Can CRISPR Slay HIV? Scientists Take a New Tack

This human T cell (blue) is under attack by HIV (yellow), the virus that causes AIDS.  (Seth Pincus, Elizabeth Fischer and Austin Athman/NIH)

Viruses like HIV and Hepatitis B—some of the world's most intractable maladies—may have a powerful foe in the gene-editing tool CRISPR. Researchers across the world are looking for the best techniques to turn CRISPR into an effective virus slayer, a role that this tool and its henchman, the Cas9 protein, play rather naturally.

"Before we adapted it to do genome editing," says George Church of Harvard University, one of the founders of the technology, "it was basically killing whichever virus it didn't like."

Now that scientists have learned to harness CRISPR/Cas9, they're hoping someday they'll be able to cure patients of HIV or hepatitis just by snipping the viral DNA out of their cells.

Research in this field has taken off. And, despite some twists and turns, many in the field seem confident that the new crop of gene-editing tools related to CRISPR will eventually prove strong enough to face down the power of viral replication.

HIV Fights Back

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"Theoretically, Cas9 is an ideal tool to do the job to cleave and remove HIV DNA," says Chen Liang of McGill University.

Yet in a study published by Liang and collaborators last month, HIV emerged from the attack able to replicate and resist further intrusions by CRISPR.

This rendering shows HIV virions (red) on bridges between an infected T cell (gold) and uninfected brain cell (blue) in vitro.
This rendering shows HIV (red) on bridges between an infected T cell (gold) and uninfected brain cell (blue) in vitro. (NIH)

In the experiment, Liang used CRISPR/Cas9 to target and snip out a section of the virus' DNA that's essential for replication. Initially, it worked. Viral replication went way down. But then, the virus began to spread again.

Their findings essentially mirrored a study published in February by researchers at the University of Amsterdam who also found CRISPR/Cas9 could target HIV, but that HIV could become resistant.

HIV is notorious for mutating and dodging attempts to kill it, so Liang says he more or less expected to see it pop up again in the T-cells.

"When we use a new approach [to kill HIV], over time, under the pressure either from drugs or from CRISPR/Cas9 the virus can develop resistance," says Liang.

But when his team sequenced the "escaped" HIV, what they found surprised them.

Let's take a look deep inside a cell, to see what Liang's team discovered.

When researchers identify the genetic sequences they want to modify, they dose the cells with the Cas9 enzyme and its guide RNA. The guide RNA contains a sequence that matches the part of DNA researchers want to cut out. Once guide RNA lines up with its mirror, Cas9 acts as a pair of DNA scissors, effectively cutting out that piece of HIV DNA.

A graphic illustration of the enzyme Cas9, in the background, clipping a strand of DNA in order to remove a mutated sequence that could cause disease.
A graphic illustration of the enzyme Cas9, in the background, clipping a strand of DNA in order to remove a mutated sequence that could cause disease. (UC Berkeley)

Sensing a break, the cell's own repair mechanisms then patch up the two ends. But that process of repairing the HIV DNA can randomly introduce mutations. Small pieces of DNA can be left out. New pieces of DNA, even just single nucleotides, can be inserted in.

Major mutations in the DNA of the virus, Liang's team found, were lethal to it. But if the repair process introduced only tiny mutations (say, a single nucleotide) the virus could often still replicate. Liang says these random mutations are "the twist in the story."

So the team sequenced the mutated HIV—the HIV that survived CRISPR and continued to replicate—to find out what was going on. And they found a bunch of mutations where Cas9 was meant to cleave the DNA. As a result, the guide RNA could no longer recognize the target viral sequence. The HIV had become effectively resistant to Cas9.

The results, Liang says, doesn't mean CRISPR/Cas9 cannot be used to fight HIV.

"Now we know what the limitation is," he says, "we can come up with ways to go around and fix the problem."

In the team's current work, they're targeting several sections of DNA at once, rather than attacking just one region.

"It's very similar to introducing a 'cocktail' therapy," he says. "If you use one drug you can only repress the disease for a short time. If you use two or three you can suppress it for a much longer time."

Liang and collaborators hope to publish their results in the fall. They're among many groups trying similar approaches.

Meanwhile, the first gene therapy for HIV could come not from CRISPR, but from an older tool that's farther along. "Zinc-finger nuclease," one of the original gene-editing techniques, has shown promise in helping patients fight HIV by rendering T cells resistant to infection. A phase 2 clinical trial is now under way.

Electron microscope image of the hepatitis B virus (HBV). Hepatitis B causes inflammation of the liver and can cause both acute and chronic disease.
Electron microscope image of the hepatitis B virus (HBV). Hepatitis B causes inflammation of the liver and can cause both acute and chronic disease. (Allain Grillet/Sanofi Pasteur/Flickr Creative Commons)

Fighting Viruses Far and Wide

Help could also be on the way for the 250 million people who are chronically infected with Hepatitis B. Last summer several groups reported they had used CRISPR/Cas9 to suppress the virus' replication in the lab.

Nor is it only human viruses that stand to be affected by the new world of gene editing.

A virus causing reproductive failure and respiratory tract illness in young pigs costs billions in losses each year around the globe. But biologists at the University of Missouri have used CRISPR to breed pigs that are resistant to porcine reproductive and respiratory syndrome virus. The edited piglets don't have the receptor protein on their cells that allows the virus in. (Most of this receptor's functions are still unclear, but carrying it isn't essential for life.)

Evidence is accumulating, says Church, that gene editing will be effective in tackling viruses, at least in the lab. But that doesn't mean, he says, that these diseases will be cured.

Church points out that the only approved gene therapy (a treatment for a rare genetic disorder that causes fat to build up in the blood) costs more than $1 million for the possibility of a permanent cure.

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For solving global viral pandemics, he says, "I think the challenge will really be more economic than technical."

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