NEW YORK (GenomeWeb) – HIV-1 can acquire resistance to editing by CRISPR/Cas9 in CD4+ SupT1 cell lines, according to the results of a new study, and in fact, it's the mechanics of the Cas9 enzyme that allows the virus to acquire benign mutations that ultimately render gene editing useless.
Led by McGill University Professor Chen Liang, the study's authors showed that while CRISPR/Cas9 can efficiently target and excise proviral DNA integrated in the host genome, gene editing can lead to mutations that allow the virus to persist in some cells.
Through deep sequencing, they determined that the mutations were caused by Cas9's penchant for cleaving DNA a few bases downstream of the PAM recognition site, which is within the target specified by the guide RNA (gRNA). Further analysis showed that small indels created by the non-homologous end joining repair pathway introduced benign mutations that are thought to make the sequence subsequently unrecognizable to the same guides originally used to excise the proviral DNA. They published their results today in Cell Reports.
"This is what is happening in tissue culture. If Cas9 is used to treat patients with HIV-1, this will happen again," Liang told GenomeWeb.
It's a sobering reminder of the challenges inherent in treating a deviously clever infection, but Liang said it wasn't insurmountable. "Knowing this is not a bad thing," he said.
In the same way that a cocktail of drugs is currently used to limit resistance to therapy, targeting multiple sites in latent HIV DNA might prevent the virus from escaping. Using different CRISPR enzymes, which cleave DNA upstream of the target region, could also prevent the virus from escaping excision. It may even be an artifact of the particular cell line used in research. But it's an important consideration for researchers going forward.
"There may be many barriers and limitations that we need to overcome, but we're confident that we will find success," he said.
The McGill study comes on the heels of a recent advance in using gene editing to excise latent HIV. Last month, scientists from Temple University reported successfully eliminating HIV-1 proviral DNA from a human T-lymphocytic cell line and reducing viral load in CD4+ primary T cells from patients infected by HIV-1. They published their paper in Scientific Reports.
Led by Kamel Khalili, the Temple study reinforced results from 2014, when they first demonstrated the ability to excise HIV provirus and prevent re-infection using multiplex CRISPR/Cas9 gene editing.
At press time, Khalili had not responded to GenomeWeb's request for comment, but he and his co-authors wrote, "Our findings show comprehensively and conclusively that the entire coding sequence of host-integrated HIV-1 was eradicated in human 2D10 T cells," and "not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection."
Using a lentivirus to deliver CRISPR/Cas9 "significantly diminished" HIV-1 replication in cell lines and "drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients," they said.
In a statement, Khalili said, "These experiments had not been performed previously to this extent. But the questions they address are critical, and the results allow us to move ahead with this technology."
It was also in 2014 that Liang's lab started a project to see whether HIV-1 could acquire a resistance to CRISPR/Cas9 gene editing. Liang had studied the virus for years, researching innate protections against it such as cellular proteins that help resist infection. After CRISPR came on the scene, Liang quickly adopted the tool. "Before we started the study, we expected HIV-1 would escape," Liang said.
They began by using CRISPR/Cas9 to inhibit HIV-1 by targeting essential gene regions in the provirus DNA. By early 2015, the group had confirmed that in some cases HIV evaded gene editing, but the surprise was in how the virus managed to wriggle out of CRISPR/Cas9's grasp.
HIV-1's reverse transcriptase is notoriously error-prone, allowing the virus to mutate rapidly and evade the host's adaptive immune response. By looking at the cells that had been treated with CRISPR/Cas9 but had remained infected, the scientists found that they had indeed acquired mutations, but, surprisingly, did not appear to be generated by the viral reverse transcriptase.
Instead, the mutations were often small indels right around the Cas9 cleavage site for some of the specific regions the scientists had purposefully targeted.
The researchers also used CRISPR/Cas9 to target the long terminal repeat regions that flank essential genes, the same targets that Khalili's team had targeted. Still, HIV was able to develop resistance to the gene-editing strategy.
Liang acknowledged that his group was working with slightly different cell types than Khalili's. The SupT1 cell line can be cultured many times, while primary cells can only be cultured so long until they die. That could have played a role, providing HIV-1 more time to develop resistance.
"Maybe they didn't culture the cells long enough," Liang said. "The virus needs time to acquire the mutations to escape."
But the virus also needs active non-homologous end joining to escape. Liang said he wasn't sure how active the NHEJ repair pathway was in primary T cells. "If this repair machinery is not active [in primary cells], then when you target the LTRs, it would give you enough time to target both sites and cleave the viral DNA from the cellular genome."
If NHEJ activity is important, it would be easy to test, Liang said, and is an important follow-up experiment that his lab or some other lab should perform.
"This idea is not hard to test because there are small inhibitors that target the NHEJ pathway," he said. "Inhibiting DNA ligase IV can really slow down the NHEJ pathway and give homology-directed repair more time to do more accurate repair."
For now, using CRISPR to treat patients with HIV remains nothing more than a promising possibility. Researchers are still not sure how to get the gene editing system into the cells that would need it. But even if CRISPR/Cas9 resistance becomes the last real concern, Liang offered some strategies to minimize it.
In the same way that current therapies provide a cocktail of drugs to increase the barrier to resistance, scientists could design gRNAs to target multiple regions of provirus.
They could also use a different CRISPR nuclease, which might help avoid the problem completely. CRISPR/Cpf1, another gene editing system, cuts upstream of the targeted region, thus it would reduce the chance of forming indels in the target region.
Liang's lab is actively pursuing both ideas in follow-up studies and has shown that Cpf1 can efficiently cut HIV provirus and inhibit infection, and it may prove to impede the virus' ability to develop resistance.
As with many CRISPR developments, visions of grand possibilities become murkier as researchers dive further down into the details.
"In the beginning everything is great," Liang said. "But when you start to know more and more, things often get more complicated."
Still he remains optimistic. "Knowing this can only move it forward," he said.