Researchers advance toward engineering ‘wildly new genome’

Contact: David Cameron david_cameron@hms.harvard.edu 617-432-0441 Harvard Medical School

In two parallel projects, researchers have created new genomes inside the bacterium E. coli in ways that test the limits of genetic reprogramming and open new possibilities for increasing flexibility, productivity and safety in biotechnology.

In one project, researchers created a novel genome—the first-ever entirely genomically recoded organism—by replacing all 321 instances of a specific “genetic three-letter word,” called a codon, throughout the organism’s entire genome with a word of supposedly identical meaning. The researchers then reintroduced a reprogramed version of the original word (with a new meaning, a new amino acid) into the bacteria, expanding the bacterium’s vocabulary and allowing it to produce proteins that do not normally occur in nature.

In the second project, the researchers removed every occurrence of 13 different codons across 42 separate E. coli genes, using a different organism for each gene, and replaced them with other codons of the same function. When they were done, 24 percent of the DNA across the 42 targeted genes had been changed, yet the proteins the genes produced remained identical to those produced by the original genes.

“The first project is saying that we can take one codon, completely remove it from the genome, then successfully reassign its function,” said Marc Lajoie, a Harvard Medical School graduate student in the lab of George Church.  “For the second project we asked, ‘OK, we’ve changed this one codon, how many others can we change?'”

Of the 13 codons chosen for the project, all could be changed.

“That leaves open the possibility that we could potentially replace any or all of those 13 codons throughout the entire genome,” Lajoie said.

The results of these two projects appear today in Science. The work was led by Church, Robert Winthrop Professor of Genetics at Harvard Medical School and founding core faculty member at the Wyss Institute for Biologically Inspired Engineering. Farren Isaacs, assistant professor of molecular, cellular, and developmental biology at Yale School of Medicine, is co-senior author on the first study.

Toward safer, more productive, more versatile biotech

Recoded genomes can confer protection against viruses—which limit productivity in the biotech industry—and help prevent the spread of potentially dangerous genetically engineered traits to wild organisms.

“In science we talk a lot about the ‘what’ and the ‘how’ of things, but in this case, the ‘why’ is very important,” Church said, explaining how this project is part of an ongoing effort to improve the safety, productivity and flexibility of biotechnology.

“These results might also open a whole new chemical toolbox for biotech production,” said Isaacs. “For example, adding durable polymers to a therapeutic molecule could allow it to function longer in the human bloodstream.”

But to have such an impact, the researchers said, large swaths of the genome need to be changed all at once.

“If we make a few changes that make the microbe a little more resistant to a virus, the virus is going to compensate. It becomes a back and forth battle,” Church said. “But if we take the microbe offline and make a whole bunch of changes, when we bring it back and show it to the virus, the virus is going to say ‘I give up.’ No amount of diversity in any reasonable natural virus population is going to be enough to compensate for this wildly new genome.”

In the first study, with just a single codon removed, the genomically recoded organism showed increased resistance to viral infection. The same potential “wildly new genome” would make it impossible for engineered genes to escape into wild populations, Church said, because they would be incompatible with natural genomes. This could be of considerable benefit with strains engineered for drug or pesticide resistance, for example. What’s more, incorporating rare, non-standard amino acids could ensure strains only survive in a laboratory environment.

Engineering and evolution

Since a single genetic flaw can spell death for an organism, the challenge of managing a series of hundreds of specific changes was daunting, the researchers said. In both projects, the researchers paid particular attention to developing a methodical approach to planning and implementing changes and troubleshooting the results.

“We wanted to develop the ability to efficiently build the desired genome and to very quickly identify any problems—from design flaws or from undesired mutations — and develop workarounds,” Lajoie said.

The team relied on number oftechnologies developed in the Church lab and the Wyss Institute and with partners in academia and industry, including next-generation sequencing tools, DNA synthesis on a chip, and MAGE and CAGE genome editing tools. But one of the most important tools they used was the power of natural selection, the researchers added.

“When an engineering team designs a new cellphone, it’s a huge investment of time and money. They really want that cell phone to work,” Church said. “With E. coli we can make a few billion prototypes with many different genomes, and let the best strain win. That’s the awesome power of evolution.”

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Funding was from Department of Energy [DE-FG02-02ER63445], NSF [SA5283-11210], NIH [NIDDK-K01DK089006], DARPA [N66001-12-C-4040, N66001-12-C-4020, N66001-12-C-4211], Arnold and Mabel Beckman Foundation, Department of Defense NDSEG Fellowship, NIH-MSTP-TG-T32GM07205, NSF graduate fellowship, NIH Director’s EarlyIndependence Award [Grant 1DP5OD009172-01], U.S. Office of Naval Research [N000141010144], Agilent Technologies, Wyss Institute, and Department of Defense NDSEG Fellowship, and Air Force Contract #FA8721-05-C-0002.

Written by JAKE MILLER

Harvard Medical School has more than 7,500 full-time faculty working in 11 academic departments located at the School’s Boston campus or in one of 47 hospital-based clinical departments at 16 Harvard-affiliated teaching hospitals and research institutes. Those affiliates include Beth Israel Deaconess Medical Center, Brigham and Women’s Hospital, Cambridge Health Alliance, Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Pilgrim Health Care, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children’s Center, Massachusetts Eye and Ear Infirmary, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Schepens Eye Research Institute, Spaulding Rehabilitation Hospital and VA Boston Healthcare System.

The Wyss Institute for Biologically Inspired Engineering at Harvard University uses Nature’s design principles to develop bioinspired materials and devices that will transform medicine and create a more sustainable world. Working as an alliance among Harvard’s Schools of Medicine, Engineering, and Arts & Sciences, and in partnership with Beth Israel Deaconess Medical Center, Brigham and Women’s Hospital, Boston Children’s Hospital, Dana Farber Cancer Institute, Massachusetts General Hospital, the University of Massachusetts Medical School, Spaulding Rehabilitation Hospital, Boston University, Tufts University, andCharité – Universitätsmedizin Berlin, the Institute crosses disciplinary andinstitutional barriers to engage in high-risk research that leads to transformative technological breakthroughs. By emulating Nature’s principles, Wyss researchers are developing innovative new engineering solutions for healthcare, energy, architecture, robotics, and manufacturing. These technologies are translated into commercial products and therapies through collaborations with clinical investigators, corporate alliances, and new start-ups. The Wyss Institute recently won the prestigious World TechnologyNetwork award for innovation in biotechnology.

Intestinal bacteria promote — and prevent — inflammatory bowel disease

2008 – re-post for filing

Contact: David Cameron
david_cameron@hms.harvard.edu
617-432-0441
Harvard Medical School

BOSTON, Mass. (May 28, 2008)—Scientists search for drug candidates in some very unlikely places. Not only do they churn out synthetic compounds in industrial-scale laboratories, but they also scour coral reefs and scrape tree bark in the hope of stumbling upon an unsuspecting molecule that just might turn into next year’s big block buster. But one region that scientists have not been searching is their guts. Literally.

Now, a team of researchers at Harvard Medical School, Brigham and Women’s Hospital, and the California Institute of Technology have demonstrated that a molecule produced by bacteria in the gut’s intestinal microflora can eliminate symptoms of inflammatory bowel disease (IBD), a condition that includes Crohn’s disease and ulcerative colitis, in animal models.

“Given the sheer number of bacteria in the gut, the potential for discovering new molecules that can treat a whole range of these diseases is promising,” says Dennis Kasper, co-lead author on the study, professor of medicine and microbiology and molecular genetics at Harvard Medical School, and director of the Channing Laboratory at Brigham and Women’s Hospital.

The study will appear as the cover story in the May 29 issue of Nature.

Scientists have known for many decades that the mammalian gut is an ecosystem teeming with approximately 1,000 different species of bacteria, species as distinct from the host as a single-cell amoeba in pond scum. Rather than causing disease, these bacteria are responsible for protecting against infection and aiding digestion. An increasing number of scientists also suspect that recent increases in asthma and even certain food allergies are caused by disruptions in the delicate balance of this intestinal ecosystem.

In 2005, Kasper and Sarkis Mazmanian, then a postdoc in Kasper’s lab and now an assistant professor of biology at the California Institute of Technology, discovered that a species of intestinal bacteria called Bacteroides fragilis could restore immune system balance in mice that were bred to lack intestinal bacteria. A particular product of B. fragilis, a sugar molecule called polysaccharide A (PSA), recovered the equilibrium of a certain subclass of immune system cells (called Th1 and Th2) whose levels became skewed when bacteria in the gut were absent. The researchers referred to PSA as a “symbiosis factor,” one that established a beneficial link between bacteria and mammals. This was the first study in which such a link was demonstrated.

Interestingly, when the study was completed, Kasper and Mazmanian found in these mice an abundance of immune system cells that were known to protect against colitis and Crohn’s disease. In the current report, the groups decided to expand these findings and explore potential links between PSA and inflammatory bowel disease.

When immunocompromised mice with a specific pathogen-free microbiota were given an intestinal bacterium called Helicobacter hepaticus, they soon developed “rip roaring” IBD, according to Kasper. However, when Helicobacter was combined with B. fragilis, the mice were fine. Further experiments revealed that PSA—the special sugar molecule—was the key factor in preventing IBD. In fact, when mice were given Helicobacter combined with PSA purified from B. fragilis bacteria, they showed no symptoms of IBD.

“But then the key question was, if PSA was essential for preventing these animals from coming down with either colitis or Crohn’s, how did it do it”” says Kasper. “What was the mechanism””

The answer came by studying a subset of interleukins, that is, molecules secreted by immune cells.

Previous studies had shown that two particular interleukins, called IL-17 and IL-23, promote intestinal inflammation and are present at high levels in IBD patients. Here, while the researchers found IL-17 and IL-23 in the guts of animals who had received Heliobacter alone, these interleukins were absent from animals who had also received both PSA-producing B. fragilis and purified PSA.

“We realized that something in PSA must be preventing the inflammation that causes colitis and Crohn’s, which would explain the reduction in IL-17 and IL-23,” says Kasper.

This hunch brought the researchers to consider a third interleukin, IL-10. The opposite of IL-17 and IL-23, IL-10 is anti-inflammatory and had previously been shown to protect against experimental colitis.

The researchers once again administered Helicobacter and PSA-active B. fragilis (the combination that had previously led to healthy mice), only this time they included an antibody that blocked IL-10. As a result, the mice all came down with IBD.

“This demonstrated for us the mechanism by which PSA protects against IBD,” says Kasper.

Indeed, the researchers deduced that PSA prompts immune system cells to secrete IL-10, which in turn suppresses the inflammation caused by IBD. In other words, PSA is an anti-inflammatory.

This research should encourage people (including many scientists) to consider the vast potential for beneficial contributions to human health by “good” bacteria. And what’s more, “This is the first time that a beneficial molecule produced by intestinal bacteria has been shown to work therapeutically in an animal model,” says Mazmanian.

The researchers caution that these findings do not promise any near-term treatments for IBD. “PSA might do the same thing in humans, and it might not,” says Kasper.

However, the mechanism that they’ve discovered should persuade scientists and drug manufacturers to consider new sources for expanding the drug pipeline.

“There is currently no effort to develop molecules that are naturally made by bacteria to use therapeutically,” continues Mazmanian. “This study opens up that possibility.”

 

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Full citation:
Nature, May 29, 2008, 453 (7195), 620-624
“A microbial symbiosis factor prevents intestinal inflammatory disease”
Sarkis K. Mazmanian(1), June L. Round(1) & Dennis L. Kasper(2,3)

1-Division of Biology, California Institute of Technology, Pasadena, California.
2-Channing Laboratory, Brigham & Women’s Hospital, Harvard Medical School, Boston, Massachusetts
3-Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts

Harvard Medical School (http://hms.harvard.edu/hms/home.asp) has more than 7,500 full-time faculty working in 11 academic departments located at the School’s Boston campus or in one of 47 hospital-based clinical departments at 18 Harvard-affiliated teaching hospitals and research institutes. Those affiliates include Beth Israel Deaconess Medical Center, Brigham and Women’s Hospital, Cambridge Health Alliance, Children’s Hospital Boston, Dana-Farber Cancer Institute, Forsyth Institute, Harvard Pilgrim Health Care, Hebrew SeniorLife, Joslin Diabetes Center, Judge Baker Children’s Center, Immune Disease Institute, Massachusetts Eye and Ear Infirmary, Massachusetts General Hospital, McLean Hospital, Mount Auburn Hospital, Schepens Eye Research Institute, Spaulding Rehabilitation Hospital, and VA Boston Healthcare System.

Brigham and Women’s Hospital (http://www.brighamandwomens.org/) is a 747-bed nonprofit teaching affiliate of Harvard Medical School and a founding member of Partners HealthCare, an integrated health care delivery network. BWH is committed to excellence in patient care with expertise in virtually every specialty of medicine and surgery. BWH is an international leader in basic, clinical and translational research on human diseases, involving more than 860 physician-investigators and renowned biomedical scientists and faculty supported by more than $416 M in funding. BWH is also home to major landmark epidemiologic population studies, including the Nurses’ and Physicians’ Health Studies and the Women’s Health Initiative.

Measles, Mumps, Rubella vaccine linked with 2-fold risk of seizures

Contact: Danielle Cass danielle.x.cass@kp.org 510-267-5354 Kaiser Permanente

Combination MMRV vaccine linked with 2-fold risk of seizures

Electronic health records study of 459,000 children sparked new CDC recommendations

Oakland, CA (June 28) – The combination vaccine for measles, mumps, rubella and chickenpox (MMRV) is associated with double the risk of febrile seizures for 1- to 2-year-old children compared with same-day administration of the separate vaccine for MMR (measles, mumps, rubella) and the varicella (V) vaccine for chicken pox, according to a Kaiser Permanente Division of Research study appearing online in the journal Pediatrics. A febrile seizure is a brief, fever-related convulsion but it does not lead to epilepsy or seizure disorders, researchers explained.

Funded by the U.S. Centers for Disease Control, the study analyzed 459,000 children 12 to 23 months old from numerous health systems across the United States receiving their first dose of measles-containing vaccine and found MMRV to be associated with a two-fold increased risk of fever and febrile seizures 7-10 days after vaccination compared with same-day administration of a separate shot for MMR and the varicella (chickenpox) vaccine. This study found that the risk for a febrile seizure after the first dose of MMRV vaccine is low, although it is higher than after MMR vaccine and varicella vaccine administered as separate injections.

The study found no evidence of an increased febrile seizure risk after any measles vaccine beyond 7-10 days post vaccination.

“Because the risk of febrile seizure is higher for the quadrivalent (combination) vaccine, providers recommending MMRV should communicate to parents that it increases the risk of fever and febrile seizure over that already associated with measles-containing vaccines,” said the study’s lead investigator Nicola Klein, MD, Ph.D., co-director of the Kaiser Permanente Vaccine Study Center. “But concerned parents should understand that the risk for febrile seizures after any measles-containing vaccine is low: less than 1 febrile seizure per 1,000 injections.”

The CDC recently recommended that either vaccine may be used for first dose for 1-2 year olds, however families without a strong preference for MMRV should receive separate MMR +V vaccines, Klein said. The CDC reiterates that providers who consider using MMRV should discuss with families and caregivers the risk and benefits.

“While this study and the resulting CDC recommendations are very important and ones our pediatricians will follow, it is also important to emphasize that it is more common for a child to have a febrile seizure caused by a simple cold than by an immunization. And though febrile seizures are a very scary event for a family, they are not dangerous and do not lead to later epilepsy or seizure disorders,” said Randy Bergen, MD, a Kaiser Permanente pediatrician and infectious disease specialist at Kaiser Permanente-Walnut Creek.

Kaiser Permanente researchers used its electronic health records and Vaccine Safety Datalink data from 2000 to 2008 to assess seizures and fever visits among children aged 12-23 months following MMRV and separate MMR +V. They compared seizure risk following MMRV to MMR +V using regression analyses and by incorporating chart-reviewed febrile seizure cases.

The Vaccine Safety Datalink project is a collaborative effort between CDC’s Immunization Safety Office and eight managed care organizations: Kaiser Permanente Northern California, Kaiser Permanente Southern California, Kaiser Permanente Colorado, Kaiser Permanente Northwest, Health Partners, Group Health Cooperative, Marshfield Clinic and Harvard Pilgrim Health Care. The VSD project was established in 1990 to monitor immunization safety and address the gaps in scientific knowledge about rare and serious events following immunization. The VSD shares electronic health records from the organizations’ health systems.

MMRV was licensed by the FDA in 2005.  MMRV was subsequently recommended by the Advisory Committee on Immunization Practices (ACIP) in 2006. Although prelicensure studies of MMRV among 1-2 year olds noted higher rates of fever and measles-like rash one to two weeks post vaccination when compared with separate MMR + V, it was unknown at the time of MMRV’s licensure whether a higher rate of fevers was similarly associated with increased risk of febrile seizures.  In February 2008, Kaiser Permanente researchers alerted the ACIP to preliminary evidence of an increased risk of febrile seizures following MMRV. This study represents additional data on twice as many vaccines.

“The Vaccine Safety Datalink, which we used to conduct this study, is a premiere example of how different managed care organizations can leverage their electronic medical records to improve vaccine safety and monitoring,” Klein said.

This is the latest in a series of Kaiser Permanente studies undertaken to better understand the protective effects and risks of vaccines. Recent published studies found children of parents who refuse vaccines are nine times more likely to get chickenpox and 23 times more likely to get whooping cough compared to fully immunized children. A study published last year found that herpes zoster, also known as shingles, is very rare among children who have been vaccinated against chicken pox. A recent study in the Journal of the American Medical Association found that the pneumococcal vaccination is not associated with a reduced risk of heart attacks or strokes in middle-aged men.

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* Requested Repost