Get Set for Clonetegration

Designer bacteria moves to the cutting edge

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From Australia to Austin, 2013 brought exciting news about designer bacteria -- produced by integrating extra pieces of genetic material into the DNA of bacteria -- leading to new and improved vaccines and other advancements.

At the University of Texas at Austin, researchers developed a menu of 61 new strains of genetically engineered bacteria that may improve the efficacy of vaccines for diseases such as flu, pertussis, cholera and HPV. Meanwhile, Australian researchers at the University of Adelaide announced a "simpler and faster way of producing designer bacteria used in biotechnology processes."

Designer bacteria are produced by integrating extra pieces of genetic material into the DNA of bacteria, in this case E. coli, so that the bacteria will make a desired product.

"E. coli strains are commonly used workhorses for biotechnology and metabolic engineering," said Keith Shearwin, PhD, the head of the research team at Adelaide's School of Molecular and Biomedical Sciences. "For example, new genes or even the genetic material for whole metabolic pathways are inserted into the bacteria's chromosome so that they produce compounds or proteins not normally produced. Insulin is an example of a therapeutic product produced in this way."

The Need for Speed
It is believed that new processes could facilitate faster development of designer bacteria used in therapeutic drug development, such as insulin and other biotechnology products.

The statistics reveal a sense of urgency for designer bacteria breakthroughs. For example, a 2012 report from the International Diabetes Federation revealed that the chronic disease kills one person every six seconds and afflicts 382 million people worldwide. The number of diabetes cases has climbed 4.4 percent over the past two years, according to new figures from the Brussels-based federation. The number of people affected by the disease is expected to climb 55 percent, afflicting 592 million, by 2035.

The journal, ACS Synthetic Biology, reported last May that researchers from the University of Adelaide - in a collaborative effort with a Stanford team, headed by Francois St. Pierre, PhD - have developed a "one-step bacterial engineering process," called "clonetegration."

"The existing process for integrating new genes is inefficient, taking several days" Shearwin added. "Our new process can be completed overnight."

He pointed to work done by Jeff Hasty, PhD, at the University of California at San Diego, whose group is developing toxin-detecting circuits, as an example where methods like "clonetegration" might prove useful.

"In the short term, unlikely; but in the longer term, biosensors based on chromosomal integration may be developed," said Shearwin. "In principle, any artificial circuit can be 'embedded' in a bacterial chromosome by 'clonetegration.'"

Nearly a year later, the push for implementation -- and quicker and more efficient laboratories -- continues.

"It is a little difficult to tell how widely our system has been taken up so far," he said. "There is always some lag time before new publications appear, containing citations to new approaches. There are a number of related technologies for optimizing expression of proteins, because the field is developing very rapidly. Similarly, it's difficult to know if the industry has taken up the system, given that publication may be less pressing."

As initially promised, the molecular tools needed for the "clonetegration" process have been available for ongoing research and development. Along with speeding up the biotech process, it will enable multiple rounds of genetic engineering on the same bacteria, and simultaneous integration of multiple genes at different specific locations.

"This will become a valuable technique for facilitating genetic engineering with sequences that are difficult to clone as well as enable the rapid construction of synthetic biological systems," said Shearwin.

Shearwin added that plasmids have been deposited with a non-profit repository, Addgene (www.addgene.org/)?.

"Addgene conducts various checks on the DNA sequences, and then sends out the plasmids to whoever requests them, for a nominal fee," he said. "I believe around 30 requests have been made since we deposited the plasmids back in July last year."

The "Dirty Little Secret"
The strains of E. coli - as described last January in the journal, Proceedings of the National Academy of the Sciences (PANS), are part of a new class of biological "adjuvants" poised to transform vaccine design. Adjuvants, termed the "dirty little secret" of immunology by Stephen Trent, PhD, associate professor of biology at the College of Natural Sciences of the University of Texas at Austin, are substances added to vaccines to boost the human immune response.

"For 70 years, the only adjuvants being used were aluminum salts," he said in last year's University of Texas at Austin release on the development. "They worked, but we didn't fully understand why, and there were limitations. Then four years ago the first biological adjuvant was approved by the FDA. I think what we're doing is a step forward from that. It's going to allow us to design vaccines in a much more intentional way."

Making History
The history of adjuvants goes back to the pioneering days of vaccines, and strictly more by error than trial. Batches of vaccine that were contaminated accidentally proved themselves to be more effective than those in their "pure" form. Researchers learned the body's immune system was "taught" to recognize the dirt adjuvant and produce antibodies in response to it. The net result was a more battle-ready immune system, armed to fight virus or bacteria, by virtue of a general alarm placing agents of the immune system in circulation in the bloodstream, allowing recognition of the key antigen.

The potentially dangerous entoxin molecules appear on the cell surface of a wide range of bacteria. As a result, humans have evolved over millions of years to detect and respond to them quickly. They trigger an immediate red alert.

"In some of its forms, an endotoxin can kill you," said Trent. "But the adjuvant, which is called MPL, is a very small, carefully modified piece of it, so it's able to trigger the immune response without overdoing it."

What Trent and his colleagues have done is expand on that basic premise. Rather than just work with an inert piece of endotoxin, they've engineered E. coli bacteria to express the endotoxin in many configurations on the cell surface.

"These 61 E. coli strains each have a different profile on their surface," said Brittany Needham, a doctoral student from Trent's lab and the first author on the paper. "In every case, the surface structure of the endotoxin is safe, but it will interact with the immune system in a range of ways. Suddenly, we have a huge potential menu of adjuvants to test out with different kinds of vaccines."

One form might work better with cholera vaccine, another with pertussis (whooping cough) and another with a future HIV vaccine. Trent, Needham and their colleagues should be able to fine-tune the adjuvants with increasing precision as more E. coli strains are engineered and tested, and as their understanding of how they interact with the immune system deepens.

"I think we're at the dawn of a new age of vaccine design," said Trent. "For a long time, vaccinology was really a trial-and-error field. It was a black box. We knew certain things worked. We knew certain vaccines had certain side effects, but we didn't entirely know why. Now that's changing."

Hurdles Ahead
In the realm of laboratory medical professionals, Shearwin was eager to explain what "clonetegration" would mean in their work environment.

"Our system is directed toward synthetic biology/metabolic engineering labs, with the aim of easily integrating sets of genes into bacterial chromosomes for the production of any compound which can be produced by fermentation," he said.  "However, in synthetic biology, I think it will make significant inroads in medical biotechnology in the area of biosensors and diagnostics, by developing custom made genetic circuits which respond in some way -- by giving off light, for example, in response to specific signals.  There are efforts underway by other research groups to produce bacteria which will 'search and destroy' other pathogenic bacteria."

He still foresees hurdles ahead, adding "work is in progress to improve versatility and efficiency" of "clonetegration."

"In the short term, 'clonetegration' should help speed up the process of optimizing the right combination of biological parts which go together to make biological circuits with desired behavior, such as sensitive and specific biosensors," he said. "Challenges include extending the range of bacteria in which the system can operate and making it even more efficient.

"There are a huge range of products which could potentially be produced, including proteins, bio-active peptides and biofuels. There is great interest in the moment in producing hydrocarbons which could supplement/replace fossil fuels.

"It is possible that protein-based vaccines could be produced with our system. Vaccines using whole, inactivated virus tend to be produced in other ways, such as tissue culture, rather than microbial cell culture."

One-Two-Three Punch
In information provided by the University of Texas at Austin, Trent said that an additional advantage of their system is that the E. coli can be engineered to express key viral and bacterial antigens along with the endotoxin. A single cell could deliver both parts of the one-two punch, or even a one-two-three punch, if antigens from multiple diseases were expressed in a single E. coli.

"It makes possible a vaccine that provides protection from multiple pathogens at the same time," said Trent.

Trent and his colleagues are working on a second round of designer E. coli. They have also filed a provisional patent on their system and are working with the university to find a corporate partner to pay for clinical trials.

"This is ready to go," said Trent. "I can't predict whether it will actually make it to the market, but it's very similar to the adjuvant that has already been approved; and my instinct is that, if a company will undertake to do the trials, it will get approved. A company could call us tomorrow, we could send them a strain and they could start working."

Gordon Glantz is a freelance writer and frequent contributor to ADVANCE.

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