The work of the new Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M) will help provide standardized production and quality testing for these living cells, which have great therapeutic potential. Standardized manufacturing techniques already exist for drug-based pharmaceuticals; the new center will help provide similar methods and standards for manufacturing therapeutic cells.

Expected to be the first of its kind in the United States, the center will include a validation facility for good manufacturing practices in cell production. In addition to The Marcus Foundation, funding will come from the Georgia Research Alliance and Georgia Tech sources for a total investment of $23 million. The center will also seek support from federal agencies, clinical research organizations and other sources.

“The aspirin you buy today from one pharmacy is essentially the same as the aspirin you buy from another pharmacy, but cell-based therapies may have different efficacy depending on the source and manufacturing processes,” said Krishnendu Roy, Robert A. Milton Chair and professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. “There are established ways to quickly assess the efficacy and safety of small-molecule drugs that are acceptable around the world. We want to develop and establish similar processes for therapeutic cell manufacturing.”

Ultimately, the growing need for these cell therapeutics could require large-scale production facilities similar to those used in today’s pharmaceutical production. But living stem cells and immune system cells are readily affected by the varying conditions under which they are grown, stored and packaged, meaning the same type of cell produced at different facilities could behave very differently. Unless those cells can be produced with consistency, in large scale and at low cost with high quality, use of the new cell therapies could be limited and their promise would not extend to large numbers of patients.

“The critical goal is to either minimize differences caused by varying manufacturing conditions, or to have a very defined characterization process so we exactly know how much the cells have changed and what specific characteristics are predictive of their efficacy in patients,” explained Roy, who will lead the new center. “That consistency will allow us to produce affordable products that can make this new technology available to the large community of people who need it.”

The new center will collaborate with research and clinical institutions around the country, especially those at which The Marcus Foundation funds research on cell-based therapies, including Duke University, the University of Miami, City of Hope, Emory University, as well as the University of Georgia and other national and international universities.

“Access to this network will provide us a huge advantage by bringing together experts to work on a common problem,” Roy said.

"Stem cell treatments and cell-based immunotherapies are, and will be, the treatment of the future,” said Bernie Marcus, who co-founded The Home Depot. “Manufacturing and characterization of stem cells and immune cells is a major first step, and that is why The Marcus Foundation chose Georgia Tech and its teams – they have the experience and the personnel to achieve key goals in this process."

The new center will be a collaboration among research groups at Georgia Tech, as well as numerous outside institutions, noted Georgia Tech President G.P. “Bud” Peterson.

“Reproducible production of high-quality therapeutic cells and understanding what markers predict cell effectiveness could give clinicians worldwide new tools in the battle against some of the most difficult human health challenges we face today,” Peterson said. “Transitioning these cells into broad clinical use will require the kind of multidisciplinary collaboration that Georgia Tech is known for. Beyond Georgia Tech, this effort will involve The Marcus Foundation, top clinical institutions, the private sector and the Georgia Research Alliance.”

The center will involve multiple research organizations at Georgia Tech, including the Institute for Electronics and Nanotechnology, the Georgia Tech Manufacturing Institute and the Parker H. Petit Institute for Bioengineering and Bioscience. Also involved will be faculty researchers from the College of Sciences, College of Computing, and various schools in the College of Engineering, which includes the Coulter Department of Biomedical Engineering operated by Georgia Tech and Emory University. The center will also work closely with the Center for Immunoengineering at Georgia Tech, the Georgia Immunoengineering Consortium, and the Regenerative Engineering and Medicine (REM) Center, a partnership between Georgia Tech, Emory University and the University of Georgia.

“There is no question that stem cell and immune cell manufacturing have the potential to significantly impact our lives, especially as we age,” said Ravi Bellamkonda, chair of the Coulter Department of Biomedical Engineering. “We are fortunate to have a visionary foundation in The Marcus Foundation, and the foresight of the Georgia Research Alliance providing leadership in this endeavor.”

Work of the center will help make new cell-based therapies more widely available to patients.

“The timing of this investment in cell manufacturing by The Marcus Foundation is absolutely critical,” said Robert E. Guldberg, executive director of Georgia Tech’s Petit Institute for Bioengineering and Bioscience. “Cell therapies are being evaluated in nearly 9,000 clinical trials worldwide, but their potential to impact human healthcare will be severely limited until we can scale up their production reproducibly and at low cost. There are currently FDA-approved, clinically effective cell therapy products sitting on the shelf and unavailable to patients because the cost of manufacturing them is simply too high.”

The cell manufacturing effort grew, in part, out of a major planning grant awarded by the National Institute of Standards and Technology (NIST) to the Georgia Research Alliance in 2014. That effort focused on developing a road map for cell manufacturing in the state of Georgia – an initiative expected to provide significant economic development benefits. Georgia Tech has been leading this road mapping effort that involves more than 30 industry partners and 16 academic institutions as well as key federal agencies.

“The NIST grant kick-started our efforts to develop a national road map for cell manufacturing,” said Michael Cassidy, president and CEO of the Georgia Research Alliance. “The cell manufacturing industry is an emerging and growing industry with annual revenues of about $1 billion. This initiative has the potential to turn scientific research into new businesses and jobs for Georgia.”

Initial funding is for five years, and ultimately the center will be expected to support itself with corporate, government and nonprofit funding, Roy said.

“This is a unique public-private philanthropic partnership to address a grand challenge,” he added. “We hope to make significant contributions to improving cell-based treatments and lowering their cost. This could provide huge benefit not only to the health of our fellow citizens, both adults and children, but as a manufacturing initiative, could be transformative to the economic development and workforce in Georgia.”

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Researchers at the Georgia Institute of Technology used high-throughput screening techniques to study the effects of titanium dioxide nanoparticles on the expression of 84 genes related to cellular oxidative stress. Their work found that six genes, four of them from a single gene family, were affected by a 24-hour exposure to the nanoparticles.

The effect was seen in two different kinds of cells exposed to the nanoparticles: human HeLa cancer cells commonly used in research, and a line of monkey kidney cells. Polystyrene nanoparticles similar in size and surface electrical charge to the titanium dioxide nanoparticles did not produce a similar effect on gene expression.

“This is important because every standard measure of cell health shows that cells are not affected by these titanium dioxide nanoparticles,” said Christine Payne, an associate professor in Georgia Tech’s School of Chemistry and Biochemistry. “Our results show that there is a more subtle change in oxidative stress that could be damaging to cells or lead to long-term changes. This suggests that other nanoparticles should be screened for similar low-level effects.”

The research was reported online May 6 in the Journal of Physical Chemistry C. The work was supported by the National Institutes of Health (NIH) through the HERCULES Center at Emory University, and by a Vasser Woolley Fellowship.

Titanium dioxide nanoparticles help make powdered donuts white, protect skin from the sun’s rays and reflect light in painted surfaces. In concentrations commonly used, they are considered non-toxic, though several other studies have raised concern about potential effects on gene expression that may not directly impact the short-term health of cells.

To determine whether the nanoparticles could affect genes involved in managing oxidative stress in cells, Payne and colleague Melissa Kemp – an associate professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University – designed a study to broadly evaluate the nanoparticle’s impact on the two cell lines.

Working with graduate students Sabiha Runa and Dipesh Khanal, they separately incubated HeLa cells and monkey kidney cells with titanium oxide at levels 100 times less than the minimum concentration known to initiate effects on cell health. After incubating the cells for 24 hours with the TiO₂, the cells were lysed and their contents analyzed using both PCR and Western Blot techniques to study the expression of 84 genes associated with the cells’ ability to address oxidative processes.

Payne and Kemp were surprised to find changes in the expression of six genes, including four from the peroxiredoxin family of enzymes that helps cells degrade hydrogen peroxide, a byproduct of cellular oxidation processes. Too much hydrogen peroxide can create oxidative stress which can damage DNA and other molecules.

The effect measured was significant – changes of about 50 percent in enzyme expression compared to cells that had not been incubated with nanoparticles. The tests were conducted in triplicate and produced similar results each time.

“One thing that was really surprising was that this whole family of proteins was affected, though some were up-regulated and some were down-regulated,” Kemp said. “These were all related proteins, so the question is why they would respond differently to the presence of the nanoparticles.”

The researchers aren’t sure how the nanoparticles bind with the cells, but they suspect it may involve the protein corona that surrounds the particles. The corona is made up of serum proteins that normally serve as food for the cells, but adsorb to the nanoparticles in the culture medium. The corona proteins have a protective effect on the cells, but may also serve as a way for the nanoparticles to bind to cell receptors.

Titanium dioxide is well known for its photo-catalytic effects under ultraviolet light, but the researchers don’t think that’s in play here because their culturing was done in ambient light – or in the dark. The individual nanoparticles had diameters of about 21 nanometers, but in cell culture formed much larger aggregates.

In future work, Payne and Kemp hope to learn more about the interaction, including where the enzyme-producing proteins are located in the cells. For that, they may use HyPer-Tau, a reporter protein they developed to track the location of hydrogen peroxide within cells.

The research suggests a re-evaluation may be necessary for other nanoparticles that could create subtle effects even though they’ve been deemed safe.

“Earlier work had suggested that nanoparticles can lead to oxidative stress, but nobody had really looked at this level and at so many different proteins at the same time,” Payne said. “Our research looked at such low concentrations that it does raise questions about what else might be affected. We looked specifically at oxidative stress, but there may be other genes that are affected, too.”

Those subtle differences may matter when they’re added to other factors.

“Oxidative stress is implicated in all kinds of inflammatory and immune responses,” Kemp noted. “While the titanium dioxide alone may just be modulating the expression levels of this family of proteins, if that is happening at the same time you have other types of oxidative stress for different reasons, then you may have a cumulative effect.”

Seed funding for the research came from the HERCULES: Exposome Research Center (NIEHS: P30 ES019776) at the Rollins School of Public Health, Emory University, NIH grant DP2OD006483-01 and a Vasser Woolley Faculty Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

CITATION: Sabiha Runa, Dipesh Khanal, Melissa L. Kemp, Christine K. Payne, “TiO2 Nanoparticles Alter the Expression of Peroxiredoxin Anti-Oxidant Genes,” (Journal of Physical Chemistry C, 2016). http://dx.doi.org/10.1021/acs.jpcc.6b01939.

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These tiny vesicles (smaller than red blood cells), discovered about 35 years ago, were initially thought of as little dumpsters for unwanted cellular material. But further study of exosomes demonstrated their role as long-distance couriers with specific messages to carry – they can transfer biomolecules (proteins, lipids, genetic material) that impact recipient cells’ functionality in a variety of physiologic and disease processes. 

It also turns out that exosomes are in the ideal size range for lymphatic transport, and this is what really interests Brandon Dixon, faculty researcher in the Petit Institute for Bioengineering and Bioscience. 

Dixon, whose research focuses on lymphatic function, attended a presentation by fellow Petit Institute researcher Fred Vannberg several years ago. Vannberg’s lab uses computer algorithms and genomics to investigate infectious diseases, and this includes the role of exosomes during infection. His presentation that day described the characteristics of exosomes, which when released in the periphery are too big to be taken up by blood vessels, but just right for lymphatic transport.

“When Fred told me that, I thought that here was a mechanism that seems to have been made to target lymphatics,” says Dixon, associate professor in the Woodruff School of Mechanical Engineering, who leads the Laboratory of Lymphatic Biology and Bioengineering (LLBB). “Exosomes are the perfect size when we think of creating contrast agents or drug delivery particles that we want to use to target lymphathics.”

So, utilizing a Petit Institute seed grant, Dixon and Vannberg brought their distinct skills together to produce a groundbreaking research paper, “Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node,” published last month in the Nature journal, Scientific Reports.

“Lymphatic vessels provide an extremely rapid route for delivery of exosomes from the tissue to the draining lymph node,” Dixon says. “Lymphatic transport has been implied in previous publications, but this is the first demonstration of immediate lymphatic transport, a feat we achieved using our non-invasive near-infrared imaging technology and fluorescently labeled exosomes.”

Their results suggest that exosome transfer via lymphatic flow (from the periphery to the lymph node) could enable a rapid exchange of infection-specific information that precedes the arrival of migrating cells, priming the node for a more effective innate immune response, or “a first warning response during infection,” Dixon explains. 

“What’s exciting about this research is, we’re kind of finding out what is happening as part of the real-time response to infection, with the innate immune system being activated in a particular way by these particles,” adds Vannberg, assistant professor in the School of Biology. “And that helps guide what’s going to happen a few days later with the adaptive immune response.”

In previous research, Vannberg has tried to quantitate the body’s ability to fight infection, focusing on tuberculosis and leprosy. He became especially interested in exosomes’ role in our immune system’s ability to detect and fight disease after reading the research of Notre Dame researcher Jeff Schorey, who identified exosomes as ideal for diagnostic development.

The collaboration between Vannberg and Dixon is like a research laboratory version of a Marvel super hero saga – individuals combining disparate skills (‘super powers’ in the movie version) to achieve a common goal. Or, as Vannberg says, “this paper helps highlight both of our areas of expertise – mine in terms of genomics and infectious diseases, while Brandon is known worldwide for his work in lymphatic biology and his understanding of kinetics.”

Dixon’s lab was able to determine how fast the particles got delivered to the node – “within a few minutes,” Dixon says. “Until now, we really had no appreciation of the time scale.”

Lead author on the paper was biology grad student Swetha Srinivasan, who is co-advised by Dixon and Vannberg. They designed the experiments and she carried out the experimental work, while all three analyzed the data, wrote and reviewed the manuscript. Their published research illustrates a potentially efficient pathway for targeted therapeutics, somewhere down the line. 

“We believe this work has far-reaching implications on how biological systems – like pathogens, immune cells and cancer cells – could utilize lymphatic transport of exosomes to rapidly manipulate the lymph node environment,” Dixon says.

An upcoming paper will help determine what actually happens when exosomes arrive in the lymph node.

“We’re interested in understanding the immune consequences of exosomes in the context of infection and immunity,” Vannberg says. “Further research will help explain how these particles can stimulate a quick response and also inform the adaptive response.”

READ THE RESEARCH PAPER HERE

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