GENE EDITITING AND REGULATION TO IMPROVE IMMUNE SYSTEM

FROM:  NATIONAL SCIENCE FOUNDATION
Rewriting genetic information to prevent disease

Breakthrough Prize winner harnesses CRISPR to improve immune system
For the last few years, scientists have been studying an ancient but only recently understood mechanism of bacterial immunity that has the potential to provide immeasurable benefits to plant and animal health.

The phenomenon known as CRISPR (for Clustered Regularly Interspaced Short Palindromic Repeats) is a natural immune system found in many bacteria with the ability to identify and destroy the genomes of invading viruses and plasmids.

Researchers are trying to harness this system for gene editing and regulation, a process that could transform "the genome of plants or animals in ways that will improve their health, or introduce genetic changes that will resist disease of climate change," says Jennifer Doudna, a Howard Hughes Medical Institute investigator and professor of biochemistry, biophysics and structural biology at the University of California, Berkeley. "The explosion of research using this technique has been amazing."

Doudna, collaborating with Emmanuelle Charpentier of Sweden's Helmholtz Center for Infection Research and Umeå University, identified how the system works and engineered it in new ways that broadened its scope. The two researchers, who described their work in a 2012 paper in the journal Science, developed a technique that enables the rewriting of genetic information and the correction of mutations that otherwise can cause disease, and also can knock out the cell's ability to make harmful proteins, she says.

"Many labs have shown in principle that this can be used to correct such mutations as those that occur in cystic fibrosis, or sickle cell disease," she says. "They are showing it in cell lines and lab animals. We're still some period of time away from using this in humans, but the pace in the field has been truly remarkable, and really exciting to see."

Many bacteria have this CRISPR-based immune system capable of identifying and destroying hostile invaders. Doudna and Charpentier showed that, in doing so, CRISPR produces the protein Cas9, a DNA-cutting enzyme guided by RNA, which relies on two short RNA guide sequences to find foreign DNA, then cleaves, or cuts, the target sequences, thereby muting the genes of the invaders.

Cas9 has evolved to provide protection against viruses that could infect the bacterium, and uses pieces of RNA derived from CRISPRS to direct its activity. The system is specific and efficient enough to stave off viral infections in bacteria.

Doudna and her colleagues programmed the process so that it can be directed by a single short RNA molecule; researchers who use it to edit genomes can customize the RNA so that it sends Cas9 to cleave, like "scissors," at their chosen location in the genome.

"When we figured out how it worked, we realized we could alter the design of RNA and program Cas9 to recognize any DNA sequence," she says. "One can therefore target Cas9 to any region of a genome simply by providing a short guide RNA that can pair with the region of interest. Once targeted, different versions of Cas9 can be used to activate or inhibit genes, as well as make target cuts within the genome. Depending on the experimental design, research can use these latter cuts to either disrupt genes or replace them with newly engineered versions."

Recently Douda and Charpentier and four other scientists received the Breakthrough Prize in life sciences, which honors transformative advances toward understanding living systems and extending human life. The prizes recognize pioneering work in physics, genetics, cosmology, neurology and mathematics, and carry a $3 million award for each researcher. The Breakthrough committee specifically cited Doudna and Charpentier for their advances in understanding the CRISPR mechanism.

Doudna has been the recipient of several National Science Foundation (NSF) grants to support her research in recent years totaling more than $1.5 million. In 2000, she received NSF's prestigious $500,000 Alan T. Waterman Award, which recognizes an outstanding young researcher in any field of science or engineering supported by NSF.

She also was a founder of the Innovative Genomics Initiative, established in 2014 at the Li Ka Shing Center for Genomic Engineering at UC Berkeley. Its goal is to promote and support genome editing research and technology in both academic and commercial research communities.

"We have a team of scientists working with various collaborative partners," she says. "We want to ensure that the technology gets into as many hands as possible, and explore ways to make it even better. We are trying to bring about fundamental change in biological and biomedical research by enabling scientists to read and write in genomes with equal ease. It's a bold new effort that embraces a new era in genomic engineering."

-- Marlene Cimons, National Science Foundation
Investigators
Jennifer Doudna
Related Institutions/Organizations
University of California-Berkeley

MUTATION AND DENGUE FEVER

Photo:  Mosquito.  Credit:  NSF/Wikipedia.

FROM: NATIONAL SCIENCE FOUNDATION"Defective" Virus Leads to Epidemic of Dengue Fever


It's 2001 in Myanmar (formerly known as Burma), a country in Southeast Asia. Almost 200 people have died, and more than 15,000 are ill--all having contracted dengue fever.

Dengue is a disease transmitted by mosquitoes and caused by four types of dengue virus. Infection may not result in symptoms, or may cause mild, flu-like illness--or hemorrhagic fever.

Dengue virus infects some 50-100 million people annually in Southeast Asia, South America and parts of the United States.

In 1998, a pandemic of dengue resulted in 1.2 million cases of dengue hemorrhagic fever in 56 countries.

In Myanmar, dengue is endemic. The disease has occurred there in three- to five-year cycles since the first recorded outbreak in 1970. Each one has been more deadly.

What caused the widespread infection in Myanmar in 2001, a disease that resulted from one type of dengue virus, DENV-1? For more than a decade, researchers have been working to solve the puzzle.

All viruses not created equal

Could the DENV-1 in Myanmar have been different in some way, perhaps "defective"?

Defective viruses result from genetic mutations or deletions that eliminate essential functions. They're generated in viruses with high mutation rates, but were believed to be unimportant.

But it now appears that defective viruses may be able to play a critical role in the spread of disease.

In a paper published this week in the journal PLoS Pathogens, scientists funded by the National Science Foundation (NSF) report a significant link between one such defective virus and the high rate of transmission of DENV-1 in Myanmar in 2001.

"The idea has always been that defective viruses are either meaningless or detrimental," says James Lloyd-Smith, an ecologist and evolutionary biologist at University of California, Los Angeles.

"We've found the opposite--that the defective virus is actually helping the normal, functional virus. It's bizarre and hard to believe, but the data are the data."

"We've shown that the defective virus not only goes with the normal virus, but increases the transmission of that virus," says scientist Ruian Ke, also of UCLA.

While defective viruses can't complete their life cycle on their own, if they're able to get into the same cell with a non-defective virus, they can "hitch-hike" with the non-defective one and propagate.

Deadly outbreak of DENV-1

The research team--James Lloyd-Smith; Ruian Ke; John Aaskov, a virologist at Queensland University of Technology in Brisbane, Australia; and Edward Holmes, a biologist at the University of Sydney--found that the presence of a defective DENV-1 virus may have led to a spike in dengue fever cases in Myanmar during 2001-2002.

"The causes of epidemics are much more complicated than we thought," says Sam Scheiner, NSF program director for the joint NSF-National Institutes of Health Ecology and Evolution of Infectious Diseases (EEID) Program. At NSF, EEID is funded by the Directorates for Biological Sciences and Geosciences.

In addition to EEID, the research was supported by NSF's Advancing Theory in Biology Program.

"Pathogens can depend on the presence of other microbial species or, as in this case, other varieties of the same species," says Scheiner. "Understanding these interactions is critical for predicting when the next epidemic might occur--and how to prevent it."

In the study, Ke designed a mathematical model to learn how the defective DENV-1 virus interacted with the normal virus.

Aaskov and Holmes collected genetic sequences from the defective viruses from 15 people sampled over an 18-month period in Myanmar. All were infected with DENV-1 virus; nine were also infected with the defective version.

Ke discovered that the lineage of defective viruses emerged between June 1998 and February 2001; it spread through the population until at least 2002.

The following year, the lineage appeared in the South Pacific island of New Caledonia, carried there by a mosquito or a person.

The scientists analyzed the genetic sequences of the defective and normal viruses to estimate how long the defective virus had been transmitting in the human population.

"We can see from the gene sequence of the defective version that it's the same lineage, and is a continued propagation of the virus," says Lloyd-Smith.

"From 2001 to 2002, it went from being quite rare to being in all nine people we sampled that year," says Lloyd-Smith. "Everyone sampled who was getting dengue fever was getting the defective version along with the functional virus.

"It rose from being rare to being very common in just one year."

Most surprisingly, say the scientists, the combination of the defective virus with the normal virus was "more fit" than the normal dengue virus alone.

"What we've shown is that this defective virus, which everyone had thought was useless or even detrimental to the fitness of the functional virus, actually appears to have made it better able to spread," Lloyd-Smith says.

Ke calculated that the defective virus makes it at least 10 percent more transmissible. "It was spreading better with its defective cousin tagging along than on its own," says Lloyd-Smith.

It takes two (viruses) to tango

The functional virus and defective virus travel in unison. The two transmit together in an unbroken chain.

"That's not just a matter of getting into the same human or the same mosquito--they need to get into the same cell inside that human or mosquito in order to share their genes, and for the defective version to continue hitchhiking," says Lloyd-Smith.

"We're gaining insights into the cellular biology of how dengue is infecting hosts. It must be the case that frequently there are multiple infections of single cells."

The defective virus appeared one to three years before the major epidemics in 2001 and 2002.

"One could imagine that if you build an understanding of this mechanism, you could measure it, see it coming and potentially get ahead of it," says Lloyd-Smith.

Defective viruses: disease transmitters beyond dengue?

Might defective viruses play a role in the transmission of the flu, measles and other diseases?

"There are a few signs that this phenomenon may be happening in other viruses," Lloyd-Smith says.

"We may be cracking open the book on the possible interactions between normal, functional viruses and the defective ones that people thought were just dead-ends.

"These supposedly meaningless viruses may be having a positive effect--positive for the virus, not for us.

"There's great variation from year to year in dengue epidemics in various locations, but we don't understand why. This is a possible mechanism."

Why would a defective virus increase transmission of a disease?

Lloyd-Smith offers two hypotheses.

One is that the presence of the defective virus with the functional virus in the same cell makes the functional virus replicate better within the cell by an unknown mechanism.

"It might give the virus flexibility in how it expresses its genes, and may make it more fit and better able to reproduce under some circumstances," Lloyd-Smith says.

A second idea is that the defective virus may be interfering with the disease-causing virus, making the disease less intense.

People then have a milder infection, and because they don't feel as sick, they're more likely to go out of their homes and spread the disease.

In conducting the research, Lloyd-Smith and Ke combined genetic sequence analyses with sophisticated mathematical models and bioinformatics.

"We were able to show that this defective virus transmitted in an unbroken chain across this population in Myanmar for a year-and-a-half," Lloyd-Smith says.

"Without gene sequencing, we wouldn't have been able to establish that."

The biologists hope their work will help turn the tide of the next deadly outbreak of dengue in Myanmar--and in other tropical countries around the globe.