to utilize the complete TCR, they have demonstrated clinical activity in solid tumors. However, this approach has also shown major limitations. TCR T-cells require tumors to express HLA to bind tumor antigens. HLA is often downregulated in cancers, preventing T-cell detection. In addition, each specific TCR-T cell therapy can only be used in patients with one of several specific HLA subtypes, limiting universal applicability of this approach and increasing the time and cost of patient enrollment in clinical trials.

In an attempt to solve this problem, researchers at TCR2 Therapeutics have developped a proprieatarry TRuC-T Cells which are designed to incorporate the best features of CAR-T and TCR-T cell therapies and overcome the limitations. The TRuC platform is a novel T cell therapy platform, which uses the complete TCR complex without the need for HLA matching.

By conjugating the tumor antigen binder to the TCR complex, the TRuC construct recognizes highly expressed surface antigens on tumor cells without the need for HLA and engage the complete TCR machinery to drive the totality of T-cell functions required for potent, modulated and durable tumor killing.

In preclinical studies, TCR2 Therapeutics TRuC T-cells technology has demonstrated superior anti-tumor activity in vivo compared to CAR T-cells therapies, while, at the same time, releasing lower levels of cytokines. These data are encouraging for the treatment of solid tumors where CAR T-cells have not shown significant clinical activity due to very short persistence and for hematologic tumors where a high incidence of severe cytokine release syndrome remains a major concern.

TCR2 Therapeutics product candidates include TC-210 and TC-110.

TC-210 is designed to targets mesothelin-positive solid tumors. While its expression in normal tissues is low, mesothelin is highly expressed in many solid tumors. Mesothelin overexpression has also been correlated with poorer prognosis in certain cancer types and plays a role in tumorigenesis. TC-210 is being developed for the treatment of non-small cell lung cancer, ovarian cancer, malignant pleural/peritoneal mesothelioma and cholangiocarcinoma.

The companys TRuC-T cell targeting CD19-positive B-cell hematological malignancies, TC-110, is being developed to improve upon and address the unmet needs of current CD19-directed CAR T-cell therapies. The clinical development TC-110 focus on the treatment of adult acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Preclinical data demonstrates that TC-110 is superior to CD19-CAR-T cells (carrying either 4-1BB or CD28 co-stimulatory domains) both in anti-tumor activity as well as the level of cytokine release which may translate into lower rates of adverse events. The development of TC-110 starts with autologous T-cells collection by leukopheresis. These T-cells undergo genetic engineering to create TRuC-T cells targeting CD19.

This strategy combines the best features of CAR T-cells and the native T-cell receptor. It is open for R/R NHL and R/R B-ALL.

AUTO1Auto1 is an autologous CD19 CAR T-cell investigational therapyis being developped by Autolus Therapeutics. The investigational drug uses a single-chain variable fragment (scFv) called CAT with a lower affinity for CD19 and a faster off-rate compared to the FMC63 scFv used in other approved CD19 CAR T-cell therapies. The investigational therapy is designed to overcome the limitations in safety while maintaining similar levels of efficacy compared to current CD19 CAR T-cell therapies.

Designed to have a fast target binding off-rate to minimize excessive activation of the programmed T-cells, AUTO1 may reduce toxicity and be less prone to T-cell exhaustion, which could enhance persistence and improve the T-cells abilities to engage in serial killing of target cancer cells.

In 2018, Autolus signed a license agreement UCL Business plc (UCLB), the technology-transfer company of UCL, to develop and commercialize AUTO1 for the treatment of B cell malignancies. AUTO1 is currently being evaluated in two Phase I studies, one in pediatric ALL and one in adult ALL.

CARPALL trialInitial results from the ongoing Phase I CARPALL trial of AUTO1 were presented during European Hematology Association 1st European CAR T Cell Meeting held in Paris, France, February 14-16, 2019.

Enrolled patients had a median age of 9 years with a median of 4 lines of prior treatment. Seventeen patients were enrolled, and 14 patients received an infusion of CAR T cells. Ten of 14 patients had relapsed post allogeneic stem cell transplant. Eight patients were treated in second relapse, 5 in > second relapse and 3 had relapsed after prior blinatumomab or inotuzumab therapy. Two patients had ongoing CNS disease at enrollment.

This data confirmed that AUTO1 did not induces severe cytokine release syndrome (CRS) (Grade 3-5). Nine patients experienced Grade 1 CRS, and 4 patients experienced Grade 2 CRS. No patients required tociluzumab or steroids. As previously reported, one patient experienced Grade 4 neurotoxicity; there were no other reports of severe neurotoxicity (Grade 3-5). The mean cumulative exposure to AUTO1 CAR T-cells in the first 28 days as assessed by AUC was 1,721,355 copies/g DNA. Eleven patients experienced cytopenia that was not resolved by day 28 or recurring after day 28: 3 patients Grades 1-3 and 8 patients Grade 4. Two patients developed significant infections, and 1 patient died from sepsis while in molecular complete response (CR).

With a single dose of CAR T cells at 1 million cells/kg dose, 12/14 (86%) achieved molecular CR. Five patients relapsed with CD19 negative disease. Event free survival (EFS) based on morphological relapse was 67% (CI 34-86%) and 46% (CI 16-72%) and overall survival (OS) was 84% (CI 50-96%) and 63% (CI 27-85%) at 6 and 12 months, respectively.

CAR T cell expansion was observed in all responding patients (N=12), with CAR T cells comprising up to 84% of circulating T cells at the point of maximal expansion. The median persistence of CAR T-cells was 215 days.

The median duration of remission in responding patients was 7.3 months with a median follow-up of 14 months. Five of 14 patients (37%) remain in CR with ongoing persistence of CAR T-cells and associated B cell aplasia.

Fate TherapeuticsFT819 is an off-the-shelf CAR T-cell therapy targeting CD19 being developed by Fate Therapeutics. The T-cells are derived from a clonal engineered master induced pluripotent stem cell line (iPSCs) with a novel 1XX CAR targeting CD19 inserted into the T-cell receptor alpha constant (TRAC) locus and edited for elimination of T-cell receptor (TCR) expression.

Patients participating in the companys clinbical trial will receive lymphodepletion with fludarabine and cyclophosphamide. Some patients will also receive IL-2. Patients with R/R ALL, R/R CLL, R/R Richter transformation, and R/R NHL are eligible. Patients with MRD+ B-ALL are eligible as well.

At the Annual Meeting of the American Societ of Hematology held in December 2019, researchers from Fate Therapeutics presented new in vivo preclinical data demonstrating that FT819 exhibits durable tumor control and extended survival. In a stringent xenograft model of disseminated lymphoblastic leukemia, FT819 demonstrated enhanced tumor clearance and control of leukemia as compared to primary CAR19 T-cells. At Day 35 following administration, a bone marrow assessment showed that FT819 persisted and continued to demonstrate tumor clearance, whereas primary CAR T cells, while persisting, were not able to control tumor growth. [2]

CAR-NK CD19Allogeneic cord blood-derived Natural Killer (NK) cells are another off-the-shelf product that does not require the collection of cells from each patient.

Unlike T-cells, NK-cells do not cause GVHD and can be given safely in the allogeneic setting. At MD Anderson Cancer Center, Katy Rezvani, M.D., Ph.D, Professor, Stem Cell Transplantation and Cellular Therapy, and her team broadly focuses their research on the role of natural killer (NK) cells in mediating protection against hematologic malignancies and solid tumors and strategies to enhance killing function against various cancer.

As part of their research, the team has developed a novel cord blood-derived NK-CAR product that expresses a CAR against CD19; ectopically produces IL-15 to support NK-cell proliferation and persistence in vivo; and expresses a suicide gene, inducible caspase 9, to address any potential safety concerns.

In this phase I and II trial researchers administered HLA-mismatched anti-CD19 CAR-NK cells derived from cord blood to 11 patients with relapsed or refractory CD19-positive cancers (non-Hodgkins lymphoma or chronic lymphocytic leukemia [CLL]). NK cells were transduced with a retroviral vector expressing genes that encode anti-CD19 CAR, interleukin-15, and inducible caspase 9 as a safety switch. The cells were expanded ex vivo and administered in a single infusion at one of three doses (1105, 1106, or 1107 CAR-NK cells per kilogram of body weight) after lymphodepleting chemotherapy. The preliminarry resilts of the trials confirmed that administration of CAR-NK cells was not associated with the development of cytokine release syndrome, neurotoxicity, or graft-versus-host disease, and there was no increase in the levels of inflammatory cytokines, including interleukin-6, over baseline.

The study results also demonstrated that of the 11 patients who were treated, 8 patients (73%) had a response. Of these patients, 7 (4 with lymphoma and 3 with CLL) had a complete remission ICR), and 1 had remission of the Richters transformation component but had persistent CLL. Noteworthy was that responses were rapid and seen within 30 days after infusion at all dose levels. The infused CAR-NK cells expanded and persisted at low levels for at least 12 months. The researchers also noted that a majority of the 11 participating patients with relapsed or refractory CD19-positive cancers had a response to treatment with CAR-NK cells without the development of major toxic effects.[3]

Note* Servier will hold ex-US commercial rights. Servier is the sponsor of the UCART19 trials.

Clinical trialsalloSHRINK Standard cHemotherapy Regimen and Immunotherapy With Allogeneic NKG2D-based CYAD-101 Chimeric Antigen Receptor T-cells NCT03692429Study Evaluating Safety and Efficacy of UCART123 in Patients With Relapsed/ Refractory Acute Myeloid Leukemia (AMELI-01) NCT03190278Study to Evaluate the Safety and Clinical Activity of UCART123 in Patients With BPDCN (ABC123) NCT03203369Study of UCART19 in Pediatric Patients With Relapsed/Refractory B Acute Lymphoblastic Leukemia (PALL) NCT02808442Dose Escalation Study of UCART19 in Adult Patients With Relapsed / Refractory B-cell Acute Lymphoblastic Leukaemia (CALM) NCT02746952Dose-escalation Study of Safety of PBCAR0191 in Patients With r/r NHL and r/r B-cell ALL NCT03666000.Dose-escalation Study of Safety of PBCAR20A in Subjects With r/r NHL or r/r CLL/SLL NCT04030195A Dose-escalation Study to Evaluate the Safety and Clinical Activity of PBCAR269A in Study Participants With Relapsed/Refractory Multiple Myeloma NCT04171843TC-110 T Cells in Adults With Relapsed or Refractory Non-Hodgkin Lymphoma or Acute Lymphoblastic Leukemia NCT04323657Phase 1/2 Trial of TC-210 T Cells in Patients With Advanced Mesothelin-Expressing Cancer NCT03907852CARPALL: Immunotherapy With CD19 CAR T-cells for CD19+ Haematological Malignancies NCT02443831Umbilical & Cord Blood (CB) Derived CAR-Engineered NK Cells for B Lymphoid Malignancies NCT03056339

Reference[1] Petti F. Broadening the Applicability of CAR-T Immunotherapy to Treat the Untreatable. OncoZine. October 24, 2019 [Article][2] Wells J, Cai T, Schiffer-Manniou C, Filipe S, Gouble A, Galetto R, Jain N, Jabbour EJ, Smith J, Konopleva M. Pre-Clinical Activity of Allogeneic Anti-CD22 CAR-T Cells for the Treatment of B-Cell Acute Lymphoblastic Leukemia Blood (2017) 130 (Supplement 1): 808. Chang C, Van Der Stegen S, Mili M, Clarke R, Lai YS, Witty A, Lindenbergh P, Yang BH, et al. FT819: Translation of Off-the-Shelf TCR-Less Trac-1XX CAR-T Cells in Support of First-of-Kind Phase I Clinical Trial. Blood (2019) 134 (Supplement_1): 4434. Liu E, Marin D, Banerjee P, Macapinlac HA, Thompson P, Basar R, Nassif Kerbauy L, Overman B, Thall P, Kaplan M, Nandivada V, Kaur I, Nunez Cortes A, Cao K, Daher M, Hosing C, Cohen EN, Kebriaei P, Mehta R, Neelapu S, Nieto Y, Wang M, Wierda W, Keating M, Champlin R, Shpall EJ, Rezvani K. Use of CAR-Transduced Natural Killer Cells in CD19-Positive Lymphoid Tumors. N Engl J Med. 2020 Feb 6;382(6):545-553. doi: 10.1056/NEJMoa1910607. PMID: 32023374; PMCID: PMC7101242.

Featured image: T-cells attacking a cancer cell. Photo courtesy: Fotolia/Adobe 2016 2020. Used with permission.

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CAR T-cell Therapies for the Treatment of Patients with Acute Lymphoblastic Leukemia - OncoZine


Study shows a molecular dance that keeps your heart beating – WSU News

Sunday, October 18th, 2020

A microscope photograph of a heart muscle cell. The regular green patterns show stained actin filaments.

By Tina Hilding, Voiland College of Engineering and Architecture

It might look like a little game at the molecular scale.

Filament-like proteins in heart muscle cells have to be exactly the same length so that they can coordinate perfectly to make the heart beat.

Another protein decides when the filament is the right size and puts a wee little cap on it. But, if that protein makes a mistake and puts the cap on too early, another protein, leiomodin, comes along and knocks the cap out of the way.

This little dance at the molecular scale might sound insignificant, but it plays a critical role in the development of healthy heart and other muscles. Reporting in the journal, Plos Biology,a WSU research team has proven for the first time how the mechanism works.

The finding could someday lead to improved diagnostics and medical treatments for serious and sometimes devastating hereditary heart conditions that come about from genetic mutations in the proteins. One of these conditions, cardiomyopathy, affects as many as one in 500 people around the world and can often be fatal or have lifetime health consequences. A similar condition called nemaline myopathy affects skeletal muscles throughout the body with often devastating consequences.

Mutations in these proteins are found in patients with myopathy, saidAlla Kostyukova, associate professor in the Gene and LindaVoiland School of Chemical Engineering and Bioengineeringand leader of the project. Our work is to prove that these mutations cause these problems and to propose strategies for treatment.

Heart muscle is made of tiny thick and thin filaments of proteins. With the help of electrical signals, the rope-like filaments bind and unbind in an intricate and precise architecture, allowing heart muscle to contract and beat.

The thin filaments are made of actin, the most abundant protein in the human body. Tropomysin, another protein, wraps itself around the actin filaments. Tropomyosin together with two other proteins, tropomodulin and leiomodin, at the end of the actin filaments act as a sort of cap and determine the filament length.

Its beautifully designed, said Kostyukova, whose research is focused on understanding protein structures.

And, tightly regulated.

To keep heart muscle healthy, the actin filaments, which are about a micron long, all have to be the exact same length. In families with cardiomyopathy, genetic mutations result in formation of filaments that are either too short or too long. Those affected can have significant heart problems that cause disability, illness and death.

In a project that spanned seven years, the researchers proved that leiomodin attaches to the end of the actin filament and kicks out the other protein, tropomodulin, to assure the actin filaments proper length.

This is the first time that this has been shown with the atomic-level precision, said Dmitri Tolkatchev, research assistant professor in the Voiland School and lead author on the paper. Previously, several laboratories attempted to solve this problem with very little success. With our data we finally have a direct proof.

The researchers used state-of-the-art approaches to make the key proteins and study them at the molecular and cellular level. The work entailed designing the molecules, constructing them at the gene level in a plasmid, and then producing them into bacterial or cardiac cells. The researchers used nuclear magnetic resonance, which works on the same physical principle as Magnetic Resonance Imaging (MRIs), to understand the proteins binding at the atomic level. They also used molecular dynamic simulation to model them.

The probability of being able to show this mechanism was not high, but the impact of the discovery is, said Tolkatchev, an expert in nuclear magnetic resonance. This was a very important problem to study and could have a significant impact in the field of muscle mechanics.

The researchers hope to continue the work, identifying additional components and molecular mechanisms that regulate thin filament architecture, whether diseased or healthy.

The multidisciplinary group included researchers from the University of Arizona led by Carol Gregorio, director of the Cellular and Molecular Medicine Department. WSUs group has expertise in protein structure, structural biochemistry, and properties of actin filaments and regulatory proteins, and UAs group has expertise in molecular, cellular and developmental biology of muscle assembly. The collaborative work was funded by the National Institutes of Health.

Study shows a molecular dance that keeps your heart beating - WSU News


Genetic Engineering Drug Market 2020 | What Is The Estimated Market Size In The Upcoming Years? – The Daily Chronicle

Wednesday, September 30th, 2020

The Global Marketers provides you regional research analysis on Genetic Engineering Drug Market and forecast to 2026. The global Genetic Engineering Drug Market report comprises a valuable bunch of information that enlightens the most imperative sectors of the Genetic Engineering Drug market. The global Genetic Engineering Drug market report provides information regarding all the aspects associated with the market, which includes reviews of the final product, and the key factors influencing or hampering the market growth.

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Main players in the Genetic Engineering Drug Market:

GeneScience Pharmaceuticals Co., LtdBeijing SL Pharmaceutical Co., LtdBiotech Pharmaceutical Co., LtdShenzhen Neptunus Interlong Bio-Technique Co., LtdJiangsu Sihuan Bioengineering Co., LtdTonghua Dongbao Pharmaceutical Co., LtdAnhui Anke Biotechnology (Group) Co., Ltd3SBio Inc.Shanghai Lansheng Guojian Pharmaceutical Co., Ltd

Some of the geographic regions examined in the overall Genetic Engineering Drug Market are:

In addition, the global Genetic Engineering Drug market report delivers brief information about federal regulations and policies that may ultimately affect market growth as well as the financial state. The situation of the global market at the global and regional levels is also described in the global Genetic Engineering Drug market report through geographical segmentation. The Genetic Engineering Drug report introduces speculation attainability evaluation, a task SWOT investigation, and venture yield evaluation.

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Monoclonal AntibodyRecombinant Human ErythropoietinRecombinant Human InterferonRecombinant Human Growth HormoneRecombinant Human Insulin

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Genetic Engineering Drug Market 2020 | What Is The Estimated Market Size In The Upcoming Years? - The Daily Chronicle


Orphan Drug Exclusivity for CRISPR/Cas-Based Therapeutics – JD Supra

Wednesday, September 30th, 2020

The prospect of genetic engineering using CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated nucleases (Cas) has long been hailed as a revolutionary development in medicine.

This technology is rapidly advancing, and several CRISPR/Cas-based drugs have entered clinical trials over the past several years. One kind of product in clinical trials is CRISPR-modified cells, such as CTX001 (CRISPR-Cas9-modified autologous hematopoietic stem cells), currently under study for the treatment of b-thalassemia and severe sickle cell anemia. Another CRISPR-based product, AGN-151587, is injected into the eye with the goal of eliminating a genetic mutation in patients with Leber congenital amaurosis 10, a leading cause of childhood blindness. In parallel, others are working to harness the CRISPR/Cas system to develop drugs for rare diseases, including bespoke therapies tailored to an individual patients needs.

Given CRISPR/Cas-based drugs potential to treat rare diseases, issues relating to orphan drug exclusivity will arise as these products are developed. In May 2020, for example, CTX001 received an orphan drug designation for transfusion-dependent b-thalassemia.

In January 2020, the FDA provided draft guidance regarding orphan drug exclusivity for gene therapy products, which includes CRISPR/Cas gene editing (Draft Guidance). This guidance focuses on the analysis of whether two gene therapy products are the same under the Orphan Drug Act. Although informative, the limited scope of the Draft Guidance invites more questions than it answers.

Same Drugs Under the Orphan Drug Act

Obtaining orphan drug exclusivity involves a two-step process. First, a sponsor requests designation of a drug for a particular rare disease or condition. See 21 C.F.R. 316.20. If this drug is the same drug as a drug already approved to treat the same rare disease or condition, the sponsor must provide a plausible hypothesis that the new drug is clinically superior to the previously-approved drug. Id. Whether two drugs are the same depends on consideration of structural features relevant to that type of drug. See id. 316.3(b)(14).

If the new drug later obtains marketing approval for a use or indication within the rare disease or condition for which it received orphan drug designation, the FDA will determine if the drug is eligible for orphan drug exclusivity. See 21 C.F.R. 316.31(a). In this situation, to receive exclusivity, the sponsor of the new drug must show that its drug is clinically superior to the same previously-approved drug for the same rare disease or condition. See id. 316.34(c). A clinical superiority determination is based on the new drugs greater efficacy, greater safety, or a major contribution to patient care. See id. 316.3(b)(3).

Highlights from Draft FDA Guidance

To determine whether one gene therapy product is the same as another, per 316.3(b)(14)(ii), the FDA will evaluate the principal molecular structural features of the two products, particularly transgenes (e.g., transgenes that encode different enzymes for treatment of the same rare disease) and vectors. For example:

Additionally, [w]hen applicable, the FDA generally intends to consider additional features of the final gene therapy product, such as regulatory elements or, in the case of genetically-modified cells, the type of cell that is transduced. It generally intends to consider requests for designation and exclusivity of gene therapy products to evaluate whether such additional features may also be considered to be principal molecular structural features.

Implications for CRISPR/Cas Therapy Exclusivity

The Draft Guidance helps answer certain high-level questions relating to whether two gene therapy products would be considered the same under the Orphan Drug Act. As various stakeholders have recognized, however, it is short on the details that meaningfully aid the process of drug research and development.

It is clear from the Draft Guidance that a new product can be considered the same as a previously-approved product even if the two products are not perfectly identical, but the guidance does not explain what would constitute a minor difference between such products, or what the scope of additional features would be.

For example, the Draft Guidance does not clarify what makes two transgenes the same. Nor does it cite to prior guidance or regulations that may answer this question. The question is significant because Cas nucleases and other parts of the CRISPR/Cas system may be modified in various ways. To address whether these modifications bar a finding of same-ness, the FDA could potentially import the kinds of considerations that govern same-ness of other kinds of large-molecule products, such as polynucleotide drugs or closely related, complex partly definable drugs with similar therapeutic intent (e.g., viral vaccines). See 21 C.F.R. 316.3(b)(14)(ii)(C), (D). However, this is not clear from the Draft Guidance.

The Draft Guidance also does not explain what will factor into the case-by-case basis assessment of whether viral vectors from the same viral class are the same. In the case of AAV2 and AAV5the two viruses identified in the guidanceresearchers have found that these viruses differ with respect to sequence analysis, tissue tropism, and heparin sensitivity. It is not clear from the guidance, however, whether a plausible hypothesis of clinical superiority will be required to seek orphan drug designation for a drug based on AAV2 if the previously-approved drug expresses the same transgene(s) but is based on AAV5.

It would be beneficial to sponsors and other stakeholders if these aspects of gene therapy drugs sameness are clarified further before they invest significant resources into the design and development of these therapeutics.

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Inventing the future for humankind | Community Perspectives – Fairbanks Daily News-Miner

Wednesday, September 30th, 2020

Back in the halcyon days, when I somehow got paid for messing with the minds of the impressionable youth of UAF, I liked to ask said minds to project themselves back in time 400 years, to take a look around and report back what, if anything, they noticed different between those times (counting back from now, for example, to the Lords Year 1620. (James I was King, if that helps) and our own times: changes in musical tastes, ethics, physics, theology or attitudes regarding leprosy, for instance.

1620 CE was earlier than heart transplants, genetic engineering and baseball. It was before George Washington and water-seal toilets. Oxygen wouldnt be invented until the mid-1700s (Really: no Periodic Table of Elements, no radioactivity). The Holy Inquisition was in practice: pious religious officials were still torturing heretics and burning witches. It was before abortion rights. Autochthonous peoples in many parts of the world had not been introduced to the blessings of European economics, religion and warfare. It was before Facebook.

Things had changed in the last 400 years. Bigly. My students always got that answer right.

Then Id ask them to project themselves 400 years into the future, to the early 25th Century, say, to look around, to report back. I asked them to pay particular attention to the way our descendants in 2420 look back on our (presumably long-obsolete) ways of doing things: our medicine, say, or our governmental systems, or our responses to global hunger, overpopulation, pollution.

This was a harder task. The problem with prognostication is that we normal people are not particularly good at it, being annoyingly set in our ways. This is not to say that we cant make predictions, but even deeply considered and finely calibrated events such as space launches, brain surgery, or steering an oil tanker around Bligh Reef occasionally go awry. Some events, like nuclear meltdowns or worldwide pandemics, can present unanticipated difficulties.

I asked my students to avoid fantasies like self-aware computers, two-way wrist radios or honest politicians. I was hoping for revolutionary ways of perceiving the world, something on the order of the atalatl, General Relativity or Akira Kurosawa. I was angling for new stuff: examples of true scientific, artistic or musical invention.

My students always protested. Were on to you, old man, you you English teacher! Youve been harping all semester about how we mortals really cant see into the future, that we make up the future with our words. Now you want us to think something no one has ever thought before!

Thats exactly what I wanted them to do, of course. To be fair, really new ideas are not particularly common. It took humans millennia to come up with the atlatl (c.20,000 BCE), even longer to invent the calculus (c.1665 CE) or germ theory (c.1840). But without inventive ways of looking at the world, humanity might still believe that malaria is caused by bad air, that light travels across a medium called luminiferous ether, or that things burn because they contain phlogiston.

Theres been much talk lately of returning to normal, but I wonder if thats really what we want. I wonder if normal isnt what got us into our present public health and economic crises. I think for a lot of people in our community normal is worrying about buying groceries, paying the rent, health care, personal safety.

In this Year of Our Trump and the Corona pandemic (known also to certain elderly cynics as the beer virus or the sniffles) the question for my students would be, Given that we really cant see into the future and given that our current pandemic is unlikely to be our last, whats our best strategy for the survival of Our People (defined however you like) for the next seven generations or so?

Id hope for some inventive thinking along the lines of how to take care of every person on Earth in honest and practical ways. Emphasizing that we have plenty to be humble be about when predicting the future, Id ask them to come up with ideas never tried before. Id suggest that food, shelter and health care need never to be money-dependent, for example. Id ask our youth for creative ways of feeding people, sheltering people, caring for people all people on this, our planetary spaceship.

Id invite them to approach the task with an honest and generous spirit.

Lynn Basham lives in Fairbanks. He taught atthe University of Alaska Fairbanks as an instructor, mostly in the English Department, for about 20 years and retired about10 years ago.

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Family seeks answers, finds hope after daughters diagnosed with rare genetic condition – Steamboat Pilot and Today

Wednesday, September 30th, 2020

STEAMBOAT SPRINGS You can hear the love in Mariah Gillaspies voice as she talks about her daughters Emma and Abby, who suffer from a rare genetic disease that causes seizures and development issues.

Emma, shes our oldest, and shell be 4 in October, Mariah said. Shes our calm, sweet little child. She has these little coos that sound like a dove. She really enjoys music, and she loves being around other kiddos her age.

Abby is our younger daughter, and shell be 2 in October, and she is our feisty little thing, Mariah continued. So, she lets you know when shes happy; she lets you know when shes not happy.

There is no question the two girls, the only two people in the world believed to have this disease, are surrounded by the love they get from Mariah and their dad Mark.

Mark grew up in Steamboat Springs and graduated from high school here in 2001. The couple now live in Centennial, but Marks parents, Jeanne and Joe Gillaspie, still live in Steamboat as does Marks older brother.

Four years ago, Mark and Mariah were overwhelmed with joy as they welcomed their first child Emma to the world, but when she was three months old, the couple started to notice she was having some strange movements, and when she started having episodes where she would hold her breath until she would turn pale, the couple took her to the doctor.

The doctor initially thought it was reflux, but when Emma stopped breathing in the doctors office, she was rushed to Childrens Hospital of Colorado for more evaluation and tests.

Throughout all this, I was convinced everything was going to be OK, Mariah said. It never crossed my mind that something was seriously wrong, and I had never considered that these were seizures.

Eventually, Emma was diagnosed with infantile spasms, which Mariah said didnt look serious on the outside but were damaging Emmas brain and impacting her development from the inside. Emma started treatment immediately, and the family was encouraged with the results. But then there was a relapse and a new medication, and then another relapse and another new medication.

Mariah said each new medicine came with a longer list of side effects, and Emmas immune system suffered. She had a bout with pneumonia that left her in the hospital for two months.

Through it all, the Gillaspies continued to search for answers.

We did a whole slew of genetic testing, and it came back inconclusive, Mariah said. They found absolutely nothing that could be the cause of her disease, and they told us this is probably some completely random condition that was caused by something that happened in utero.

They also told the Gillaspies that Emmas condition was rare, and there was less than a 3% chance of it happening again. So after extensive genetic testing, they decided to have a second child.

When Abby arrived two years later, they were thrilled, but at about six weeks, they noticed their youngest daughter was displaying the same movements that Emma had shown prior to her diagnosis. So it was back to the doctors, and it was confirmed through genetic testing that Abby and Emma shared the same mutated gene THAP12.

After discovering their daughters were suffering from the same condition, the family embarked on a grassroots effort to drive research about the rare genetic disease, which led to the creation of a foundation, Lightning and Love, a name that was chosen because the family believes lightning struck their family twice in the form of two daughters with the same rare disease.

The doctors would say, Im sorry, theres nothing we can do. We have to wait for science to catch up,' Mariah said. Every doctor that weve encountered has really been amazing and done their very best for us. Its just unfortunate science hasnt caught up to the girls, yet. Thats kind of, whereas parents, were passionate enough to move science along a little faster.

The nonprofit organization is supported by a GoFundMePage, and tax-deductible donations can be made through the Lightning and Love website.

The latest research funded by the foundation involved genetically engineering a zebrafish model to see if it showed symptoms of disease, specifically seizures. The zebrafish did have seizures, which Mariah said was a major breakthrough toward the ultimate goal of finding a gene replacement cure for her daughters.

But the journey for Mark and Mariah has proven to be more than just research and discovery.

What were realizing is the more we talk about it, and the more we do to get our story out there, the more were realizing that theres a lot of other parents that are going through tough times with their kids, too, Mark said. In an odd twist, or an ironic twist, this tough hand that weve been dealt has actually been a very positive light to a lot of other people out there. For me, that is just as important as the research.

The familys story was recently featured on the podcast, Go Shout Love.

The couples positive message is guiding them along the road they hope will lead to a better life for their family. But in the meantime, Mark and Mariah will continue to put smiles ontheir daughters faces the same way most other parents do by offering their love, support and opportunities to find happiness.

For Emma, that means being tossed into the air and caught by her daddy, and for Abby, it is time in her sensory room and being around her dad and her family.

Emma loves very big movements. Shes not mobile, and she cant walk, so when we kind of throw her around in the air or fly her around the room, she absolutely loves it, Mariah said. Abby loves her daddy. She gives big old smiles when he walks into the room.

To reach John F. Russell, call 970-871-4209, email or follow him on Twitter @Framp1966.

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Family seeks answers, finds hope after daughters diagnosed with rare genetic condition - Steamboat Pilot and Today


COVID-19 Vaccine and Therapeutics Pipeline Analysis Report 2020: The Race to Market as Clinical Trials Move Up a Gear – -…

Wednesday, September 30th, 2020

DUBLIN--(BUSINESS WIRE)--The "COVID-19 Vaccine and Therapeutics Pipeline Analysis 2020" report has been added to's offering.

The report covers market characteristics, size and growth, segmentation, regional and country breakdowns, competitive landscape, market shares, trends and strategies for this market. It traces the market's historic and forecast market growth by geography. It places the market within the context of the wider COVID-19 vaccine & therapeutics pipeline analysis 2020 market, and compares it with other markets.

Major players in the COVID-19 vaccine and therapeutics pipeline analysis market are CanSino Biologics, Moderna, Inovio Pharmaceuticals, Regeneron, Gilead Sciences, GlaxoSmithKline, Medicago Inc., Sanofi, University of Oxford, and Altimmune.

The COVID-19 vaccine and therapeutics pipeline analysis market covered in this report is segmented by product type into small molecules, biologics, blood & plasma derivatives, monoclonal antibodies, vaccines, others. It is also segmented by the phase of development into preclinical therapeutics & vaccines, clinical studies, by treatment mechanism & route of administration, and by type of sponsor into pharma/biotech company, academic research/institution, others.

The COVID-19 vaccine and therapeutics pipeline analysis market report provides an analysis of the coronavirus (COVID-19) therapeutics and vaccines under development. The report includes existing vaccines developed against MERS-CoV and SARS-CoV. The novel coronavirus-2019 (nCoV-19) has been named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) due to its genetic similarity with the coronavirus responsible for the 2003 SARS outbreak. Currently, government agencies, international health authorities and institutions and biopharmaceutical companies worldwide are focusing on developing vaccines/drugs to prevent or treat the COVID-19 infection.

Ever since the coronavirus hit the world as a global pandemic, many key vaccine developers are collaborating to develop potential COVID-19 vaccine against coronavirus.

Most recently, on 21st May 2020, CanSino Biologics Inc. and Precision NanoSystems announced a co-development agreement of an mRNA lipid nanoparticle (mRNA-LNP) vaccine against COVID-19. The parties will leverage Precision NanoSystems's proprietary RNA vaccine platform, comprising of lipid nanoparticle delivery system and the NanoAssemblr manufacturing technology, to rapidly advance a COVID-19 mRNA-LNP vaccine candidate towards human clinical testing and pursuant to regulatory approvals, and commercialization in different regions. Precision NanoSystems will be responsible for the development of the mRNA-LNP vaccine and CanSinoBIO will be responsible for pre-clinical testing, human clinical trials, regulatory approval and commercialization.

Similarly, on May 19, 2020, IPharmaJet, the maker of innovative, needle-free injection technology announced that its Needle-free Injection System technology will be used to deliver a messenger RNA (mRNA) vaccine against SARS-CoV-2. The vaccine is being developed by Abnova Corporation, the world's largest antibody manufacturer, based in Taiwan.

The development of potential drugs and vaccines for COVID-19 is progressing quickly. There is a massive increase in COVID-19 drugs and vaccines pipeline owing to the urgent need to contain the spread of disease. Government agencies, global health authorities and institutes, and biopharmaceutical companies are focusing on remedies to treat the patients and control the infection spread. Increasing every day, 450+ potential therapeutic candidates are under investigation. While two-thirds of the pipeline account for therapeutic drugs, the remaining one-third accounts for vaccines.

Of the confirmed active vaccine candidates, nearly 70% are being developed by private/industry developers, with the remaining 30% of projects being led by the academic, public sector and other non-profit organizations. Most COVID-19 vaccine development activity is in North America, with around 36 (46%) developers of the confirmed active vaccine candidates. China constitutes 18% with 14 developers, while, Asia excluding China and Europe also constitute 18% each with 14 developers in each region, respectively.

The long and costly drug development process is anticipated to limit the growth of the COVID-19 vaccine & therapeutics. According to the Pharmaceutical Research and Manufacturers of America (PhRMA), the average cost of research and development of a new drug is approximately $2.6 billion. Moreover, the stringent regulations imposed by the various regulatory authorities such as European Medicines Agency and the US Food and Drug Administration (FDA) in regards with clinical trials during the COVID-19 outbreak attributing to the safety of trial participants, maintaining compliance with good clinical practice, and minimizing risks to trial integrity is a major challenge faced by the COVID-19 vaccine and therapeutics market.

The compounds and medications that are under investigation can be grouped into three broad categories - antivirals, immune-system based, and vaccines. The anti-virals including Darunavir, Favipiravir, Hydroxychloroquine and chloroquine, Lopinavir, and Remdesivir (GS-5734), immune system-related therapies including Tocilizumab, Tocilizumab, and Vitamin C, and other medications are currently being evaluated as therapies. Three key drugs are currently in phase III, of which are two small molecule-based drugs, Remdesivir by Gilead Sciences Inc. and Favipiravir by Fujifilm Toyama Chemical Co Ltd, and Sarilumab, a monoclonal antibody by Regeneron Pharmaceutical. With regards to the prophylactic vaccine pipeline, more than 90% are in early-stage development (discovery and preclinical), and only three in Phase II. These three COVID-19 vaccines are being developed by Sinovac Biotech Ltd, the University of Oxford, and the third vaccine, named CIGB-2020, is being developed by the Center for Genetic Engineering and Biotechnology.

According to the European Centre for Disease Prevention and Control, worldwide, there are over 10.8 million cases of COVID-19. Globally, R&D spending has increased to find a potential drug or vaccine to combat this pandemic. Currently, there is no approved targeted therapy for patients with COVID-19. However, an array of drugs approved for other indications as well as several new investigational drugs are being studied in several hundred clinical trials. The increased R&D spending has contributed to the invention/discovery of more than 400 unique drugs to treat COVID-19 among which 298 are therapeutic drugs and 140 prophylactic vaccines that are spread across all stages of development (Discovery, Preclinical, Phase I, Phase II, and Phase III). As of June 2020, over 2,341 clinical trials are investigating potential therapies for COVID-19, of which nearly 800 are interventional trials.

Other Collaborations:

Key Topics Covered:

1. Executive Summary

2. Disease Overview

2.1. Novel Coronavirus Etiology and Pathogenesis

2.2. Novel Human Coronavirus (ClOVID-19) Clinical Features-Signs and Symptoms

3. Disease Epidemiology and Epidemic Statistics for Major countries

4. Global Pipeline Analysis of COVID-19 Therapeutics and Vaccines

4.1 Global Pipeline Analysis, By Product Type

4.1.1 Small Molecules

4.1.2 Biologics Blood & Plasma Derivatives Monoclonal Antibodies Vaccines Others

4.2 Global Pipeline Analysis, By Phase of development

4.2.1 Preclinical Therapeutics & Vaccines

4.2.2 Clinical Studies Clinical Phase I, II, III

4.3 Global Pipeline Analysis, By Treatment Mechanism & Route of Administration

4.3.1 Mechanism of Action Viral Replication Inhibitors Protease Inhibitors Immunostimulants Other Mechanism of Action

4.3.2 Route of Administration Oral Intravenous Subcutaneous Other Route of Administration

4.4 Global Pipeline Analysis, By Type of Sponsor

4.4.1 Pharma/Biotech Company

4.4.2 Academic Research/Institution

4.4.3 Others such as Government Organizations and CROs

5. Competitive Landscape for Late Stage Therapeutics and Vaccine

5.1 Company Overview

5.2 Product Description

5.3 Research and Development

5.3.1 Non-Clinical Studies

5.3.2 Clinical Studies

5.3.3 Highest/Late Stage Development Activities

5.4 Licensing and Collaboration Agreements

5.5 Milestones & Future Plans

6. Regulatory Framework for COVID-19 Therapeutics and Vaccines Marketing Approvals

6.1 Regulatory Framework in the USA

6.2 Regulatory Framework in EU and Other Countries

7. Recommendations & Conclusion

Companies Mentioned

For more information about this report visit

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COVID-19 Vaccine and Therapeutics Pipeline Analysis Report 2020: The Race to Market as Clinical Trials Move Up a Gear - -...


Thermofluidic heat exchangers for actuation of transcription in artificial tissues – Science Advances

Wednesday, September 30th, 2020


Cells transform noisy environmental signals into spatial and dynamic gene expression patterns that guide biological form and function. Information describing how these transcriptional networks are patterned is exploding because of revolutions in single-cell RNA sequencing and spatial transcriptomics. Recapitulating this spatiotemporal information transfer in three-dimensional (3D) tissue settings remains a pivotal yet elusive goal of diverse fields, such as tissue engineering (1), synthetic biology (2, 3), and developmental biology (4, 5).

To control gene expression, biologists have developed diverse technologies to rewire cells at the genetic level, such as gene knockout, inhibition, overexpression, and editing (68). To further enable spatial and dynamic control of gene expression, several of these tools have been adapted to be triggered by exogenous stimuli such as light (e.g., optogenetic transcriptional control) (9, 10). Light-based actuation of gene expression patterning has been especially useful in 2D culture or optically transparent settings. However, the inherently poor penetration of light in densely populated tissues (11), long exposure times needed to activate molecular switches, and corresponding challenges in patterning light delivery have limited widespread adoption of light-based patterning of gene expression in 3D settings (12).

We hypothesized that we could overcome these challenges by exploiting more penetrant forms of energy to drive gene patterning. In particular, mild heating is an attractive option for 3D patterning across length scales, as heat can be targeted locally and penetrate tissues at depth. Furthermore, heat can diffuse through tissues to establish thermal gradients in predictable and controllable patterns that are dictated by established rules of heat transfer (13). Last, advances in molecular engineering have led to proliferation of thermal molecular bioswitches to regulate gene expression (14, 15), with mammalian systems activating in the mild hyperthermia range (~38 to 45C).

Heat transfer has a long industrial history, as heat is often added, removed, or moved between processes using heat exchangers, which transfer heat between fluidic networks. Recently, heat exchanger fabrication has undergone a radical shift due to developments in advanced manufacturing (e.g., 3D printing). Predating its history in industry, biological organisms have also long used heat exchanger design principles for thermoregulation. We reasoned that instead of building heat exchangers from hard materials, developing methods to build heat exchangers in materials compatible with living cells could facilitate volumetric heat patterning in artificial tissues.

We introduce a thermofluidic method for mesoscale spatiotemporal control of gene expression in artificial tissues that exploits volumetric fluid-based heat transfer, which we call heat exchangers for actuation of transcription (HEAT; Fig. 1A). HEAT leverages our open-source projection stereolithography bioprinting technology (16) to fabricate topologically complex fluidic channels of user-defined geometries in hydrogels (Fig. 1B, top and middle). 3D printed hydrogels are laden with genetically engineered heat-inducible cells during the printing process (Fig. 1A). Encased channel networks are perfused with precisely heated fluid from a power-supplied heating element. During perfusion, tissue temperature is monitored in real-time using an infrared camera (Fig. 1A). We find that thermofluidic perfusion facilitates heat transfer from the channels into the bulk hydrogel and enables architectural heat patterning in hydrogels (Fig. 1B, bottom).

(A) Schematic of thermofluidic workflow. A biocompatible fluid flows around a power supplied heating element to preheat the fluid before entry in perfusable channel networks within hydrogel tissue constructs laden with heat-sensitive cells. During perfusive heating, hydrogel temperature is continuously monitored using an infrared camera. (B) Perfusable channel networks of varying spatial geometries can be bioprinted within biocompatible 3D hydrogels. Top: 3D rendering of network architectures. Middle: Hydrogel channels infused with tonic water fluoresce when imaged under ultraviolet backlight. Bottom: Infrared thermography of heat-perfused hydrogels demonstrates that during perfusion, heat traces the path of fluid flow and dissipates into the bulk hydrogel. Scale bars, 5 mm.

Most mammalian thermally inducible gene switches require exposure to mild hyperthermia (39 to 45C) for prolonged periods of ~15 to 60 min to activate transcription (15, 17). We therefore tested whether this approach could precisely regulate tissue temperature over prolonged periods of time by maintaining steady-state thermal profiles in perfused hydrogels. To do this, we first printed hydrogels that contained a single channel (Fig. 2A). We then perfused precisely heated fluid through this channel while tracking hydrogel temperature in real-time using infrared thermography (Fig. 2B). Upon initiating perfusion, we observed that hydrogel temperature underwent an initial ramp-up phase (~5 min) followed by a steady-state plateau in which temperature deviated by <0.4C/min at three separate regions measured across the hydrogel (Fig. 2B, right).

(A) Photograph of a single-channel bioprinted hydrogel used for initial thermal characterization. Scale bar, 5 mm. (B) Representative infrared images from controlled perfusion of heated fluid through the channel over time (left). Scale bars, 5 mm. (C) Representative finite-element modeling images depicting steady-state predictions on the surface of perfused hydrogels at varying flow rates and constant heater power (left; full dataset in fig. S1B). Computational modeling predicts that flow rate can achieve maximal hydrogel temperatures in the mild hyperthermia temperature range (right, gray shading denotes mild hyperthermia range). (D) Hydrogels were experimentally perfused at flow rates of 0.5 and 1.0 ml min1 and imaged using infrared thermography. Scale bars, 5 mm. (E) Hydrogel temperature plotted orthogonal (x) to the flow direction at inlet and outlet positions show agreement between thermal gradients in computational and experimental measurements (computational, dashed lines; experimental, solid lines). (F) Hydrogel temperature plotted parallel (y) to flow direction demonstrates a larger temperature drop from inlet to outlet (y) during flow at 0.5 ml min1 (T0.5) compared to flow at 1.0 ml min1 (T1.0) in computational and experimental models (computational, dashed lines; experimental, solid lines; n = 5, data are mean temperature standard error, **P < 0.01 by Students t test). Photo credit: Daniel Corbett, University of Washington.

During perfusion, heat is transferred from fluidic channels to the bulk through convection and conduction, resulting in thermal gradients throughout the bulk volume (18). The perfusate input temperature is known to govern the rate and magnitude of heat transfer, while fluid flow rate influences the thermal profile (18). To determine the relative effects of perfusate temperature and flow rate on hydrogel heating at biologically relevant temperatures, we sought to develop a finite element model of heated hydrogel perfusion for mild hyperthermia that incorporated thermal and flow parameters from our heating system. To derive these parameters, we first incrementally increased flow rate over a range of heating element powers and measured fluid temperature at the point of heater outflow (i.e., hydrogel inlet; fig. S1). We then implemented perfusate temperature values observed from each flow rate at 13.5-W heater power into a computational model of single-channel hydrogel heating (Fig. 2C and fig. S1B). Computational simulations predicted that hydrogel temperatures in the range for mild hyperthermia were achievable using flow rates from 0.4 to 1.6 ml min1, but not for slower or faster flow rates (Fig. 2C and fig. S1B). Within this window, we observed that flow rates of 0.5 and 1.0 ml min1 produced subtle differences in the shape of thermal profiles, despite roughly equivalent input temperatures (Fig. 2C and fig. S1B). Thus, these flow rates provided a set of conditions to further examine the effects of flow rate on heat transfer.

We therefore performed experimental validation studies of perfused single-channel hydrogels at 0.5 or 1.0 ml min1 and analyzed the steady-state thermal profiles from infrared images (Fig. 2D). Experimental temperature measurements (solid lines) and computational simulation predictions (dashed lines) showed agreement when measured both orthogonal (Fig. 2E) and parallel (Fig. 2F) to channel flow. Both physical measurements and simulations demonstrated thermal gradients in the hydrogel. Temperature along the channel was better maintained under flow at 1.0 ml min1 compared to flow at 0.5 ml min1 (**P < 0.01; Fig. 2, E and F), and flow at 0.5 ml min1 promoted more heat transfer at the channel inlet (fig. S2A). Addition of cells to single-channel hydrogels did not affect temperature profile after thermofluidic perfusion (fig. S2B) nor did differences in hydrogel weight percent in ranges commonly used for 3D printing of cellularized hydrogels [i.e., 10 to 20 weight % (wt %); fig. S2C] (16). Stiffer hydrogel formulations (i.e., 25 wt %) did exhibit different temperatures at the hydrogel edge, although these formulations are less commonly used for bioprinting due to their limited support of cell viability (16).

These findings led us to further computationally explore the potential spatial design space for a single-channel system. To do this, we assessed how varying channel length and ambient temperature affect the thermal profile in our model. Predictions showed that single channels up to 30 mm long achieved hyperthermic temperatures (40 to 45C) along their entire length, with outlet temperatures falling out of the hyperthermic range at greater lengths (fig. S3A). Spatial heat distribution was only marginally affected within the ambient temperature range used in our studies here (20 to 22C; fig. S3B), but more substantive increases in ambient temperature (e.g., to 30, 37C) produced wider spatial gradients in hyperthermic range (fig. S3B). Together, these studies showed that the rules of heat transfer could be leveraged to predict thermal spatial profiles in perfused hydrogels and that these profiles could be finely tuned by varying parameters such as flow rate, channel length, and input and ambient temperature.

We next aimed to genetically engineer heat-inducible cells that activate gene expression upon exposure to mild hyperthermia. To do this, we implemented a temperature-responsive gene switch-based on the human heat shock protein 6A (HSPA6) promoter, which exhibits a low level of basal activity and a high degree of up-regulation in response to mild heating (19). This promoter activates heat-regulated transcription through consensus pentanucleotide sequences (5-NGAAN-3) called heat shock elements, which are binding sites for heat shock transcription factors (19). We transduced human embryonic kidney (HEK) 293T cells with a lentiviral construct in which a 476base pair (bp) region of the HSPA6 promoter containing eight canonical heat shock elements was placed upstream of a firefly luciferase (fLuc) reporter gene (Fig. 3A). Initial characterization of temperature-sensitive promoter activity in engineered cells in 2D tissue culture demonstrated a temperature-dose dependent up-regulation of luciferase activity in the range of mild hyperthermia (fig. S4A). Statistically significant up-regulation was observed in heated cells compared to nonheated controls after hyperthermia for 30 min at 45C or 60 min from 43 to 45C, while peak bioluminescence occurred after 60 min at 44C (292 26-fold increase in bioluminescence relative to 37C controls). Bioluminescent signal was first detected 8 hours after heat shock, peaked at 16 hours (110 30-fold increase), and fell back to baseline by 2 days (fig. S4B). Administration of a second heat shock stimulus 3 days later reinduced bioluminescent signal (fig. S4C). Thus, gene activation with this promoter system is transient but can be reactivated with pulsing.

(A) HEK293T cells were engineered to express fLuc under the HSPA6 promoter. (B) Schematic of thermofluidic activation of encapsulated cells. (C) Single-channel tissue used for 3D heat activation (left). Scale bar, 3 mm. Transmittance image of cellularized hydrogel after printing (middle). Scale bar, 500 m. HEK293T cells in bioprinted tissues stained with calcein-AM (live, green) and ethidium homodimer (dead, red; right). Scale bars, 200 m. (D) Representative infrared images of thermofluidic perfusion in single-channel hydrogels. Scale bars, 2 mm. (E) Hydrogel temperatures are tuned by changing heater power at constant flow rate (n = 3, mean temperature standard error). (F) Representative bioluminescence images of hydrogels (top; scale bars, 2 mm) and intensity traces at three positions (A to C) across the width (x) of the hydrogel after 30 min of perfused heating. (G) Fold change in bioluminescence after 30 min of heating relative to 25C controls. (H) Representative bioluminescence images of hydrogels (top; scale bars, 2 mm) and intensity traces after 60 min of perfused heating (bottom; scale bars, 2 mm). (I) Fold change in bioluminescence after 60 min of heating demonstrates a temperature-dependent dosage response in gene expression [(G and I); n = 3, mean fold luminescence standard error; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Dunnetts multiple comparison test]. (J) Temperature-expression response curve (black) shows mean bioluminescent radiance across temperature; shaded regions (gray) indicate SD. n = 3. Photo credit: Daniel Corbett, University of Washington.

We observed that our highest heat exposure (45C for 60 min) led to a tradeoff between bioluminescence and cell integrity, as indicated by reduced cell metabolic activity and substrate detachment (fig. S5A). These findings suggested that fine control of heat would be needed for thermofluidics to be useful in cellularized applications. We therefore rigorously characterized the effect of heating on HEK293T cells embedded in the hydrogel formulation used for our thermofluidic studies. Similar to 2D studies, cell viability fell significantly only after exposure to our highest temperature, 45C (fig. S5B). Together, these studies demonstrate engineering of human cells with a heat-sensitive gene switch and identification of a tight window of thermal exposure parameters that both differentially up-regulate gene bioluminescence and maintain cell integrity.

We sought to determine whether thermofluidic heating could be used to induce gene expression in heat-inducible cells encased within 3D artificial tissues (Fig. 3B). To do this, we encapsulated heat-inducible cells in the bulk of bioprinted constructs that contained a single perfusable channel (Fig. 3, B and C). Since tissue constructs were printed from biocompatible materials without ultraviolet light cross-linking, most cells remained viable upon encapsulation, similar to our previous studies (16) (Fig. 3C). To determine whether our heat-inducible cells could be activated using thermofluidics, we perfused channels at 0.5 ml min1 using thermal exposure parameters identified in 2D culture (Fig. 3, D and E). Similar to 2D, we observed that thermal dose-dependent luciferase up-regulation (Fig. 3, F to J) was statistically significant after 30 min of heating to a target hydrogel temperature of 44C or after 60 min of heating to temperatures of 43 and 44C by whole-gel bioluminescent output (71 22-fold and 169 44-fold increase relative to controls, respectively; Fig. 3, H and I). To more finely characterize how bioluminescent intensity correlates with temperature, infrared and bioluminescence images were overlaid to map individual pixels and generate temperature-bioluminescence response curves. The shape of temperature-response curves appeared similar in shape across various target temperatures (Fig. 3J, all data overlaid; fig. S6, individual response curves). Similar to whole-gel analyses, greater target temperatures generated the most robust activation (Fig. 3J and fig. S6). In initial studies, we noted that leakage at the hydrogel inlet or outlet could activate cells. Subsequent improvements to fluidic connectivity with a custom-printed perfusion apparatus led to higher precision thermal patterning (fig. S7; see link to open source perfusion apparatus design in Methods). Last, multiperspective imaging and bioluminescence quantification of single-channel perfused hydrogels from both top-down and cross-sectional perspectives demonstrated that reporter gene activation had a 3D radial gradient topology around each channel (fig. S8). Together, these results illustrate that thermofluidics can be used to activate varying levels of gene expression in 3D artificial tissues.

Spatial patterns of gene expression within native tissues vary widely in magnitude, scale, and spatial complexity. While we achieved variation in magnitude in our signal-channel studies, the expression profile geometry across the hydrogel remained similar at various perfusion temperatures. This raised the question of how to design heat delivery schemes that enable more spatially complex expression patterns across the hydrogel. Our thermal characterization (Fig. 2) revealed flow rate as one parameter that we could use, but changing flow rate alone imparted only subtle differences to the spatial thermal profile (Fig. 2, D to F). To identify a more perturbative and user-defined means of affecting heat distribution across the hydrogel, we turned to industrial heat transfer applications, in which heat exchangers are optimized to transfer heat between fluids by controlling parameters such as channel placement and flow pattern.

We mimicked a double pipe heat exchanger design within cellularized hydrogels by printing two channels at varying distances from one another (Fig. 4A, narrow versus wide). We then perfused hydrogels under different conditions for flow direction (concurrent versus countercurrent) and fluid temperature [hot (44C) versus cold (25C)]. Similar to our single-channel characterization, double-channel tissues showed close matching between thermal and bioluminescence profiles (Fig. 4A). Concurrent flow in narrow spaced channels created elongated spatial plateaus of heat and bioluminescence between the channels. Conversely, widely spaced hot channels generated mirror-imaged thermal and bioluminescent profiles, with distinct spatial separation between channels. Countercurrent flow patterns generated parallelogrammic thermal and bioluminescent profiles in both channel spacings. Substituting a hot channel for a cold channel attenuated bioluminescence in a manner that depended on channel spacing (Fig. 4A). Computational models of a similar bifurcating channel geometry further demonstrated how simple changes to parameters such as channel spacing can alter spatial thermal profile (fig. S9).

(A) Heat exchanger inspired designs for various flow directions, fluid temperatures, and channel architectures (schematics; left and center). Representative thermal (middle) and bioluminescent (right) images demonstrate spatial tunability of thermal and gene expression patterning. Scale bars, 5 mm. (B) Photographic image of four-armed clock-inspired hydrogel used for dynamic activation (top; channel filled with red dye). Each inlet is assigned to a local region (A to D). Schematic shows the spatial and dynamic heating pattern for the 4-day study (bottom). (C) Representative infrared (top) and bioluminescence expression (bottom) images for dynamic hydrogel activation at each day during the time course. (D) Quantification of local bioluminescent signals from regions of interest corresponding to each day of heating. Across all 4 days, regions corresponding to perfused arms had higher bioluminescent signals than nonperfused arms (n = 5, data are mean luminescence standard error; *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukeys post hoc test).

As biological gene expression patterns are transient and fluctuating, we next tested whether thermofluidics could dynamically localize regions of gene expression over time. To do this, we printed clock-inspired constructs, in which four separate inlets converged on a circular channel (Fig. 4B, top). We then perfused heated fluid through each inlet over four consecutive days (Fig. 4B, bottom) and imaged tissues for bioluminescence. Bioluminescent images demonstrated statistically significant luciferase up-regulation for regions surrounding heated inlets compared to nonheated inlet regions on all 4 days (Fig. 4, C and D.) Together, our results illustrate that by exploiting heat transfer design principles, thermofluidics enables user-defined spatial and dynamic patterning of mesoscale gene expression patterns in 3D artificial tissues.

To test whether gene patterning could be maintained after engraftment of artificial tissues in vivo, we stimulated tissues with HEAT and implanted these tissues into athymic mice. All tissues contained HEK293T cells expressing fLuc under the control of the heat-inducible HSPA6 promoter. All tissue constructs contained a single channel and were stimulated in one of three ways: (i) thermofluidic perfusion at 44C for 60 min, (ii) bulk heating in a cell culture incubator at 44C for 60 min, or (iii) bulk exposure in a cell culture incubator to 37C. Tissues were implanted into mice immediately after heating, and bioluminescence imaging was performed 24 hours later. We found that thermofluidic spatial control of gene expression was maintained after in vivo tissue engraftment (Fig. 5A and movie S1).

(A) Artificial tissues with embedded heat-inducible fLuc HEK293T cells received 44C thermofluidic heating (channel heat, n = 5), 44C global heating (bulk heat, n = 3), or remained at 37C (no heat, n = 3) for 1 hour before immediate implantation into athymic mice. (B) Bioluminescence from implanted hydrogels (dashed lines) showed region specific signal only in channel heated hydrogels. (C) Average line profiles (top) across the width (x) of the hydrogel for inlet, middle, and outlet positions show that only channel heated gels induced a spatially coordinated response that was statistically significant (bottom) between the center (position B) and edges of the hydrogel (position A and C; channel heat, n = 5; bulk heat, n = 3; no heat, n = 3; data are mean luminescence standard error; **P < 0.01, by one-way ANOVA.

We next sought to demonstrate the modularity of our system for spatially regulating expression of the Wnt/-catenin signaling pathway, which directs diverse aspects of embryonic development, tissue homeostasis, regeneration, and disease (20). We engineered heat-inducible constructs to drive expression of three genes in the Wnt/-catenin signaling pathway: (i) R-spondin-1 (RSPO1), a potent positive regulator of Wnt/-catenin signaling (21); (ii) -catenin, a critical transcriptional coregulator that translates to the nucleus upon canonical Wnt signaling (22); and (iii) Wnt-2, a ligand that binds to membrane-bound receptors to activate the Wnt/-catenin signaling pathway. The Wnt-2 gene was also tagged with V5 (23). We engineered lentiviral constructs in which RSPO1, -catenin, or Wnt2-V5 is driven by the heat-inducible HSPA6 promoter, and mCherry is driven by a constitutive promoter [spleen focus-forming virus (SFFV); Fig. 6A]. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of each engineered cell line for mCherry expression relative to GAPDH expression suggested lentiviral integration (fig. S10A). We then printed artificial tissues containing heat-inducible -catenin, RSPO1, or Wnt2 HEK293T cells and a single fluidic channel (Fig. 6B). Constructs were heated fluidically and then sliced into longitudinal zones (Fig. 6, A and B) to analyze expression of the Wnt family gene expression by RT-qPCR. Representative artificial tissues contained mCherry+ cells across the tissue (Fig. 6C). Immunostaining for the V5 tag fused to Wnt2 appeared higher near the heated channel compared to the gel periphery (Fig. 6C). RSPO1, -catenin, or Wnt2 expression was highest in the zone surrounding the heated channel (Fig. 6D). These results show that HEAT can be leveraged to activate expression of various family members of the Wnt/-catenin signaling pathway.

(A) Schematics of lentiviral constructs (left) and thermofluidic HEK293T tissue experiments (right). (B) Transmittance image of cellularized construct after printing (left; zones indicated by dashed lines). Infrared image of construct during heating (right). Scale bars, 1 mm. (C) mCherry+ HEK293T cells in printed tissues (left). Scale bars, 1 mm. Images of thermofluidically heated Wnt2 constructs after immunostaining for V5 tag (coexpressed with Wnt2; right; images taken near the tissues channel and periphery as indicated by insets). Scale bars, 200 m. (D) Wnt family genes were up-regulated in zone 3 of thermofluidically perfused gels compared to controls (n = 4, mean fold change standard error; *P < 0.05 and **P < 0.01 by two-way ANOVA followed by Tukeys multiple comparison test). (E) Differentiated HepaRG cells were engineered with a heat-inducible RSPO1 construct (schematic, top) and printed in single-channel hydrogels (photograph, left). Scale bars, 1 mm. After heating (infrared), HepaRGs remained viable in printed constructs (calcein). Scale bar, 200 m. (F) Thermofluidically heated RSPO-1 HepaRG hydrogels were dissected into zones 1 to 3 based on distance from the heat channel for RT-qPCR analysis at 1, 24, and 48 hours after heating. Expression fold change was normalized to no heat control samples. qPCR analysis of RSPO-1 across dissected zones (n = 5 to 10, data are mean fold change standard error; *P < 0.05 by one-way ANOVA followed by Tukeys multiple comparison test). (G) RT-qPCR analysis of pooled RNA across all zones at each time point for pericentral associated genes, glutamine synthetase, CYP1A2, CYP1A1, CYP2E1, and CYP3A4, and periportal/midzonal genes, Arg1 and E-cadherin (n = 15 to 30, data are mean fold change standard error, **P < 0.01 and *P < 0.05 by one-way ANOVA followed by Tukeys multiple comparison test). Photo credit: Daniel Corbett, University of Washington. n.s., not significant.

We reasoned that the ability to activate expression of Wnt/-catenin signaling pathway members could be useful for the emerging human organ-on-a-chip field by affecting functional cellular phenotypes in vitro. To test this, we turned to the liver, which performs hundreds of metabolic functions essential for life, including central roles in drug metabolism. To carry out these functions, hepatocytes divide the labor, with hepatocytes in different spatial locations performing different functions, a phenomenon called liver zonation. Recent studies have shown that liver zonation is regulated at the molecular level by Wnt/-catenin signaling (22), with higher Wnt activity associated with a pericentral vein phenotype and lower Wnt activity characteristic of a periportal phenotype. However, the extent to which different members of this pathway affect human zonated hepatic phenotypes remains unclear. A better understanding of this process would accelerate development of zonated human liver models for hepatotoxicity and drug metabolism studies.

We hypothesized that thermofluidic activation of RSPO1 in human hepatic cells would be sufficient to activate zonated hepatic gene expression profiles, as ectopic expression of RSPO1 in mouse liver has recently been shown to induce a pericentral zonation phenotype in vivo (24). To test this hypothesis, we transduced human HepaRG cells, an immortalized human hepatic cell line that retains characteristics of primary human hepatocytes, with our lentiviral construct in which HSPA6 drives RSPO1, and SFFV drives mCherry (Fig. 6E). Transduced human hepatic cells were then printed in artificial tissues with a single fluidic channel, to mimic central lobular placement of the central vein (Fig. 6E). Constructs were heated fluidically and then sliced into zones (Fig. 6A), and gene expression was measured by RT-qPCR (Fig. 6F). Fold up-regulation values were normalized to identically fabricated control artificial tissues maintained at 37C. We found that RSPO1 expression increased in a dose-dependent and spatially defined manner, with expression in zone 3 nearest the channel (central vein) 10-fold higher than in zone 1 by 1 hour after heating. RSPO1 expression was transient, falling with each day after heating, similar to our luciferase studies (Fig. 4C and fig. S5C). Thermofluidic activation of RSPO1 induced expression of key pericentral marker genes, including glutamine synthetase, an enzyme involved in nitrogen metabolism, and the cytochrome P450 (CYP) drug-metabolizing enzymes CYP1A2, CYP1A1, and CYP2E1 relative to control tissues that were not heated, although with varied timing and without spatial localization in this study (Fig. 6G and fig. S10). Expression of pericentral drug-metabolizing enzyme CYP3A4 was not induced with heating, consistent with other studies in which adding Wnt3a ligand to primary human hepatocyte cultures did not alter CYP3A4 expression (25). Periportal marker E-cadherin was not induced, but periportal/midzonal gene Arg1 increased at 48 hours, especially in the zone 2 midzonal region (fig. S10). Together, these studies contribute a fundamental understanding of how various liver zonation genes are induced by RSPO1 activation in human hepatic cells.

In this study, we demonstrate that thermal patterning via bioprinted fluidics can directly pattern gene expression in 3D artificial tissues. A key advantage of the HEAT method is that it leverages the recent explosion in accessible additive manufacturing tools (16, 26, 27) by using open-source bioprinting methods that are readily available to the broader community. Furthermore, the entire patterned network is stimulated nearly simultaneously (as opposed to sequentially by time-intensive rastering), and this parallel stimulation can be sustained for exposure times required to trigger gene expression. Together, the sheer rapidity and highly parallel nature of this process enable spatial and dynamic genetic patterning at length scales and depths not previously possible in 3D artificial tissues.

Most previous methods to elicit cellular signaling in artificial tissues have focused on tethering extracellular cues to hydrogels (28, 29). Innovations in stimuli-responsive or smart biomaterials enabled activation of these chemistries by exogenous physical stimuli, such as light, to control the spatial position and timing of extracellular cues (30, 31). Although useful, these material-focused methods are unlikely to provide complete control even in fully defined starting environments because cells rapidly remodel their microenvironments (32). Moreover, these technologies offer an imprecise means to control downstream transcription because many, often unknown, intermediary steps modify intracellular signal transduction before gene activation. Our thermofluidic approach provides a complementary new technology to these methods that target extracellular signals by facilitating spatiotemporal control at the intracellular genetic level.

While our studies here reveal the potential power of HEAT for gene patterning, the first-generation system presented here does have limitations in its ability to fully control heat transfer both spatially and temporally. In our studies here, we found that channels up to 30 mm long (but no longer) could achieve hyperthermic temperature ranges along the entire channel length. Furthermore, the effect of heat-mediated stimulation on gene expression was transient. These limits could be overcome through a variety of design modifications. For example, the hydrogel or perfusates thermal conductivity could be increased by materials engineering to extend patterning area or length, such as by cross-linking metal nanoparticles into the polymer backbone as has been done before for other applications (33). To achieve different activation temperatures or dynamics, further genetic engineering of the heat shock promoter or other heat-activatable gene switches could be used (14). Thus, we envision that our initial system here will establish an important foundation that leads to a new family of studies that will ultimately describe a far greater design space for thermofluidic patterning.

To fully realize the vision of precision-controlled 3D artificial tissues, a diverse toolkit of orthogonal physical delivery and molecular remote control agents will likely be needed (34, 35). Thermofluidics could be coupled with other tissue engineering strategies that program extracellular (3, 2931) or intracellular (10, 14) signal presentation, cell patterning (36), or tissue curvature (37). Thermofluidics could also be used orthogonally with other remote control agents, such as those leveraging small-molecule (12), ultrasound (38), radio wave (39), magnetic (40), or light-based activation (41). Coupled with rapid advances in gene editing (10), synthetic morphogenesis (2, 3), and stem cell technology (4, 5), thermofluidics could be useful for spatially and temporally activating genes across tissues to drive cell proliferation, fate, or assembly decisions. While we demonstrate utility for activating Wnt/-catenin signaling pathway genes here, this approach could be rapidly adapted to activate any gene of interest. In our studies, we demonstrate one application of this approach by driving human hepatic cells toward a more pericentral liver phenotype in 3D artificial tissues. In doing so, we gain fundamental insights into how activation of Wnt agonist RSPO1 regulates expression of various metabolic zonation genes. These findings have important implications for developing both organ-on-chip systems for pharmacology and hepatotoxicity, as well as artificial tissues for human therapy. By blurring the interface between the advanced fabrication and biological realms, thermofluidics creates a new avenue for bioactive tissues with applications in both basic and translational biomedicine.

Poly(ethylene glycol) diacrylate (PEGDA; 6000 Da) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were prepared as previously described (16, 42). Gelatin methacrylate (GelMA) was synthesized as previously described, with slight modifications (43). Methacrylic anhydride was added dropwise to gelatin dissolved in carbonate-bicarbonate buffer at 50C for 3 hours, followed by precipitation in ethanol. The precipitate was allowed to dry, dissolved in phosphate-buffered saline (PBS), frozen at 80C, and then lyophilized for up to 1 week. GelMA was stored at 20C until use. Tartrazine (Sigma-Aldrich T0388, St. Louis, MO, USA) was added to prepolymer solutions as a photoabsorber to increase print resolution as previously described (16). Prepolymer mixtures for all cellular studies contained 7.5 wt % 6 K PEGDA, 7.5 wt % GelMa with 17 mM LAP, and 1.591 mM tartrazine. For characterization of heat transfer with respect to gel density, the overall polymer weight percent was varied while holding the ratio of 6 K PEGDA to GelMA constant at 50:50 (for example, 20 wt % = 10 wt % 6 K PEGA + 10 wt % GelMa).

Hydrogels with perfusable channel networks were designed in an open-source 3D computer graphics software Blender 2.7 (Blender Foundation, Amsterdam, Netherlands) or in SolidWorks (Dassault Systemes SolidWorks Corp., Waltham, MA).

Our stereolithography apparatus for tissue engineering bioprinting system was used in this study (16). Briefly, the system contains three major components: (i) a Z-axis with stepper motor linear drive, (ii) an open-source RepRap Arduino Mega Board (UltiMachine, South Pittsburg, TN) microcontroller for Z-axis control of the build platform, and (iii) a projection system consisting of a DLP4500 Optical Engine with a 405-nm light-emitting diode output (Wintech, Carlsbad, CA) connected to a laptop for photomask projection and motor control. The projector is placed in front of the Z-axis, and a mirror is positioned at 45 to the projection light path to reflect projected images onto the build platform. A sequence of photomasks based on a 3D model is prepared using Creation Workshop software (, which also controls the Z-axis movement of the build platform. Printing is achieved by curing sequential model layers of the photosensitive prepolymer. All printing was conducted in a sterile tissue culture hood. For visualization of channel networks, we perfused open channels with ultraviolet fluorescent tonic water or India ink dyes (P. Martins, Oceanside, CA).

To control temperature distribution in perfused hydrogels, an in-line fluid heater was developed to prewarm perfusate solutions before infusion in hydrogel channel networks. The fluid heater consists of four components: (i) an adjustable dc Power Supply (Yescom USA Inc., City of Industry, CA), (ii) a cylindrical cartridge heater (Uxcell, Hong Kong), (iii) perfusate tubing (peroxide-cured silicone tubing, Cole Parmer, Vernon Hills, IL), and (iv) a syringe pump (Harvard Apparatus, Holliston, MA). To construct the in-line fluid heater, perfusate tubing was connected to the syringe pump for flow rate control, while the cartridge heater was connected to the power supply for heating control. Perfusate tubing was then wounded around the cylindrical cartridge heater, allowing for heat transfer from the heater into the flowing perfusate. The temperature of the fluid was then controlled by changing the flow rate or heater power. In all studies, we used PBS (Thermo Fisher Scientific, Hampton, NH) for the perfusate solution.

To establish a fluidic connection between the heating system and hydrogel channel networks, we used custom-designed 3D printed perfusion chips printed on a MakerGear M2 3D printer (MakerGear, Beachwood, OH) in consumer-grade poly(lactic acid) plastic filament. Perfusion chips were fabricated with (i) an open cavity to insert 3D bioprinted hydrogels and (ii) attachment ports for fluid-dispensing nozzles. The outflow of the fluid heater was fitted with a male luer hose barb (Cole Parmer) connected to a flexible tip, polypropylene nozzle (Nordson EFD, East Providence, RI) and inserted into 3D printed attachment ports. Hydrogels were then inserted to perfusion chips, and proper fluidic connections were ensured before beginning perfusion. Model files for 3D printed perfusion holders are provided in the open repository data of our previously published work (16).

Fluid temperature and heat distribution were measured in perfused hydrogels by infrared thermography. Images were acquired by an uncooled microbolometer-type infrared camera (FLIR A655sc, Wilsonville, OR) that detects a 7.5- to 14.0-m spectral response with a thermal sensitivity of <0.05C and analyzed for temperature values using the FLIR ResearchIR software (Wilsonville, OR).

We built finite element models of perfused hydrogels in COMSOL 4.4 software (COMSOL AB, Burlington, MA). Simulations were run under transient conditions using the Conjugate heat transfer module and 3D printed hydrogel and housing geometries to predict the temperature distribution. The model was based on (i) forced convective heat transfer from the perfusion channel to the hydrogel volume and (ii) conductive heat transfer within the hydrogel volume.

Equation for (i): Heat transfer in a fluidCTt+CuT=pT(pAt+upA)+:S+(kT)+

Where is the fluid density, T is the temperature, C is the heat capacity at constant pressure, u is the velocity field, is the thermal expansion coefficient, pA is the absolute pressure, is the viscous stress tensor, S is the strain rate tensor, k is the fluid thermal conductivity, and Q is the heat content.

Equation for (ii)CpTt=(kT)+Q

Where is the hydrogel density, T is the temperature, k is the hydrogel thermal conductivity, and Q is the heat content.

Material properties of both the hydrogel and perfusate were modeled as water. Heat flux boundary conditions were included to model heat loss to the ambient environment, heat transfer coefficients of 5 and 30 W/(m2 * K) were applied to the sides and upper boundaries of the hydrogel, respectively, with an infinite temperature condition of 22.0C applied for all boundaries. Boundary temperature and fluid inflow conditions at the channel inlet were used to simulate the effect of changing perfusate temperature and flow rate, respectively. Model geometry was manipulated for studies on channel length and channel branching. Prescribed external temperature was varied for ambient temperature studies.

HEK293T cells were maintained in Dulbeccos modified Eagles medium (DMEM; Corning, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco) and 1% (v/v) penicillin-streptomycin (GE Healthcare Life Sciences, WA, USA). Differentiated HepaRG cells (Fisher Scientific) were maintained at confluence in six-well plates at a density of 2 106 cells per well in Williams E media (Lonza, MD, USA) supplemented with 5 HepaRG Thaw, Plate & General Purpose Medium Supplement (Fisher), and 1% (v/v) Glutamax (Fisher).

A vector containing a 476-bp version of the human HSPA6 promoter driving expression of fLuc reporter gene (gift of R. Schez Shouval from the Weizmann Institute of Science) was packaged into lentivirus using helper plasmids pMDLg/pRRE (Addgene no. 12251), pMD2.G (Addgene no. 12259), and pRSV-Rev (Addgene no. 12253) by cotransfection into HEK293T cells. Crude viral particles were harvested after 48 hours of transfection. For viral transduction, crude lentivirus was diluted 1:20 in DMEM containing polybrene (6 g/ml; Invitrogen), added to competent HEK293T cells in six-well tissue culture plates, and incubated overnight (Corning). The next day, virus-containing media was removed and replaced with fresh DMEM containing 10% FBS. After transduction, cells were heat-activated (see below) and flow-sorted to obtain a pure cell population.

To activate transgene expression under the HSPA6 promoter, engineered HEK293T cells were exposed to varying levels of hyperthermia in 2D and 3D. For 2D heat treatment studies, cells were seeded at 8 104 cells/cm2 in tissue culture plates 1 day before heat treatment. The next day, tissue culture plates were exposed to indicated heat treatments in thermostatically controlled cell culture incubators. Temperature was verified with a secondary method by a thermocouple placed inside the incubator. Upon completion of heat treatment, cells were returned to a 37C environment and sorted or analyzed at later time points. For the luminescent transient studies in fig. S4B, cells were lysed in TE buffer [100 mM tris and 4 mM EDTA (pH 7.5)] and stored at 4C until imaging. For the pulsed activation studies in fig. S4C, cells received two heat shocks as described previously at days 0 and 3. Luminescence was quantified across days 1 to 4 and normalized to cell counts from tissue culture plates that were processed in parallel according to each experimental temperature. For 3D heat shock studies, cells were encapsulated and printed in 3D perfusable hydrogels (see below) 1 day before heating. 3D hydrogels were then heat-perfused in a room temperature environment. Hydrogel temperature was monitored continuously with the infrared camera, and small adjustments to heater power were made as necessary to maintain a stable temperature profile. During perfused heating, outlet medium was continuously discarded. Upon completion of perfused heating, hydrogels were dismounted from the perfusion chips and returned to a cell culture incubator.

Cultured HEK293T cells were detached from tissue culture plates with 0.25% trypsin solution (Corning), counted, centrifuged at 1000 rpm for 5 min, and resuspended in liquid prepolymer (7.5 wt % 6 K PEGDA, 7.5 wt % GelMA, 17 mM LAP, and 1.591 mM tartrazine). For characterization of heat transfer with respect to cell density, cells were encapsulated in prepolymer mixtures at final densities from 0 to 24 106 cells ml1 before printing. For HEK293T expression studies, cells were encapsulated at a final density of 6 106 cells ml1. For HepaRG studies, cells were encapsulated at a final density of 2.5 106 cells ml1. Printing was performed as previously described under DLP light intensities ranging from 17 to 24.5 mW cm2, with bottom layer exposure times from 30 to 35 s and remaining layer exposure times from 12 to 17.5 s. Upon print completion, fabricated hydrogels were removed from the platform with a sterile razor blade and allowed to swell in cell culture media. Hydrogels were changed to fresh media 15 min after swelling and allowed to incubate overnight. Media was replaced the following morning. We tested the viability of both HEK293T and HepaRG cells following 3D printing by incubating cell-laden hydrogels with Live/Dead viability/cytotoxicity kit reagents (Life Technologies, Carlsbad, CA) according to manufacturers instructions. Fluorescence imaging was performed on a Nikon Eclipse Ti inverted epifluorescent microscope, and images were quantified using ImageJs built-in particle analyzer tool [National Institutes of Health, Bethesda, Maryland].

To visualize the magnitude and spatial localization of heat-induced luciferase expression, bioluminescence imaging was performed on heated cells and hydrogels using the in vivo imaging system (IVIS) Spectrum imaging system (PerkinElmer, Waltham, MA). Immediately before bioluminescence imaging, cell culture media was changed to media containing d-luciferin (0.15 mg/ml; PerkinElmer), and images were taken every 2 min until a bioluminescent maximum was reached. Images were analyzed using Living Image software (PerkinElmer). Luminescent imaging was performed from a top-down view (perspective orthogonal to hydrogel channel axis) for most studies. For cross-sectional images in fig. S8, hydrogels were manually sliced, incubated in luciferin containing media, and imaged under cross-section view (perspective parallel to hydrogel channel axis).

Data for the expression versus temperature plot was obtained by aligning thermal and bioluminescent images using MATLAB. To align the images, four reference points corresponding to the corners of the hydrogel were manually selected on both thermal and bioluminescence images. Then, an orthogonal transformation was performed on each image to align the corners of the hydrogel, after which the areas outside the selection were cropped. Pixel values from each image were then plotted against each other to produce the expression versus temperature plot.

Heat-inducible cells were generated as previously described and embedded into 3D-printed artificial tissues with single channels before being placed at 37C overnight. The next day, artificial tissues received either thermofluidic heat stimulation via flow of 44C biocompatible fluid at 1.0 ml min1 for 60 min (n = 5), global heat stimulus by being placed in a 44C tissue culture incubator for 60 min (n = 3), or were maintained in a 37C tissue culture incubator (n = 3). The artificial tissues were then immediately implanted subcutaneously on the ventral side of female NCr nude mice aged 8 to 12 weeks old (Taconic). Twenty-four hours after implantation, mice were anesthetized and injected with luciferin (15 mg/ml; PerkinElmer, Waltham, MA). Bioluminescence was then recorded via the IVIS Spectrum Imaging System (PerkinElmer). For 3D images, a custom 3D imaging unit developed by A. D. Klose and N. Paragas (44) (InVivo Analytics, New York, NY) was used. Briefly, anesthetized mice were placed into body-fitting animal shuttles and secured into the custom 3D imaging unit that uses a mirror gantry for multiview bioluminescent imaging. Collected images were then compiled and overlaid onto a standard mouse skeleton for perspective.

Line profiles in the x-direction across the inlet, middle, and outlet of 2D IVIS projection images from artificial gels were generated using Living Systems software (PerkinElmer, Waltham, MA). The three line profiles (inlet, middle, and outlet) from each artificial tissue were then averaged together with the average line profiles from the other artificial gels within each respective group (experimental group, n = 5; positive control group, n = 3; negative control group, n = 3). The average line profile of each group was then plotted, and average radiance values from positions 0.75 cm from the center of the channel (denoted positions A and C) were then statistically compared to the average radiance value at the center of the channel (position B) within each group by one-way analysis of variance (ANOVA).

Lentiviral constructs in which the HSPA6 promoter drives a Wnt family gene were subcloned using Gibson assembly by the UW BioFab facility. Human -catenin pcDNA3 was a gift from E. Fearon (Addgene plasmid no. 16828;; RRID: Addgene_16828) (45). Active Wnt2-V5 was a gift from X. He (Addgene plasmid no. 43809;; RRID:Addgene_43809) (46). RSPO1 was subcloned using a complementary DNA (cDNA) clone plasmid. (Sino Biological, Beijing, China). All plasmids contained a downstream cassette in which a constitutive promoter (SFFV) drives the reporter gene mCherry (gift from G. A. Kwong, Georgia Institute of Technology). Lentivirus was generated by cotransfection of HEK293Ts with HSPA6Wnt transfer plasmids with third-generation packaging plasmids (pMDLg/pRRE, pMD2.G, pRSV-REV) in DMEM supplemented with 0.3% Xtreme Gene Mix (Sigma-Aldrich). Crude virus was harvested starting the day after initial transfection for four consecutive days. For viral transduction, HEK293Ts at 70% confluency and HepaRGs at 100% confluency were treated with crude virus containing polybrene (8 g/ml; Sigma-Aldrich) for 24 hours. Five days following viral transduction, mCherry+ HEK293Ts were sorted from the bulk population by flow cytometry at the UW Flow analysis facility. HepaRGs were not sorted by flow cytometry. mCherry expression in positive HEK293T cell populations was performed using RT-qPCR.

To quantify Wnt regulator levels in HEAT-treated gels, HEK293Ts and HepaRGs for a given construct were encapsulated and heated in 3D hydrogels as previously described. No heat control samples remained at 37C in tissue culture incubators until RNA isolation. One to 48 hours following heat treatment, hydrogels were manually sliced into corresponding zones (1 to 3) and RNA was isolated using phenol-chloroform extraction (47). cDNA was synthesized using the Superscript III First-Strand Synthesis Kit (Thermo Fisher Scientific), and qPCR was performed using iTaq Universal SYBR Green Supermix (Biorad, Hercules, CA) on the 7900HT Real Time PCR System (Applied Biosystems, Waltham, MA). Primers for Wnt and housekeeping genes were designed and synthesized by Integrated DNA Technologies (Coraville, IA). Relative gene expression was normalized against the housekeeping gene 18S RNA calculated using the Ct method. Data are presented as the mean relative expression SEM. Data for HEK293T studies were normalized to relative expression of the Wnt target in 2D culture at 37C. Data for HEK293T mCherry expression were normalized to 18 s RNA and compared to GAPDH (also normalized to 18S RNA) expression levels. Data for HepaRG studies were normalized by relative expression of the Wnt target or pericentral/periportal gene marker to no heat control samples.

HSPA6Wnt2/V5 gels were fixed in 4% paraformaldehyde 24 hours postheating. For staining, samples are blocked overnight at room temperature in 1% bovine serum albumin, 1% normal donkey serum, 0.1 M tris, and 0.3% Triton X-100 with agitation. After blocking, samples are incubated in Anti-V5 tag antibody (Abcam, ab27671) diluted 1:100 in fresh blocking buffer and 5% dimethyl sulfoxide for 24 hours at 37C and agitation. Samples are washed and then incubated in secondary antibody diluted 1:500 in fresh blocking buffer and 5% dimethyl sulfoxide overnight at 37C and agitation. After incubation, samples are washed in PBS + 0.2% Triton X-100 + 0.5% 1-thioglycerol three times at room temperature and agitation, changing fresh buffer every 2 hours. To begin clearing, samples are incubated in clearing enhanced 3D (Ce3D) (48) solution at room temperature overnight with agitation protected from light. 4,6-Diamidino-2-phenylindole is diluted 1:500 in the Ce3D solution to counter stain for nuclei. To 3D image the cleared samples, the gels are placed on glass-bottom dishes and imaged overnight on an SP8 Resonant Scanning Confocal Microscope.

Data in graphs are expressed as the SE or SEM SD, as denoted in figure legends. Statistical significance was determined using two-tailed Students t test for two-way comparisons or one-way ANOVA or two-way ANOVA followed by Dunnetts, Sidaks, or Tukeys multiple comparison test.

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Thermofluidic heat exchangers for actuation of transcription in artificial tissues - Science Advances


The Global CRISPR Technology Market Size Is Seeing Exponential Growth Due To The Application Of CRISPR Technology In Treating COVID-19 – GlobeNewswire

Wednesday, September 30th, 2020

LONDON, Sept. 24, 2020 (GLOBE NEWSWIRE) -- (Companies Included: Crispr Therapeutics, Thermo Fisher Scientific, Intellia Therapeutics, Horizon Discovery, and Synthego Corporation)

In another instance, in early May, the US Food and Drug Administration (FDA) granted Sherlock Biosciences an emergency use authorization (EUA) for its COVID-19 diagnostic assay, beating out other companies and academic groups trying to use the powerful gene-editing technology to figure out who is infected with the novel coronavirus. Sherlocks test is the first FDA-authorized use of CRISPR technology for anything. Sherlocks test is a molecular diagnostic, intended to identify people who have acute SARS-CoV-2 infection. It capitalizes on a CRISPR-based technology developed in the lab of Feng Zhang, a scientist at Broad Institute of MIT and Harvard and a cofounder of Sherlock.

The Business Research Companys report titled CRISPR Technology Global Market Report 2020-30: Covid 19 Growth And Change covers the CRISPR market 2020, CRISPR technology market share by company, global CRISPR technology market analysis, global CRISPR technology market size, and CRISPR technology market forecasts. The report also covers the global CRISPR technology market and its segments. The CRISPR technology market share is segmented by product type into Cas9 and gRNA, design tool, plasmid and vector, and other delivery system products. The CRISPR technology market share is segmented by end-user into biopharmaceutical companies, agricultural biotechnology companies, academic research organizations, and contract research organizations (CROs). By application, it is segmented into biomedical, agriculture, diagnostics, and others.

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The global CRISPR technology market value is expected to grow from $685.5 million in 2019 to $1,654.2 million in 2023 at a compound annual growth rate (CAGR) of 24.6%. The application of CRISPR technology as a diagnostic tool is expected to boost CRISPR technology market growth during the period. The Sherlock CRISPR SARS-CoV-2 kit is the first diagnostic kit based on CRISPR technology for infectious diseases caused due to COVID-19. In May 2020, the US FDA (Food and Drug Administration) announced emergency use authorization of Sherlock BioSciences Inc.s Sherlock CRISPR SARS-CoV-2 kit, which is a CRISPR-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) diagnostic test.

This test helps in specifically targeting RNA or DNA sequences of the SARS-CoV-2 virus from specimens or samples such as nasal swabs from the upper respiratory tract, and fluid in the lungs from bronchoalveolar lavage specimens. This diagnostic kit has high specificity and sensitivity, and does not provide false negative or positive results. Widening the application of CRISPR technology for the diagnosis of infectious diseases will further increase the demand for CRISPR technology products and services and drive the CRISPR market 2020.

Several advancements in CRISPR technology are trending in the market. Advancements in technology will help in reducing errors, limiting unintended effects, improving the accuracy of the tool, widening its applications, developing gene therapies, and more. Scientists, researchers and companies are increasingly developing advanced CRISPR technologies for more precise editing and to get access to difficult to reach areas of human genome. For instance, in March 2020, scientists at University of Toronto developed CHyMErA, a CRISPR-based tool for more versatile genome editing. Similarly, in March 2020, researchers at New York genome center developed a new CRISPR screening technology to target RNA, including RNA of novel viruses like COVID.

In November 2019, researchers at ETH Zurich, Switzerland, swapped CAS9 enzyme for Cas 12a, that allowed the researchers to edit genes in 25 target sites. It is also estimated that hundreds of target sites can be modified using the above method. In October 2019, a team from MIT and Harvard developed new CRISPR genome editing approach called prime editing by combining CRISPR-Cas9 and reverse transcriptase into a single protein. The prime editing has the potential to directly edit human cells with high precision and efficiency.

The CRISPR technology market share consists of sales of CRISPR technology products and services, which is a gene-editing technology that allows researchers to alter DNA sequences and modify gene function. The revenue generated by the market includes the sales of products such as design tools, plasmid & vector, Cas9 & gRNA, and libraries & delivery system products and services that include design & vector construction, screening and cell line engineering. These products and services are used in genome editing/genetic engineering, genetically modifying organisms, agricultural biotechnology and others, which include gRNA database/gene library, CRISPR plasmid, and human stem cell & cell line engineering.

CRISPR Technology Global Market Report 2020-30: Covid 19 Growth And Change is one of a series of new reports from The Business Research Company that provide market overviews, analyze and forecast market size and growth for the whole market, CRISPR technology market segments and geographies, CRISPR technology market trends, CRISPR technology market drivers, CRISPR technology marketrestraints, CRISPR technology market leading competitors revenues, profiles and market shares in over 1,000 industry reports, covering over 2,500 market segments and 60 geographies. The report also gives in-depth analysis of the impact of COVID-19 on the market. The reports draw on 150,000 datasets, extensive secondary research, and exclusive insights from interviews with industry leaders. A highly experienced and expert team of analysts and modellers provides market analysis and forecasts. The reports identify top countries and segments for opportunities and strategies based on market trends and leading competitors approaches.

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The Global CRISPR Technology Market Size Is Seeing Exponential Growth Due To The Application Of CRISPR Technology In Treating COVID-19 - GlobeNewswire


Novavax Initiates Phase 3 Efficacy Trial of COVID-19 Vaccine in the United KingdomClinical trial to enroll up to 10000 volunteers across the UK to…

Wednesday, September 30th, 2020

GAITHERSBURG, Md., Sept. 24, 2020 (GLOBE NEWSWIRE) -- Novavax, Inc. (Nasdaq: NVAX), a late stage biotechnology company developing next-generation vaccines for serious infectious diseases, today announced that it has initiated its first Phase 3 study to evaluate the efficacy, safety and immunogenicity of NVX-CoV2373, Novavax COVID-19 vaccine candidate. The trial is being conducted in the United Kingdom (UK), in partnership with the UK Governments Vaccines Taskforce, and is expected to enroll and immunize up to 10,000 individuals between 18-84 (inclusive) years of age, with and without relevant comorbidities, over the next four to six weeks.

With a high level of SARS-CoV-2 transmission observed and expected to continue in the UK, we are optimistic that this pivotal Phase 3 clinical trial will enroll quickly and provide a near-term view of NVX-CoV2373s efficacy, said Gregory M. Glenn, M.D., President, Research and Development at Novavax. The data from this trial is expected to support regulatory submissions for licensure in the UK, EU and other countries. We are grateful for the support of the UK Government, including from its Department of Health and Social Care and National Institute for Health Research, to advance this important research.

NVX-CoV2373 is a stable, prefusion protein made using Novavax recombinant protein nanoparticle technology that includes Novavax proprietary MatrixM adjuvant. The vaccine has a favorable product profile that will allow handling in an unfrozen, liquid formulation that can be stored at 2C to 8C, allowing for distribution using standard vaccine channels.

Novavax has continued to scale-up its manufacturing capacity, currently at up to 2 billion annualized doses, once all capacity has been brought online by mid-2021.

About the Phase 3 Study

Consistent with its long-standing commitment to transparency and in order to enhance information-sharing during the worldwide pandemic, Novavax will be publishing its UK study protocol in the coming days.

The UK Phase 3 clinical trial is a randomized, placebo-controlled, observer-blinded study to evaluate the efficacy, safety and immunogenicity of NVX-CoV2373 with Matrix-M in up to 10,000 subjects aged 18 to 84 years. Half the participants will receive two intramuscular injections of vaccine comprising 5 g of protein antigen with 50 g MatrixM adjuvant, administered 21 days apart, while half of the trial participants will receive placebo.

The trial is designed to enroll at least 25 percent of participants over the age of 65 as well as to prioritize groups that are most affected by COVID-19, including racial and ethnic minorities. Additionally, up to 400 participants will also receive a licensed seasonal influenza vaccine as part of a co-administration sub-study.

The trial has two primary endpoints. The first primary endpoint is first occurrence of PCR-confirmed symptomatic COVID-19 with onset at least 7 days after the second study vaccination in volunteers who have not been previously infected with SARS-CoV-2. The second primary endpoint is first occurrence of PCR-confirmed symptomatic moderate or severe COVID-19 with onset at least 7 days after the second study vaccination in volunteers who have not been previously infected with SARS-CoV-2. The primary efficacy analysis will be an event-driven analysis based on the number of participants with symptomatic or moderate/severe COVID-19 disease. An interim analysis will be performed when 67% of the desired number of these cases has been reached.

For further information, including media-ready images, b-roll, downloadable resources and more, click here.

About NVX-CoV2373

NVXCoV2373 is a vaccine candidate engineered from the genetic sequence of SARSCoV2, the virus that causes COVID-19 disease. NVXCoV2373 was created using Novavax recombinant nanoparticle technology to generate antigen derived from the coronavirus spike (S) protein and contains Novavax patented saponin-based Matrix-M adjuvant to enhance the immune response and stimulate high levels of neutralizing antibodies. NVX-CoV2373 contains purified protein antigens and cannot replicate, nor can it cause COVID-19. In preclinical trials, NVXCoV2373 demonstrated indication of antibodies that block binding of spike protein to receptors targeted by the virus, a critical aspect for effective vaccine protection. In its the Phase 1 portion of its Phase 1/2 clinical trial, NVXCoV2373 was generally well-tolerated and elicited robust antibody responses numerically superior to that seen in human convalescent sera. NVX-CoV2373 is also being evaluated in two ongoing Phase 2 studies, which began in August; a Phase 2b trial in South Africa, and a Phase 1/2 continuation in the U.S. and Australia. Novavaxhas secured$2 billionin funding for its global coronavirus vaccine program, including up to$388 millionin funding from theCoalition for Epidemic Preparedness Innovations(CEPI).

About Matrix-M

Novavax patented saponin-based Matrix-M adjuvant has demonstrated a potent and well-tolerated effect by stimulating the entry of antigen-presenting cells into the injection site and enhancing antigen presentation in local lymph nodes, boosting immune response.

About Novavax

Novavax, Inc.(Nasdaq:NVAX) is a late-stage biotechnology company that promotes improved health globally through the discovery, development, and commercialization of innovative vaccines to prevent serious infectious diseases.Novavaxis undergoing clinical trials for NVX-CoV2373, its vaccine candidate against SARS-CoV-2, the virus that causes COVID-19. NanoFlu, its quadrivalent influenza nanoparticle vaccine, met all primary objectives in its pivotal Phase 3 clinical trial in older adults. Both vaccine candidates incorporate Novavax proprietary saponin-based Matrix-M adjuvant in order to enhance the immune response and stimulate high levels of neutralizing antibodies.Novavaxis a leading innovator of recombinant vaccines; its proprietary recombinant technology platform combines the power and speed of genetic engineering to efficiently produce highly immunogenic nanoparticles in order to address urgent global health needs.

For more information, visit and connect with us on Twitter and LinkedIn.

Novavax Forward-Looking Statements

Statements herein relating to the future ofNovavaxand the ongoing development of its vaccine and adjuvant products are forward-looking statements.Novavaxcautions that these forward-looking statements are subject to numerous risks and uncertainties, which could cause actual results to differ materially from those expressed or implied by such statements. These risks and uncertainties include those identified under the heading Risk Factors in the Novavax Annual Report on Form 10-K for the year endedDecember 31, 2019, and Quarterly Report on Form 8-K for the period endedJune 30, 2020, as filed with theSecurities and Exchange Commission(SEC). We caution investors not to place considerable reliance on forward-looking statements contained in this press release. You are encouraged to read our filings with theSEC, available, for a discussion of these and other risks and uncertainties. The forward-looking statements in this press release speak only as of the date of this document, and we undertake no obligation to update or revise any of the statements. Our business is subject to substantial risks and uncertainties, including those referenced above. Investors, potential investors, and others should give careful consideration to these risks and uncertainties.



InvestorsSilvia Taylor and Erika Trahanir@novavax.com240-268-2022

MediaBrandzone/KOGS CommunicationEdna Kaplankaplan@kogspr.com617-974-8659

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Novavax Initiates Phase 3 Efficacy Trial of COVID-19 Vaccine in the United KingdomClinical trial to enroll up to 10000 volunteers across the UK to...


Genome Editing/Genome Engineering Market Research Report by Technology, by Application – Global Forecast to 2025 – Cumulative Impact of COVID-19 -…

Sunday, September 20th, 2020

Genome Editing/Genome Engineering Market Research Report by Technology (Antisense, Crispr, Talen, and Zfn), by Application (Cell Line Engineering, Diagnostic Applications, Drug Discovery & Development, and Genetic Engineering) - Global Forecast to 2025 - Cumulative Impact of COVID-19

New York, Sept. 18, 2020 (GLOBE NEWSWIRE) -- announces the release of the report "Genome Editing/Genome Engineering Market Research Report by Technology, by Application - Global Forecast to 2025 - Cumulative Impact of COVID-19" -

The Global Genome Editing/Genome Engineering Market is expected to grow from USD 4,901.67 Million in 2019 to USD 14,012.67 Million by the end of 2025 at a Compound Annual Growth Rate (CAGR) of 19.13%.

Market Segmentation & Coverage:This research report categorizes the Genome Editing/Genome Engineering to forecast the revenues and analyze the trends in each of the following sub-markets:

Based on Technology, the Genome Editing/Genome Engineering Market studied across Antisense, Crispr, Talen, and Zfn.

Based on Application, the Genome Editing/Genome Engineering Market studied across Cell Line Engineering, Diagnostic Applications, Drug Discovery & Development, and Genetic Engineering. The Genetic Engineering further studied across Animal Genetic Engineering and Plant Genetic Engineering.

Based on Geography, the Genome Editing/Genome Engineering Market studied across Americas, Asia-Pacific, and Europe, Middle East & Africa. The Americas region surveyed across Argentina, Brazil, Canada, Mexico, and United States. The Asia-Pacific region surveyed across Australia, China, India, Indonesia, Japan, Malaysia, Philippines, South Korea, and Thailand. The Europe, Middle East & Africa region surveyed across France, Germany, Italy, Netherlands, Qatar, Russia, Saudi Arabia, South Africa, Spain, United Arab Emirates, and United Kingdom.

Company Usability Profiles:The report deeply explores the recent significant developments by the leading vendors and innovation profiles in the Global Genome Editing/Genome Engineering Market including Creative Biogene, Crispr Therapeutics, Editas Medicine, Epigenie, Eurofins Scientific SE, Genscript Biotech, Horizon Discovery Group PLC, Integrated DNA Technologies, Inc., Intellia Therapeutics, Inc., Lonza Group AG, Merck & Co., Inc., New England Biolabs, OriGene Technologies, Inc., Oxford Genetics Ltd., Precision Biosciences, Sangamo Therapeutics, Synthego Corporation, Thermo Fisher Scientific Inc., Transposagen Biopharmaceuticals, Inc., and Vigene Bioscience Inc..

FPNV Positioning Matrix:The FPNV Positioning Matrix evaluates and categorizes the vendors in the Genome Editing/Genome Engineering Market on the basis of Business Strategy (Business Growth, Industry Coverage, Financial Viability, and Channel Support) and Product Satisfaction (Value for Money, Ease of Use, Product Features, and Customer Support) that aids businesses in better decision making and understanding the competitive landscape.

Competitive Strategic Window:The Competitive Strategic Window analyses the competitive landscape in terms of markets, applications, and geographies. The Competitive Strategic Window helps the vendor define an alignment or fit between their capabilities and opportunities for future growth prospects. During a forecast period, it defines the optimal or favorable fit for the vendors to adopt successive merger and acquisition strategies, geography expansion, research & development, and new product introduction strategies to execute further business expansion and growth.

Cumulative Impact of COVID-19:COVID-19 is an incomparable global public health emergency that has affected almost every industry, so for and, the long-term effects projected to impact the industry growth during the forecast period. Our ongoing research amplifies our research framework to ensure the inclusion of underlaying COVID-19 issues and potential paths forward. The report is delivering insights on COVID-19 considering the changes in consumer behavior and demand, purchasing patterns, re-routing of the supply chain, dynamics of current market forces, and the significant interventions of governments. The updated study provides insights, analysis, estimations, and forecast, considering the COVID-19 impact on the market.

The report provides insights on the following pointers:1. Market Penetration: Provides comprehensive information on the market offered by the key players2. Market Development: Provides in-depth information about lucrative emerging markets and analyzes the markets3. Market Diversification: Provides detailed information about new product launches, untapped geographies, recent developments, and investments4. Competitive Assessment & Intelligence: Provides an exhaustive assessment of market shares, strategies, products, and manufacturing capabilities of the leading players5. Product Development & Innovation: Provides intelligent insights on future technologies, R&D activities, and new product developments

The report answers questions such as:1. What is the market size and forecast of the Global Genome Editing/Genome Engineering Market?2. What are the inhibiting factors and impact of COVID-19 shaping the Global Genome Editing/Genome Engineering Market during the forecast period?3. Which are the products/segments/applications/areas to invest in over the forecast period in the Global Genome Editing/Genome Engineering Market?4. What is the competitive strategic window for opportunities in the Global Genome Editing/Genome Engineering Market?5. What are the technology trends and regulatory frameworks in the Global Genome Editing/Genome Engineering Market?6. What are the modes and strategic moves considered suitable for entering the Global Genome Editing/Genome Engineering Market?Read the full report:

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