Category Archives: Genetic Engineering

This year, TechCrunch is proudly hosting the Extreme Tech Challenge Global Finals on July 22. The event is among the worlds largest purpose-driven startup competitions that are aiming to solve global challenges based on the United Nations 17 sustainability goals.

If you want to catch an array of innovative startups across a range of categories, all of them showcasing what theyre building, you wont want to miss our must-see pitch-off competition.

You can also catch feature panels hosted by TechCrunch editors, including one of the most highly anticipated discussions of the event, a talk on going green with guest speakers Shilpi Kumar, Jenny Rooke, and Albert Wenger, all of whom are actively investing in climate startups that are targeting big opportunities

Shilpi Kumar is a partner with Urban Us, an investment platform focused on urban tech and climate solutions. She previously led go-to-market and early sales efforts at Filament, a startup focused on deploying secure wireless networks for connected physical assets. As an investor, Shilpi has also focused on hardware, mobility, energy, IoT, and robotics, having worked previously for VTF Capital, First Round Capital, and Village Global.

Jenny Rooke is the founder and managing director of Genoa Ventures, but Rooke has been deploying capital into innovative life sciences opportunities for years, including at Fidelity Biosciences and later the Gates Foundation, where she helped managed more than $250 million in funding, funneling some of that capital into genetic engineering, diagnostics, and synthetic biology startups. Rooke began independently investing under the brand 5 Prime Ventures, ultimately establishing among the largest life sciences syndicates on AngelList before launching Genoa.

Last but not least, Albert Wenger, has been a managing partner at Union Square Ventures for more than 13 years. Before joining USV, Albert was the president of del.icio.us through the companys sale to Yahoo and an angel investor, including writing early checks to Etsy and Tumblr. He previously founded or co-founded several companies, including a management consulting firm and an early hosted data analytics company. Among his investments today is goTenna, a company trying to advance universal access to connectivity by building a scalable mobile mesh network.

Sustainability is the key to our planets future and our survival, but its also going to be incredibly lucrative and a major piece of our world economy. Hear from these seasoned investors about how VCs and startups alike are thinking about Greentech and how that will evolve in the coming years.

Join us on July 22 to find out how the most innovative startups are working to solve some of the worlds biggest problems. And best of all, tickets are free book yours today!

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Hear top VCs Albert Wegner, Jenny Rooke, and Shilpi Kumar talk green bets at the Extreme Tech Challenge finals - TechCrunch

For Baric, that research started in the late 1990s. Coronaviruses were then considered low risk, but Barics studies on the genetics that allowed viruses to enter human cells convinced him that some might be just a few mutations away from jumping the species barrier.

That hunch was confirmed in 200203, when SARS broke out in southern China, infecting 8,000 people. As bad as that was, Baric says, we dodged a bullet with SARS. The disease didnt spread from one person to another until about a day after severe symptoms began to appear, making it easier to corral through quarantines and contact tracing. Only 774 people died in that outbreak, but if it had been transmitted as easily as SARS-CoV-2, we would have had a pandemic with a 10% mortality rate, Baric says. Thats how close humanity came.

As tempting as it was to write off SARS as a one-time event, in 2012 MERS emerged and began infecting people in the Middle East. For me personally, that was a wake-up call that the animal reservoirs must have many, many more strains that are poised for cross-species movement, says Baric.

By then, examples of such dangers were already being discovered by Shis team, which had spent years sampling bats in southern China to locate the origin of SARS. The project was part of a global viral surveillance effort spearheaded by the US nonprofit EcoHealth Alliance. The nonprofitwhich has an annual income of over $16 million, more than 90% from government grantshas its office in New York but partners with local research groups in other countries to do field and lab work. The WIV was its crown jewel, and Peter Daszak, president of EcoHealth Alliance, has been a coauthor with Shi on most of her key papers.

By taking thousands of samples from guano, fecal swabs, and bat tissue, and searching those samples for genetic sequences similar to SARS, Shis team began to discover many closely related viruses. In a cave in Yunnan Province in 2011 or 2012, they discovered the two closest, which they named WIV1 and SHC014.

Shi managed to culture WIV1 in her lab from a fecal sample and show that it could directly infect human cells, proving that SARS-like viruses ready to leap straight from bats to humans already lurked in the natural world. This showed, Daszak and Shi argued, that bat coronaviruses were a substantial global threat. Scientists, they said, needed to find them, and study them, before they found us.

Many of the other viruses couldnt be grown, but Barics system provided a way to rapidly test their spikes by engineering them into similar viruses. When the chimera he made using SHC014 proved able to infect human cells in a dish, Daszak told the press that these revelations should move this virus from a candidate emerging pathogen to a clear and present danger.

To others, it was the perfect example of the unnecessary dangers of gain-of-function science. The only impact of this work is the creation, in a lab, of a new, non-natural risk, the Rutgers microbiologist Richard Ebright, a longtime critic of such research, told Nature.

To Baric, the situation was more nuanced. Although his creation might be more dangerous than the original mouse-adapted virus hed used as a backbone, it was still wimpy compared with SARScertainly not the supervirus Senator Paul would later suggest.

In the end, the NIH clampdown never had teeth. It included a clause granting exceptions if head of funding agency determines research is urgently necessary to protect public health or national security. Not only were Barics studies allowed to move forward, but so were all studies that applied for exemptions. The funding restrictions were lifted in 2017 and replaced with a more lenient system.

If the NIH was looking for a scientist to make regulators comfortable with gain-of-function research, Baric was the obvious choice. For years hed insisted on extra safety steps, and he took pains to point these out in his 2015 paper, as if modeling the way forward.

The CDC recognizes four levels of biosafety and recommends which pathogens should be studied at which level. Biosafety level 1 is for nonhazardous organisms and requires virtually no precautions: wear a lab coat and gloves as needed. BSL-2 is for moderately hazardous pathogens that are already endemic in the area, and relatively mild interventions are indicated: close the door, wear eye protection, dispose of waste materials in an autoclave. BSL-3 is where things get serious. Its for pathogens that can cause serious disease through respiratory transmission, such as influenza and SARS, and the associated protocols include multiple barriers to escape. Labs are walled off by two sets of self-closing, locking doors; air is filtered; personnel use full PPE and N95 masks and are under medical surveillance. BSL-4 is for the baddest of the baddies, such as Ebola and Marburg: full moon suits and dedicated air systems are added to the arsenal.

There are no enforceable standards of what you should and shouldnt do. Its up to the individual countries, institutions, and scientists.

In Barics lab, the chimeras were studied at BSL-3, enhanced with additional steps like Tyvek suits, double gloves, and powered-air respirators for all workers. Local first-responder teams participated in regular drills to increase their familiarity with the lab. All workers were monitored for infections, and local hospitals had procedures in place to handle incoming scientists. It was probably one of the safest BSL-3 facilities in the world. That still wasnt enough to prevent a handful of errors over the years: some scientists were even bitten by virus-carrying mice. But no infections resulted.

In 2014, the NIH awarded a five-year, $3.75 million grant to EcoHealth Alliance to study the risk that more bat-borne coronaviruses would emerge in China, using the same kind of techniques Baric had pioneered. Some of that work was to be subcontracted to the Wuhan Institute of Virology.

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Inside the risky bat-virus engineering that links America to Wuhan - MIT Technology Review

ROME, July 2 A United Nations-backed scientific research centre hasteamedupwith an Italian tech firm to explore whetherlaserlight can be used to kill coronavirusparticles suspended in the air and help keep indoor spaces safe.

The joint effort between the International Centre for Genetic Engineering and Biotechnology (ICGEB) of Trieste, a city in the north ofItaly, and the nearby Eltech K-Lasercompany, was launched last year as COVID-19 was battering the country.

They created a device that forces air through a sterilization chamber which contains alaserbeam filter that pulverizesviruses and bacteria.

I thoughtlasers were more for a shaman rather than a doctor but I have had to change my mind. The device proved able to kill theviruses in less than 50 milliseconds, said Serena Zacchigna, groupleader for Cardiovascular Biology at the ICGEB.

Healthy indoor environments with a substantially reduced pathogen count are deemed essential for public health in the post COVID-19 crisis, a respiratory infection which has caused more than four million deaths worldwide in barely 18 months.

Zacchigna hookedupwith Italian engineer Francesco Zanata, the founder of Eltech K-Laser, a firm specialised in medicallasers whose products are used by sports stars to treat muscle inflammation and fractures.

Some experts have warned against the possible pitfalls of using light-based technologies to attack thevirusthat causes COVID-19.

A study published by the Journal of Photochemistry & Photobiology in November 2020 highlighted concerns ranging from potential cancer risks to the cost of expensive light sources.

But Zacchigna and Zanata dismissed any health issues, saying thelasernever comes into contact with human skin.

Our device uses nature against nature. It is 100% safe for people and almost fully recyclable, Zanata told Reuters.

The technology, however, does not eliminateviruses and bacteria when they drop from the air onto surfaces or the floor. Nor can it prevent direct contagion when someone who is infected sneezes or talks loudly in the proximity of someone else.

Eltech K-Laserhas received a patent from Italian authorities and is seeking to extend this globally.

The portable version of the invention is some 1.8 metres (5.9 ft) high and weighs about 55 lb. The company said the technology can also be placed within air-conditioning units.

In the meantime, the first potential customers are liningup, including Germanys EcoCare, a service provider of testing and vaccination solutions.

The company aims to license the technology for German and UAE markets, an EcoCare spokesperson said in an email to Reuters.

Excerpt from:
Science, industry team up in Italy to zap COVID with laser - New York Post

A rendering of an air purifier prototype developed by Italian tech company Eltech K-laser is seen in this image obtained by Reuters on June 30, 2021. Eltech K-Laser/Handout via REUTERS

ROME, July 2 (Reuters) - A United Nations-backed scientific research centre has teamed up with an Italian tech firm to explore whether laser light can be used to kill coronavirus particles suspended in the air and help keep indoor spaces safe.

The joint effort between the International Centre for Genetic Engineering and Biotechnology (ICGEB) of Trieste, a city in the north of Italy, and the nearby Eltech K-Laser company, was launched last year as COVID-19 was battering the country.

They created a device that forces air through a sterilization chamber which contains a laser beam filter that pulverizes viruses and bacteria.

"I thought lasers were more for a shaman rather than a doctor but I have had to change my mind. The device proved able to kill the viruses in less than 50 milliseconds," said Serena Zacchigna, group leader for Cardiovascular Biology at the ICGEB.

Healthy indoor environments with a substantially reduced pathogen count are deemed essential for public health in the post COVID-19 crisis, a respiratory infection which has caused more than four million deaths worldwide in barely 18 months.

Zacchigna hooked up with Italian engineer Francesco Zanata, the founder of Eltech K-Laser, a firm specialised in medical lasers whose products are used by sports stars to treat muscle inflammation and fractures.

Some experts have warned against the possible pitfalls of using light-based technologies to attack the virus that causes COVID-19.

A study published by the Journal of Photochemistry & Photobiology in November 2020 highlighted concerns ranging from potential cancer risks to the cost of expensive light sources.

But Zacchigna and Zanata dismissed any health issues, saying the laser never comes into contact with human skin.

"Our device uses nature against nature. It is 100% safe for people and almost fully recyclable," Zanata told Reuters.

The technology, however, does not eliminate viruses and bacteria when they drop from the air onto surfaces or the floor. Nor can it prevent direct contagion when someone who is infected sneezes or talks loudly in the proximity of someone else.

Eltech K-Laser has received a patent from Italian authorities and is seeking to extend this globally.

The portable version of the invention is some 1.8 metres (5.9 ft) high and weighs about 25 kg (55 lb). The company said the technology can also be placed within air-conditioning units.

In the meantime, the first potential customers are lining up, including Germany's EcoCare, a service provider of testing and vaccination solutions.

"The company aims to license the technology for German and UAE markets," an EcoCare spokesperson said in an email to Reuters.

Reporting by Giselda Vagnoni; Editing by Crispian Balmer, William Maclean

Our Standards: The Thomson Reuters Trust Principles.

View original post here:
Science, industry team up in Italy to zap virus with laser - Reuters

Amid the devastating Covid-19 pandemic, two researchers are proposing a drastic way to stop future pandemics: using a technology called a gene drive to rewrite the DNA of bats to prevent them from becoming infected with coronaviruses.

The scientists aim to block spillover events, in which viruses jump from infected bats to humans one suspected source of the coronavirus that causes Covid. Spillover events are thought to have sparked other coronavirus outbreaks as well, including SARS-1 in the early 2000s and Middle East respiratory syndrome (MERS).

This appears to be the first time that scientists have proposed using the still-nascent gene drive technology to stop outbreaks by rendering bats immune to coronaviruses, though other teams are investigating its use to stop mosquitoes and mice from spreading malaria and Lyme disease.

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The scientists behind the proposal realize they face enormous technical, societal, and political obstacles, but want to spark a fresh conversation about additional ways to control diseases that are emerging with growing frequency.

With a very high probability, we are going to see this over and over again, argues entrepreneur and computational geneticist Yaniv Erlich of the Interdisciplinary Center Herzliya in Israel, who is one of two authors of the proposal, titled Preventing COVID-59.

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Maybe our kids will not benefit, maybe our grandchildren will benefit, but if this approach works, we could deploy the same strategy against many types of viruses, Erlich told STAT.

As the Covid-19 pandemic has killed more than 3.9 million people and triggered $16 trillion in economic losses, scientists, public health officials, ecologists, and many others have called for deeper investments in longstanding pandemic prevention measures.

Such measures include boosting global health funding, reducing poverty and health inequity, strengthening disease surveillance networks and community education, preventing deforestation, controlling the wildlife trade, and beefing up investments in infectious disease diagnostics, treatments, and vaccines.

Erlich and his co-author, immunologist Daniel Douek at the U.S. National Institute of Allergy and Infectious Diseases, now propose an additional measure: creating a gene drive to render wild horseshoe bats immune to the types of coronavirus infections that are thought to have triggered the SARS, MERS, and Covid-19 pandemics. They shared the proposal Wednesday on the Github publishing and code-sharing platform.

Though there is heated debate about whether the Covid-19 virus originated in a lab, most scientists say the virus is most likely to have originated in wild animals. There is strong evidence, for instance, that horseshoe bats carry the coronavirus that caused the SARS outbreak.

A gene drive is a technique for turbocharging evolution and spreading new traits throughout a species faster than they would spread through natural selection. It involves using a gene editing technology such as CRISPR to modify an organisms genome so that it passes a new trait to its offspring and throughout the species.

The idea of making a gene drive in bats faces such enormous scientific, technical, social, and economic obstacles that scientists interviewed by STAT called it folly, far-fetched, and concerning. Among other objections, they worried about unintended consequences with so radically tampering with nature.

We have other ways of preventing future Covid-19 outbreaks, argued Natalie Kofler, a trained molecular biologist and bioethicist and founder of Editing Nature, a group focused on inclusive decision-making about genetic technologies.

We need to be thinking about changing the unhealthy relationship of humans and nature, not to gene drive a wild animal so that we can continue our irresponsible and unsustainable behavior that is going to come back to bite us in the ass in the future.

Coming from anyone else, the idea might be laughed off.

But Erlich has a reputation as a visionary. In 2014, for instance, he and another scientist predicted that genetic genealogy databases might one day be used to reveal peoples identities. Four years later, that happened, when law enforcement officials used the method to identify a former California police officer as the notorious Golden State Killer. Erlich has since become chief scientific officer of the genetic genealogy company MyHeritage and he is also founder of a biotech startup, Eleven Therapeutics.

Now, Erlich says, its worth thinking about how a gene drive could work in bats.

Erlich proposes to modify bat genomes so that they would block coronavirus infections. He would create a genetic element, called a shRNA, that targets and destroys coronaviruses. He would then use CRISPR to insert this element into the bat genome. The insertion would also contain a component that pushes bats to preferentially pass the shRNA to their offspring, so that entire bat populations would soon resist coronavirus infection.

Its almost like creating a self-propagating vaccine in these bats, Erlich said.

The idea is intriguing, said geneticist and molecular engineer George Church of the Wyss Institute for Biologically Inspired Engineering at Harvard University.

Most of the proposals Ive heard involving gene drives have seemed quite attractive, and this is probably the most attractive, he said.

Creating a gene drive in bats would be enormously difficult, and perhaps impossible, other scientists say. Researchers have created gene drives in mosquitoes and mice in the lab, but none has been released in the wild. The most advanced gene drive projects intended for field use involve modifying mosquitoes to prevent the spread of malaria and attempting to engineer mice to stop them from causing ecological damage.

But its been difficult to engineer effective gene drives in mammals. Developmental geneticist Kim Cooper and her team at the University of California, San Diego, engineered a gene drive that spread a genetic variant through 72% of mouse offspring in her lab. That isnt efficient enough to quickly spread the desired trait in the wild.

Whats more, creating a gene drive in bats would be much harder than it is in mice, because bat researchers lack the genetic tools available in mice, said Paul Thomas, a developmental geneticist at the University of Adelaide in Australia, who is trying to engineer mouse gene drives.

And unlike mice, which can breed at 6 to 8 weeks of age, bats take two years to reach sexual maturity, so it would take much longer for a trait to spread throughout wild bat populations than in lab mouse populations.

They say the proposal is not an easy feat from a technical standpoint, and I think that underplays how hard it might be, Cooper said.

Biologists also say that Erlichs proposal is unlikely to work in the wild even if researchers get bat gene drives to work in a lab because bats are incredibly diverse.

There are 1,432 bat species, including multiple horseshoe bat species that carry coronaviruses and pass them among each other.

Wild viruses similar to the human Covid-19 virus have been found in bats across Asia, and in pangolins. And in June, Weifeng Shi of the Shandong First Medical University & Shandong Academy of Medical Sciences in Taian, China, found 24 coronavirus genomes in bat samples taken from in and around a botanical garden in Yunnan province, in southern China.

Engineering one gene drive in just one bat species would not solve the problem, biologists say.

Youd have to develop systems for entire bat communities, said evolutionary biologist Liliana Dvalos of Stony Brook University. Its the job of visionaries to come up with creative ideas, but this is a giant blind spot in their thinking.

Biologists are also concerned about focusing on bats themselves, because they may not be the most important source of human epidemics. No one has found the exact bat analog to the human Covid-19 virus, or definitively proven that spillover from bats did start the pandemic. Coronaviruses have also been found in other species, including palm civets, pangolins, and camels.

Further, nobody knows how eliminating coronaviruses might affect bats.

We dont know the implications of wiping out coronaviruses in bat populations, because we dont know how bats have evolved to coexist with these viruses, said virologist Arinjay Banerjee of the Vaccine and Infectious Disease Organization at the University of Saskatchewan in Saskatoon, Canada.

Some scientists, though, welcomed Erlichs proposal, hoping that it will focus attention on what it would take to create successful mammalian gene drive systems.

Royden Saah, for instance, coordinates the Genetic Biocontrol of Invasive Rodents (GBIRd) program, which is trying to engineer gene drives in mice to prevent island bird extinctions. He wants to see more funding to help scientists solve the technical obstacles to such projects, and involve more communities in discussions about these ideas.

I would be concerned if this proposal detracted from the need to fund public health infrastructure, said Saah. But with that caveat, he added, I think this proposal could make people think, OK, if we were to use this technology in this animal in this system, what would we need to do? There would need to be a foundation of ethical development, of clear understanding, of social systems and trust, and technology built in a stepwise manner.

Virologist Jason Kindrachuk of the University of Manitoba said that there are numerous technical and political challenges to a bat gene drive project, and that preventing future outbreaks should mainly involve tackling the challenges that drive spillover events, such as underfunded public health systems, poverty, food insecurity and climate-change-driven ecological disruption. But, he said, given the enormous economic and human toll of Covid-19 and other recent outbreaks, scientists and public health officials might also need to consider new approaches.

In the past, maybe we were blinded a little bit by our belief that we would just be able to increase surveillance and identify these pathogens prior to them spilling over, Kindrachuk said. We now realize that this is going to take a lot of different efforts, so theres an aspect from a research standpoint where we continue to look at things like this, and say, what are the top 5 to 10 things we should invest in.

Erlich acknowledges the obstacles to his proposal, but thinks they arent insurmountable. He thinks the project would require an international investment involving a multidisciplinary consortium.

While we totally agree about the technical complexities, technology advances at exponential rates, Erlich said. Things that are nearly impossible now can be totally reachable within a decade or so.

He also thinks a gene drive could be a better alternative than culling bats, which has been tried (unsuccessfully) in communities around the world, and that scientists could monitor for negative impacts on bat populations.

Lets discuss the idea and think about what we can do to identify a very rigorous and cautious way to test this approach, Erlich said. We dont like to mess with nature, but the current situation is not sustainable.

Go here to see the original:
Could editing the genomes of bats prevent future pandemics? - STAT - STAT

SOCOMs Human Performance Program includes innovating capabilities for physical training, injury mitigation and performance nutrition.

WASHINGTON: Special Operations Command expects to move into clinical trials next year of a pill that may inhibit or reduce some of the degenerative affects of aging and injury part of a broader Pentagon push for improved human performance.

The pill has the potential, if it is successful, to truly delay aging, truly prevent onset of injury which is just amazingly game changing, Lisa Sanders, director of science and technology for Special Operations Forces, acquisition, technology & logistics (SOF AT&L), said Friday.

We have completed pre-clinical safety and dosing studies in anticipation of follow-on performance testing in fiscal year 2022, Navy Cmdr. Tim Hawkins, a SOCOM spokesperson, said.

SOCOM is usingOther Transaction Authority (OTA) funds to partner with private biotech laboratory Metro International Biotech, LLC (MetroBiotech) in the pills development, which is based on what is called a human performance small molecule, he explained.

These efforts are not about creating physical traits that dont already exist naturally. This is about enhancing the mission readiness of our forces by improving performance characteristics that typically decline with age, Hawkins said.Essentially, we are working with leading industry partners and clinical research institutions to develop a nutraceutical, in the form of a pill that is suitable for a variety of uses by both civilians and military members, whose resulting benefits may include improved human performance like increased endurance and faster recovery from injury.

Hawkins said SOCOM has spent $2.8 million on this effort since its launch in 2018.

A small molecule in biology is a low molecular weight organic compound, many of which regulate biological processes and often form the basis for drugs, i.e. pharmaceuticals. A nutraceutical, by contrast, is a food containing health-giving additives and having medicinal benefit, according to the Oxford Dictionary in essence a dietary supplement.

But in the case of the SOCOM program, the pill in question is the result of biotechnology.

MetroBiotech did not respond to a request for comment. However, its website explains that the firm has developed a number of proprietary precursor compounds for nicotinamide adenine dinucleotide (NAD+) which is critically important to the function of all living cells.

The website explains that reduced levels of NAD+ are linked to aging and numerous diseases, including mitochondrial dysfunction, inflammation and a variety of associated diseases. These levels decline as humans age and remain depleted during disease states. Preclinical evidence suggests disease- and age-related functional decline can be mitigated by boosting NAD+, which supports the Metro International Biotech hypothesis that maintaining optimal NAD+ levels may allow humans to lead longer and healthier lives.

Sanders told the Defense One Defense Tech Summit that SOCOMs ability to use OTAs and Middle Tier Acquisition authorities has helped the command explore things in this burgeoning sector of biotechnology. Those authorities have allowed SOCOM to enter into partnerships with industry, research institutes and labs to spur commercial research that could result in health benefits for the troops, she explained.

SOCOM has stayed out of long-term genetic engineering that makes people very very uncomfortable, Sanders said, but theres a huge commercial marketplace for things that can avoid injury, that can slow down aging, that can improve sleep.

Indeed, SOCOM has been working to bolster its relationships with small businesses and innovative companies involved in emerging tech, including biotech and artificial intelligence. Its innovation arm, SOFWERX, launched a campaign in May to speed contracting with non-traditional DoD suppliers.

Original post:
SOCOM To Test Anti-Aging Pill Next Year - Breaking Defense

A year ago, the term plasmid may have generated little interest among people. And nobody would have associated DNA with vaccines. But on July 1, 2021, this changed, even if momentarily, when Ahmedabad-based Zydus Cadila announced that it had applied for emergency use authorisation of its COVID-19 vaccine, ZyCoV-D, which is set to become the worlds first plasmid DNA vaccine for human use.

There are many platformsnew and oldthat are currently being used to develop COVID-19 vaccines. These include viral vector, inactivated virus, RNA, DNA, sub-unit and protein-based vaccines. The currently licensed COVID-19 vaccines include RNA-based vaccines (Moderna and Pfizer-BioNTech); viral-vectored (Oxford-AstraZeneca; Sputnik) and inactivated virus-based (Covaxin) vaccines, among many others. As of now, no DNA-based vaccine has been licensed for human use, nor for any disease.

The RNA and DNA vaccines together are called genetic vaccines or nucleic acid-based vaccines. These vaccines deliver one or more of the SARS-CoV-2 genes into the human cells to provoke an immune response.

DNA vaccines involve direct injection of a plasmid containing the DNA sequence encoding the antigen(s) against which an immune response is sought, into appropriate tissues.

Plasmids are circular pieces of DNA, which are found in many bacteria. These plasmids store and share genes, which are not essential for the bacterium but may play a role in its survival. One of the characteristics of plasmids is that they replicate independent of the main chromosomal DNA. Therefore, they can be a simple tool for transferring genes between cells. It is for this reason that plasmids are widely used in genetic engineering.

The plasmid DNA has the unique property of self-replication, a reason why it is used in different kinds of molecular genetic research, such as gene therapy, gene transfer and recombinant DNA technology. A very good analogy of plasmid is a computer flash/pen drive. Pen drive improves the functionality but is not essential for the functioning of a laptop or computer. And that is exactly what a plasmid isuseful but not essential.

One of the first steps in developing a DNA vaccine is identifying the antigenic section in virus, following which the DNA encoding of the antigen is chemically synthesized. Thereafter, it is inserted into an identified bacterial plasmid with the help of specific enzymes. Then, multiple copies of the plasmid are produced within giant vats of rapidly dividing bacteria, followed by isolation and purification. This material, after the standardized process, becomes the vaccine material.

The plasmid DNA, which carries an identified sequence of spike protein of the SARS-CoV-2, enters the host cell and then its nucleus, instructing the cell to make the messenger RNA. (Essentially, it is engaging human cells to do a task which they do not do on a routine.) Thereafter, the messenger RNA will carry the sequence to where protein is synthesized. The genetic material needs to be read by human cells protein-making machinery. Once protein is synthesized (which mimics the spike protein), these need to appear on the surface of human cells. It is at this stage the host immune system gets activated and starts producing antibodies and mounts a cell-based immune response.

The plug and play technology here means that the antigenic part of SARS-CoV-2 can be identified and easily packaged as plasmid to modify the vaccine, if need be. Both plasmid DNA and RNA vaccines work on this science. The advantage: the vaccine material can be easily adapted to deal with the mutations in the virus and emerging variants.

The technology for producing DNA vaccines is simple and rapid. They offer a number of potential advantages over traditional approaches that include stimulation of both B and T cell responses. There are no live components; therefore, there is no risk of vaccine- triggered disease. The DNA molecule (in comparison of RNA) is stable, has a long shelf life, and does not require a strict cold chain for distribution. RNA vaccines, in contrast, need to be stored at low or ultra-low temperature. The plasmid DNA platform provides ease of manufacturing with minimal bio-safety requirements. DNA and RNA vaccines are considered cost-effective, and it is relatively easy to manufacture them at large scale.

However, there are known challenges as well. DNA vaccines find it harder to get inside the cell and be accepted by the cells protein-making system. Developing a plasmid DNA vaccine is considered slightly more complicated compared to an RNA vaccine, which can be synthesized in a laboratory.

On July 1, Zydus Cadila reported the interim findings of its plasmid DNA-based COVID-19 vaccine. The manufacturer reported that Phase-3 clinical trials of the vaccine were carried out on 28,000 volunteers at 50 different trial sites across India. Of them, around 1,000 participants were in the 12-18 age group. The vaccine is being developed in partnership with the Department of Biotechnology and the Indian Council of Medical Research, Government of India.

The interim analysis has found the three-dose vaccine showing a 66.6 per cent efficacy, with 4-week interval between each dose. (Although the manufacturer has reported that a two-dose schedule, 3mg per dose, is equally effective). Zydus Cadila has applied to the Indian Drug regulatorthe Central Drugs Standard Control Organization (CDSCO)and sought emergency use authorization of the vaccine for 12 year olds and above. The vaccine can be stored at 2-8 degrees Celsius and at 25 degrees Celsius for up to three months. Once approved, it will be an intra-dermal (between skin and muscles) vaccine administered through a specialized needle-free injector. The currently licensed COVID-19 vaccines are administered intra-muscularly.

The subject expert committee (SEC) under CDSCO is yet to take a decision on the plasmid DNA-based vaccine developed by Zydus Cadila. However, if and when it is approved for emergency use authorization, it may become the worlds first DNA vaccine for human use. It is already the first Indian vaccine to have completed clinical trials in the 12-17 age group and could well become the first vaccine in India to be licensed for adolescents. Once approved, the vaccine is likely to be available in the next 6-8 weeks.

Vaccines have reignited everyones interest in science. Vaccine development on newer platforms is challenging as well as exciting. Even a year ago, who would have thought that plasmid would become a near-household term in India!

Disclaimer:Dr Chandrakant Lahariya is a vaccines, public policy and health systems expert. He writes a column HealthHacks for News18, which appears every alternate Saturday. He tweets at @DrLahariya. Views expressed are personal.

Read all the Latest News, Breaking News and Coronavirus News here

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ZyCoV-D: Decoding the Science behind Indias Plasmid DNA Vaccine & What Makes it Special - News18

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Genetic Engineering for Food Security to Have Strong Impact on Oilseed and Grain Farming Businesses | Discover Company Insights on BizVibe -...

On December 14, 2020, the US Food and Drug Administration (FDA) approved GalSafe pigs, which are genetically modified (GM) for use in food production and medical products. At the time, the agency noted in its Consumer Q&A that intentional genomic alterations (IGAs) in animals would be regulated by FDA to ensure that it is safe for the animal, safe for anyone that consumes food from the animal, and that it is effective, i.e., it does what the developer claims it will do. The agency also explained that IGAs would be subject to premarket oversight whether they are intended to be used for food or to produce pharmaceuticals or other useful products (emphasis added), with the US Department of Agriculture (USDA) being responsible for the labeling of food from GM animals.

However, on yet another show of intra-agency conflict during the Trump administration, just several weeks later the USDA moved to wrest the oversight of GM animals intended for food production from FDA by issuing an Advanced Notice of Proposed Rulemaking (ANPRM), titled Regulation of the Movement of Animals Modified or Developed by Genetic Engineering. Under the ANPRM, the USDA would be responsible for:

Notably, FDA would continue to regulate GM seafood. This proposed regulatory framework is intended to operate under a Memorandum of Understanding (MOU) between the USDA and the US Department of Health and Human Services (HHS).

The MOU was signed by the two agencies on January 13, 2021, just mere days before the change in administration. The MOU transfers the oversight of GM animals intended for agricultural purposes (i.e., human food, fiber, and labor) from FDA to the USDA under authorities granted to the USDA by the Animal Health Protection Act, the Federal Meat Inspection Act, and the Poultry Products Inspection Act. Under the MOU, FDA will continue to have authority over IGAs intended for any purpose other than agricultural use, including biopharma, xenotransplantation, and gene therapies. Importantly, if a specific GM animal species is intended for human food supply, FDA must consult with the USDA on the food safety review to promote consistent food safety reviews and monitoring for all amenable species intended for human food as part of USDA's new program.

Where Are We Now?

Considering the strong interest in the proposed change in agency oversight by both industry and consumers alikeas well as the Biden-Harris administrations likely desire to gauge the support and opposition for the plan before making a decisionUSDA reopened the comment period for the ANPRM in early March, which was extended to May 7, 2021. Despite strong support from industry, however, animal welfare, public health, and environmental advocates have signed letters urging both Tom Vilsack, Agriculture Secretary, and Xavier Becerra, HHS Secretary, to allow FDA to retain its oversight over GM animals intended for food production, claiming the MOU weakens FDAs authority to protect public health.

In the meantime, FDA continues to regulate GM animals for both agricultural and medical purposes. Whether USDAs effort to retain jurisdiction over GM meat intended for the food supply will be successful is unclear. However, it seems there would be some amount of duplication in determining whether a genetic modification to an animal is safe for purposes of producing food, drugs, new cells, or tissue structures for use in humans. Presumably the agencies will share their relevant scientific expertise in assessing the use of this novel technology and its possible effects on humans. Because state agencies are also heavily involved in the regulation of livestock, it is likely that states will have a view on which federal agency they believe is more capable to set appropriate standards and police activity. It remains unclear when a decision on the ANPRM will be issued and whether the Biden-Harris administration will support the proposed rule.

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USDA's Proposal to Take Back Regulatory Oversight of GM Animals from FDA Remains Viable Despite Change in Administration - Lexology

Abstract

As numerous diseases are associated with increased local inflammation, directing drugs to the inflamed sites can be a powerful therapeutic strategy. One of the common characteristics of inflamed endothelial cells is the up-regulation of vascular cell adhesion molecule1 (VCAM-1). Here, the specific affinity between very late antigen4 (VLA-4) and VCAM-1 is exploited to produce a biomimetic nanoparticle formulation capable of targeting inflammation. The plasma membrane from cells genetically modified to constitutively express VLA-4 is coated onto polymeric nanoparticle cores, and the resulting cell membranecoated nanoparticles exhibit enhanced affinity to target cells that overexpress VCAM-1 in vitro. A model anti-inflammatory drug, dexamethasone, is encapsulated into the nanoformulation, enabling improved delivery of the payload to inflamed lungs and significant therapeutic efficacy in vivo. Overall, this work leverages the unique advantages of biological membrane coatings to engineer additional targeting specificities using naturally occurring target-ligand interactions.

The chemical and physiological changes associated with inflammation are an important part of the innate immune system (1). Proinflammatory processes can lead to the release of cytokines such as interleukin-6 (IL-6) and tumor necrosis factor, which are capable of effecting vascular changes to improve immune responses at a site of stress or injury (2). These may include vasodilation and an increase in vascular permeability, which can promote more efficient immune cell recruitment (3, 4). On the cellular level, proinflammatory cytokines cause the up-regulation of specific surface markers, including vascular cell adhesion molecule1 (VCAM-1) or intercellular adhesion molecule1 (ICAM-1), which allow for immune cell adhesion at the site of inflammation (5, 6). Although inflammation is an integral process that is required for survival, a dysregulated immune system is implicated in a wide range of disease states (7, 8). The disease relevance of inflammation is further supported by the fact that inflammatory markers such as cellular adhesion molecules are often implicated in pathogenesis (9, 10), and these have been explored as therapeutic and diagnostic targets.

Nanoparticle-based platforms, especially those functionalized with active targeting ligands, have the potential to serve as powerful tools for managing a wide range of diseases associated with inflammation (11). Along these lines, the targeted delivery of anti-inflammatory agents to the vasculature of affected sites via cell adhesion molecules represents a promising strategy (1214). Using inflammation as the cue, a diverse range of nanodelivery systems have been designed to target up-regulated markers such as VCAM-1 and ICAM-1 (1520), and this approach has been leveraged to treat conditions such as cancer and cardiovascular diseases (2123). More recently, cell membrane coating technology has garnered considerable attention in the field of nanomedicine (24, 25). From erythrocytes to cancer cells, virtually any type of cell membrane can be coated onto the surface of nanoparticles, resulting in nanoformulations with enhanced functionality that can be custom-tailored to specific applications (26, 27). In particular, cell membranecoated nanoparticles have proven to be effective drug delivery systems owing to their extended circulation times and disease-homing capabilities (2628). The targeting ability of these biomimetic nanoparticles is often mediated by proteins that are expressed on the source cells, and this bestows the nanoparticles with the ability to specifically interact with various disease substrates. For example, nanoparticles coated with the membrane derived from platelets were shown to specifically target bacteria as well as the exposed subendothelium in damaged vasculature (29). A similar platform was shown to target the lungs in a murine model of cancer metastasis (30). On top of the natural biointerfacing capabilities of cell membranecoated nanoparticles, their traits can be further enhanced by introducing exogenous moieties onto the membrane surface. One way to achieve this is to tether targeting ligands via a lipid anchor, which can then be inserted into the cell membrane (31, 32). Red blood cell membranecoated nanoparticles, which exhibit prolonged blood circulation, have been functionalized in this manner to enhance their cancer targeting ability.

Instead of relying on post-fabrication methods to introduce additional functionality, cell membranecoated nanoparticles can be developed using the membrane from genetically engineered source cells (33). A wide range of tools are available to introduce or up-regulate the expression of specific surface markers (34, 35), and this approach enables researchers to augment the functionality of cell membranebased nanodelivery platforms based on application-specific needs (36, 37). In this study, we genetically engineered cell membranecoated nanoparticles to specifically target sites of inflammation (Fig. 1). Inflamed endothelial cells are known to up-regulate the expression of VCAM-1 to recruit immune cells such as leukocytes that express its cognate ligand, very late antigen4 (VLA-4) (38). To exploit this interaction, we genetically modified a source cell line to stably express VLA-4 and harvested the engineered membrane to coat polymeric nanoparticle cores. A potent anti-inflammatory drug, dexamethasone (DEX), was used as a model payload to be loaded for the treatment of inflammation. The ability of the final nanoformulation to target inflamed cells without compromising the activity of DEX was first tested in vitro. Then, therapeutic efficacy was evaluated in vivo using a murine model of endotoxin-induced lung inflammation.

Wild-type cells were genetically engineered to express VLA-4, which is composed of integrins 4 and 1. Then, the plasma membrane from the genetically engineered cells was collected and coated onto dexamethasone-loaded nanoparticle cores (DEX-NP). The resulting VLA-4expressing cell membranecoated DEX-NP (VLA-DEX-NP) can target VCAM-1 on inflamed lung endothelial cells for enhanced drug delivery.

VLA-4 is a heterodimer that is formed by the association of integrin 4 with integrin 1 (39). To generate a cell line constitutively displaying the full complex, we elected to modify wild-type C1498 cells (C1498-WT), which were confirmed to express high levels of integrin 1 but lack integrin 4 (Fig. 2A). Following viral transduction of C1498-WT to introduce the integrin 4 gene, a subpopulation of the resulting engineered cells (referred to as C1498-VLA) was found to express both VLA-4 components (Fig. 2B). After successfully establishing C1498-VLA, the cells were harvested and their membrane was derived by a process involving cell lysis and differential centrifugation. The cell membrane was then coated onto poly(lactic-co-glycolic acid) (PLGA) nanoparticle cores that were prepared by a single emulsion method. Membrane-coated nanoparticles prepared with the membrane from C1498-WT and C1498-VLA (referred to as WT-NP and VLA-NP, respectively) both had an average diameter of approximately 175 nm, which was slightly larger than the uncoated PLGA cores (Fig. 2C). In terms of zeta potential, the membrane-coated nanoparticles exhibited a surface charge of approximately 20 mV, which was less negative than the PLGA cores (Fig. 2D). Both the size and zeta potential data suggested proper membrane coating, which was further verified for VLA-NP by transmission electron microscopy, which clearly showed a membrane layer surrounding the core (Fig. 2E). Western blotting analysis was used to probe for the two components of VLA-4 on the nanoformulations (Fig. 2F). As expected, both integrins 4 and 1 were found on VLA-NP, whereas only integrin 1 was present on WT-NP. To evaluate long-term stability of the membrane-coated nanoparticles, they were suspended in 10% sucrose solution at 4C, and their size was monitored over the course of 8 weeks (Fig. 2G). Neither nanoparticle sample exhibited a significant increase in size during this period.

(A and B) Expression of integrins 4 and 1 on C1498-WT (A) and C1498-VLA (B) cells was confirmed by flow cytometry. (C and D) The average diameter (C) and surface zeta potential (D) of PLGA cores, WT-NP, and VLA-NP were confirmed by dynamic light scattering (n = 3, mean + SD). (E) Representative transmission electron microscopy image of VLA-NP (scale bar, 100 nm). (F) Western blots for integrins 4 and 1 on WT-NP and VLA-NP. (G) Size of WT-NP and VLA-NP when stored in solution over a period of 8 weeks (n = 3, mean SD).

The binding of VLA-NP was assessed in two different in vitro experiments. First, C1498-WT transduced to constitutively express high amounts of VCAM-1 (referred to as C1498-VCAM) was used as a model target cell. The expression of VCAM-1 on C1498-VCAM was confirmed via flow cytometry (Fig. 3A). Whereas the C1498-WT cells did not show any expression, the C1498-VCAM cells yielded a signal that was over an order of magnitude higher than the isotype control. To evaluate binding, fluorescent dyelabeled WT-NP or VLA-NP were incubated with either C1498-WT or C1498-VCAM (Fig. 3, B and C). For each pairing, the incubation was performed either with or without antiVCAM-1 to block the specific interaction between VLA-4 and VCAM-1. For the samples with blocking, cells were first incubated with the antibody for 30 min before nanoparticle treatment. After incubating with the nanoparticles for 30 min, the cells were washed twice and were analyzed by flow cytometry. The data revealed that there was significant nanoparticle binding only when VLA-NP were paired with C1498-VCAM. The level of binding was reduced back to baseline levels in the presence of antiVCAM-1, thus confirming the specificity of the interaction. In contrast, there was no evidence of specific binding when VLA-NP were paired with C1498-WT, which does not express the cognate receptor for VLA-4. The same held true for the WT-NP paired with either cell type, where antibody blocking had no impact on the relative nanoparticle binding.

(A) Expression of VCAM-1 on C1498-WT and C1498-VCAM cells (gray, isotype antibody; green, antiVCAM-1). (B and C) Binding of WT-NP (B) or VLA-NP (C) to C1498-WT or C1498-VCAM cells; blocking was performed by preincubating cells with antiVCAM-1 (n = 3, mean + SD). ****P < 0.0001, Students t test. (D) Expression of VCAM-1 on untreated or LPS-treated bEnd.3 cells (gray, isotype antibody; green, antiVCAM-1). (E and F) Binding of WT-NP (E) or VLA-NP (F) to untreated or LPS-treated bEnd.3 cells; blocking was performed by preincubating cells with antiVCAM-1 (n = 3, mean + SD). **P < 0.01, Students t test.

Next, we elected to study the nanoparticle binding to endothelial cells, which represent a more biologically relevant target compared to the artificially engineered C1498-VCAM cells. For this purpose, we used a murine brain endothelial cell line, bEnd.3, whose VCAM-1 expression can be up-regulated in the presence of proinflammatory signals (40). To induce an inflamed state, bEnd.3 cells were treated with bacterial lipopolysaccharide (LPS), and the level of VCAM-1 expression was evaluated using flow cytometry (Fig. 3D). Whereas expression of VCAM-1 was near baseline levels for the untreated bEnd.3 cells, those that were treated with LPS exhibited a distinct population with elevated VCAM-1. As we observed in the previous experiment with C1498-VCAM cells, enhanced nanoparticle binding was only observed when VLA-NP were paired with inflamed bEnd.3 cells, and antibody blocking reduced the levels back to baseline (Fig. 3, E and F). When incubating with noninflamed bEnd.3 cells, there was no evidence of specific binding interactions, and the same held true for the control WT-NP paired with bEnd.3 cells regardless of their inflammatory status. The data in these two studies confirmed the successful engineering of membrane-coated nanoparticles with the ability to target inflammation based on the interaction between VLA-4 and VCAM-1.

As a model anti-inflammatory payload, we selected DEX, which was loaded into the PLGA core by a single emulsion method before coating with either C1498-WT or C1498-VLA membrane to yield DEX-loaded WT-NP or VLA-NP (referred to as WT-DEX-NP or VLA-DEX-NP, respectively). When the drug content was measured by high-performance liquid chromatography (HPLC), it was determined that the encapsulation efficiency and drug loading yield were approximately 11 and 2 weight % (wt %), respectively (Fig. 4A). To evaluate drug release, VLA-DEX-NP was dialyzed against a large volume of phosphate-buffered saline (PBS), and the amount of drug retained within the nanoparticles was quantified over time (Fig. 4B). The results revealed an initial burst, where approximately 80% of the drug payload was released in the first hour, followed by a sustained release. The release profile was in agreement with previous reports on DEX-loaded PLGA formulations (41, 42), and the data showed a good fit with the Peppas-Sahlin model with a regression coefficient of 0.978 (43). To evaluate the biological activity of the DEX loaded within the nanoparticles, we used an in vitro assay based on the LPS treatment of DC2.4 dendritic cells, which causes an elevation in the levels of proinflammatory cytokines such as IL-6 (Fig. 4C). DC2.4 cells were first treated with either free DEX or VLA-DEX-NP for 2 hours, followed by incubation with LPS overnight. The supernatant was then collected to measure the concentration of IL-6 by an enzyme-linked immunosorbent assay (ELISA). It was shown that both free DEX and VLA-DEX-NP were able to attenuate IL-6 secretion in a drug concentrationdependent manner (Fig. 4D). Although free DEX more efficiently lowered IL-6 levels at drug concentrations of 0.01 and 0.1 M, the level of inflammation was reduced to levels near baseline for both free DEX and VLA-DEX-NP at 1 M of drug. The data indicated that the activity of the drug payload was retained after being loaded inside of VLA-NP. It was confirmed that neither PLGA cores nor VLA-NP without DEX loading had an impact on the level of IL-6 production by the DC2.4 cells (Fig. 4E).

(A) Drug loading (DL) and encapsulation efficiency (EE) of dexamethasone (DEX) into VLA-NP (n = 3, mean + SD). (B) Drug release profile of VLA-DEX-NP (n = 3, mean SD). The data were fitted using the Peppas-Sahlin equation (dashed line). (C) Secretion of IL-6 by LPS-treated DC2.4 cells (n = 3, mean + SD). UD, undetectable. (D) Secretion of IL-6 by LPS-treated DC2.4 cells preincubated with DEX in free form or loaded into VLA-NP (n = 3, mean SD). (E) Relative inflammatory response, as measured by IL-6 secretion, of DC2.4 cells treated with LPS only, LPS and PLGA nanoparticles, LPS and VLA-NP, PLGA nanoparticles only, or VLA-NP only; all of the nanoparticles were empty without DEX loading (n = 3, mean + SD). NS, not significant (compared to the LPS-only group), one-way analysis of variance (ANOVA).

After confirming the biological activity of the VLA-DEX-NP formulation in vitro, we next sought to evaluate the formulation in vivo using a murine model of lung inflammation. The model was established by intratracheal injection of LPS directly into the lungs of BALB/c mice. To evaluate targeting ability, fluorescently labeled WT-NP or VLA-NP were injected intravenously after the induction of lung inflammation. After 6 hours, major organs, including the heart, lungs, liver, spleen, kidneys, and blood, were collected to assess nanoparticle biodistribution (Fig. 5A). The majority of the nanoparticles accumulated in the liver and spleen. Notably, a significant increase in accumulation of VLA-NP was observed in the lungs compared to WT-NP. This in vivo targeting result was in agreement with the in vitro findings where VLA-NP were able to specifically bind to inflamed cells. The safety of the formulation was assessed by monitoring the plasma levels of creatinine, a marker of kidney toxicity that was previously studied in the context of DEX nanodelivery (44). After 9 days of repeated daily administrations of free DEX or VLA-DEX-NP into healthy mice, it was shown that the creatinine concentration in mice receiving VLA-DEX-NP remained consistent with baseline levels, whereas it was significantly elevated in mice administered with free DEX (Fig. 5B).

(A) Biodistribution of WT-NP or VLA-NP in a lung inflammation model 6 hours after intravenous administration (n = 3, mean + SD). *P < 0.05, Students t test. AU, arbitrary units. (B) Creatinine levels in the plasma of mice after repeated daily administrations for 9 days with free DEX or VLA-DEX-NP (n = 3, mean + SD). *P < 0.05, one-way ANOVA. (C) IL-6 levels in the lung tissue of mice intratracheally challenged with LPS and then treated intravenously with vehicle solution, free DEX, WT-DEX-NP, or VLA-DEX-NP (n = 3, mean SD). ***P < 0.001, ****P < 0.0001 (compared to VLA-DEX-NP), one-way ANOVA. (D) Representative hematoxylin and eosinstained lung histology sections of mice intratracheally challenged with LPS and then treated intravenously with vehicle solution, free DEX, WT-DEX-NP, or VLA-DEX-NP (scale bar, 100 m).

The therapeutic efficacy of VLA-DEX-NP was then evaluated following the same experimental design as the targeting study. After 6 hours, the lungs were collected and homogenized, and the homogenate was then clarified by centrifugation and filtered through a 0.22-m porous membrane before measuring the concentration of IL-6 by ELISA. As shown in Fig. 5C, the VLA-DEX-NP formulation was able to completely abrogate lung inflammation, while both free DEX and WT-DEX-NP did not have any discernable effect. The fact that WT-DEX-NP were not able to significantly reduce lung IL-6 levels suggested that systemic exposure to DEX was not a major contributor to the efficacy observed with VLA-DEX-NP. The efficacy of the formulation against lung inflammation was further confirmed by analyzing lung sections stained with hematoxylin and eosin (Fig. 5D). Leukocyte recruitment and peribronchial thickening, which are hallmarks of lung inflammation (45, 46), were prominent in the lungs of mice receiving no treatment, free DEX, or WT-DEX-NP. In contrast, minimal leukocyte recruitment and no peribronchial thickening were observed for the group treated with VLA-DEX-NP, and there were no other signs of toxicity present in these lung sections. Overall, the results from the in vivo studies confirmed the benefit of targeted delivery to inflamed lungs using VLA-NP as a drug nanocarrier.

In conclusion, we have engineered cell membranecoated nanoparticles that can be used to specifically target and treat localized lung inflammation via systemic administration. A host cell positive for integrin 1 was modified to express integrin 4. Together, the two protein markers formed VLA-4, which specifically interacts with VCAM-1, a common marker for inflammation found on vascular endothelia. Nanoparticles fabricated using the membrane from these genetically engineered cells were able to leverage this natural affinity to target inflamed sites, including in a murine model of LPS-induced lung inflammation. When the nanoparticles were loaded with DEX, an anti-inflammatory drug, significant therapeutic efficacy was achieved in vivo. Future studies will comprehensively evaluate the safety profile of the VLA-DEX-NP formulation, obtain additional lung-specific efficacy readouts, elucidate the optimal time window for treatment, and assess clinical relevance using additional animal models of severe inflammatory disease. As pathological inflammation is heavily implicated in a number of important disease conditions (7, 47), the reported biomimetic platform could be leveraged to improve the in vivo activity of various therapeutic payloads through enhanced targeting. Notably, VCAM-1 up-regulation has been observed in renal pathologies as well as in inflamed cerebral vasculature (48, 49). In addition, DEX has been shown to be effective at managing the inflammation associated with COVID-19 (50), and a targeted formulation capable of localizing the drug to the lungs may help to further boost its therapeutic profile. In this work, we specifically engineered the nanoparticles to display VLA-4, which is a complex, multicomponent membranebound ligand that would otherwise be infeasible to incorporate using traditional synthetic strategies. This highlights the advantages of using genetic engineering techniques to expand the wide-ranging utility of cell membrane coating technology. In particular, the generalized application of this approach would enable researchers to streamline the development of new targeted nanoformulations by using target-ligand interactions that occur in nature. Combined with the biocompatibility and biointerfacing characteristics that are inherent to cell membrane coatings, the work presented here could initiate a new wave of biomimetic nanomedicine with finely crafted functionalities.

Wild-type C1498 mouse leukemia cells (TIB-49, American Type Culture Collection) were cultured at 37C in 5% CO2 with Dulbeccos modified Eagles medium [DMEM; with l-glutamine, glucose (4.5 g/liter), and sodium pyruvate; Corning] supplemented with 10% bovine growth serum (BGS; Hyclone) and 1% penicillin-streptomycin (Pen-Strep; Gibco). Engineered C1498-VCAM cells were cultured with DMEM supplemented with 10% U.S. Department of Agriculture (USDA) fetal bovine serum (FBS; Omega Scientific), 1% Pen-Strep, and hygromycin B (400 g/ml; InvivoGen). Engineered C1498-VLA cells were cultured with DMEM supplemented with 10% USDA FBS, 1% Pen-Strep, and puromycin (1 g/ml; InvivoGen). bEnd.3 mouse brain endothelial cells (CRL-2299, American Type Culture Collection) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep. AmphoPhoenix cells (obtained from the National Gene Vector Biorepository) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep. DC2.4 mouse dendritic cells (SCC142, Sigma-Aldrich) were cultured with DMEM supplemented with 10% BGS and 1% Pen-Strep.

Engineered C1498-VLA and C1498-VCAM cells were created by transducing C1498-WT. Briefly, the genes for integrin 4 (MG50049-M, Sino Biological) and VCAM-1 (MG50163-UT, Sino Biological) gene were cloned into pQCXIP and pQCXIH plasmids (Clontech), respectively, using an In-Fusion HD cloning kit (Clontech) following the manufacturers protocol, yielding pQCXIP-4 and pQCXIH-VCAM-1. AmphoPhoenix cells were plated onto 100-mm tissue culture dishes containing 10 ml of medium at 3 105 cells/ml and cultured overnight. The cells were transfected with pQCXIP-4 or pQCXIH-VCAM-1 using Lipofectamine 2000 (Invitrogen) following the manufacturers instructions. The supernatant of the transfected AmphoPhoenix was collected and used to resuspend C1498-WT cells, which were then centrifuged at 800g for 90 min. After the spin, the transduced cells were incubated for 4 hours before the media were changed with fresh media. Fluorescently labeled antibodies, including FITC (fluorescein isothiocyanate) anti-mouse CD49d (R1-2, BioLegend), Alexa647 anti-mouse/rat CD29 (HM1-1, BioLegend), or PE (phycoerythrin) anti-mouse CD106 (STA, BioLegend), were used to assess the expression levels of VLA-4 or VCAM-1. Data were collected using a Becton Dickinson FACSCanto-II flow cytometer and analyzed using FlowJo software. All of the engineered cells were sorted using a Becton Dickinson FACSAria-II flow cytometer to select for cells expressing high levels of VLA-4 or VCAM-1.

The membranes from C1498-WT and engineered C1498-VLA cells were derived using a previously described method with some modifications (51). First, the cells were harvested and washed in a starting buffer containing 30 mM tris-HCl (pH 7.0) (Quality Biological) with 0.0759 M sucrose (Sigma-Aldrich) and 0.225 M d-mannitol (Sigma-Aldrich). The washed cells were resuspended in an isolation buffer containing 0.5 mM ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (Sigma-Aldrich), a phosphatase inhibitor cocktail (Sigma-Aldrich), and a protease inhibitor cocktail (Sigma-Aldrich). Then, the cells were homogenized using a Kinematica Polytron PT 10/35 probe homogenizer at 70% power for 15 passes. The homogenate was first centrifuged at 10,000g in a Beckman Coulter Optima XPN-80 ultracentrifuge for 25 min. The supernatant was then collected and centrifuged at 150,000g for 35 min. The resulting pellet of cell membrane was washed and stored in a solution containing 0.2 mM ethylenediaminetetraacetic acid (USB Corporation) in UltraPure DNase-free/RNase-free distilled water (Invitrogen). Total membrane protein content was quantified by a BCA protein assay kit (Pierce).

Polymeric cores were prepared by a single emulsion process using carboxyl-terminated 50:50 PLGA (0.66 dl/g; LACTEL absorbable polymers). For DEX-loaded PLGA cores, 500 l of PLGA (50 mg/ml) in dichloromethane (DCM; Sigma-Aldrich) was mixed with 500 l of DEX (10 mg/ml) in acetone. This mixture was added to 5 ml of 10 mM tris-HCl (pH 8) and sonicated using a Thermo Fisher Scientific 150E Sonic Dismembrator at 70% power for 2 min. The sonicated mixture was added to 10 ml of 10 mM tris-HCl (pH 8) and was magnetically stirred at 700 rpm overnight. For 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD, ex/em = 644/663 nm; Biotium) labeling, 500 l of PLGA (50 mg/ml) in DCM was mixed with 500 l of DiD (20 g/ml) in DCM. This mixture was added to 5 ml of 10 mM tris-HCl (pH 8) and sonicated using a Thermo Fisher Scientific 150E Sonic Dismembrator at 70% power for 2 min. The sonicated mixture was added to 10 ml of 10 mM tris-HCl (pH 8) and was magnetically stirred at 700g for 3 hours. Empty PLGA core preparation followed the same procedure, except substituting the DiD solution for 500 l of neat DCM. To coat the polymeric cores with cell membranes, the nanoparticle cores were first centrifuged at 21,100g for 8 min. The pellets were resuspended in solution containing membranes derived from C1498-WT or C1498-VLA. The mixture was sonicated in a 1.5-ml disposable sizing cuvette (BrandTech Scientific Inc.) using a Thermo Fisher Scientific FS30D bath sonicator at a frequency of 42 kHz and a power of 100 W for 3 min. For the in vitro studies, UltraPure water and sucrose were added to adjust the polymer concentration to 1 mg/ml and the sucrose concentration to 10%. For the in vivo studies, UltraPure water and sucrose were added to adjust the polymer concentration to 10 mg/ml and the sucrose concentration to 10%.

The size and surface zeta potential of WT-NP and VLA-NP were measured by dynamic light scattering using a Malvern ZEN 3600 Zetasizer. For electron microscopy visualization, a VLA-NP sample was negatively stained with 1 wt % uranyl acetate (Electron Microscopy Sciences) on a carbon-coated 400-mesh copper grid (Electron Microscopy Sciences) and visualized using a JEOL 1200 EX II transmission electron microscope. The presence of VLA-4 on WT-NP and VLA-NP was determined using western blotting. First, the samples were adjusted to 1 mg/ml protein content, followed by the addition of NuPAGE 4 lithium dodecyl sulfate sample loading buffer (Novex) and heating at 70C for 10 min. Then, 25 l was loaded into the wells of 12-well Bolt 4 to 12% Bis-Tris gels (Invitrogen) and ran at 165 V for 45 min in MOPS running buffer (Novex). The proteins were transferred for 60 min at a voltage of 10 V onto 0.45-m nitrocellulose membranes (Pierce) in Bolt transfer buffer (Novex). Nonspecific interactions were blocked using 5% milk (Genesee Scientific) in PBS (Thermo Fisher Scientific) with 0.05% Tween 20 (National Scientific). The blots were probed using anti-integrin 4 antibody (B-2, Santa Cruz Biotechnology) or anti-integrin 1 antibody (E-11, Santa Cruz Biotechnology). The secondary staining was done using the corresponding horseradish peroxidaseconjugated antibodies (BioLegend). Membranes with stained samples were developed in a dark room using ECL western blotting substrate (Pierce) and an ImageWorks Mini-Medical/90 Developer. Long-term stability of WT-NP and VLA-NP in 10% sucrose solution was tested by storing the particles at 4C for 2 months with weekly size measurements.

The expression level of VCAM-1 on C1498-WT, C1498-VCAM, untreated bEnd.3 cells, and bEnd.3 cells treated overnight with LPS (1 g/ml) from Escherichia coli K12 (LPS; InvivoGen) was evaluated as described above. For the first binding study, 5 104 cells, either C1498-WT or C1498-VCAM, were collected and resuspended in 160 l of DMEM containing 0.5% USDA FBS, 1% bovine serum albumin (BSA; Sigma-Aldrich), and 1 mM MnCl2 (Sigma-Aldrich). For blocking, anti-mouse CD106 antibody was added to the cells, followed by incubation at 4C for 30 min. Then, 40 l of DiD (1 mg/ml)labeled WT-NP or VLA-NP was added, and the mixture was incubated at 4C for another 30 min. After washing the cells twice with PBS, the fluorescent signals from the cells were detected using flow cytometry. For the second study, 5 104 bEnd.3 cells were plated and then either left untreated or pretreated with LPS overnight. The media were then removed and replaced with 160 l of DMEM containing 0.5% USDA FBS, 0.8% BSA, and 1 mM MnCl2. For blocking, anti-mouse CD106 antibody was added to the cells, followed by incubation at 4C for 30 min. Then, 40 l of DiD (1 mg/ml)labeled WT-NP or VLA-NP was added, and the mixture was incubated at 4C for another 30 min. After washing the cells twice with PBS, the cells were detached by scraping, and the fluorescent signals from the cells were detected using flow cytometry. All data were collected using a Becton Dickinson FACSCanto-II flow cytometer and analyzed using FlowJo software.

Drug loading and encapsulation efficiency were measured using HPLC on an Agilent 1220 Infinity II gradient liquid chromatography system equipped with a C18 analytical column (Brownlee). VLA-DEX-NP samples were dissolved overnight in 80% acetonitrile (ACN; EMD Millipore) and then centrifuged at 21,100g for 8 min to collect the supernatant for analysis. The solutions were run through the column at a flow rate of 0.3 ml/min and DEX was detected at a wavelength of 242 nm. The DEX release profile was obtained by loading 200 l of VLA-DEX-NP (1 mg/ml) into Slide-A-Lyzer MINI dialysis devices (10K molecular weight cutoff; Thermo Fisher Scientific) and floating them on 1 liter of PBS stirred at 150 rpm. At each time point, dialysis cups were retrieved, and their contents were centrifuged at 21,100g for 8 min. The pellets were dissolved in 80% ACN overnight and processed as described above for HPLC analysis.

The biological activity of DEX was evaluated in vitro using a test system involving the LPS treatment of DC2.4 dendritic cells. To validate the system, DC2.4 cells were first plated onto a 24-well tissue culture plate at 5 104 cells per well and cultured overnight with or without LPS at a concentration of 1 g/ml. Then, supernatant was collected, and the concentration of IL-6 was measured using a mouse IL-6 ELISA kit (BioLegend) according to the manufacturers protocol. To compare free DEX and VLA-DEX-NP, the two formulations were first added to the culture medium at final drug concentrations of 0.01, 0.1, and 1 M, followed by 2 hours of incubation. For free DEX, 1000 stock solutions were prepared at 0.01, 0.1, and 1 mM in dimethyl sulfoxide. Then, the cells were treated with LPS overnight before measuring the concentration of IL-6 in the supernatant. To test the effect of empty nanoparticles, either PLGA cores or VLA-NP at a final concentration of 1 g/ml were first incubated with the cells for 2 hours, followed by an overnight incubation either with or without LPS before measuring IL-6 levels.

All animal experiments were performed in accordance with the National Institutes of Health (NIH) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California San Diego. To induce lung inflammation in mice, 30 l of LPS (400 g/ml) in PBS was injected intratracheally into male BALB/c mice (Charles River Laboratories). At 1 hour after LPS injection, 100 l of DiD (10 mg/ml)labeled WT-NP or VLA-NP was administered intravenously. After 6 hours, the heart, lungs, liver, spleen, kidneys, and blood were collected. All solid tissues were washed with PBS and suspended in 1 ml of PBS before being homogenized with a Biospec Mini-Beadbeater-16. The homogenates and blood were then diluted 4 with PBS and added to a 96-well plate, and fluorescence was measured using a BioTek Synergy Mx microplate reader. For each sample, the background signal measured from the corresponding organ or blood of control mice that did not receive any treatment was subtracted.

Male BALB/c mice were intravenously injected with 100 l of free DEX or VLA-DEX-NP, each at a drug concentration of 200 g/ml, daily for the first 7 days. Then, for the next 2 days, the dosage was doubled by injecting 200 l of each formulation at the same drug concentration. At 24 hours after the last injection, blood was collected by submandibular puncture and collected into tubes containing sodium heparin (Sigma-Aldrich). Plasma samples were obtained by taking the supernatant of the blood after centrifuging at 800g for 10 min. Creatinine levels were measured using a creatinine colorimetric assay kit (Cayman Chemical Company) according to the manufacturers protocol.

To treat lung inflammation, male BALB/c mice were first intratracheally challenged with 30 l of LPS (400 g/ml) in PBS. At 1 hour after the challenge, 100 l of free DEX, WT-DEX-NP, and VLA-DEX-NP, each at a drug concentration of 200 g/ml, was injected intravenously. After 6 hours, the lungs were collected and homogenized as described above. The homogenates were centrifuged at 10,000g, and the supernatants were filtered through 0.22-m polyvinylidene difluoride syringe filters (CELLTREAT). The concentration of IL-6 was measured using a mouse IL-6 ELISA kit according to the manufacturers protocol. For histology analysis, the lungs were collected after 6 hours and fixed in 10% phosphate-buffered formalin (Fisher Chemical) for 24 hours. The fixed lungs were sectioned, followed by hematoxylin and eosin (Sakura Finetek) staining. Histology slides were prepared by the Moores Cancer Center Tissue Technology Shared Resource (Cancer Center Support Grant P30CA23100). Images were obtained using a Hamamatsu NanoZoomer 2.0-HT slide scanner and analyzed using the NanoZoomer Digital Pathology software.

Acknowledgments: Funding: This work was supported by the National Institutes of Health under award no. R01CA200574 and the Defense Threat Reduction Agency Joint Science and Technology Office for Chemical and Biological Defense under grant no. HDTRA1-18-1-0014. J.H.P. was supported by a National Institutes of Health 5T32CA153915 training grant from the National Cancer Institute. Author contributions: J.H.P., Y.J., R.H.F., and L.Z. conceived and designed the experiments. J.H.P., Y.J., J.Z., H.G., A.M., and J.H. performed all experiments. All authors analyzed and discussed the data. J.H.P., A.M., R.H.F., and L.Z. wrote the paper. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper.

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