Gene therapy—CRISPR starts to move forward against HIV

One approach to gene therapy that has much potential is called CRISPR (clustered regularly interspaced short palindromic repeats). CRISPR was discovered several decades ago in bacteria, where it served as part of a defence system against viruses that infect bacteria. In these bacteria, invading viruses insert key parts of their genetic material into the genetic material of the bacteria. These inserted viral genes then produce proteins and other molecules that hijack the cell and turn it into a mini virus factory, continuing the life cycle of the invading virus and eventually destroying the bacteria.

CRISPR contains small pieces of the virus’ genetic material that can be used to screen the bacteria’s genes for the presence of DNA from a virus. That is, CRISPR is used to screen the bacteria’s DNA for signals of a viral infection—the presence of viral DNA. Once the viral DNA is detected, CRISPR then uses an enzyme called Cas9 (CRISPR-associated protein 9) to remove or edit the unwanted viral DNA. By removing the viral DNA, the bacteria are protected from the effects of viral infection.

The combination of CRISPR-Cas has many potential applications. In particular, CRISPR-Cas has the potential to treat inherited disorders such as hemophilia and some infectious diseases such as HIV. For the rest of our reports on CRISPR-Cas, we will simply refer to it as CRISPR. Before further discussion of CRISPR, we provide some background on HIV’s infection of a cell, the virus’ distribution in the body and some effects of ART.

Removing HIV

Like the earlier example of the virus that infected bacteria, a broadly similar process affects cells of the immune system targeted by HIV. Cells that display the receptor CD4 on their surface (as well as the co-receptors CCR5 and CXCR4) can be infected by HIV. Such cells mainly include certain T lymphocytes (T-cells) commonly called CD4 T-cells (or simply CD4+ cells), as well as cells called monocytes (in their mature form these are called macrophages) and cells related to macrophages (such as dendritic cells in the skin and mucosal tissues and microglia in the brain). After HIV inserts its genetic material into target cells, these can become activated, converting into mini virus factories and eventually dying.

ART and the HIV reservoir

HIV treatment (ART) is highly effective when taken as directed and prescribed. As a result, scientists increasingly expect that many ART users will have near-normal life expectancy. However, ART does not cure HIV. The reasons for this are not certain but some experiments suggest that despite good adherence to ART and an undetectable viral load in the blood, small amounts of HIV remain deep within the body—in lymph nodes, the brain, parts of the gut, fatty tissue, testicles and so on. Scientists refer to these parts of the body where small amounts of HIV still lurk despite the use of ART as sanctuaries or reservoirs.


For at least a decade, researchers at the University of Nebraska have been developing what they call nano-formulations of ART—very tiny crystals of drugs surrounded by a sphere of fat. These medicine-filled fat balls have been found to penetrate a range of tissues in laboratory and animal experiments where they have good anti-HIV activity. However, short-term experiments have found that nano-ART by itself does not cure HIV infection in lab mice.

Back to CRISPR

Experiments with mice infected with HIV have found that CRISPR can remove much of HIV’s DNA from infected cells. However, CRISPR by itself does not cure HIV infection in mice.

Combining nano-ART and CRISPR

In recent experiments, scientists at the University of Nebraska have collaborated with other scientists at Temple University in Philadelphia who have expertise with CRISPR. They performed a series of very complex and detailed experiments with HIV-infected mice that received one of the following interventions:

  • nano-ART
  • nano-ART + CRISPR
  • no intervention

The nano-ART used in these experiments was made from three widely used anti-HIV medicines as follows:

  • dolutegravir + 3TC + abacavir

An immediate-release formulation of all three drugs is sold as a pill called Triumeq and is taken once daily.

CRISPR was used to search for key pieces of HIV’s genetic material in cells of the immune system of the mice.

Extensive analysis of blood and tissues from the mice suggested that a combination of nano-ART and CRISPR was able to remove HIV from about 30% of the mice. There was no obvious toxicity.

The results from the combination of nano-ART and CRISPR are exciting but must be considered preliminary. They suggest that one day such a combination might be able to cure some monkeys infected with SIV (simian immunodeficiency virus)—a virus that causes an AIDS-like condition in susceptible monkeys. The results also suggest the possibility that CRISPR + nano-ART could be used to try to cure HIV infection in people. However, there are still many steps that lie ahead and issues that need to be explored before nano-ART and CRISPR are ready for use in people. Below are some of these issues.

CRISPR—safety and other issues

When reporting on exciting news about work done in a research laboratory concerning cures for HIV or other catastrophic conditions, it must be stressed that mice are not people. That is, if every experimental therapy that did not harm mice also worked safely and effectively in people, we would have many more highly effective therapies available for different conditions.

In addition to removing HIV’s genetic material from the mice, it is possible that CRISPR can inadvertently remove useful DNA from cells. This has happened in some experiments done by other scientists but not in the current collaboration. Removal of useful DNA by CRISPR is called an “off-target” effect by scientists. So far, the team collaborating on the use of nano-ART and CRISPR with mice has found no off-target effects.

CRISPR is also being used to remove SIV from some infected monkeys. The preliminary results of these experiments appear promising. However, as some species of monkey are more genetically similar to people than mice, long-term observation and close clinical monitoring of monkeys that have been treated with CRISPR (and nano-ART) are needed to assess the potential for any long-term side effects. Long-term studies are important because the off-target consequences of CRISPR may not become apparent for some time—perhaps years. Such monitoring is also essential because it is currently impossible to assess every cell in the body to find out if its DNA has been inadvertently injured by CRISPR.

It is not clear how CRISPR can be turned off in people who receive it. It may not be safe to leave CRISPR constantly on and activated in a living person.

In the experiments conducted by the Nebraska-Philadelphia team, it appears that mice were infected with a harmless virus that was modified to enable CRISPR. It is not clear if the same technology would work in people.

Nano-ART—safety and other issues

The formulations of ART used by the Nebraska-Philadelphia team of scientists were developed in a lab on a small scale and meant for use in mice. If large numbers of monkey and human experiments with such formulations are planned, scientists will have to manufacture nano-ART on a relatively large scale. They will also have to ensure that such nano-ART is free from any contaminants. Next, they will need to assess its short- and long-term safety in monkeys and people. Nano-ART will be able to penetrate and concentrate in reservoirs in the body, including the brain, gut, lymph nodes, testicles and so on. The effect of nano-ART on the health of these tissues needs to be assessed and fully understood. After it has passed initial safety experiments, nano-ART will need to be tested for both long-term safety and effectiveness at achieving viral suppression. Nano-ART by itself is unlikely to cure HIV.

Another issue with nano-ART is determining the ideal way to get this formulation into the body. Is regular intravenous infusion best? Or will other methods, such as intramuscular injection, work equally well?

CRISPR currently in clinical trials

Clinical trials are currently underway with CRISPR in HIV-negative people in the following countries for the following conditions:

United States

  • cancer of certain white blood cells – one person
  • cancer that appears near joints – one person


  • non-small cell lung cancer that has spread to other organs – 12 people

Preliminary results from these experiments in people suggest that, so far, CRISPR is safe. More time is needed before doctors know if CRISPR will work in these cases. However, some scientists and doctors have warned that gene editing could inadvertently enable the spread of cancerous cells in some of these people. Therefore, long-term monitoring of CRISPR-treated participants in the above studies will be necessary.

Bear in mind

It is important to note that about 30% of the HIV-infected mice in the Nebraska-Philadelphia study were cured of HIV with the combined use of nano-ART and CRISPR. This is an incredible scientific achievement. However, cure rates need to increase in future experiments. CRISPR and nano-ART therapy are still in their infancy and much research lies ahead before they can be tested in large numbers of HIV-positive people.

To achieve a cure rate higher than the 30% cure rate reported in the present study, it is at least plausible that in the future CRISPR may need to be refined. Intensifying CRISPR’s editing capacity may inadvertently increase the risk of vital human genes being deleted.

Another possibility is that, in addition to CRISPR and nano-ART, more experimental therapies will have to be used. Using multiple experimental therapies in people living with HIV could increase potential problems, particularly side effects. This is yet another reason for close laboratory and clinical monitoring of animals and people who undergo CRISPR, both over the short- and long-term.

There is also another issue associated with CRISPR and other potential HIV cure therapies that we discuss in the next article in this issue of TreatmentUpdate.


TreatmentUpdate 231

The Canadian HIV Cure Enterprise (CanCURE)

—Sean R. Hosein


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