Scientists have demonstrated the potential of CRISPR-Cas9 technology to remove the HIV genome from the DNA of infected cells. They have also identified some of the consequences of doing so and begun to investigate how these side-effects may be mitigated.
In one study, Dr Michele Lai and colleagues at the University of Pisa demonstrated the capacity of CRISPR-Cas9 to remove HIV genetic material from infected cells but also investigated whether the excised sections of DNA may reintegrate and start replicating again.
In another, Dr Jonathan Herskovitz and team at the University of Nebraska showed that when CRISPR-Cas9 is manipulated to target multiple sites at two of HIV’s most important genes, viral replication in infected cells almost completely stopped – with no immediately obvious damage to cellular DNA.
In humans, HIV works by attaching to a cell and releasing its genetic material that is then turned into DNA by the cell it has invaded. This DNA is then integrated into the host’s DNA and, if not inhibited by antiretroviral therapy (ART), will continue to make further copies of the virus.
CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR Associated Protein 9) is a molecular tool adapted from a technique that bacteria use to remove viral genetic material that has been integrated into their own DNA. It essentially allows scientists to pick a sequence of known genetic material (for example part of the sequence of the HIV genome) and instruct an enzyme to go and cut this material out of a sequence of DNA. Theoretically, this means the HIV genetic material that is stored in the DNA of people living with HIV could be removed.
What happens to the excised viral DNA?
The researchers at Pisa tested this notion in the laboratory (in vitro), and also investigated what happened to the viral DNA that was cut out. First they took a group of cells and, using a retroviral vector similar to HIV, inserted a modified strand of HIV material into the cells. The cells then began to produce a protein that the researchers could measure, suggesting that, as with human HIV infection where the virus releases its p24 protein, the HIV material had been integrated into the DNA of the cells.
They then introduced a CRISPR-Cas9 complex that was targeted toward a specific part of the HIV material, in the hope that it would then cut the HIV viral DNA from the infected cells, and so stop making the protein.
The area they targeted was the long terminal repeat (LTR) – areas at the end of each strand of viral genetic material that ‘switch on’ the host cell’s machinery so it starts to produce viral proteins. The researchers looked for signs that active production of HIV protein was happening in the cells exposed to CRISPR-Cas9 and found that it had indeed almost completely stopped after a couple of days.
They also wanted to see what would happen to all the excised viral genetic material. They found that it can form small circles of circular DNA that persist in the cells for at least two weeks following introduction of the CRISPR-Cas9 complex.
It was thought that eventually these DNA fragments would break down or be diluted through repeated cell division. Sometimes, however, the researchers found, they joined back together and formed complete strands of the HIV genetic material.
In certain manipulated lab conditions created by the investigators, this genetic material had the potential to make some of the building blocks of the HIV virus and so, in theory, could produce infectious virus, providing a potential barrier to the use of CRISPR-Cas9 as a complete cure for HIV. This issue however might be abated by the concurrent, hopefully temporary, use of antiretroviral therapy.
Targeting different HIV genes
The Nebraska researchers explored some of the other challenges that CRISPR-Cas9 technology faces. Given there are thousands of strains of HIV virus with great genetic diversity and that HIV continues to mutate after infection, they wanted to ensure that the technology would not become redundant due to the large number of strains, each having slightly different genetic material. In addition, they looked at how best such a treatment would be delivered to human cells in real life conditions (in vivo).
"Hurdles exist that if not overcome could confine CRISPR-CAS9 to just being an intriguing lab tool."
This group targeted multiple and different regions of the HIV genome to be excised, rather than the single region targeted by the first group of researchers.
HIV’s tat and rev genes code for proteins that cause infected cells to rapidly produce much greater quantities of the HIV virus. They remain relatively unchanged across varying viral strains. The researchers found that when they targeted these genes at the same time with a CRISPR-Cas9 complex called TatDE it cut the viral DNA in multiple places. They were able to almost completely block viral reproduction in HIV- infected cells with less viral rebound than when targeting LTR alone, as in the Pisa study.
As for how CRISPR-Cas9 could feasibly be delivered to human cells, the Nebraska group used a variety of methods to see what would be effective.
The researchers tested mRNA (messenger RNA) technology similar to that used in the recent COVID-19 Pfizer and Moderna vaccines. This introduces genetic material via small fat droplets into cells, which then start producing the CRISPR-Cas9 complex. They found this a highly effective way of implementing CRISPR-Cas9.
Given its proven track record of safety as a delivery method, mRNA does provide an exciting potential avenue for the translation of this technology from lab to human.
One further concern that has been raised regarding CRISPR-Cas9 is that of 'off-target' edits: could the Cas9 enzyme, though targeted at viral DNA that has been inserted into the DNA of infected (host) cells, mistakenly cut out some of the host cell’s own genetic material?
The authors of the paper analysed the cells that had had been exposed to CRISPR-Cas9 treatment, and in particular looked at areas in the human genome that the Cas9 system might be most likely to mistake for HIV DNA. They found that there was no such 'off-target' editing in any of these regions. The authors did not study the whole of the cells’ remaining DNA however and so there remains a possibility that off-target edits could exist but just haven’t been found.
Before any human study can feasibly begin, studies of the whole genome must take place; deletion of a critical gene could have dire consequences for anybody taking CRISPR-Cas9 treatment.
CRISPR-Cas9 technology is constantly evolving. These studies go some way to proving that, in the lab dish, it is possible to remove viral DNA from that of infected human cells. They demonstrate that through testing different targets for the Cas9 enzyme, they can improve its effectiveness, and have explored feasible and safe ways of delivering such a treatment to cells.
But before people living with HIV and the clinicians treating them rejoice at the prospect of a cure it is important to note that these studies have taken place in controlled laboratory conditions with a narrow range of cell lines. For this treatment to be effective in humans it would have to reach and be effective in every cell that has integrated HIV DNA. Whole genome sequencing of multiple cell lines needs to take place before it can be fully determined that there are no harmful or even potentially fatal 'off-target' edits.
Even with this in place, other questions need to be answered, such as how long to continue antiretroviral therapy after treatment with CRISPR-Cas9. Though these findings are promising, these studies currently still provide only a proof of concept. Further research will focus on how to evolve CRISPR-Cas9 technology into an effective and safe treatment for HIV, but hurdles exist that if not overcome could confine CRISPR-CAS9 to just being an intriguing lab tool.
Lai M et al. CRISPR/CAS9 Ablation of integrated HIV-1 accumulates proviral DNA circles with reformed long terminal repeats. Journal of Virology; 95 (23), 2021 (open access).
Herskovitz J et al. CRISPR-Cas9 mediated exonic disruption for HIV-1 elimination. EBioMedicine; 73 (103678). 2021 (open access).