HIV has been one of the largest public health challenges facing the world in the past few decades. Approximately 36.7 million people were infected with HIV, as reported by the World Health Organization, at the end of 2015. Despite the public attention and large population impacted by this virus, developing a cure for HIV has been particularly difficult. To successfully cure an infected individual the virus would need to be fully eradicated from their body, which is difficult because HIV is extraordinarily good at hiding in a person’s cells. In fact, part of the virus’ replication cycle is to insert itself into the host’s DNA. Once there, HIV appears to be undetectable and it can stay in the cells for the lifetime of the cell. In this state, the virus is termed “latent.”
Despite the development of anti-retroviral therapy (ART) drugs, which are very effective at keeping viral infection below detection limits, the drugs cannot reach these hiding places, or “latent reservoirs.” One strategy to clear out latent reservoirs is termed “shock and kill” or “kick and kill.” It involves treating patients with drugs known to activate latent HIV, pushing it out of the host DNA and forcing it to replicate. Essentially, the drugs are supposed to push HIV out of its hiding spots. The idea is that in the presence of these activating drugs, plus ART, the activated virus will be pushed out of the cell and unable to continue the infection. While these techniques have had some success (Kaminski et al., 2016), they haven’t yet resulted in a cure.
A newer strategy is to directly target HIV DNA sequences that are hiding in the host’s DNA and cut them out using gene editing techniques, such as the CRISPR-Cas9 system. Researchers attempted to snip the HIV genome out of a cell line that is known to have a latent copy of HIV in the host’s DNA (Kaminski et al., 2016). Targeting two highly conserved HIV sequences with the CRISPR-Cas9 system, they were able to effectively remove HIV from these cells. To confirm that the virus was actually removed, they went in and sequenced the entire genome of the cells. They were only able to find the remnants of the virus; they couldn’t find an intact HIV genome.
The next step was to take cells from healthy individuals and infect those cells with 2 different HIV strains in the lab. The cells were then treated with the CRISPR-Cas9 system the researchers had developed. The results showed that one of the strains was completely removed, but the other was not. While there was approximately a 50% decrease in HIV levels, the virus was still able to hide in about half of the cells. To try their approach in a more “real world” scenario, the researchers then collected cells from two individuals that were already HIV positive to see if they could remove latent HIV from their cells. While they were able to remove a great deal of the latent virus, approximately 10-20% of the cells still carried the virus in their host’s DNA.
Other researchers have utilized a slightly different gene editing technique, directed Cre recombinase activity (Karpinski et al., 2016). While the specifics of the editing are different, the functional result is the same. A specific section of DNA can be removed from a larger chunk of DNA. In this study, the researchers started with an expression vector that would glow either red or green depending on if their target sequences were removed. They inserted this expression vector into both bacterial and mammalian cells and found that their system could efficiently remove the target DNA.
Next, the researchers wanted to see if their technique would work in a system that mimics a HIV-infected person. The researchers took a special type of mouse that can accept human immune cells and use the human cells at the mouse’s own immune system. This “humanized mouse” was populated with the immune cells from an HIV-infected individual; essentially resulting in a mouse that is carrying human cells that are infected with HIV. The cells travel to the same places in the mouse that they would be expected to travel to in the human. This ends up being a relatively good system for testing out certain hypotheses since it looks similar to a human immune system. In this case, the mouse was mimicking an HIV-infected individual. When the researchers applied their recombinase treatment, they were able to dramatically reduce the amount of HIV-bearing human cells. Unfortunately, similar to the previously mentioned study, they didn’t completely remove the virus from all host cells.
These are exciting studies, but there are still many hurdles to overcome. One of the biggest hurdles is the target sequence chosen. While it’s true that both studies selected highly conserved sequences, both couldn’t completely recognize viruses that were found “in the wild.”
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Kaminski, R., Chen, Y., Fischer, T., Tedaldi, E., Napoli, A., Zhang, Y., Karn, J., Hu, W., and Khalili, K. (2016). Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing. Scientific reports 6, 22555.
Karpinski, J., Hauber, I., Chemnitz, J., Schafer, C., Paszkowski-Rogacz, M., Chakraborty, D., Beschorner, N., Hofmann-Sieber, H., Lange, U.C., Grundhoff, A., et al. (2016). Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nature biotechnology 34, 401-409.