Category Archives: Genetic Engineering

--Focusing on neuroblastoma, the researchers used a multi-omics approach to identify tumor-specific peptides and then used genetic engineering to harness the immune system to destroy tumors--

PHILADELPHIA, Nov. 3, 2021 /PRNewswire/ -- In a breakthrough for the treatment of aggressive solid cancers, researchers at Children's Hospital of Philadelphia (CHOP) have developed a novel cancer therapy that targets proteins inside cancer cells that are essential for tumor growth and survival but have been historically impossible to reach. Using the power of large data sets and advanced computational approaches, the researchers were able to identify peptides that are presented on the surface of tumor cells and can be targeted with "peptide-centric" chimeric antigen receptors (PC-CARs), a new class of engineered T cells, stimulating an immune response that eradicates tumors.

Senior author John M. Maris, MD, pediatric oncologist and Giulio D'Angio Chair in Neuroblastoma Research at CHOP

The discovery, which was described today in Nature, opens the door to treating a broader array of cancers with immunotherapy as well as applying each therapy across a greater proportion of the population.

"This research is extremely exciting because it raises the possibility of targeting very specific tumor molecules, expanding both the cancers that can be treated with immunotherapy and the patient population who can benefit," said Mark Yarmarkovich, PhD, an investigator in the Maris Laboratory at Children's Hospital of Philadelphia and first author of the paper. "By using a multi-omics approach, we were able to identify peptides specific to neuroblastoma tumors, but this method could be used in any cancer, allowing for a more personalized approach to cancer treatment."

The development of CAR T cell-based cancer immunotherapy marked a breakthrough in the treatment of leukemia, but the approach has not yet made significant strides against solid tumors due, at least in part, to a lack of tumor-specific targets. In these cancers, most of the proteins responsible for tumor growth and survival are in the nuclei of tumor cells, not on the cell surface, where they would generally be accessible to CAR T cells. Instead, fragments of these proteins may be presented on the tumor cell surface through the presentation of peptides on the major histocompatibility complex (MHC), which evolved to present viral and bacterial peptides to the immune system. Cancer cells can also present intracellular proteins on MHC, and if these are mutant peptides, they may be recognized as foreign. However, all pediatric cancers and many adult malignancies have few mutations and are rather driven by other factors like dysregulated developmental pathways.

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Neuroblastoma is an explosively aggressive pediatric cancer that is driven by modifications of gene expression that promote uncontrolled tumor growth. Historically, neuroblastoma has been treated with chemotherapy, surgery, and radiation therapy, but patients often relapse with forms of the disease that are chemotherapy resistant. Additionally, the low mutational burden of the cancer, combined with its low MHC expression, have made it difficult to target with immunotherapies.

Despite these obstacles, the researchers hypothesized that some of the peptides presented on the surface of neuroblastoma tumor cells come from proteins that are essential for tumor growth and survival and could be targeted with synthetic CARs. These PC-CARs would allow for direct targeting and killing of tumor cells. The challenge was differentiating tumor-specific peptides from other, similar looking peptides or peptides that exist in normal tissues to avoid cross-reactivity and lethal toxicity.

To do so, the researchers stripped the MHC molecules off neuroblastoma cells and determined which peptides were present and at what abundance. They used a large genomic dataset that the Maris lab has generated to determine which peptides were unique to neuroblastoma and not expressed by normal tissues. They prioritized peptides that were derived from genes essential to the tumor and had characteristics required to engage the immune system. To weed out any potential antigens that might have cross reactivity with normal tissue, the researchers filtered the remaining tumor peptides against a database of MHC peptides on normal tissues, removing any peptide with a parent gene represented in normal tissue.

Using this multi-omics approach, the researchers pinpointed an unmutated neuroblastoma peptide that is derived from PHOX2B, a neuroblastoma dependency gene and transcriptional regulator that was previously identified and characterized at CHOP. The next major hurdle was developing a PC-CAR that specifically recognized just the peptide, which makes up 2-3% of the peptide-MHC complex. In collaboration with antibody-discovery company Myrio Therapeutics, the researchers developed a PC-CAR targeting this peptide and showed that these PC-CARs recognized the tumor-specific peptide on different HLA types, meaning the treatment could be applied to patients of diverse genetic lineages.

Taking the research a step further, the team tested the PC-CARs in mice and found that the treatment led to complete and targeted elimination of neuroblastoma tumors.

"We are excited about this work because it allows us to now go after essential cancer drivers that have been considered 'undruggable' in the past. We think that PC-CARS have the potential to vastly expand the pool of immunotherapies and significantly widen the population of eligible patients," said senior author John M. Maris, MD, pediatric oncologist and Giulio D'Angio Chair in Neuroblastoma Research at CHOP. "Thanks to the Acceleration grant we received through the Cell and Gene Therapy Collaborative at CHOP, we will bring our PHOX2B PC-CAR to a clinical trial at CHOP in late 2022 or early 2023."

Yarmarkovich et al. "Therapeutic Targeting of Intracellular Oncoproteins with Peptide-Centric CAR T Cells," Nature, November 3, 2021, DOI: 10.1038/s41586-021-04061-6

About Children's Hospital of Philadelphia:Children's Hospital of Philadelphia was founded in 1855 as the nation's first pediatric hospital. Through its long-standing commitment to providing exceptional patient care, training new generations of pediatric healthcare professionals, and pioneering major research initiatives, Children's Hospital has fostered many discoveries that have benefited children worldwide. Its pediatric research program is among the largest in the country. In addition, its unique family-centered care and public service programs have brought the 595-bed hospital recognition as a leading advocate for children and adolescents. For more information, visit http://www.chop.edu

Contact: Jennifer LeeChildren's Hospital of Philadelphia(267) 426-6084LEEJ41@chop.edu

Cision

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SOURCE Children's Hospital of Philadelphia

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CHOP Researchers Develop a New Class of CAR-T Cells that Target Previously Untargetable Cancer Drivers - Yahoo Finance

PHILADELPHIA, Nov. 04, 2021 (GLOBE NEWSWIRE) -- Century Therapeutics (NASDAQ: IPSC), an innovative biotechnology company developing induced pluripotent stem cell (iPSC)-derived cell therapies in immuno-oncology, today announced that preclinical data from the Companys CNTY-101 program and CAR-iT platform will be presented in two posters at the 63rd American Society of Hematology (ASH) Annual Meeting & Exposition, on December 11-14, 2021 in Atlanta, Georgia and virtually.

The Company also announced today that it will host a virtual research & development update on Thursday, December 16, 2021 from 8:00 AM - 9:30 AM EST to share progress on its iPSC technology platform and pipeline. Eduardo Sotomayor, M.D., director of the Cancer Institute at Tampa General Hospital, will discuss the current treatment paradigm for B-cell malignancies. For additional information on how to access the event, please visit the Events & Presentations section of Centurys website.

Details of the two poster presentations are as follows:

Abstract Number: 1729Title: Development of Multi-Engineered iPSC-Derived CAR-NK Cells for the Treatment of B-Cell MalignanciesSession Name: 703. Cellular Immunotherapies: Basic and Translational: Poster I Session Date: Saturday, December 11, 2021 Session Time: 5:30 PM - 7:30 PM Presenter: Luis Borges, Chief Scientific Officer, Century Therapeutics

Abstract Number: 2771Title: Induced Pluripotent Stem Cell-Derived Gamma Delta CAR-T Cells for Cancer ImmunotherapySession Name: 703 Cell Therapies: Basic and TranslationalSession Date: Sunday, December 12, 2021Session Time: 6:00 PM 8:00 PMPresenter: Mark Wallet, Vice President, Immuno-Oncology, Century Therapeutics

Full abstracts are currently available through the ASH conference website.

About Century Therapeutics

Century Therapeutics (NASDAQ: IPSC) is harnessing the power of adult stem cells to develop curative cell therapy products for cancer that we believe will allow us to overcome the limitations of first-generation cell therapies. Our genetically engineered, iPSC-derived iNK and iT cell product candidates are designed to specifically target hematologic and solid tumor cancers. We are leveraging our expertise in cellular reprogramming, genetic engineering, and manufacturing to develop therapies with the potential to overcome many of the challenges inherent to cell therapy and provide a significant advantage over existing cell therapy technologies. We believe our commitment to developing off-the-shelf cell therapies will expand patient access and provide an unparalleled opportunity to advance the course of cancer care. For more information on Century Therapeutics please visit http://www.centurytx.com.

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Century Therapeutics Forward-Looking Statement

This press release contains forward-looking statements within the meaning of, and made pursuant to the safe harbor provisions of, The Private Securities Litigation Reform Act of 1995. All statements contained in this press release, other than statements of historical facts or statements that relate to present facts or current conditions, including but not limited to, statements regarding our clinical development plans, are forward-looking statements. These statements involve known and unknown risks, uncertainties and other important factors that may cause our actual results, performance, or achievements to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements. In some cases, you can identify forward-looking statements by terms such as may, might, will, should, expect, plan, aim, seek, anticipate, could, intend, target, project, contemplate, believe, estimate, predict, forecast, potential or continue or the negative of these terms or other similar expressions. The forward-looking statements in this presentation are only predictions. We have based these forward-looking statements largely on our current expectations and projections about future events and financial trends that we believe may affect our business, financial condition, and results of operations. These forward-looking statements speak only as of the date of this press release and are subject to a number of risks, uncertainties and assumptions, some of which cannot be predicted or quantified and some of which are beyond our control, including, among others: our ability to successfully advance our current and future product candidates through development activities, preclinical studies, and clinical trials; our reliance on the maintenance of certain key collaborative relationships for the manufacturing and development of our product candidates; the timing, scope and likelihood of regulatory filings and approvals, including final regulatory approval of our product candidates; the impact of the COVID-19 pandemic on our business and operations; the performance of third parties in connection with the development of our product candidates, including third parties conducting our future clinical trials as well as third-party suppliers and manufacturers; our ability to successfully commercialize our product candidates and develop sales and marketing capabilities, if our product candidates are approved; and our ability to maintain and successfully enforce adequate intellectual property protection. These and other risks and uncertainties are described more fully in the Risk Factors section of our most recent filings with the Securities and Exchange Commission and available at http://www.sec.gov. You should not rely on these forward-looking statements as predictions of future events. The events and circumstances reflected in our forward-looking statements may not be achieved or occur, and actual results could differ materially from those projected in the forward-looking statements. Moreover, we operate in a dynamic industry and economy. New risk factors and uncertainties may emerge from time to time, and it is not possible for management to predict all risk factors and uncertainties that we may face. Except as required by applicable law, we do not plan to publicly update or revise any forward-looking statements contained herein, whether as a result of any new information, future events, changed circumstances or otherwise.

For More Information: Company: Elizabeth Krutoholow investor.relations@centurytx.comInvestors: Melissa Forst/Maghan Meyers century@argotpartners.comMedia: Joshua R. Mansbach century@argotpartners.com

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Century Therapeutics to Present at the 63rd American Society of Hematology Annual Meeting and Host Virtual Research & Development Update - Yahoo...

Some cases of heart failure have root causes surprisingly similar to diseases like Alzheimers, Huntingtons and ALS.

Over time, sometimes quite rapidly, the heart's thick strong muscle tissue becomes thin and weak, causing the left ventricle to swell like a balloon. This makes the heart less able to squeeze efficiently, which can lead to blood clots, irregular heartbeats, and sometimes sudden death when the malfunctioning heart simply stops beating. The origins of cardiomyopathy are diverse, including viral infections, autoimmune diseases, toxic drug exposures, and dozens of gene mutations.

Now, a multi-disciplinary team of clinicians and researchers has deciphered the function of a specific genetic mutation that causes cardiomyopathy. Their findings,published Nov. 3, 2021, in Nature Communications, were made possible by growing gene-edited human heart tissue from induced pluripotent stem cells and measuring the activity, location and binding of this mutant protein.

The team was led by co-corresponding authors Charles Murry, MD, PhD, a regenerative medicine expert at the University of Washington; Bruce Conklin, MD, a genetic engineering expert with the Gladstone Institutes in California, and Nathan Salomonis, PhD, a computational genomics expert at Cincinnati Children's.

"We hope this study will lead to broader insights that could lead to improved heart failure therapies," Conklin says.

Cutting-edge experiments expose more of the heart's inner workings

Over the last several decades, the research community has made many discoveries that have led to improved medications and medical devices that can dramatically extend life by slowing down the progression of heart failure. However, we still lack proven cures.

This study reveals a new mechanism of cardiomyopathy initiation by the RNA binding motif protein 20 (RBM20). This protein helps control RNA splicing in the heart, the process by which RNAs are sliced and diced to give rise to different proteins in different tissues. Normally, RBM20 splices RNAs to make proteins that enable the heart to adapt to stress and contract regularly throughout a person's entire life. But a class of mutations in RBM20 result in severe cardiomyopathy in adulthood.

"We and others had previously studied RBM20's function during heart development, but we had little to no clue of why it stops working in disease. We needed to step up our game if our research was to have a clinical impact," says Alessandro Bertero, PhD, who contributed to the work while at the University of Washington and now leads an Armenise-Harvard Laboratory at the University of Turin in Italy.

Discovering this protein's role was especially complex because knocking out this gene in animal models does not mimic the damaging effects seen in people. Instead, the work required editing the genome of healthy cells and engineering human heart tissue from these cells in a lab dish. Only by producing heart tissue similar to that found in humans could the authors understand the contractile defects and molecular mechanisms underlying this gene's function in a controlled manner.

"That was exactly what we intended when we started this project by genome-editing induced pluripotent stem cells," says co-leading author Yuichiro Miyaoka, PhD, of the Tokyo Metropolitan Institute of Medical Science.

First, the team observed that the engineered muscle tissue carrying the mutant form of RBM20 did not function like tissue engineered with normal RBM20 or lacking the protein all together. The mutated muscle fibers contracted with significantly less force and upstroke velocity, much like a heart affected by cardiomyopathy.

Then, at the single-cell level, the team detected another important clue. Normally, RBM20 is located exclusively within the cell nucleus. However, the mutated form localizes almost entirely out of the nucleus, in the cell's cytoplasm.

This, by itself, did not mean muchuntil the cell was exposed to heavy stress. When that occurred, the mutant protein was detected within tiny "stress granules" made of protein and RNA that cells rapidly produce as a reaction to stress. In contrast, RBM20 in healthy cells remained within the nucleus and distinct from stress granules. This suggests there are additional cellular mechanisms, along with changes in splice-activity, leading to RBM20 cardiomyopathy.

"When the RNA binding landscape of mutant RBM20 was revealed by a technology called enhanced CLIP, it mimicked the binding of other splicing factors that have been implicated in neurodegenerative diseases. These factors, when mutated, also change their activity from RNA splicing to RNA aggregation outside the nucleus," says co-author Gene Yeo, PhD, MBA, a member of the Department of Cellular and Molecular Medicine at the University of California San Diego.

"Over time, such aggregates play havoc with other cell functions, ultimately leading to the tissue-weakening of heart muscle during cardiomyopathy," Salomonis says.

"It is intriguing to note the parallels between our observations with RBM20 and recent findings in neuro-degeneration," the paper states. "Indeed, recent work has hypothesized cytoplasmic RBM20 may be similar to the cytoplasmic RNP granules associated with neurodegeneration (Schneider et al., 2020), such as TAU for Alzheimer s disease, Huntingtin for Huntington s disease, and FUS for amyotrophic lateral sclerosis (ALS)."

Next steps

Co-authors for this study also included scientists from the University of Cincinnati Department of Electrical Engineering and Computer Science, Sana Biotechnology, and the University of California San Francisco.

The co-authors say the 3D heart tissue model they've developed has the potential to be used to test new drugs to block the formation of cytoplasmic granules as a possible treatment for cardiomyopathy, even those without RBM20 mutations.

"RBM20 has been a frustrating protein to study, as animal models don't fully recapitulate human disease pathology," says lead author Aidan Fenix, PhD. "It's exciting to now have an in vitrohuman cell model of RBM20 cardiomyopathy that shows the major clinical feature of dilated cardiomyopathy--reduced contractile force. We hope these models will speed the discovery of therapies to treat RBM20 dilated cardiomyopathy."

About this study

This work was supported by grants from the National Heart, Lung, and Blood Institute (U01 HL099997, P01 HL089707, R01 HL130533, F32 HL156361-01, HL149734, R01 HL128362, R01 HL128368, R01 HL141570, R01 HL146868); the National Institute of Diabetes and Digestive and Kidney (U54DK107979-05S1); the National Science Foundation (NSF CMMI-1661730); a JSPS Grant-in-Aid for Young Scientists, and grants from NOVARTIS, the Mochida Memorial Foundation, SENSHIN Medical Research Foundation, Naito Foundation, Uehara Memorial Foundation, a Gladstone-CIRM Fellowship, and the A*STAR International Fellowship.

SOURCE Cincinnati Children's Hospital Medical Center

http://www.cincinnatichildrens.org

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Heart Tissue in a Dish Reveals New Links Between Neurodegeneration and Heart Disease - PRNewswire

Nov 1st 2021

DISQUIET IS growing about gain-of-function (GOF) research, a form of genetic manipulation on micro-organisms. Some anxiety stems from the idea that such work was responsible for creating SARS-CoV-2, the virus that causes covid-19. This remains unproven. But, more broadly, there is unease that Americas National Institutes of Health (NIH), which funds research, may have supported GOF work in China. Recently there was further controversy when the NIH said that a recipient of one of its grants, EcoHealth Alliance, failed to report its work in 2018 on making a mouse coronavirus more virulent. (EcoHealth Alliance says this information was reported.) Many have been alarmed to discover that, before 2019, Chinese and American researchers often genetically tinkered with SARS-like viruses. What exactly is gain-of-function research, and is it cause for alarm?

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Researchers have been modifying viruses for decadesand not always in benign ways. Twenty years ago Australian scientists changed a mousepox virus during an attempt to create a mouse contraceptive. The idea was to stimulate the production of antibodies against mouse eggs. But the gene that was inserted switched off the mouses immune system, making the virus more lethal.

In 2015 Ralph Baric and colleagues from the University of North Carolina in Chapel Hill took the virus that caused the first SARS outbreak and added a surface protein from another SARS-like virus, known as SHC014 and found in horseshoe bats. The newly created virus was able to infect human airway cells. This caused much alarm. The researchers themselves pointed out that their work showed, usefully, that the ingredients for future outbreaks of SARS-like viruses circulated in nature.

Some virus-tinkering is useful and no cause for worry, for example to create a covid-19 adenoviral vaccine, or to deliver a novel gene as a drug. But biosecurity experts worry when a pathogen has its ability to infect, or cause disease, enhanced. Agents of concern include Ebola, anthrax and respiratory viruses with the potential to cause pandemics, such as influenza and coronaviruses. A new term, gain-of-function research of concern (GOFROC), has emerged to distinguish this sort of work, although it is not widely used.

In July the debate about what constitutes GOF reached Americas Senate. Rand Paul, a Republican senator from Kentucky, tussled with Anthony Fauci, the director of the National Institute of Allergy and Infectious Diseases, which is part of the NIH. Mr Paul is angry that the research agency funded GOF work at the Wuhan Institute of Virology (close to where covid-19 was first spotted in late 2019). The results of the work were published in 2017. Spike proteinsthe parts of the virus that help it bind to its hostfrom eight different coronaviruses were put into the genome of another coronavirus called WIV1, and these new genetic innovations could infect human cells. Dr Fauci says the work does not qualify as GOF. The counter argument is that, although the work certainly invented new viruses, it did not enhance the ability of those viruses to infect humans because the original virus was also able to infect human cells. There is a bit of linguistic wriggling here. It may be true in technical terms that the NIH has not supported specific gain-of-function research, as it claims. But it remains possible that the work it funded ended up creating gains of function on agents of concern unintentionally.

It is still unclear whether SARS-CoV-2 originated from a petri dish or a pothole. But as more details emerge of the work that was done at the Wuhan Institute of Virology, there is growing unease about whether genetic tinkering with agents of concern should be done at all. One argument for continuing such research would be to predict the mutations in SARS-CoV-2 that might in future cause trouble for humans. An argument against this is that the virus will not necessarily behave in the wild as it does in the lab. The debate about GOF work will continue long after the interest in the origins of SARS-CoV-2 has subsided.

More from The Economist explains:What are DNA vaccines?Why official covid-19 deaths do not capture the pandemics true tollWhy are so few pregnant women vaccinated against covid-19?

Read more:
What is gain-of-function research? - The Economist

The cows in Farmer Johns pasture lead an idyllic life. They roam through tree-shaded meadows, tearing up mouthfuls of clover while nursing their calves in tranquility. Tawny brown, compact and muscular, they are Limousins, a breed known for the quality of its meat and much sought-after by the high-end restaurants and butchers in the nearby food mecca of Maastricht, in the southernmost province of the Netherlands. In a year or two, meat from these dozen cows could end up on the plates of Maastrichts better-known restaurants, but the cows themselves are not headed for the slaughterhouse. Instead, every few months, a veterinarian equipped with little more than a topical anesthetic and a scalpel will remove a peppercorn-size sample of muscle from their flanks, stitch up the tiny incision and send the cows back to their pasture.

Limousin cows in Farmer Johns pasture. Mosa Meat will cultivate their cells in a lab to grow into hamburger that is genetically identical, no slaughter required

Ricardo Cases for TIME

The biopsies, meanwhile, will be dropped off at a lab in a nondescript warehouse in Maastrichts industrial quarter, five miles away, where, when I visit in July, cellular biologist Johanna Melke is already working on samples sent in a few days prior. She swirls a flask full of a clear liquid flecked with white filamentsstem cells isolated from the biopsy and fed on a nutrient-dense growth medium. In a few days, the filaments will thicken into tubes that look something like short strands of spaghetti. This is fat, says Melke proudly. Fat is really important. Without fat, meat doesnt taste as good.

Mosa Meat has recruited a global team of lab technicians and biologists to develop, build and run its scaled-up operations. Rui Hueber, checks the health of recent cell samples.

Ricardo Cases for TIME

On the opposite side of the building, other scientists are replicating the process with muscle cells. Like the fat filaments, the lean muscle cells will be transferred to large bioreactorstemperature- and pressure-controlled steel vesselswhere, bathed in a nutrient broth optimized for cell multiplication, they will continue to grow. Once they finish the proliferation stage, the fat and the muscle tissue will be sieved out of their separate vats and reunited into a product resembling ground hamburger meat, with the exact same genetic code as the cows in Farmer Johns pasture. (The farmer has asked to go by his first name only, in order to protect his cows, and his farm, from too much media attention.)

That final product, identical to the ground beef you are used to buying in the grocery store in every way but for the fact that it was grown in a reactor instead of coming from a butchered cow, is the result of years of research, and could help solve one of the biggest conundrums of our era: how to feed a growing global population without increasing the greenhouse-gas emissions that are heating our planet past the point of sustainability. What we do to cows, its terrible, says Melke, shaking her head. What cows do to the planet when we farm them for meat? Its even worse. But people want to eat meat. This is how we solve the problem.

Once stem cells are isolated from the biopsy and fed a nutrient-dense growth medium, they thicken into filaments of fat. Once mature, they can be blended with cultivated muscle cells to create a product similar to ground beef.

Ricardo Cases for TIME

When it comes to the importance of fat in the final product, Melke admits to a slight bias. She is a senior scientist on the Fat Team, a small group of specialists within the larger scientific ecosystem of Mosa Meat, the Maastricht-based startup whose founders introduced the first hamburger grown from stem cells to the world eight years ago. That burger cost $330,000 to produce, and now Melkes Fat Team is working with the Muscle Team, the (stem cell) Isolation Team and the Scale Team, among others, to bring what they call cell-cultivated meat to market at an affordable price.

They are not the only ones. More than 70 other startups around the world are courting investors in a race to deliver lab-grown versions of beef, chicken, pork, duck, tuna, foie gras, shrimp, kangaroo and even mouse (for cat treats) to market. Competition is fierce, and few companies have allowed journalists in for fear of risks to intellectual property. Mosa Meat granted TIME exclusive access to its labs and scientists so the process can be better understood by the general public.

Livestock raised for food directly contributes 5.8% of the worlds annual greenhouse-gas emissions, and up to 14.5% if feed production, processing and transportation are included, according to the U.N. Food and Agriculture Organization. Industrial animal agriculture, particularly for beef, drives deforestation, and cows emit methane during digestion and nitrous oxide with their manure, greenhouse gases 25 and 298 times more potent than carbon dioxide, respectively, over a 100-year period.

Read More: Dinner As We Know it Is Hurting the Planet. But What If We Radically Rethink How We Make Food?

In 2019, the U.N.s International Panel on Climate Change issued a special report calling for a reduction in global meat consumption. The report found that reducing the use of fossil fuels alone would not be enough to keep planetary temperature averages from going beyond 1.5C above preindustrial levels, at which point the floods, droughts and forest fires we are already starting to see will negatively impact agriculture, reducing arable land while driving up costs. Yet global demand for meat is set to nearly double by 2050, according to the World Resources Institute (WRI), as growing economies in developing nations usher the poor into the meat-eating middle class.

The Mosa team. From left: Peter Verstrate, co-founder and chief operating officer; Maarten Bosch, CEO; Mark Post, chief scientific officer, at their headquarters in Maastricht, Netherlands, in July

Ricardo Cases for TIME

Growing meat in a bioreactor may seem like an expensive overcorrection when just reducing beef intake in high-consuming nations by 1.5 hamburgers per week, per person, could achieve significant climate gains, according to the WRI. But denying pleasure, even in the pursuit of a global good, is rarely an effective way to drive change. Earlier this year the U.N. published the largest ever opinion poll on climate change, canvassing 1.2 million residents of 50 countries. Nearly two-thirds of the respondents view the issue as a global emergency. Nonetheless, few favored plant-based diets as a solution. For 50 years, climate activists, global health experts and animal-welfare groups have been begging people to eat less meat, but per capita consumption is higher than ever, says Bruce Friedrich, head of the Good Food Institute, a nonprofit organization promoting meat alternatives. The reason? It tastes too good, he says. Our bodies are programmed to crave the dense calories. Unfortunately, current production methods are devastating for our climate and biodiversity, so its a steep price were paying for these cravings. The best solution, says Friedrich, is meat alternatives that cost the same or less, and taste the same or better. Melke and her fellow scientists at Mosa say they are getting very close.

According to Mark Post, the Dutch scientist who midwifed the first lab-grown hamburger into existence, and who co-founded Mosa Meat in 2015, one half-gram biopsy of cow muscle could in theory create up to 4.4 billion lb. of beefmore than what Mexico consumes in a year. For the moment, however, Mosa Meat is aiming for 15,000 lb., or 80,000 hamburgers, per biopsy. Even by those modest metrics, Farmer Johns little herd could supply about 10% of the Netherlands annual beef consumption. Eventually, says Post, we would need only some 30,000 to 40,000 cows worldwide, instead of the 300 million we slaughter every year, without the environmental and moral consequences of large-scale intensive cattle farming. I admire vegetarians and vegans who are disciplined enough to take action on their principles, says Post. But I cant give up meat, and most people are like me. So I wanted to make the choice for those people easier, to be able to keep on eating meat without all the negative externalities.

Even as it sets out to change everything about meat production, cellular agriculture, as the nascent industry is called, will in theory change nothing about meat consumption. This presents a tantalizing opportunity for investors, who have thrown nearly $1 billion at cultivated-meat companies over the past six years. Participating in the high-profile stampede to invest in the industry: Bill Gates, Richard Branson, Warren Buffett and Leonardo DiCaprio. Plant-based burger companies such as Impossible and Beyond already paved the way by proving that the market wants meat alternatives. Cellular agriculture promises to up that game, providing the exact same experience as meat, not a pea-protein facsimile.

While private investment has been vital for getting the industry off the ground, it is not enough given the immense benefits that the technology could provide the world were it developed at large scale, says Friedrich of the Good Food Institute. Cultivated-meat production could have as much impact on the climate crisis as solar power and wind energy, he argues. Just like renewable energy and electric vehicles have been successful because of government policies, we need the same government support for cultivated meat.

Read More: How China Could Change the World by Taking Meat Off the Menu

In the meantime, regulatory approval helps. In December 2020, GOOD Meat, the cultivated-meat division of California-based food-technology company Eat Just Inc., was granted regulatory approval to sell its chicken product to the public in Singapore, a global first. Later that month, a tasting restaurant for cell-based chicken produced by Israeli startup SuperMeat opened in Israel. Cultivated meat could be a $25 billion global industry by 2030, accounting for as much as 0.5% of the global meat supply, according to a new report from consulting firm McKinsey & Co. But to get there, many technological, economic and social hurdles must be tackled before cultivated cutlets fully replace their predecessors on supermarket shelves.

When Austrian food-trends analyst Hanni Rtzler appeared onstage to taste Mark Posts burger at its public debut in London, on Aug. 5, 2013, her biggest fear was that it might taste so bad she would spit it out on the live video broadcast. But once the burger started sizzling in the pan and the familiar scent of browning meat hit her nose, she relaxed. It was closer to the original than I even expected, she says. At the tasting, she pronounced it close to meat, but not that juicy. That was to be expected, says Mosa co-founder, COO and food technologist Peter Verstratethe burger was 100% lean meat. And without fat, burgers dont work. In fact, without fat, he says, youd be hard-pressed to tell the difference between a piece of beef and a cut of lamb. Fat isnt necessarily harder to create than muscle. Its just that as with protein cells, getting the process right is time-consuming, and Verstrate and Post prioritized protein. The technology itself is relatively straightforward and has been used for years in the pharmaceutical industry to manufacture insulin from pig pancreases: identify and isolate the stem cellsthe chameleon-like building blocks of animal biologyprod them to create the desired tissue, and then encourage them to proliferate by feeding them a cell-culture medium made up of amino acids, sugars, salts, lipids and growth factors. Scientists have been trying for years to use the same process to grow artificial organs, arteries and blood vessels, with mixed results.

Post, a vascular cardiologist, used to be one of those scientists. He jokes that stem-cell meat, unlike organs, doesnt have to function. On the other hand, it has to be produced in massive amounts at a reasonable cost, and pharmaceutical companies have spent decades and billions of dollars attemptingand largely failingto scale up stem-cell production to a fraction of what it would take to make cultivated meat affordable. If cellular-agriculture companies succeed where so many others have failed, it could unlock a completely new way of feeding human beings, as radical a transformation as the shift from hunting to domesticating animals was thousands of years ago. Despite investor enthusiasm, thats still a big if; Eat Just, the company closest to market, is producing only a couple hundred pounds of cultivated chicken a year.

Read More: I Tried Lab-Grown Fish Maw. Heres Why It Could Help Save Our Oceans

Many of the scientists at Mosa reflexively attribute sentience to the cells they are working with, discussing their likes and dislikes as they would those of a family pet. Fat tissue can handle temperature swings and rough handling; muscle is more sensitive and needs exercise. Its like producing cows on a really microscopic scale, says Laura Jackisch, the head of the Fat Team. We basically want to make the cells as comfortable as possible. That means fine-tuning their cell-culture medium in the same way you would regulate a cows feed to maximize growth and health. For one biopsy to reach the 4.4 billion lb. of meat in Posts theoretical scenario, it would have to double 50 times. So far, Jackischs team has made it to the mid-20s.

Laura Jackisch in front of the analytics lab, where Mosa measures the safety of products.

Ricardo Cases for TIME

A lot of that has to do with the quality of the growth medium. Until recently, most cultivated-meat companies used a cell culture derived from fetal bovine serum (FBS), a pharmaceutical-industry staple that comes from the blood of calf fetuses, hardly a viable ingredient for a product that is supposed to end animal slaughter. The serum is as expensive as it is controversial, and Jackisch and her fellow scientists spent most of the past year developing a plant-based alternative. They have identified what, exactly, the cells need to thrive, and how to reproduce it in large amounts using plant products and proteins derived from yeast and bacteria. What we have done is pretty breathtaking, she says. Figuring out how to make a replacement [for FBS] thats also affordable means that we can actually sell this product to the masses. In May, the Fat Team fried up a couple of teaspoons. Though they could tell from the cell structure and lipid profile that they had created a near identical product, they were still astonished by the taste. It was so intense, a rich, beefy, meaty flavor, says Jackisch, a vegan of six years. It was an instant flashback to the days when I used to eat meat. I started craving steak again. She nearly picked up a couple on her way home from the lab that night.

For all the successes that cultivated-meat companies have broadcast over the past few years, biotechnologist Ricardo San Martin, research director for the UC Berkeley Alternative Meats Lab, is skeptical that lab-bench triumphs will translate into mass-market sales anytime soon, if at all. Not one of the companies currently courting investment has proved it can manufacture products at scale, he says. They bring in all the investors and say, Here is our chicken. And yes, it is really chicken, because there are chicken cells. But not very many. And not enough for a market.

The skepticism is justifiedvery few people outside of Israel and Singapore have actually been able to try cultivated meat. (Citing a pending E.U. regulatory filing, Mosa declined to let TIME try its burger. Eat Just offered a tasting but would not allow access to its labs.) And the rollout of Eat Justs chicken nuggets in Singapore raises as many questions as it answers. At the moment, the cost to produce cultivated meat hovers around $50 a pound, according to Michael Dent, a senior technology analyst at market-research company IDTechEx. Eat Justs three-nugget portion costs about $17, or 10 times as much as the local McDonalds equivalent. CEO Josh Tetrick admits that the company is losing a lot on every sale, but argues that the current production cost per pound is just not relevant. At this point, says Dent, making a profit isnt the point. It is not in itself a viable product. But its been very, very successful at getting people talking about cultured meat. And its been very successful in getting [Eat] Just another round of investments.

Read More: Why We Must Revolutionize Food Systems to Save Our Planet

On Sept. 20, Eat Just announced that its GOOD Meat division had secured $97 million in new funding, adding to an initial $170 million publicized in May. The company also recently announced that it was partnering with the government of Qatar to build the first ever cultivated-meat facility in the Middle East outside of Israel. In June, Tetrick confirmed that the company, which also produces plant-based egg and mayonnaise products, was mulling a public listing in late 2021 or early 2022, with a possible $3 billion valuation. But all that investment still isnt enough to scale the production process to profitability, let alone to make a dent in the conventional meat industry, says Tetrick. You can make the prettiest steak in the world in the lab, but if you cant make this stuff at large scale, it doesnt matter.

The biggest obstacle to getting the cost per pound of cell-cultivated meat below that of chicken, beef or pork, says Tetrick, is the physical equipment. GOOD Meat is currently using 1,200- and 5,000-liter bio-reactors, enough to produce a few hundred pounds of meat at a time. To go large scale, which Tetrick identifies as somewhere north of 10 million lb. per facility per year, where my mom could buy it at Walmart and my dad could pick it up at a fast-food chain, would require 100,000-liter bioreactors, which currently do not exist. Vessels that big, he says, are an engineering challenge that may take as long as five years to solve. GOOD Meat has never been able to test the capacity of cell proliferation to that extent, but Tetrick is convinced that once he has the necessary bioreactors, it will be a slam dunk.

San Martin, at UC Berkeley, says Tetricks confidence clashes with the basics of cellular biology. Perpetual cell division may work with yeasts and bacteria, but mammalian cells are entirely different. At a certain point, you enter the realm of physical limitations. As they grow they excrete waste. The viscosity increases to a point where you cannot get enough oxygen in and they end up suffocating in their own poo. The only way San Martin could see cellular agriculture working on the kind of scale Tetrick is talking about is if there were a breakthrough with genetic engineering. But I dont know anyone whos gonna eat a burger made out of genetically modified lab-grown cells, he says. Mosa Meat, based in the GMO-phobic E.U., has absolutely ruled out genetic modification, and Tetrick says his current products dont use GMOs either.

That said, his rush to market has led him to rely on technologies that go against the companys slaughter-free (or cruelty-free) ethos. Not long after the companys cultivated chicken nugget was released for sale in Singapore, Tetrick revealed that FBS had been used in the production process, even though he concedes that it is self-evidently antithetical to the idea of making meat without needing to harm a life. The company has since developed an FBS-free version, but it is not yet in use, pending regulatory review.

Eat Justs initial bait and switch left a bad taste, says Dent. Cell-cultured meat technology may be sound, but if consumers start having doubts about the product and whats in it, there could be a backlash against the industry as a whole, particularly if FBS continues to be used. The first products are what everybody will judge the whole industry on, says Dent. He points to the botched rollout of genetically modified seeds in the 1990s as a precedent. Despite the science pointing to GMOs being a safer, more reliable option for agriculture, theyre still [a] pariah. It could go the same way with cultured meat. If they get it wrong now, in 20 years, people will still be saying, Cultured meats, uh-uh, freak meats, we arent touching it.

For the moment, Mosa is focused on re-creating ground beef instead of whole cuts. A ground product is easier, and cheaper, to makethe fat and muscle come out of the bioreactor as an unstructured mass, already fit for blending. Other companies, like Israels Aleph Farms, have opted to go straight for the holy grail of the cellular-agriculture worlda well-marbled steakby 3-D printing the stem cells onto a collagen scaffold, the same process medical scientists are now using to grow artificial organs. So far, Aleph has only managed to produce thin strips of lean meat, and while the technology is promising, a market-ready rib eye is still years away.

Small thin slabs are exactly what Michael Selden, co-founder and CEO of the Berkeley-based startup Finless Foods, which is producing cell-cultivated tuna, wants. Few people would pay $50 for a pound of cultivated beef15 times the cost of the conventional versionbut consumers are already paying more for high-grade sushi. Bluefin tuna sells in restaurants for $10 to $20 for two pieces of sashimi. Thats $200 a pound, he says. Sashimi, with its thin, repeatable strips and regular fat striations, is much easier to create than a thick marbled steak, and Selden says Finless Foods has already produced something close to perfect. His cell-cultivated bluefin tuna is nearly identical to the original in terms of nutrition and taste profile, he says, but the texture still needs work. Its just a little bit crunchier than we want it to be. But hes confident that by the time the product makes it through the regulatory processhes hoping by the end of the year or early 2022his team will have perfected the texture. If they do, it could be the first cultivated meat product on the U.S. market.

Cell-cultivated luxury products could be the ideal thin end of the wedge for the market, attracting conscientiousand well-heeledconsumers who want an environmentally friendly product, and thus creating space for the technological advances that will bring down the cost of commodity meat alternatives like cultivated beef and chicken. People who are buying ethical food right now are doing the right thing, but the vast majority of people are never going to convert when its only about doing the right thing, says Selden. So we want to make stuff that competes not on morals or ethicsalthough it holds those valuesbut competes on taste, price, nutrition and availability. Assuming they can, it will revolutionize the meat business.

If I was in the beef industry, I would be shaking in my boots, because theres no way that conventionally grown beef is going to be able to compete with whats coming, says Anthony Leiserowitz, director of the Yale Program on Climate Change Communication. There are many reasons people eat meat, ranging from the taste to religious and cultural traditions. But the bulk of meat consumption is not cultural, says Verstrate of Mosa Meat. Its just your average McDonalds every day. And if for that type of consumption, if you can present an alternative that is not just similar but the same, without all those downsides that traditional meat has, then it simply makes no sense to kill animals anymore.

Read More: How Eating Less Meat Could Help Protect the Planet From Climate Change

Four of the worlds five largest meat companies (JBS, Cargill, Tyson and BRF) are already embracing the technology. From a market point of view, it makes sense, says Friedrich of the Good Food Institute. These companies want to feed high-quality protein to as many people as possible, as profitably as possible. That is their entire business model. If they can make meat from plants that satisfies consumers, if they can cultivate meat from cells that tastes the same and costs less, they will shift.

A transition to a lab-grown meat source doesnt necessarily mean the end of all cows, just the end of factory farming. Ground beef makes up half the retail beef market in the U.S., and most of it comes from the industrial feedlots that pose the greatest environmental threats. Eliminating commodity meat, along with its ugly labor issues, elevated risks of zoonotic disease spread and animal-welfare concerns, would go a long way toward reining in the outsize impact of animal-meat production on the planet, says Friedrich. The meat that people eat because it is cheap and convenient is what needs to be replaced. But there will always be the Alice Waterses of the worldand there are lots of themwho will happily pay more for ethically ranched meat from live animals.

Small herds like Farmer Johns could provide both. John feeds his cows on pasture for most of the yearrather than on cattle feed, which is typically more environmentally intensiveand rotates them through his orchards in order to supplement the soil with their manure, a natural fertilizer. When he needs to feed them in the winter, he uses leftover hay from his wheat and barley crops. Its a form of regenerative agriculture that is impossible to replicate on the large scale that industrial meat production requires to overcome its smaller margins. We want good food for everybody. But if we do this [the old] way, we only have good food for some people, John says. Thats why hes willing to embrace the new technology, even if it is a threat to his way of life. This is the future, and Im proud that my cows are part of it.

Its likely to be more than a year before John can finally taste the lab-grown version of meat from his cows. Mosa is in the process of applying for regulatory approval from the E.U. In the meantime, the company is already expanding into a new space with roughly 100,000 liters of bioreactor capacity, enough to produce several tons of meat every six to eight weeks. Richard McGeown, the chef who cooked Posts first burger on the live broadcast, is already dreaming about how he will cook and serve the next one at his restaurant in southern England. Hed like to pair it with an aged cheddar, smoky ketchup and house-made pickles. It would do great, he says. Everyone loves a good burger. More important, hed love to serve something that is as good for the environment as it is good to eat.

Josias Mouafo stands in front of a CNC (computer numerical control) machine which makes custom made parts for Mosa's processes.

Ricardo Cases for TIME

But for those in the $386 billion-a-year cow business, a battle is brewing. As production moves from feedlot to factory, cattle ranchers stand to lose both jobs and investments. Like coal country in the era of clean energy, entire communities are at risk of being left behind, and they will fight. The cattle industry will do everything they can to call lab-grown meat into question, says Leiserowitz. Because once it breaks through to grocery stores, theyre competing on basic stuff, like taste and price. And they know they wont be able to win.

The U.S. Cattlemens Association has already petitioned the U.S. Department of Agriculture to limit the use of the terms beef and meat exclusively to products derived from the flesh of a [bovine] animal, harvested in the traditional manner. A decision is pending, but if it comes down in the favor of the cattle industry, it could create a significant barrier to market adoption of cell-cultured meat, says Dent. For a new product that consumers dont know and dont trust, the terms you can use make a critical difference. Whos going to buy something called lab-grown cell-protein isolates?

Its meat, says Tetrick. Even down to the genetic level, it is meat. Its just made in a different way. Tetrick, who won a similar naming battle in 2015 when his company, then known as Hampton Creek, successfully maintained the right to call its eggless mayonnaise substitute Just Mayo, says the U.S. Cattlemens Associations complaint is as senseless as if the U.S. automotive industry had argued that Tesla couldnt use the word car to describe its electric vehicles, on the basis that they lacked an internal combustion engine. Still, he says, naming is critically important. As the technology has gathered speed over the past several years, terms including cell-cultured, cultivated, slaughter-free, cell-based, clean, lab-grown and synthetic have been variously used, but consensus is gathering around cultivated meat, which is Tetricks term of choice.

Verstrate, at Mosa, is ambivalent. Ultimately were going to produce a hamburger that is delicious. We can call it meat or we can call it Joe, but if a meat lover consumes it and has the same experience as when consuming a great Wagyu burger, then were good to go.

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Lab-Grown Meat Could Feed the Planet - TIME

Dr Laura Moriarty, senior marketing manager at Bio-Rad, looks at the impressive immuno-therapeutic potential of bispecific antibodies (bsAbs).

Bispecific antibodies (bsAbs) are designed to recognise two different epitopes and, compared to current monovalent antibody therapeutics, bsAbs have enhanced binding, specificity, and efficacy, making them exciting candidates for more targeted cancer treatments. In 2019, there were 57 bsAb candidates in clinical trials, against both haematological and solid tumours.

BsAbs physically link two distinct epitopes in a dependency that can be temporal, with binding events occurring sequentially, or spatial, with binding events occurring simultaneously a novel functionality which extends the mechanistic range of antibody-mediated therapies, such as linking an effector to a target cell, or engaging two molecules on the membrane of one cell. Though their bispecific nature complicates large-scale production and purification workflows, with challenges such as antibody chain mispairing, bsAbs have come a long way since first developed. They provide clinicians with an off-the-shelf approach to cancer treatment, and they are typically cheaper and easier to produce than cell-based therapies.

Extending the reach of immunotherapy

Compared to haematological cancers, solid tumours have proven more difficult to target with current antibody and cell-based therapies, typically due to lower antigen expression levels and immunosuppressive tumour microenvironments. The more targeted, bispecific functionality of bsAbs broadens the reach of antibody-mediated therapies against both solid and haematological cancers, creating novel approaches that can induce a tumour-specific immune response, target immune checkpoints, or improve payload delivery to tumour cells (Fig. 1).

Clinical interest in bsAbs gained momentum following the promising data and market success of blinatumomab (BLINCYTO, Amgen), a fragment-based bispecific T cell engager (BiTE), which first received FDA approval in 2014 and EMA approval in 2015. The BiTE platform is an innovative technology designed to engage the immune system against numerous stages and types of cancers. BiTE molecules function by inducing proximity at the cellular level, bringing T cells together with tumour cells to initiate an immune response with a cytotoxic effect. Acapatamab, for example, is an anti-prostate-specific membrane antigen x anti-CD3 BiTE molecule, and is currently being investigated for the treatment of prostate cancer and non-small cell lung cancer.

BiTE molecules offer more versatility in potential cancer treatments than current cell-based therapies. They eliminate the need to extract T cells for manipulation, expanding the accessibility of immuno-oncology to patients and clinical areas currently unmet by cell-based therapies, such as CAR T-cell treatments. They are also designed to have a short in vitro half-life, meaning that they will be cleared from a patients system within several hours. In the pipeline, Amgen has BiTE molecules targeting seven types of cancer in clinical trials, as well as further structures in the design stage.

Further to T cell engagement, bsAbs offer promise in targeting immune checkpoints to advance solid tumour therapeutics. For example, one target under clinical assessment is CD47, an immune checkpoint that is upregulated in solid and haematological cancers as an immune evasion technique. CD47 is ubiquitously expressed and, as a result, antibody-mediated therapies to date have been unable to distinguish between CD47-expressing tumour and healthy cells. To overcome this, bsAbs can be designed to have affinity for both CD47 and tumour associated antigens (TAAs), and thus selectively bind CD47-expressing tumour cells.

BsAbs also have the potential to improve the delivery of payloads, an isotype or drug, to tumour cells. Payload delivery using monovalent antibodies, such as radioimmunotherapy and antibody-drug conjugates, relies on the direct coupling of a payload to the antibody, which then binds directly at the tumour site for delivery. However, prolonged exposure of healthy tissue to the drug or isotype can incur adverse, toxic effects. BsAbs that bind both the payload and TAAs eliminate the need for direct coupling and enable pre-targeted delivery, achieved by injecting the bsAb and payload sequentially, to reduce payload exposure to healthy cells. Five bsAbs delivering payloads entered clinical trials in 2019, four of which targeted solid tumours.

Fig. 1. Simplified schematic overview of the proposed mechanisms of action for bsAbs. 1. Engagement of immune cells to the tumour cell. 2. Targeted delivery of payloads. 3. Targeting immune checkpoint signalling.

Solving the chain-association issue

The archetypal asymmetric format of bsAbs requires the expression of four unique chains. This format seeks to closely resemble the architecture of native antibodies, to preserve favourable properties such as stability and solubility, as well as lowering the potential for immunogenicity. To obtain a functional asymmetric bsAb, each heavy (H) chain must pair with its cognate light (L) chain, and the two H chains must heterodimerise.

The co-expression of two distinct H and two distinct L chains results in a complex of 16 potential H2L2 combinations, and thus one of the initial challenges in development is obtaining the functional bsAb from this mixture. Though most HL-chain pairs hold a preference for their cognate partner, undesirable mispairings often occur; this challenge is commonly referred to as the chain-association issue. Using chimeric quadromas, common light chains, and recombinant proteins in bsAb production offers solutions to this issue by limiting the options for association, thus promoting H chain heterodimerisation and forcing cognate HL-chain pairing.

Chimeric quadromas, a hybridisation of two cell types of different species origin, reduce the number of mispaired H2L2 combinations through species-restricted HL-chain pairing. Recombinant proteins popular for their flexibility regarding origin, composition, and production systems can be utilised to force the correct association of HL-chains and H chain heterodimerisation, through several mechanisms. For instance, in the knob-in-hole approach, one H chain is engineered to consist of relatively large amino acids and the other to consist of relatively small amino acids, to facilitate cognate pairing.

Another approach to solving the chain-association issue lies in the design of alternative bsAb formats. For example, a fragment-based format is a minimalistic approach that combines multiple antigen-binding moieties into one molecule without an Fc region, and thereby circumvents the chain-association issue. This lack of complexity also promotes simpler production workflows, offering the advantage of reduced costs and high yields. An additional bsAb design that alleviates chain mispairing is a symmetric format, which incorporates both specificities in a single HL pair or polypeptide chain. Unlike the fragment-based format, the Fc region is retained, which improves pharmacokinetic properties and effector functions.

Overcoming challenges in large-scale production and purification

Despite the exciting potential for more targeted cancer treatments using bsAbs, the challenges presented in monovalent antibody discovery and development workflows are effectively doubled due to their bispecific nature. Rapid, accurate and highly sensitive screening, production and purification methods are therefore crucial for large-scale bsAb development.

Flow cytometry is a high-throughput screening technique performed directly on antibody-expressing cells, and allows a quick, multiplexed analysis for candidate selection. Once a bsAb candidate is identified, a cell line, such as the previously mentioned chimeric hybridomas, must be used to produce it reliably and consistently. Though advances in genetic engineering have greatly supported high-producing cell lines, the successful development of bsAbs at scale relies on the genetic stability of such lines. Droplet digital PCR (ddPCR) technology provides a sensitive, accurate, and absolute quantification of gene edit confirmation in cell line development, and the gene copy number can be reproducibly determined to assure safety and stability. To improve custom cell engineering workflows, ddPCR offers a screening strategy to characterise the expected homologous recombination frequency, as clones containing heterozygous and homozygous edits can be easily distinguished.

Therapeutic bsAbs also require a highly selective and easy-to-scale system to obtain a suitable purity for the final product. Resin-based chromatography is typically used during antibody purification workflows, and there are several novel modifications to this familiar technique that can improve bsAb purification. For example, mixed mode resins have the unique ability to combine different types of interactions such as hydrophobic, ion exchange, and metal affinity into a single support matrix, to improve selectivity, binding capacity, and salt tolerance. Incorporating mixed mode resins into chromatography systems allows bsAbs to be effectively purified according to their distinct epitope affinities, whilst removing product-related impurities to near-negligible levels.

Next steps for bispecific antibodies

There is an increasingly dynamic preclinical and clinical landscape for bsAbs, with a range of novel targets, formats, and mechanisms of action in the pipeline. Their capability to improve treatments for both solid and haematological cancer makes them an exciting addition to the immuno-oncology toolbox. Nevertheless, innovative methods to overcome challenges throughout bsAb discovery and development workflows are essential for their full potential to be realised, and to support continued advances in the field.

About the author

Dr Laura Moriarty is senior marketing manager at Bio-Rad.

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Using bispecific antibodies to advance cancer immunotherapy - - pharmaphorum

Patients with nonsmall cell lung cancer (NSCLC), the most common lung cancer in humans, are frequently treated with an immunotherapy called immune checkpoint blockade (ICB). This therapy induces a population of tumor-infiltrating T cells called CD8 positive T cells to secrete interferon gamma which in turn induces the expression of programmed cell death ligand 1 (PD-L1).

PD-L1 expression in the tumor microenvironment indicates the T cells are poised to kill tumor cells and patients with PD-L1 positive T-cell infiltrated tumors are most likely to respond to ICB. However, only about 35% of NSCLC patients respond to ICB therapy. Not all CD8 positive T cells in lung tumors express PD-L1 and respond to ICB and little is known about the mechanisms that govern ICB resistance in T cells within NSCLC.

In a new study published in Science Immunologytitled, Lack of CD8+ T cell effector differentiation during priming mediates checkpoint blockade resistance in nonsmall cell lung cancer, Stefani Spranger, PhD, professor at the MIT department of biology, and her colleagues uncover what causes some T cells in animal models of NSCLC to fail to respond to ICB, offering a potential way around it.

Jeffrey Bluestone, PhD, professor of metabolism and endocrinology at the University of California, San Francisco, who was not involved with the paper said, The study provides a potential opportunity to rescue immunity in the NSCLC non-responder patients with appropriate combination therapies.

It has been generally held that the continuous fight against tumor cells exhausts T cells which causes them to stop working. The rationale behind ICB therapy, therefore, has been to reinvigorate the exhausted T cells that pass into the tumors microenvironment.

However, experiments conducted by Brendan Horton, PhD, postdoctoral fellow in Sprangers lab, showed some ICB-resistant T cells stop working before they even enter the tumor, indicating exhaustion is not the cause behind their dysfunction.

Instead, the authors found that gene expression in these T cells is altered during their activation in lymph nodes which causes them to stop functioning. Once activated, T cells specialize into different subtypes with distinct functions that can be detected by specific genetic signatures.

According to Spranger, the idea that the dysfunctional state leads to ICB resistance arises before T cells enter the tumor is quite novel.

We show that this state is actually a preset condition, and that the T cells are already nonresponsive to therapy before they enter the tumor, she said. As a result, she explained, ICB therapies that work by reinvigorating exhausted T cells within the tumor are less likely to be effective. This suggests that combining ICB with other forms of immunotherapy that target T cells differently might be a more effective approach to help the immune system combat this subset of lung cancer.

To determine why some tumors are resistant to ICB, the team studied T cells in mouse models of NSCLC. They sequenced mRNA from responsive and non-responsive T cells and used a technique called Seq-Well, developed in the lab of fellow Koch Institute member, J. Christopher Love, PhD, professor of chemical engineering, and a co-author of the study. The technique allows rapid gene expression profiling of single cells. T cells responsive and nonresponsive to ICB show different gene expression patterns at specialized functional states, the single-cell sequencing analysis showed. For instance, nonresponsive T cells express low levels of some cytokinesproteins that control immunity.

Armed with the differential gene expression pattern, the team sought to convert ICB-resistant T cells into ICB-responsive T cells. The researchers treated lung tumors in mouse models with cytokines IL-2 and IL-12. This led the previously nonresponsive T cells to fight cancer cells in the mouse NSCLC.

This is potentially something that could be translated into a therapeutic that could increase the therapy response rate in non-small cell lung cancer, Horton said.

Spranger and Horton suspect cytokine therapy could be used in combination with ICB, although current clinical practices avoid cytokine treatments due to potential adverse side effects, including a condition called cytokine storm that can be fatal.

Spranger feels this work will help researchers develop more innovative cancer therapies, refocusing their efforts from reversing T-cell exhaustion to earlier states of T-cell specialization.

If T cells are rendered dysfunctional early on, ICB is not going to be effective, and we need to think outside the box, she said. Theres more evidence, and other labs are now showing this as well, that the functional state of the T cell actually matters quite substantially in cancer therapies.

To Spranger, this means that cytokine therapy might be a therapeutic avenue for NSCLC patients beyond ICB.

Read more from the original source:
Lung Cancer Unresponsive to Immunotherapy and a Potential Solution - Genetic Engineering & Biotechnology News

Genetic Engineering additionally called genetic modification or genetic manipulation is the immediate control of a living being's genes using biotechnology. It is an arrangement of innovations used to change the hereditary forms of cells, including the exchange of qualities inside and across species limits to create enhanced or novel living beings.

Genetic Engineering has been connected in various fields including research, medicine, industrial biotechnology and agriculture. In research, GMOs are utilized to contemplate quality capacity and articulation through loss of function, gain of function, tracking and expression experiments. By thumping out genes responsible for specific conditions it is possible to create animal model organisms of human diseases. And in addition to producing hormones, immunizations and different drug genetic engineering can possibly fix hereditary diseases through quality treatment. Similar strategies that are utilized to create medications can likewise have mechanical applications, for example, producing enzymes for detergents, cheeses and different products.

The ascent of commercialised genetically modified crops has given a financial advantage to agriculturists in a wide range of nations, however, has additionally been the wellspring of a large portion of the debate encompassing the innovation. This has been available since its initial implementation, the primary field trials were destroyed by anti-GM activists. In spite of the fact that there is a logical accord that at presently accessible sustenance got from GM crops represents no more serious hazard to human wellbeing than regular nourishment, GM sustenance security is the main concern with critics.

Genetic engineering is the study of genes and the science of heredity. Genetic engineers or geneticists study living organisms ranging from human being to crops and even bacteria.

These professionals also conduct researches which is a major part of their work profile. The experiments are conducted to determine the origin and governing laws of a particular inherited trait. These traits include medical condition, diseases etc. The study is further used to seek our determinants responsible for the inherited trait.

Genetic engineers or Geneticists keep on finding ways to enhance their work profile depending on the place and organization they are working with. In manufacturing, these professionals will develop new pharmaceutical or agricultural products while in a medical setting, they advise patients on the diagnosed medical conditions that are inherited and also treat patients on the same.

Skill sets for Genetic engineers or Geneticists

Strong understanding of scientific methods and rules

complex problem solving and critical thinking

ability to use computer-aided design (CAD)

graphics or photo imaging

PERL, Python

word processing software programs

excellent mathematical, deductive and inductive reasoning, reading, writing, and oral comprehension skills

ability to use lasers spectrometers, light scattering equipment, binocular light compound microscopes, bench top centrifuges, or similar laboratory equipment

Typical responsibilities of a Genetic Engineering or Geneticist includes:

When a genetic engineer gains a year of experience, one of the regions they can indulge into is hereditary advising, which includes offering data, support and counsel on hereditary conditions to your patients.

An individual aspiring to pursue a professional degree in Genetic Engineering can begin the BTech course after his/her 10+2 Science with Physics, Chemistry, Maths and Biology.

Admission to BTech in Genetic Engineering is made through entrance tests conducted in-house by various universities or through the scores of national engineering entrance examination like JEE for IITs/NITs & CFTIs across the country.

Genetic Engineering professionals require a bachelors or masters degree in Genetic Engineering or Genetic Sciences for entry-level careers. In any case, a doctoral qualification is required for those looking for free research professions. Important fields of study in Genetic Engineering incorporate natural chemistry, biophysics or related fields.

Genetic Engineers require a solid comprehension of logical techniques and guidelines, and in addition complex critical thinking and basic reasoning aptitudes. Phenomenal scientific, deductive and inductive thinking aptitudes, and in addition perusing, composing, and oral cognizance abilities are additionally expected to work in this field.

A semester- wise breakup of the course is tabulated below

SEMESTER I

SEMESTER II

Mathematics 1

Mathematics 2

English

Material Science

Physics

Principles of Environmental Science

Chemistry

Biochemistry

Basic Engineering 1

Basic Engineering 2

-

Cell Biology

-

Value Education

SEMESTER III

SEMESTER IV

Enzyme Technology

Basic Molecular Techniques

Genetics & Cytogenetics

Molecular Biology

Immunology

Stoichiometry and Engineering Thermodynamics

Microbiology

Bio-press Principles

Mechanical Operations & heat Transfer

Biostatistics

German Language Phase 1/French Language Phase 1/Japanese Language Phase 1

German Language Phase 2/Japanese Language Phase 2/French Language Phase 2

-

SEMESTER V

SEMESTER VI

Advanced Molecular Techniques

Recombinant DNA Technology

Functional Genomics and Microarray Technology

Bioinformatics

Momentum Transfer

Chemical Reaction Engineering

Bioprocess Engineering

Gene Therapy

Biophysics

Biosensors and Biochips

Plant Tissue Culture and Transgenic Technology

-

Personality Development

-

SEMESTER VII

SEMESTER VIII

Bio-separation Technology

Project Work

Animal Cell Culture and Transgenic Technology

Bio-Safety, Bio-ethics, IPR & Patients

Nano-biotechnology in Healthcare

-

Stem Cell Biology

-

Aspirants who wish to join the engineering industry as a genetic engineer can apply for the following jobs profiles available:

JOB PROFILE

JOB DESCRIPTION

Genetic Engineer

They apply their knowledge ofengineering, biology, and biomechanical principles into the design, development, and evaluation of biological and health systems and products, such as artificial organs, prostheses, instrumentation, medical information systems, and health care and management.

Lecturer/Professor

They teach at undergraduate and graduate level in areas allocated and reviewed from time to time by the Head of Department.

Research Scientist

They are responsible for designing, undertaking and analyzing information from controlled laboratory-based investigations, experiments and trials.

Scientific/Medical Writer

The research, prepare and coordinate scientific publications. The medical writer is responsible for researching, writing and editing clinical/statistical reports and study protocols, and summarizing data from clinical studies.

Most of the engineering educational institutes shortlist candidates for admission Into BTech in Genetic Engineering course on the basis of engineering entrance exams. These entrance exams are either conducted at the national level like JEE or held in-house by various engineering institutes in the country.

Some of the popular engineering entrance examinations aspirants should consider appearing for admissions to UG and PG level Automobile engineering courses are:

Q. Which college is best for genetic engineering?

A. SRM University Chennai Tamil Nadu, Bharath University Chennai Tamil Nadu, Aryabhatta Knowledge University Patna Bihar, Jawaharlal Nehru Centre for Advanced Scientific Research Bangalore are some of the institutes offering genetic engineering

Q. Is Jee required for genetic engineering?

A. NITs and IITs across India does not offer genetic engineering. But there are 23 collages which take admission on the basis of JEE main

Q. What is the qualification for genetic engineering?

A. For admission to BTech Genetic Engineering course, the candidate is needed to have passed the Higher Secondary School Certificate (10+2) examination from a recognized Board of education with Biology, Physics and Chemistry as main subjects with a minimum aggregate score of 60%.

Q. Does IIT offer genetic engineering?

A. No, IIT directly does not offer genetic engineering. Candidates have to take Life Sciences in graduation or Biotechnology from any engineering college in India.

Link:
Genetic Engineering - Courses, Subjects, Eligibility ...

Can Vet J. 2011 May; 52(5): 544550.

Canadian Council on Animal Care, 1510-130 Albert Street, Ottawa, Ontario K1P 5G4 (Ormandy, Dale, Griffin); The University of British Columbia, Animal Welfare Program, 2357 Main Mall, Vancouver, British Columbia V6T 1Z4 (Ormandy)

The genetic engineering of animals has increased significantly in recent years, and the use of this technology brings with it ethical issues, some of which relate to animal welfare defined by the World Organisation for Animal Health as the state of the animalhow an animal is coping with the conditions in which it lives (1). These issues need to be considered by all stakeholders, including veterinarians, to ensure that all parties are aware of the ethical issues at stake and can make a valid contribution to the current debate regarding the creation and use of genetically engineered animals. In addition, it is important to try to reflect societal values within scientific practice and emerging technology, especially publicly funded efforts that aim to provide societal benefits, but that may be deemed ethically contentious. As a result of the extra challenges that genetically engineered animals bring, governing bodies have started to develop relevant policies, often calling for increased vigilance and monitoring of potential animal welfare impacts (2). Veterinarians can play an important role in carrying out such monitoring, especially in the research setting when new genetically engineered animal strains are being developed.

Several terms are used to describe genetically engineered animals: genetically modified, genetically altered, genetically manipulated, transgenic, and biotechnology-derived, amongst others. In the early stages of genetic engineering, the primary technology used was transgenesis, literally meaning the transfer of genetic material from one organism to another. However, with advances in the field, new technology emerged that did not necessarily require transgenesis: recent applications allow for the creation of genetically engineered animals via the deletion of genes, or the manipulation of genes already present. To reflect this progress and to include those animals that are not strictly transgenic, the umbrella term genetically engineered has been adopted into the guidelines developed by the Canadian Council on Animal Care (CCAC). For clarity, in the new CCAC guidelines on: genetically-engineered animals used in science (currently in preparation) the CCAC offers the following definition of a genetically engineered animal: an animal that has had a change in its nuclear or mitochondrial DNA (addition, deletion, or substitution of some part of the animals genetic material or insertion of foreign DNA) achieved through a deliberate human technological intervention. Those animals that have undergone induced mutations (for example, by chemicals or radiation as distinct from spontaneous mutations that naturally occur in populations) and cloned animals are also considered to be genetically engineered due to the direct intervention and planning involved in creation of these animals.

Cloning is the replication of certain cell types from a parent cell, or the replication of a certain part of the cell or DNA to propagate a particular desirable genetic trait. There are 3 types of cloning: DNA cloning, therapeutic cloning, and reproductive cloning (3). For the purposes of this paper, the term cloning is used to refer to reproductive cloning, as this is the most likely to lead to animal welfare issues. Reproductive cloning is used if the intention is to generate an animal that has the same nuclear DNA as another currently, or previously existing animal. The process used to generate this type of cloned animal is called somatic cell nuclear transfer (SCNT) (4).

During the development of the CCAC guidelines on: genetically- engineered animals used in science, some key ethical issues, including animal welfare concerns, were identified: 1) invasiveness of procedures; 2) large numbers of animals required; 3) unanticipated welfare concerns; and 4) how to establish ethical limits to genetic engineering (see Ethical issues of genetic engineering). The different applications of genetically engineered animals are presented first to provide context for the discussion.

Genetic engineering technology has numerous applications involving companion, wild, and farm animals, and animal models used in scientific research. The majority of genetically engineered animals are still in the research phase, rather than actually in use for their intended applications, or commercially available.

By inserting genes from sea anemone and jellyfish, zebrafish have been genetically engineered to express fluorescent proteins hence the commonly termed GloFish. GloFish began to be marketed in the United States in 2003 as ornamental pet fish; however, their sale sparked controversial ethical debates in California the only US state to prohibit the sale of GloFish as pets (5). In addition to the insertion of foreign genes, gene knock-out techniques are also being used to create designer companion animals. For example, in the creation of hypoallergenic cats some companies use genetic engineering techniques to remove the gene that codes for the major cat allergen Fel d1: (http://www.felixpets.com/technology.html).

Companion species have also been derived by cloning. The first cloned cat, CC, was created in 2002 (6). At the time, the ability to clone mammals was a coveted prize, and after just a few years scientists created the first cloned dog, Snuppy (7).

With the exception of a couple of isolated cases, the genetically engineered pet industry is yet to move forward. However, it remains feasible that genetically engineered pets could become part of day-to-day life for practicing veterinarians, and there is evidence that clients have started to enquire about genetic engineering services, in particular the cloning of deceased pets (5).

The primary application of genetic engineering to wild species involves cloning. This technology could be applied to either extinct or endangered species; for example, there have been plans to clone the extinct thylacine and the woolly mammoth (5). Holt et al (8) point out that, As many conservationists are still suspicious of reproductive technologies, it is unlikely that cloning techniques would be easily accepted. Individuals involved in field conservation often harbour suspicions that hi-tech approaches, backed by high profile publicity would divert funding away from their own efforts. However, cloning may prove to be an important tool to be used alongside other forms of assisted reproduction to help retain genetic diversity in small populations of endangered species.

As reviewed by Laible (9), there is an assorted range of agricultural livestock applications [for genetic engineering] aimed at improving animal productivity; food quality and disease resistance; and environmental sustainability. Productivity of farm animal species can be increased using genetic engineering. Examples include transgenic pigs and sheep that have been genetically altered to express higher levels of growth hormone (9).

Genetically engineered farm animals can be created to enhance food quality (9). For example, pigs have been genetically engineered to express the 12 fatty acid desaturase gene (from spinach) for higher levels of omega-3, and goats have been genetically engineered to express human lysozyme in their milk. Such advances may add to the nutritional value of animal-based products.

Farm species may be genetically engineered to create disease-resistant animals (9). Specific examples include conferring immunity to offspring via antibody expression in the milk of the mother; disruption of the virus entry mechanism (which is applicable to diseases such as pseudorabies); resistance to prion diseases; parasite control (especially in sheep); and mastitis resistance (particularly in cattle).

Genetic engineering has also been applied with the aim of reducing agricultural pollution. The best-known example is the EnviropigTM; a pig that is genetically engineered to produce an enzyme that breaks down dietary phosphorus (phytase), thus limiting the amount of phosphorus released in its manure (9).

Despite resistance to the commercialization of genetically engineered animals for food production, primarily due to lack of support from the public (10), a recent debate over genetically engineered AquAdvantageTM Atlantic salmon may result in these animals being introduced into commercial production (11).

Effort has also been made to generate genetically engineered farm species such as cows, goats, and sheep that express medically important proteins in their milk. According to Dyck et al (12), transgenic animal bioreactors represent a powerful tool to address the growing need for therapeutic recombinant proteins. In 2006, ATryn became the first therapeutic protein produced by genetically engineered animals to be approved by the Food and Drug Administration (FDA) of the United States. This product is used as a prophylactic treatment for patients that have hereditary antithrombin deficiency and are undergoing surgical procedures.

Biomedical applications of genetically engineered animals are numerous, and include understanding of gene function, modeling of human disease to either understand disease mechanisms or to aid drug development, and xenotransplantation.

Through the addition, removal, or alteration of genes, scientists can pinpoint what a gene does by observing the biological systems that are affected. While some genetic alterations have no obvious effect, others may produce different phenotypes that can be used by researchers to understand the function of the affected genes. Genetic engineering has enabled the creation of human disease models that were previously unavailable. Animal models of human disease are valuable resources for understanding how and why a particular disease develops, and what can be done to halt or reverse the process. As a result, efforts have focused on developing new genetically engineered animal models of conditions such as Alzheimers disease, amyotrophic lateral sclerosis (ALS), Parkinsons disease, and cancer. However, as Wells (13) points out: these [genetically engineered animal] models do not always accurately reflect the human condition, and care must be taken to understand the limitation of such models.

The use of genetically engineered animals has also become routine within the pharmaceutical industry, for drug discovery, drug development, and risk assessment. As discussed by Rudmann and Durham (14): Transgenic and knock out mouse models are extremely useful in drug discovery, especially when defining potential therapeutic targets for modifying immune and inflammatory responsesSpecific areas for which [genetically engineered animal models] may be useful are in screening for drug induced immunotoxicity, genotoxicity, and carcinogenicity, and in understanding toxicity related drug metabolizing enzyme systems.

Perhaps the most controversial use of genetically engineered animals in science is to develop the basic research on xenotrans-plantation that is, the transplant of cells, tissues, or whole organs from animal donors into human recipients. In relation to organ transplants, scientists have developed a genetically engineered pig with the aim of reducing rejection of pig organs by human recipients (15). This particular application of genetic engineering is currently at the basic research stage, but it shows great promise in alleviating the long waiting lists for organ transplants, as the number of people needing transplants currently far outweighs the number of donated organs. However, as a direct result of public consultation, a moratorium is currently in place preventing pig organ transplantation from entering a clinical trial phase until the public is assured that the potential disease transfer from pigs to humans can be satisfactorily managed (16). According to Health Canada, xenotransplantation is currently not prohibited in Canada. However, the live cells and organs from animal sources are considered to be therapeutic products (drugs or medical devices)No clinical trial involving xenotransplantation has yet been approved by Health Canada (see http://www.hc-sc.gc.ca for details).

Ethical issues, including concerns for animal welfare, can arise at all stages in the generation and life span of an individual genetically engineered animal. The following sections detail some of the issues that have arisen during the peer-driven guidelines development process and associated impact analysis consultations carried out by the CCAC. The CCAC works to an accepted ethic of animal use in science, which includes the principles of the Three Rs (Reduction of animal numbers, Refinement of practices and husbandry to minimize pain and distress, and Replacement of animals with non-animal alternatives wherever possible) (17). Together the Three Rs aim to minimize any pain and distress experienced by the animals used, and as such, they are considered the principles of humane experimental technique. However, despite the steps taken to minimize pain and distress, there is evidence of public concerns that go beyond the Three Rs and animal welfare regarding the creation and use of genetically engineered animals (18).

The generation of a new genetically engineered line of animals often involves the sacrifice of some animals and surgical procedures (for example, vasectomy, surgical embryo transfer) on others. These procedures are not unique to genetically engineered animals, but they are typically required for their production.

During the creation of new genetically engineered animals (particularly mammalian species) oocyte and blastocyst donor females may be induced to superovulate via intraperitoneal or subcutaneous injection of hormones; genetically engineered embryos may be surgically implanted to female recipients; males may be surgically vasectomized under general anesthesia and then used to induce pseudopregnancy in female embryo recipients; and all offspring need to be genotyped, which is typically performed by taking tissue samples, sometimes using tail biopsies or ear notching (19). However, progress is being made to refine the genetic engineering techniques that are applied to mammals (mice in particular) so that less invasive methods are feasible. For example, typical genetic engineering procedures require surgery on the recipient female so that genetically engineered embryos can be implanted and can grow to full term; however, a technique called non-surgical embryo transfer (NSET) acts in a similar way to artificial insemination, and removes the need for invasive surgery (20). Other refinements include a method referred to as deathless transgenesis, which involves the introduction of DNA into the sperm cells of live males and removes the need to euthanize females in order to obtain germ line transmission of a genetic alteration; and the use of polymerase chain reaction (PCR) for genotyping, which requires less tissue than Southern Blot Analysis (20).

Many of the embryos that undergo genetic engineering procedures do not survive, and of those that do survive only a small proportion (between 1% to 30%) carry the genetic alteration of interest (19). This means that large numbers of animals are produced to obtain genetically engineered animals that are of scientific value, and this contradicts efforts to minimize animal use. In addition, the advancement of genetic engineering technologies in recent years has lead to a rapid increase in the number and varieties of genetically engineered animals, particularly mice (21). Although the technology is continually being refined, current genetic engineering techniques remain relatively inefficient, with many surplus animals being exposed to harmful procedures. One key refinement and reduction effort is the preservation of genetically engineered animal lines through the freezing of embryos or sperm (cryopreservation), which is particularly important for those lines with the potential to experience pain and distress (22).

As mentioned, the number of research projects creating and/or using genetically engineered animals worldwide has increased in the past decade (21). In Canada, the CCACs annual data on the numbers of animals used in science show an increase in Category D procedures (procedures with the potential to cause moderate to severe pain and distress) at present the creation of a new genetically engineered animal line is a Category D procedure (23). The data also show an increase in the use of mice (24), which are currently the most commonly used species for genetic engineering, making up over 90% of the genetically engineered animals used in research and testing (21). This rise in animal use challenges the Three Rs principle of Reduction (17). It has been reasoned that once created, the use of genetically engineered animals will reduce the total number of animals used in any given experiment by providing novel and more accurate animal models, especially in applications such as toxicity testing (25). However, the greater variety of available applications, and the large numbers of animals required for the creation and maintenance of new genetically engineered strains indicate that there is still progress to be made in implementation of the Three Rs principle of Reduction in relation to the creation and use of genetically engineered animals (21).

Little data has been collected on the net welfare impacts to genetically engineered animals or to those animals required for their creation, and genetic engineering techniques have been described as both unpredictable and inefficient (19). The latter is due, in part, to the limitations in controlling the integration site of foreign DNA, which is inherent in some genetic engineering techniques (such as pro-nuclear microinjection). In such cases, scientists may generate several independent lines of genetically engineered animals that differ only in the integration site (26), thereby further increasing the numbers of animals involved. This conflicts with efforts to adhere to the principles of the Three Rs, specifically Reduction. With other, more refined techniques that allow greater control of DNA integration (for example, gene targeting), unexpected outcomes are attributed to the unpredictable interaction of the introduced DNA with host genes. These interactions also vary with the genetic background of the animal, as has frequently been observed in genetically engineered mice (27). Interfering with the genome by inserting or removing fragments of DNA may result in alteration of the animals normal genetic homeostasis, which can be manifested in the behavior and well-being of the animals in unpredictable ways. For example, many of the early transgenic livestock studies produced animals with a range of unexpected side effects including lameness, susceptibility to stress, and reduced fertility (9).

A significant limitation of current cloning technology is the prospect that cloned offspring may suffer some degree of abnormality. Studies have revealed that cloned mammals may suffer from developmental abnormalities, including extended gestation; large birth weight; inadequate placental formation; and histological effects in organs and tissues (for example, kidneys, brain, cardiovascular system, and muscle). One annotated review highlights 11 different original research articles that documented the production of cloned animals with abnormalities occurring in the developing embryo, and suffering for the newborn animal and the surrogate mother (28).

Genetically engineered animals, even those with the same gene manipulation, can exhibit a variety of phenotypes; some causing no welfare issues, and some causing negative welfare impacts. It is often difficult to predict the effects a particular genetic modification can have on an individual animal, so genetically engineered animals must be monitored closely to mitigate any unanticipated welfare concerns as they arise. For newly created genetically engineered animals, the level of monitoring needs to be greater than that for regular animals due to the lack of predictability. Once a genetically engineered animal line is established and the welfare concerns are known, it may be possible to reduce the levels of monitoring if the animals are not exhibiting a phenotype that has negative welfare impacts. To aid this monitoring process, some authors have called for the implementation of a genetically engineered animal passport that accompanies an individual animal and alerts animal care staff to the particular welfare needs of that animal (29). This passport document is also important if the intention is to breed from the genetically engineered animal in question, so the appropriate care and husbandry can be in place for the offspring.

With progress in genetic engineering techniques, new methods (30,31) may substantially reduce the unpredictability of the location of gene insertion. As a result, genetic engineering procedures may become less of a welfare concern over time.

As pointed out by Lassen et al (32), Until recently the main limits [to genetic engineering] were technical: what it is possible to do. Now scientists are faced with ethical limits as well: what it is acceptable to do (emphasis theirs). Questions regarding whether it is acceptable to make new transgenic animals go beyond consideration of the Three Rs, animal health, and animal welfare, and prompt the discussion of concepts such as intrinsic value, integrity, and naturalness (33).

When discussing the nature of an animal, it may be useful to consider the Aristotelian concept of telos, which describes the essence and purpose of a creature (34). Philosopher Bernard Rollin applied this concept to animal ethics as follows: Though [telos] is partially metaphysical (in defining a way of looking at the world), and partially empirical (in that it can and will be deepened and refined by increasing empirical knowledge), it is at root a moral notion, both because it is morally motivated and because it contains the notion of what about an animal we ought to at least try to respect and accommodate (emphasis Rollins) (34). Rollin has also argued that as long as we are careful to accommodate the animals interests when we alter an animals telos, it is morally permissible. He writes, given a telos, we should respect the interests which flow from it. This principle does not logically entail that we cannot modify the telos and thereby generate different or alternative interests (34).

Views such as those put forward by Rollin have been argued against on the grounds that health and welfare (or animal interests) may not be the only things to consider when establishing ethical limits. Some authors have made the case that genetic engineering requires us to expand our existing notions of animal ethics to include concepts of the intrinsic value of animals (35), or of animal integrity or dignity (33). Veerhoog argues that, we misuse the word telos when we say that human beings can change the telos of an animal or create a new telos that is to say animals have intrinsic value, which is separate from their value to humans. It is often on these grounds that people will argue that genetic engineering of animals is morally wrong. For example, in a case study of public opinion on issues related to genetic engineering, participants raised concerns about the nature of animals and how this is affected (negatively) by genetic engineering (18).

An alternative view put forward by Schicktanz (36) argues that it is the human-animal relationship that may be damaged by genetic engineering due to the increasingly imbalanced distribution of power between humans and animals. This imbalance is termed asymmetry and it is raised alongside ambivalence as a concern regarding modern human-animal relationships. By using genetically engineered animals as a case study, Schicktanz (36) argues that genetic engineering presents a troubling shift for all human-animal relationships.

Opinions regarding whether limits can, or should, be placed on genetic engineering are often dependent on peoples broader worldview. For some, the genetic engineering of animals may not put their moral principles at risk. For example, this could perhaps be because genetic engineering is seen as a logical continuation of selective breeding, a practice that humans have been carrying out for years; or because human life is deemed more important than animal life. So if genetic engineering creates animals that help us to develop new human medicine then, ethically speaking, we may actually have a moral obligation to create and use them; or because of an expectation that genetic engineering of animals can help reduce experimental animal numbers, thus implementing the accepted Three Rs framework.

For others, the genetic engineering of animals may put their moral principles at risk. For example costs may always be seen to outweigh benefits because the ultimate cost is the violation of species integrity and disregard for the inherent value of animals. Some may view telos as something that cannot or should not be altered, and therefore altering the telos of an animal would be morally wrong. Some may see genetic engineering as exaggerating the imbalance of power between humans and animals, whilst others may fear that the release of genetically engineered animals will upset the natural balance of the ecosystem. In addition, there may be those who feel strongly opposed to certain applications of genetic engineering, but more accepting of others. For example, recent evidence suggests that people may be more accepting of biomedical applications than those relating to food production (37).

Such underlying complexity of views regarding genetic engineering makes the setting of ethical limits difficult to achieve, or indeed, even discuss. However, progress needs to be made on this important issue, especially for those genetically engineered species that are intended for life outside the research laboratory, where there may be less careful oversight of animal welfare. Consequently, limits to genetic engineering need to be established using the full breadth of public and expert opinion. This highlights the importance for veterinarians, as animal health experts, to be involved in the discussion.

Genetic engineering also brings with it concerns over intellectual property, and patenting of created animals and/or the techniques used to create them. Preserving intellectual property can breed a culture of confidentiality within the scientific community, which in turn limits data and animal sharing. Such limits to data and animal sharing may create situations in which there is unnecessary duplication of genetically engineered animal lines, thereby challenging the principle of Reduction. Indeed, this was a concern that was identified in a recent workshop on the creation and use of genetically engineered animals in science (20).

It should be noted that no matter what the application of genetically engineered animals, there are restrictions on the methods of their disposal once they have been euthanized. The reason for this is to restrict the entry of genetically engineered animal carcasses into the natural ecosystem until the long-term effects and risks are better understood. Environment Canada (http://www.ec.gc.ca/) and Health Canada (http://www.hc-sc.gc.ca/) offer specific guidelines in this regard.

As genetically engineered animals begin to enter the commercial realm, it will become increasingly important for veterinarians to inform themselves about any special care and management required by these animals. As animal health professionals, veterinarians can also make important contributions to policy discussions related to the oversight of genetic engineering as it is applied to animals, and to regulatory proceedings for the commercial use of genetically engineered animals.

It is likely that public acceptance of genetically engineered animal products will be an important step in determining when and what types of genetically engineered animals will appear on the commercial market, especially those animals used for food production. Veterinarians may also be called on to inform the public about genetic engineering techniques and any potential impacts to animal welfare and food safety. Consequently, for the discussion regarding genetically engineered animals to progress effectively, veterinarians need to be aware of the current context in which genetically engineered animals are created and used, and to be aware of the manner in which genetic engineering technology and the animals derived from it may be used in the future.

Genetic engineering techniques can be applied to a range of animal species, and although many genetically engineered animals are still in the research phase, there are a variety of intended applications for their use. Although genetic engineering may provide substantial benefits in areas such as biomedical science and food production, the creation and use of genetically engineered animals not only challenge the Three Rs principles, but may also raise ethical issues that go beyond considerations of animal health, animal welfare, and the Three Rs, opening up issues relating to animal integrity and/or dignity. Consequently, even if animal welfare can be satisfactorily safeguarded, intrinsic ethical concerns about the genetic engineering of animals may be cause enough to restrict certain types of genetically engineered animals from reaching their intended commercial application. Given the complexity of views regarding genetic engineering, it is valuable to involve all stakeholders in discussions about the applications of this technology.

The authors thank the members of the Canadian Veterinary Medicine Association Animal Welfare Committee for their comments on the draft, and Dr. C. Schuppli for her insight on how the issues discussed may affect veterinarians.

Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (gro.vmca-amvc@nothguorbh) for additional copies or permission to use this material elsewhere.

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Genetic engineering of animals: Ethical issues, including ...

Have you heard? A revolution has seized the scientific community. Within only a few years, research labs worldwide have adopted a new technology that facilitates making specific changes in the DNA of humans, other animals, and plants. Compared to previous techniques for modifying DNA, this new approach is much faster and easier. This technology is referred to as CRISPR, and it has changed not only the way basic research is conducted, but also the way we can now think about treating diseases [1,2].

CRISPR is an acronym for Clustered Regularly Interspaced Short Palindromic Repeat. This name refers to the unique organization of short, partially palindromic repeated DNA sequences found in the genomes of bacteria and other microorganisms. While seemingly innocuous, CRISPR sequences are a crucial component of the immune systems [3] of these simple life forms. The immune system is responsible for protecting an organisms health and well-being. Just like us, bacterial cells can be invaded by viruses, which are small, infectious agents. If a viral infection threatens a bacterial cell, the CRISPR immune system can thwart the attack by destroying the genome of the invading virus [4]. The genome of the virus includes genetic material that is necessary for the virus to continue replicating. Thus, by destroying the viral genome, the CRISPR immune system protects bacteria from ongoing viral infection.

Figure 1 ~ The steps of CRISPR-mediated immunity. CRISPRs are regions in the bacterial genome that help defend against invading viruses. These regions are composed of short DNA repeats (black diamonds) and spacers (colored boxes). When a previously unseen virus infects a bacterium, a new spacer derived from the virus is incorporated amongst existing spacers. The CRISPR sequence is transcribed and processed to generate short CRISPR RNA molecules. The CRISPR RNA associates with and guides bacterial molecular machinery to a matching target sequence in the invading virus. The molecular machinery cuts up and destroys the invading viral genome. Figure adapted from Molecular Cell 54, April 24, 2014 [5].

Interspersed between the short DNA repeats of bacterial CRISPRs are similarly short variable sequences called spacers (FIGURE 1). These spacers are derived from DNA of viruses that have previously attacked the host bacterium [3]. Hence, spacers serve as a genetic memory of previous infections. If another infection by the same virus should occur, the CRISPR defense system will cut up any viral DNA sequence matching the spacer sequence and thus protect the bacterium from viral attack. If a previously unseen virus attacks, a new spacer is made and added to the chain of spacers and repeats.

The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps [5]:

Step 1) Adaptation DNA from an invading virus is processed into short segments that are inserted into the CRISPR sequence as new spacers.

Step 2) Production of CRISPR RNA CRISPR repeats and spacers in the bacterial DNA undergo transcription, the process of copying DNA into RNA (ribonucleic acid). Unlike the double-chain helix structure of DNA, the resulting RNA is a single-chain molecule. This RNA chain is cut into short pieces called CRISPR RNAs.

Step 3) Targeting CRISPR RNAs guide bacterial molecular machinery to destroy the viral material. Because CRISPR RNA sequences are copied from the viral DNA sequences acquired during adaptation, they are exact matches to the viral genome and thus serve as excellent guides.

The specificity of CRISPR-based immunity in recognizing and destroying invading viruses is not just useful for bacteria. Creative applications of this primitive yet elegant defense system have emerged in disciplines as diverse as industry, basic research, and medicine.

In Industry

The inherent functions of the CRISPR system are advantageous for industrial processes that utilize bacterial cultures. CRISPR-based immunity can be employed to make these cultures more resistant to viral attack, which would otherwise impede productivity. In fact, the original discovery of CRISPR immunity came from researchers at Danisco, a company in the food production industry [2,3]. Danisco scientists were studying a bacterium called Streptococcus thermophilus, which is used to make yogurts and cheeses. Certain viruses can infect this bacterium and damage the quality or quantity of the food. It was discovered that CRISPR sequences equipped S. thermophilus with immunity against such viral attack. Expanding beyond S. thermophilus to other useful bacteria, manufacturers can apply the same principles to improve culture sustainability and lifespan.

In the Lab

Beyond applications encompassing bacterial immune defenses, scientists have learned how to harness CRISPR technology in the lab [6] to make precise changes in the genes of organisms as diverse as fruit flies, fish, mice, plants and even human cells. Genes are defined by their specific sequences, which provide instructions on how to build and maintain an organisms cells. A change in the sequence of even one gene can significantly affect the biology of the cell and in turn may affect the health of an organism. CRISPR techniques allow scientists to modify specific genes while sparing all others, thus clarifying the association between a given gene and its consequence to the organism.

Rather than relying on bacteria to generate CRISPR RNAs, scientists first design and synthesize short RNA molecules that match a specific DNA sequencefor example, in a human cell. Then, like in the targeting step of the bacterial system, this guide RNA shuttles molecular machinery to the intended DNA target. Once localized to the DNA region of interest, the molecular machinery can silence a gene or even change the sequence of a gene (Figure 2)! This type of gene editing can be likened to editing a sentence with a word processor to delete words or correct spelling mistakes. One important application of such technology is to facilitate making animal models with precise genetic changes to study the progress and treatment of human diseases.

Figure 2 ~ Gene silencing and editing with CRISPR. Guide RNA designed to match the DNA region of interest directs molecular machinery to cut both strands of the targeted DNA. During gene silencing, the cell attempts to repair the broken DNA, but often does so with errors that disrupt the geneeffectively silencing it. For gene editing, a repair template with a specified change in sequence is added to the cell and incorporated into the DNA during the repair process. The targeted DNA is now altered to carry this new sequence.

In Medicine

With early successes in the lab, many are looking toward medical applications of CRISPR technology. One application is for the treatment of genetic diseases. The first evidence that CRISPR can be used to correct a mutant gene and reverse disease symptoms in a living animal was published earlier this year [7]. By replacing the mutant form of a gene with its correct sequence in adult mice, researchers demonstrated a cure for a rare liver disorder that could be achieved with a single treatment. In addition to treating heritable diseases, CRISPR can be used in the realm of infectious diseases, possibly providing a way to make more specific antibiotics that target only disease-causing bacterial strains while sparing beneficial bacteria [8]. A recent SITN Waves article discusses how this technique was also used to make white blood cells resistant to HIV infection [9].

Of course, any new technology takes some time to understand and perfect. It will be important to verify that a particular guide RNA is specific for its target gene, so that the CRISPR system does not mistakenly attack other genes. It will also be important to find a way to deliver CRISPR therapies into the body before they can become widely used in medicine. Although a lot remains to be discovered, there is no doubt that CRISPR has become a valuable tool in research. In fact, there is enough excitement in the field to warrant the launch of several Biotech start-ups that hope to use CRISPR-inspired technology to treat human diseases [8].

Ekaterina Pak is a Ph.D. student in the Biological and Biomedical Sciences program at Harvard Medical School.

1. Palca, J. A CRISPR way to fix faulty genes. (26 June 2014) NPR < http://www.npr.org/blogs/health/2014/06/26/325213397/a-crispr-way-to-fix-faulty-genes> [29 June 2014]

2. Pennisi, E. The CRISPR Craze. (2013) Science, 341 (6148): 833-836.

3. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A., and Horvath, P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 17091712.

4. Brouns, S.J., Jore, M.M., Lundgren, M., Westra, E.R., Slijkhuis, R.J., Snijders, A.P., Dickman, M.J., Makarova, K.S., Koonin, E.V., and van der Oost, J. (2008). Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960964.

5. Barrangou, R. and Marraffini, L. CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity (2014). Molecular Cell 54, 234-244.

6. Jinkek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. (2012) 337(6096):816-21.

7. CRISPR reverses disease symptoms in living animals for first time. (31 March 2014). Genetic Engineering and Biotechnology News. <http://www.genengnews.com/gen-news-highlights/crispr-reverses-disease-symptoms-in-living-animals-for-first-time/81249682/> [27 July 2014]

8. Pollack, A. A powerful new way to edit DNA. (3 March 2014). NYTimes < http://www.nytimes.com/2014/03/04/health/a-powerful-new-way-to-edit-dna.html?_r=0> [16 July 2014]

9. Gene editing technique allows for HIV resistance? <http://sitn.hms.harvard.edu/flash/waves/2014/gene-editing-technique-allows-for-hiv-resistance/> [13 June 2014]

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