CRISPR-Cas9 technology shows promise in ID, presents ethical quandary

Issue: August 2016

Researchers are racing to discover new applications of clustered regularly interspaced short palindromic repeats, or CRISPR, an increasingly popular gene-editing technology that could be used to develop novel antimicrobial therapies, vector control strategies and other infectious disease-focused implementations.

CRISPR is an immune system found in bacteria and archaea that uses a protein and short RNA molecules to cut the DNA of an invading virus. In 2012, Jennifer Doudna, PhD, professor of chemistry and molecular cell biology at the University of California, Berkeley; Emmanuelle Charpentier, PhD, director at the Max Planck Institute for Infection Biology, Berlin; and colleagues described a method of engineering RNA to guide the Cas9 (CRISPR-associated protein 9) nuclease and allow cleavage of specific DNA sequences. A few months later, Feng Zhang, PhD, professor of biomedical engineering at Massachusetts Institute of Technology; Luciano Marraffini, PhD, associate professor and head of the laboratory of bacteriology at Rockefeller University; and colleagues repurposed CRISPR-Cas9 for expression in mammalian cells and demonstrated the technology’s capacity to modify human genes.

Since these initial breakthroughs, researchers have used CRISPR-Cas9 to modify the genomes of fish, insects, animals and plants. Further, interest in curing genetic diseases has led Chinese researchers to perform genome editing in human pre-implantation embryos, and more recently prompted an NIH advisory committee to greenlight a trial using CRISPR-Cas9 to boost cancer therapy — the first human clinical trial involving the new technology.

Despite the abundance of multidisciplinary applications, it is clear that CRISPR-Cas9 also will make its mark on the treatment and prevention of infectious diseases, Rachel J. Whitaker, PhD, associate professor of microbiology at the University of Illinois School of Molecular and Cellular Biology, told Infectious Disease News.

“You can use CRISPR-Cas to engineer genes in the human, you can use CRISPR-Cas to engineer or study microbial populations, or you can use it to control the vector population,” Whitaker said. “Those are three distinct, direct applications of CRISPR-Cas technology to infectious disease.”

New approaches on the horizon

According to Marraffini, the most direct application of CRISPR-Cas9 in infectious disease would target the pathogen itself. By programing the nuclease to cleave key portions of a pathogen’s genetic material, he explained, the technology can be applied as an antimicrobial.

This sort of approach — which Marraffini and colleagues demonstrated in a 2014 study — could be especially valuable in clinical situations where the use of a standard broad-spectrum antibiotic may have unintended consequences.

“Currently, broad-spectrum antibiotics ... target every bacterium in our flora, and more and more it is becoming evident that this is not very good,” Marraffini told Infectious Disease News. “The CRISPR antibiotic will be, as we call it, a ‘smart’ antibiotic or a sequence-specific antibiotic that will kill only pathogenic bacteria and spare the good bacteria in the microbiota.”

Separate in vitro studies have also demonstrated CRISPR-Cas9’s effectiveness in clearing latent infection and halting viral replication in human cells. According to Marraffini, however, clinical implementation of these methods will not be immediate. He explained that several issues facing the technique — such as how to effectively deliver the Cas9 to the infection site, or how to transmit the large protein past a bacterium’s protective cell wall — have yet to be solved and could require several years to overcome.

“How do you deliver the Cas9 nuclease inside not only one bacterium, but inside of lots of bacteria that are propagating and causing disease?” he said. “That’s the main problem of the technology. We have a way to do that, but it’s not efficient enough, so a lot of the ongoing research is to try to deliver this Cas9 nuclease efficiently into every bacterium that we have in our bodies.”

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Whitaker agreed that many of these direct antimicrobial strategies still linger on the horizon, but other CRISPR-based advances in genomic surveillance could be coming soon.

“Say you have this strain that cannot be infected by a virus — that’s going to affect the traits of the bacteria,” Whitaker said. “Sometimes virulence, toxins and antibiotic resistance are carried on mobile elements, [so we could] either use CRISPRs as a way of tracking those elements, or to predict which strains are most susceptible to those elements. That can be used in epidemiology of these traits and strategies to stop their spread.”

CRISPR-Cas9 is already being employed in conjunction with other genomic technologies to better understand how some pathogens depend on human cells. In a recent study, Abraham L. Brass, MD, PhD, assistant professor in the microbiology and physiological systems department at the University of Massachusetts Medical School, and colleagues performed functional genomic screens based on CRISPR-Cas9 technology, which allowed them to discover previously unknown host factors of Zika and dengue viruses.

While this type of work may not have any immediate clinical impact, Marraffini said it will be vital to treating and preventing diseases in the long run.

“If you systematically knock out every gene in the human genome, you can find out which genes are important for, let’s say, immunity against a bacterial pathogen,” he said. “That is not a direct clinical application, but once you understand how a bacterial pathogen spreads and how our immune system deals with it or cannot deal with it, then you can design some sort of therapy.”

CRISPR-Cas9 genome editing spurs debate

Although these and other applications promise substantial benefits to human health, safety concerns and the potential for unintended consequences have made CRISPR-Cas9 genome editing a topic of ethical debate.

“While CRISPR-Cas9 genome editing technology holds promise to personalized medicine, human genetic modification and the development of new drugs, the technology has raised caution flags,” Michael O. Otieno, of the Hekima University College in Nairobi, Kenya, wrote in the Journal of Clinical Research and Bioethics. “Genome editing technology is a cautionary tale. We can easily get caught up in the glamour of scientific and technological advancement while at the same time oblivious to the ethical ramification of such scientific and technological advancement.”

According to Arthur L. Caplan, PhD, director of the division of medical ethics at New York University School of Medicine, many of these concerns also are relevant to infectious disease. With several novel CRISPR-Cas9 therapies and prevention approaches under investigation, he stressed the importance of extensive safety testing when modifying a host organism’s genome.

“How do you design a trial where you’re going to test out those agents?” Caplan told Infectious Disease News. “I think the answer is that you’re going to take the sickest people first, but you need to do extensive animal work whenever possible.

“With CRISPR, you really want to make sure that you can hit your targets, and that you don’t cause any later-in-life problems. It’s super controversial because the risks are high, but the benefits are also high.”

These concerns are compounded when genetic modifications could have a multigenerational impact, Caplan said. For instance, a recent study by Andrew M. Hammond, a PhD student in the department of life sciences at Imperial College London, and colleagues described a CRISPR-Cas9 construct that could function as a gene drive in Anopheles gambiae, a common vector of malaria. By conferring a recessive female-sterility phenotype, the technology could greatly reduce mosquito populations within a few years and limit the transmission of malaria in endemic regions.

Both Whitaker and Caplan noted the substantial benefits of this vector-driven approach to using CRISPR-Cas9, which the latter argued would far outweigh the minor ecological cost.

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“What are you going to do if the thing causes unexpected environmental impacts, if you wipe out a species that you didn’t realize had some value in the ecosystem?” Caplan said. “I think we know enough about some of the mosquitoes carrying, in particular, malaria, dengue and Zika, that those subspecies could be eliminated without anyone having a funeral for them. If we had malaria in the U.S., for example, I think we’d be pushing much harder to try gene drives — it’s a horrible disease.”

Of greater concern will be prevention methods requiring direct human genome modification, Caplan said. Although he said these procedures are still hypothetical, such conversations are inevitable.

“The biggest concern is trying to use CRISPR on embryos to try to design resistance in the future generations. Should we try to engineer traits into our descendants that would confer disease resistance?” Caplan asked. “With things like immunity and problems of infectious disease, whether it’s MRSA, [cytomegalovirus] or HIV, I can’t imagine that we’re not going to try to do that someday. But it’s going to take a long course of animals and somatic genetic engineering before we get there. I don’t think it’s going to be the problem of any of your readers. It will be the problem of their children.” – by Dave Muoio

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Disclosures: Marraffini is the founder of Avivo BioSciences. Caplan and Whitaker report no relevant financial disclosures. Please see the full studies for a list of all other authors’ relevant financial disclosures.