NIDA Notes

Antiretroviral Therapy Combined With CRISPR Gene Editing Can Eliminate HIV Infection in Mice

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By Chris Palmer, Ph.D., NIDA Notes Contributing Writer

This study reported that:

  • HIV-1 could be eliminated in mice using a combination of two antiviral technologies—long-acting viral reservoir–targeted antiretroviral therapy and CRISPR/Cas-9 gene editing.
  • HIV was undetectable in 9 out of 23 mice that received the combination treatment. HIV was not eliminated in any of the mice that were given either treatment alone.

NIDA-supported research led by Dr. Kamel Khalili at Temple University and Dr. Howard E. Gendelman at the University of Nebraska Medical Center has resulted in a novel treatment approach for HIV infection that, if confirmed in further studies, could eventually lead to a cure for the disease. Currently, treatment with a combination of antiretroviral drugs allows people with HIV to live a relatively normal life. However, these drugs must be taken regularly and for life because they cannot fully eradicate the virus. If antiretroviral therapy (ART) is stopped, HIV symptoms typically return within a matter of weeks. "Our new research merging two very disparate technologies—long-acting viral reservoir–targeted antiretroviral therapy and CRISPR/Cas-9 viral excision—shows that an established HIV-1 infection can be eliminated," says Dr. Gendelman, "This is the first time, to my knowledge, that any group has shown viral eradication is possible in a live animal model." 

Attacking HIV From Two Directions

Virologists have understood for years that HIV integrates into the genome of some of the host's cells, where it can lie dormant and beyond the reach of antiretroviral drugs. They concluded that to eliminate the virus permanently, treatment likely would require a gene-editing component. This is the approach pursued by Dr. Khalili and his team, who recently demonstrated that the gene-editing tool CRISPR can remove HIV DNA from host cell genomes. CRISPR uses one or more sequence-specific guide RNAs to direct a scissors-like bacterial enzyme (Cas9) to cut out or replace disease-causing DNA sequences, such as integrated HIV proviral DNA. However, the Temple researchers found that this technique could not remove every last bit of HIV from the host. In the current study, in fact, CRISPR alone only excised 60–80 percent of the HIV DNA.

Meanwhile, Dr. Gendelman's team was developing long-acting slow-effective release antiretroviral therapy (LASER ART). Unlike the commonly used ART drugs that are water soluble and quickly processed and excreted by the body, LASER ART combines modified versions of the medications rilpivirine, dolutegravir, lamivudine, and abacavir and packages the prodrugs within very small fat-soluble particles. This delivery system allows the drug cocktail to be released slowly over time, increasing its effectiveness. 

Figure 1. Timeline of the Treatment Mice were humanized (i.e., carried human T cells) and then infected with HIV-1. Once the infection was established, some of the animals were treated with LASER ART for 4 weeks. At 3 weeks after the end of this treatment, some of the treated animals and some of the untreated control animals were treated once with CRISPR/Cas-9 gene editing. The animals were then observed for another 5 weeks before their tissues were harvested. See full text description at end of article.

The two teams then combined their treatments to test them in mice transplanted with human T cells (i.e., "humanized" mice) that can be infected with HIV-1. The HIV-infected mice were first treated with LASER ART to suppress viral replication. Three weeks after discontinuing the therapy, the researchers used CRISPR gene editing to snip out specific portions of HIV proviral DNA lurking in the genome of the human T cells in the mice's spleen, bone marrow, lymph nodes, brain, kidneys, and other tissues (Figure 1).

The researchers conducted three independent sets of experiments in which humanized and HIV-infected mice were treated with LASER ART, CRISPR gene editing, or both. They found that across the three experiments, 9 out of 23 mice receiving the combination treatment showed no detectable signs of HIV DNA, RNA, or proteins, indicating that the virus had been completely eradicated in these animals. Even after several weeks, there was no rebound of the HIV infection. In contrast, HIV was not eliminated in any of the mice that were given either treatment alone (for the results from one of the experiments, see Figure 2).

Figure 2. Combination Treatment With LASER ART and CRISPR/Cas-9 Restores CD4+ T Cells and Fully Eliminates HIV-1 in Some Animals In one experiment, a total of 29 mice were infected with HIV, out of which 6 received no treatment (red), 10 were treated with LASER ART only (blue), 6 were treated with CRISPR/Cas-9 gene editing only (black), and 7 received both LASER ART and CRISPR/Cas-9 (green). (top panel) Levels of CD4+ T cells, which are the main target of HIV-1, were measured at different times after HIV-1 infection. Animals that received no treatment or those that received only CRISPR/Cas-9 treatment showed declining CD4+ T-cell levels. Animals that received LASER ART alone or in combination with CRISPR/Cas-9 maintained high CD4+ T-cell levels. (bottom panel) Levels of virus particles in the blood (plasma viral load) were measured in HIV-infected mice given no treatment (upper red) and mice given both LASER ART and CRISPR/Cas-9 treatment (green). Two of seven mice given the combination treatment showed no evidence of viral rebound at Week 14. In the other five animals, virus levels increased again after the CRISPR/Cas-9 treatment (lower red). See full text description at end of article.

To confirm that the seemingly virus-free mice indeed carried no more active HIV-1, the team extracted immune cells from some of these animals and transferred them into previously uninfected humanized mice. None of the recipient animals developed HIV infection. In contrast, cells from animals only treated with LASER ART could still cause infection in previously uninfected animals. Finally, the researchers established that the CRISPR treatment caused no unintended gene edits.

A First Step

Although the study provides a proof of concept that it is possible to fully eradicate HIV-1 from an infected organism, there are still many limitations and concerns that need to be overcome before researchers can begin to consider what this may mean for people with HIV infection.

One limitation is the small number of animals tested with the dual therapy. The findings will need to be replicated in a much larger sample and in other species, such as nonhuman primates, before human testing. Researchers also need to learn more about how exactly the therapies work in tandem (and why they don't work in all test subjects) before trying them in humans.

Also, the mice were studied for 5 weeks after CRISPR treatment; therefore, it is possible that given more time, the virus might have rebounded. Another important issue is the specificity of the CRISPR gene editing. Because HIV mutates rapidly, the virus may change to a form that could elude the CRISPR guide RNAs. This may be a particular problem in humans, where the virus is usually only detected long after the initial infection and therefore has plenty of time to mutate into numerous different variants. In these cases, CRISPR may not be able to eliminate all variants, and even one virus that escapes the LASER ART and CRISPR could regrow an entire viral population.

Finally, in the study, the mice's immune systems were severely weakened so that they could tolerate the human immune cells. This also means that they may respond to HIV in ways that differ from human disease progression.

Nevertheless, Dr. Khalili finds the results promising. "After years of being considered a fatal disease, then a chronic disease, today we are looking at HIV-1/AIDS as a potentially curable disease," he says.

Dr. Gendelman's team is now working to increase the efficiency of the dual treatment by improving the delivery of both LASER ART and CRISPR approaches.

"The next step will be using a nonhuman primate model to test this strategy, with the long-term goal of bringing this technology to clinical trials," adds Dr. Khalili. "In addition, we hope that this strategy will become useful in developing treatments for other infectious diseases."

This study was supported by NIDA grants DA037830, DA013137, and DA42706.

Text Description of Figure 1

This figure shows the experimental design to determine the effects of LASER ART and CRISPR/Cas-9 gene editing alone or in combination on HIV-1 levels. The horizontal black line shows the time in weeks, from -18 weeks to +14 weeks. A green vertical arrow at Week -18 indicates that the mice were humanized (i.e., received human T cells). A small vertical red arrow indicates that the mice were infected with HIV-1 at Week 0. A horizontal blue line with two vertical arrows represents LASER ART treatment from Weeks 2 to 6. A black horizontal arrow above the timeline represents LASER ART withdrawal from Weeks 6 to 16. A small vertical black arrow at Week 9 indicates treatment with CRISPR/Cas-9 gene editing. A small vertical purple arrow at Week 14 indicates the time when the animals’ tissues were evaluated for HIV-1 DNA, RNA, and proteins.

Text Description of Figure 2

This figure illustrates the effects of no treatment, LASER ART, and CRISPR/Cas-9 gene editing on CD4+ T cell levels and HIV-1 viral RNA particles (plasma viral load) in the blood of humanized and HIV-1–infected mice. Red curves represent untreated animals, black curves represent animals treated with CRISPR gene editing alone, blue curves represent animals treated with LASER ART alone, and green curves represent animals treated with both LASER ART and CRISPR. A red triangle above Week 0 indicates infection with HIV-1, a blue horizontal line above Weeks 2 to 6 indicates LASER ART treatment, and a black triangle above Week 9 indicates CRISPR/Cas9 gene editing.

Top Panel: The graph shows the number of CD4+ T cells throughout the experimental period. The horizontal x-axis shows the time in weeks on a scale from -1 to +14 weeks. The vertical y-axis shows the CD4+ T cells as percent from the CD3 gate on a scale from 0 to 100 percent. For the HIV-1–infected untreated animals, the percentage of CD4+ T cells declined from about 75 percent at Week 0 to about 70 percent at Week 2, about 65 percent at Week 7, about 55 percent at Week 9, and less than 6 percent at Week 14. For the HIV-1–infected animals treated only with CRISPR/Cas-9 gene editing, the percentage of CD4+ T cells was about 75 percent at Week 0, about 65 percent at Week 2, about 50 percent at Week 7, about 45 percent at Week 9, and about 15 percent at Week 14. For the HIV-1–infected mice treated with LASER ART only, the percentage of CD4+ T cells was about 75 percent at Week 0, about 65 percent at Week 2, about 80 percent at Week 7, about 75 percent at Week 9, and about 68 percent at Week 14. For the HIV-1–infected mice treated with both LASER ART and CRISPR/Cas9, the percentage of CD4+ T cells was about 75 percent at Week 0, about 70 percent at Week 2, about 85 percent at Week 7, about 80 percent at Week 9, and about 90 percent at Week 14.

Bottom Panel: The graphs show the plasma viral load for animals infected with HIV-1 that were either untreated or received both LASER ART and CRISPR/Cas-9 gene editing. The horizontal x-axis shows the time in weeks from -1 to 14 weeks. The vertical y-axis shows the viral plasma load in RNA copies/mL on a logarithmic scale from 102 to 106. A horizontal gray dotted line between 102 and 103 indicates the level of detection of viral RNA copies. For the HIV-1–infected, untreated animals (top red line), viral RNA copies increased from between 104 and 105 at Week 2 to above 105 by Week 6 and remained at that level up to Week 14. For the HIV-1–infected animals treated with LASER ART plus CRISPR/Cas9 (green line), viral RNA copies decreased from about 105 at Week 2 to between 103 and 104 by Week 6 and just above the level of detection by Week 9. For two of the animals, viral RNA decreased further, below the level of detection by Week 14, indicating no rebound of HIV-1; for five of the animals (lower red curve), viral RNA levels increased to almost 105 by Week 14, indicating a rebound of HIV-1.

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