Scientists wield gene editing with CRISPR in the fight against HIV

The global epidemic of Human Immunodeficiency Virus (HIV) has been a leading public health concern for almost four decades. Left untreated, HIV eventually infects and destroys T cells—a cornerstone of the human immune system—leading to Acquired Immune Deficiency Syndrome (AIDS) and the inability of the body to fight off routine infections. Presently there is no cure for HIV, but gene editing technology could soon change that.

According to UNAIDS, an estimated 37 million people are currently infected with HIV, and the virus has claimed about the same number of lives. Thanks to awareness, research, and treatment, the number of new infections is declining each year and the global rate of AIDS-related deaths has fallen by almost 50 percent since 2010. However, there were still 1.8 million new HIV infections worldwide in 2016, including 160,000 children.

Modern treatments are very effective at halting the progression of HIV to AIDS and are largely responsible for the increased survival of HIV/AIDS patients, but many infected people still do not have access to these life-saving treatments and those who do must take them for the rest of their lives. The application of gene editing—specifically, technologies made possible by the CRISPR-Cas9 system—to combat HIV has recently splashed into national headlines and provided new hope in the search for an HIV cure. In just a few short years, CRISPR-Cas9-based HIV treatments have advanced to being tested in mice, and human clinical trials are likely on the horizon. A one-time treatment to cure HIV is finally becoming a tangible possibility, and while there is still much work to be done, it’s well worth getting excited about.

Decades of research bring AIDS treatment, but no cure 

HIV was formally discovered in 1983, although we now know that it could have been infecting humans as early as 1910. Specific antiviral drugs were quickly developed that were able to target critical parts of the viral life cycle and stop the production of new HIV viruses.

Like all viruses, HIV contains genetic information that serves as a set of instructions for how to make more copies of itself. The HIV genome is passed between hosts as RNA, which is similar to the DNA that carries our genetic information but is slightly different chemically, making it more unstable and prone to degradation. When HIV first infects a cell, an HIV enzyme called reverse transcriptase (RT) converts the RNA genome into DNA. This DNA version of the virus is incorporated into the human genome within the infected cell. From the integrated viral DNA—now called a provirus—more copies of HIV are made.

When HIV infects a new cell, it first binds to a receptor on the cell surface, which allows the virus to release its RNA genome and other HIV proteins into the interior of the healthy human cell (1). The HIV reverse transcriptase (RT) enzyme converts the RNA genome into a DNA molecule (2). The newly made HIV DNA genome becomes integrated into the DNA of the host human cell, where it can become dormant and hide from the immune system (3). However, this HIV DNA—now called a provirus—can reanimate at any time, turning this infected cell into a virus factory that makes new HIV RNA, which can then go on to infect other healthy human cells (4). The RT enzyme and the integration of the provirus are common targets of highly active antiretroviral treatment (HAART). Illustration by Allison Terbush.

Researchers took advantage of the essential function of HIV RT and developed drugs to stop the enzyme from converting HIV’s RNA genome into DNA. The first of these drugs was commercially available in 1987, but RT inhibitors did not provide long-lasting protection from the virus because the virus developed resistance. Eventually, more drugs were developed to target other HIV life cycle stages, and research showed that combinations of these drugs were much more effective at stopping HIV than the single RT inhibitor is alone. As such, modern highly active antiretroviral therapy (HAART, also called cART)—the best treatment we have now—consists of multiple HIV inhibitors.

HAART has transformed HIV/AIDS from a devastating illness to a manageable chronic condition. The treatment is so successful that in the United States, the average life expectancy of HIV-infected patients who take HAART is no different from that of the general population. HAART is so powerful that it may be able to cure babies born to HIV-infected mothers when the treatment is given shortly after birth. HAART can also be taken daily as a preventive measure and can be up to 90% effective in preventing the virus from taking hold in the body after an exposure.

The development of HAART is an incredible success story, but it does not address the root of the HIV infection: the HIV provirus buried within the genes of infected patients. HIV remains a lifelong infection because some of the provirus lies dormant, not making new HIV, and is unaffected by HAART. An infected cell can’t tell the difference between the provirus and its own genome, so the provirus is replicated during cell division and maintained just like one of our own genes. Dormant provirus can become active again at any time, causing periodic resurgences of HIV and continued erosion of the immune system.

While we have made enormous strides in managing HIV/AIDS, there is still a dire need for an HIV cure. Those with access to HAART still face the burden of managing a chronic illness, and those without access to HAART—about half of all HIV-infected people—have no HIV treatment or management options available.

The search for a cure 

Scientists have made several efforts to find a cure for HIV. Within the last decade, the “kick and kill” approach has gained popularity and has been explored in animal models and clinical trials. In the kick and kill strategy, drugs are used to reactivate the dormant HIV provirus, and then HIV inhibitors fight the reawakened HIV while the immune system kills off the last of the infected cells. However, there are concerns that the drugs used to reanimate the provirus are quite toxic and still may not be able to eradicate all of the dormant HIV within a patient.

More recently, two gene-editing technologies have been developed and tested as HIV cures. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are enzymes that cut DNA strands at a programmable location. Both ZFNs and TALENs can effectively target the provirus and HIV-involved human genes, and ZFN-based treatments are being tested in the clinic.

Gene editing shows immense promise, but both ZFN and TALEN are difficult to program to target relevant locations within DNA. CRISPR-Cas9 is a simpler gene-editing technology that burst onto the biotech stage in 2012. The principle enzyme at work in the CRISPR system—Cas9—pairs with a programmable RNA molecule called a guide that contains a sequence matching the sequence of targeted DNA. The RNA guide finds and pairs with its matching stretch of DNA, and the Cas9 enzyme that was brought along with the RNA guide makes a cut through the DNA. When the cell tries to repair this damaged DNA, it does so in an imperfect manner and often introduces changes in the DNA called mutations, which often disable the affected genes.

CRISPR was discovered by studying how simple one-celled organisms like bacteria manage to fight off viruses. After an infection, CRISPR serves as a memory of the invading viral genetic material and helps prevent the virus from being able to replicate again during a subsequent attack. CRISPR-Cas9-based strategies to fight HIV are based on this principle: targeting CRISPR to HIV proviral DNA or a human gene the virus depends on to replicate and disabling them.

Purging HIV with CRISPR

One strategy to use CRISPR to cure HIV is to either mutate or cut out the provirus itself. There are currently two methods to do so. One is to direct CRISPR-Cas9 toward integrated DNA proviruses, where it makes cuts in critical genes and lets the repair mechanisms of the cell introduce crippling mutations that render the provirus nonfunctional. The provirus is not removed but is mutated such that it will never make HIV virus.

A second method focuses on using CRISPR-Cas9 to actually cut out the provirus. HIV genomes are surrounded on both sides by stretches of RNA (or DNA in the provirus) called long terminal repeats (LTRs). By directing CRISPR-Cas9 toward these two identical LTR sequences, DNA is cleaved on either side of the provirus, releasing the provirus and letting the surrounding DNA be repaired, provirus-free, in its place.

CRISPR can act directly on the HIV provirus in two ways. First, a guide RNA may target any unique part of the virus, like a gene that is critical for HIV function (A). This guide RNA leads the Cas9 enzyme to this location where Cas9 makes a cut in the DNA (1). The cell repairs this but doesn’t preserve the original DNA sequence and introduces mutations in the provirus that cause it to no longer be able to make new HIV viruses (2). A guide RNA may also target the HIV long terminal repeats (LTRs)—identical stretches of DNA that surround the provirus (B). In this case, Cas9 makes one cut on either side of the provirus, releasing the provirus from the surrounding human DNA (1). The cell destroys the released provirus and the two far ends of the DNA are brought together again and repaired (2). Illustration by Allison Terbush.

Since 2013, the effectiveness of both HIV gene- and LTR-targeted CRISPR-Cas9 in both cell culture systems and animal models have been tested in many studies, and in general researchers found the techniques were successful in reducing HIV infection in cells with no detectable off-target effects. To test the first method, several research groups administered Cas9 and guides targeted to essential HIV genes and were able to detect a wide variety of gene-breaking mutations. To test the second method, multiple research groups also directed Cas9 to the LTRs and found that HIV provirus was actually removed from the host DNA, as predicted.

Despite its effectiveness, HIV-targeted CRISPR-Cas9 strategies are still quite susceptible to failure due to the high mutation rate of HIV, which allows it to evolve quickly, and the fact that although mutations in the viral genome can disable genes, they can also change the genes in ways favorable for the virus. The remarkable pace at which HIV evolves is largely responsible for the inadequacy of the original RT inhibitor therapy because the virus can quickly evolve a resistance to a single drug. In the recent CRISPR-Cas9 studies, Cas9 was directed toward the parts of the HIV genome that are least likely to change without being detrimental to the virus, making it likely that almost all HIV viruses—even mutants—would be susceptible to the treatment. Still, two research groups reported that, if given enough time, HIV would eventually mutate to become resistant to every single guide sequence that was tested, even in the regions of the viral genome that seem most stable and essential.

The natural mutation rate of HIV also presents a serious challenge to bringing these treatment strategies into the clinic. In a 2014 study done at Drexel University College of Medicine, researchers sequenced infected patients’ HIV genomes and found that only 50 percent of the cohort could have their personal HIV strains targeted by a combination of ten CRISPR guides. But this obstacle may not be a death knell for the budding therapy. HIV evolves quickly, even within individual patients, but since it’s relatively easy to program guides for CRISPR-Cas9 therapy, it still may be possible to take a personalized approach to design custom anti-HIV guides for every patient.

Generating HIV-resistant cells with CRISPR

The second strategy to use CRISPR to cure HIV is to turn CRISPR-Cas9 on the proteins that allow the HIV virus to access cells. HIV gains entry into cells by binding to the human protein CCR5, and HIV is not able to infect cells that do not make CCR5. Because AIDS progresses as HIV slowly infects and kills helper T cells—cells that are crucial to fight off HIV infection—it may be possible to stop AIDS and cure an HIV infection if helper T cells lacking CCR5 are given to a patient. These helper T cells would be able to fight HIV without the risk of becoming infected by HIV. In fact, the only patient known to have been cured of HIV received a transplant of immune system stem cells that had a natural mutation in the CCR5 gene.

CRISPR can also be directed toward human proteins. HIV needs to infect cells and survive. This strategy has been focused on CCR5—the receptor on the surface of human cells that allows HIV to enter. By targeting the CCR5 gene with CRISPR (1), it is possible to shut down the production of CCR5 in cells (2). Cells that lack CCR5 are resistant to HIV infection. Illustration by Allison Terbush.

Since this extraordinary case, there has been an avalanche of research investigating how to therapeutically shut down the production of CCR5 in stem cells that can be given as a transplant, and with the introduction of CRISPR-Cas9, shutting off CCR5 has become even easier. CRISPR can be used to edit CCR5 and generate HIV-resistant helper T cells that can fight HIV and replace their HIV-vulnerable counterparts that have been destroyed by the virus. Since human genes are encoded and replicated as DNA—a type of molecule less prone to damage and mutations than RNA is—this strategy would eliminate the need for personalized CRISPR-Cas9 guides because unlike HIV, the CCR5 DNA sequence is almost the same in all humans. The CCR5-disruption strategy also overcomes the problem of needing to deliver gene-editing treatment to every HIV-infected cell within an infected patient. Stem cells can be removed from the body, edited in a lab, and re-injected into a patient without needing to target multiple HIV-infected cells at once.

Several groups have demonstrated efficient CRISPR-Cas9-directed editing of the CCR5 gene in a broad range of different human cells, including T cells and stem cells. While mutating CCR5 has been shown to make these cells resistant to HIV, it is still possible that HIV could evolve a new way to enter cells to overcome the lack of CCR5. In addition, there are concerns that artificially disrupting CCR5 could negatively impact a patient’s immune system. Despite these concerns, many CCR5-editing therapies have already entered clinical trials, and there is little doubt that CRISPR-based CCR5-editing will soon follow.

The last mile

CRISPR-Cas9-based HIV treatments have been shown to work in cells in the laboratory and animal studies are currently underway, but there are considerable barriers to navigate before they have a chance of being used regularly on human patients. HIV-targeted gene editing will likely need to be tailored to each individual patient, and the delivery of therapy remains a serious challenge. The effectiveness of the CCR5-targeted approach will depend on whether the HIV-resistant cells can be deployed into the body in a way that does not trigger graft-versus-host disease—a common, dangerous complication of transplants in which the body attacks foreign material—but still eradicates the existing reservoir of cells containing the provirus. Still, both of these strategies may be the closest we’ve ever come to an HIV cure, and there is significant scientific, political, and financial drive to ensure these CRISPR-Cas9-based options are thoroughly developed and evaluated.

We must also consider how a CRISPR-Cas9 HIV cure, if approved for use in humans, would fit into the existing frameworks of HIV treatment worldwide. The patients who are most vulnerable to developing uncontrolled AIDS and dying from HIV/AIDS-related conditions are also those most desperately in need of a cure, but it is not clear if they would gain access to a cure if one were to become available. However, a functional HIV cure could alleviate the burden of a daily, lifelong HAART regimen for those who can access it. The availability of such a cure could also spark more investment in medical infrastructure by countries with high prevalence of HIV, as decreasing the number of people with HIV/AIDS-related health costs saves a considerable amount of money in the long term.

In just the past five years, CRISPR-based gene-editing technologies have revolutionized biomedical science, and the HIV field is no exception. Although researchers will need to follow up on these promising early studies, the fact that multiple groups have now been able to eliminate HIV—rather than just suppress it—in cells and even animal models is monumental.

Featured image credit: CDC/ C. Goldsmith, P. Feorino, E. L. Palmer, W. R. McManus.

Leave a Reply