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Genome Editing Cuts Out HIV

Researchers use the CRISPR/Cas9 method to remove the virus from the host genome in human cell lines.

By | July 21, 2014

WIKIMEDIA, C. GOLDSMITHLike other retroviruses, the genetic material of HIV wedges itself into the genome of its human host. While antiretroviral therapies are effective at repressing HIV, they don’t eliminate the integrated virus, which can lie low in a latent state and reactivate if the treatment is stopped. In a study published today (July 21) in PNAS, researchers take advantage of the snipping precision of a genome-editing technique to cut HIV out of the human genome.

“They looked at it in several different systems,” said Daniel Stone, a staff scientist at the Fred Hutchinson Cancer Research Center in Seattle, Washington. “It’s really convincing that the approach has promise. The next question is, how do you deliver this?”

The researchers, led by Kamel Khalili at Temple University in Philadelphia, Pennsylvania, used the CRISPR/Cas9 genome-editing system to excise HIV from several human cell lines, including microglia and T cells. They targeted both the 5’ and 3’ ends of the virus, called the long terminal repeats (LTRs), so that the entire viral genome was removed.

“We were extremely happy with the outcome,” Khalili told The Scientist. “It was a little bit . . . mind-boggling how this system really can identify a single copy of the virus in a chromosome, which is highly packed DNA, and exactly cleave that region.”

His team showed that not only could Cas9 excise one copy of the HIV genome, but—operating in the same cell—it could also clip out another copy lurking in a different chromosome. Often, Khalili said, a cell can have several copies of latent HIV distributed across various chromosomes. “Most likely the technology is going to clean up the viral DNA” in a cell, he said.

Additionally, Khalili’s group reported that the gene-editing method also prevented subsequent HIV infection. That’s something “nobody has shown before,” Stone told The Scientist.

The work follows on a study published last year by Yoshio Koyanagi and colleagues at Kyoto University that also used CRISPR/Cas9 to disrupt HIV. Khalili’s approach of using two guide RNAs to cleave both LTR ends “appears to more efficiently induce insertion/deletion gene mutations (indels) for HIV-1 LTR and excision of HIV-1 proviral DNA than single gRNA expression strategy with Cas9 nuclease that we reported last year,” Koyanagi told The Scientist in an e-mail.

One limitation of the CRISPR/Cas9 approach is that it can chop up unintended regions of the genome, producing so-called off-target effects. Khalili’s group performed whole-genome sequencing to look for off-target effects, but didn’t find any. T.J. Cradick, the director of the protein engineering core facility at Georgia Tech, said that a more thorough analysis of potential off-target effects is still required to make sure nothing has been overlooked. Nonetheless, “latent HIV provirus is a very exciting target and . . . a very promising way forward,” said Cradick, who did not participate in the study.

Koyanagi’s group is now working on a different CRISPR approach to “flush out” HIV, one in which the system activates the latent HIV, which is later eliminated with antiretroviral therapies.

The challenge to any kind of CRISPR approach is the delivery. “Latently infected cells are one in a million,” said Premlata Shankar, who studies HIV at the Texas Tech University Health Sciences Center. It may be difficult to administer a genome-editing-based therapy that could find those cells. Shankar said that the technology looks promising and that the data are “amazing,” but “you need a delivery strategy.”

Khalili has now set his sights on that particular challenge. He said his group is working to develop a nanoparticle delivery vehicle, and he hopes to be able to test it in a mouse model soon. “We have to optimize the system,” he said. “I think we have enough in vitro cell culture data and expertise to justify moving on to the next step.”

W. Hu et al., “RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection,” PNAS, doi:10.1073/pnas.1405186111, 2014.

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Avatar of: mightythor

mightythor

Posts: 43

July 22, 2014

The delivery problem is uniquely difficult in this case.  Most gene therapists struggle to achieve 90% delivery.  To cure AIDS, you need to achieve 100% delivery to infected cells.  A few untreated cells will eventually activate the virus and trigger a relapse.  The bar for clinical success is impossibly high.

The alternative approach would seem to be more practical: use the same technology to knock out the host cell CCR5 co-receptor for the virus.  The advantage of this strategy is that you only need to achieve a few percent delivery, because the disease provides an automatic selection mechanism.  The virus will eventually kill all the target cells that retain the host cell receptor, leaving a population of resistant cells.  You need to use a little clinical jiu jitzu.  Make the virus work against itself. 

 

Avatar of: Recensere

Recensere

Posts: 1

Replied to a comment from mightythor made on July 22, 2014

July 23, 2014

I'm interested in the alternative approach hypothesis. I was wondering if durring the selection mechanism, do you think that the virus could achieve CCR5 independent integration, and if this could be selected against?  

Avatar of: mightythor

mightythor

Posts: 43

Replied to a comment from Recensere made on July 23, 2014

July 30, 2014

The short answer is, apparently not. CCR5 independence does not seem to be readily accessible to the virus.  Look up The Berlin Patient on Wikipedia.  For more detailed information, check out references 11 - 18 therein.  This is an active area of research with great potential, which, as my high school football coach was fond of pointing out, is a fancy way of saying "ain't done it yet".

Avatar of:

Posts: 1

September 16, 2014

I'd be interested to know if this technique is specific for HIV itself, or could potentially also excise ancient endogenous retroviral elements accumulated in the human genome. That would be a bit worrisome, since there are many of these elements, often in critical coding regions. This is some very cool science, though!

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