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Enlisting CRISPR in the Quest for an HIV Cure

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Today, thanks to remarkable advances in antiretroviral drugs, most people with the human immunodeficiency virus (HIV) can expect to live an almost normal lifespan. But that means staying on medications for life. If those are stopped, HIV comes roaring back in just weeks. Finding a permanent cure for HIV infection, where the virus is completely and permanently eliminated from the body, has proven much tougher. So, I’m encouraged by recent work that shows it may be possible to eliminate HIV in a mouse model, and perhaps—with continued progress—someday we will actually cure HIV in humans.

This innovative approach relies on a one-two punch: drugs and genetic editing. First, HIV-infected mice received an experimental, long-acting form of antiretroviral therapy (ART) that suppresses viral replication. This step cleared the active HIV infection. But more was needed because HIV can “hide” by inserting its DNA into its host’s chromosomes—lying dormant until conditions are right for viral replication. To get at this infectious reservoir, researchers infused the mice with a gene-editing system designed to snip out any HIV DNA still lurking in the genomes of their spleen, bone marrow, lymph nodes, and other cells. The result? Researchers detected no signs of HIV in more than one-third of mice that received the combination treatment.

The new study in Nature Communications is the product of a collaboration between the NIH-funded labs of Howard Gendelman, University of Nebraska Medical Center, Omaha, and Kamel Khalili, Temple University, Philadelphia [1]. A virologist by training, Khalili years ago realized that HIV’s ability to integrate into the genomes of its host’s cells meant that the disease couldn’t be thought of only as a typical viral infection. It had a genetic component too, suggesting that an HIV cure might require a genetic answer.

At the time, however, the tools to remove HIV DNA from human cells without harming the human genome weren’t available. That’s changed in recent years with the discovery and subsequent development of a very precise gene-editing tool known as CRISPR/Cas9.

CRISPR/Cas9 editing systems rely on a sequence-specific guide RNA to direct a scissor-like, bacterial enzyme (Cas9) to just the right spot in the genome, where it can be used to cut out, replace, or repair disease-causing mutations. Efforts are underway to apply CRISPR/Cas9 to the treatment of sickle cell disease, muscular dystrophy, and more.

Could CRISPR/Cas9 also remove HIV DNA from infected cells and eliminate the infection for good? Such an approach might be particularly helpful for people on ART who have persistent HIV DNA in the cells of their cerebrospinal fluid. A recent NIH-funded study in Journal of Clinical Investigation found that an association between this HIV reservoir and neurocognitive difficulties [2]

Earlier work by Khalili’s team showed that CRISPR could indeed remove HIV DNA from the genomes of host cells [3]. The problem was that, when delivered on its own, CRISPR couldn’t snip out every last bit of viral DNA from all cells as needed to get rid of HIV completely and permanently. It was crucial to reduce the burden of HIV genomes to the lowest possible level.

Meanwhile, Gendelman’s lab had been working to develop a new and more effective way to deliver ART. Often delivered in combinations, standard ART drugs are effective in suppressing HIV replication. However, people need to take their oral medications daily without fail. Also, most ART triple therapy drugs are water soluble, which means its cocktail of medications are swiftly processed and excreted by the body without reaching many places in the body where HIV hides.

In his quest to make ART work more effectively with fewer doses, Gendelman’s team altered the chemical composition of antiretroviral medicines, generating fat-soluble drug nanocrystals. The nanocrystals were then packaged into nanoparticles and delivered by intramuscular injection. The new drug formulation, known as long-acting slow-effective release (LASER) ART, reaches lymph nodes, spleen, gut, and brain tissues where HIV lurks [4]. Once there, it’s stored and released slowly over time. Still, like conventional ART, LASER ART can never completely cure HIV.

So, Gendelman teamed up with Khalili to ask: What would happen if LASER ART was followed by a round of CRISPR/Cas9? In a series of studies, the researchers tested LASER ART and CRISPR/Cas9, both alone and in combination. A total of 23 HIV-infected mice engineered to have some “humanized” immune features received the experimental combination therapy.

As expected, neither LASER ART nor CRISPR/Cas9 by itself proved sufficient to eradicate HIV in the mice. However, when LASER ART and CRISPR/Cas9 were delivered sequentially, the results were much different. Researchers found no evidence of HIV in the spleens or other tissues of more than one-third of the sequentially treated animals.

It’s important to note that this gene-editing approach to eradicating HIV is being applied to non-reproductive cells (somatic). The NIH does not support the use of gene-editing technologies in human embryos (germline) [5].

Of course, mice, even with humanized immune systems, are not humans. More research is needed to replicate these findings and to figure out how to make this approach to HIV treatment more effective in animal models before we can consider moving into human clinical trials. Still, these findings do provide a new reason for increased hope that an actual cure may ultimately be found for the tens of millions of people in the United States and around the globe now living with HIV.

References:

[1] Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice. Dash PK, Kaminski R, Bella R, Su H, Mathews S, Ahooyi TM, Chen C, Mancuso P, Sariyer R, Ferrante P, Donadoni M, Robinson JA, Sillman B, Lin Z, Hilaire JR, Banoub M, Elango M, Gautam N, Mosley RL, Poluektova LY, McMillan J, Bade AN, Gorantla S, Sariyer IK, Burdo TH, Young WB, Amini S, Gordon J, Jacobson JM, Edagwa B, Khalili K, Gendelman HE. Nat Commun. 2019 Jul 2;10(1):2753.

[2] Spudich S et al. Persistent HIV-infected Cells in Cerebrospinal Fluid are Associated with Poorer Neurocognitive Performance. J Clin Invest. 2019. DOI: 10.1172/JCI127413 (2019).

[3] In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Yin C, Zhang T, Qu X, Zhang Y, Putatunda R, Xiao X, Li F, Xiao W, Zhao H, Dai S, Qin X, Mo X, Young WB, Khalili K, Hu W. Mol Ther. 2017 May 3;25(5):1168-1186.

[4] Creation of a nanoformulated cabotegravir prodrug with improved antiretroviral profiles. Zhou T, Su H, Dash P, Lin Z, Dyavar Shetty BL, Kocher T, Szlachetka A, Lamberty B, Fox HS, Poluektova L, Gorantla S, McMillan J, Gautam N, Mosley RL, Alnouti Y, Edagwa B, Gendelman HE. Biomaterials. 2018 Jan;151:53-65.

[5] Statement on Claim of First Gene-Edited Babies by Chinese Researcher. The NIH Director, NIH. 2018 November 28.

Links:

HIV/AIDS (National Institute of Allergy and Infectious Diseases/NIH)

HIV Treatment: The Basics (U.S. Department of Health and Human Services)

Fast Facts (HIV.gov)

Global Statistics (HIV.gov)

Kamel Khalili (Temple University, Philadelphia, PA)

Howard Gendelman (University of Nebraska Medical Center, Omaha)

NIH Support: National Institute of Mental Health; National Institute of Neurological Disorders and Stroke; National Institute of Allergy and Infectious Diseases; National Institute on Aging; National Institute on Drug Abuse; Common Fund


Snapshots of Life: Finding Where HIV Hides

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HIV

Credit: Nadia Roan, University of California, San Francisco

Researchers have learned a tremendous amount about how the human immunodeficiency virus (HIV),  which causes AIDS, infects immune cells. Much of that information comes from studying immune cells in the bloodstream of HIV-positive people. Less detailed is the picture of how HIV interacts with immune cells inside the lymph nodes, where the virus can hide.

In this image of lymph tissue taken from the neck of a person with uncontrolled HIV infection, you can see areas where HIV is replicating (red) amid a sea of immune cells (blue dots). Areas of greatest HIV replication are associated with a high density of a subtype of human CD4 T-cells (yellow circles) that have been found to be especially susceptible to HIV infection.


Simplifying HIV Treatment: A Surprising New Lead

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CD4+ cells in the gut

Caption: PET/CT imaging reveals a surprisingly high concentration (yellow, light green) of key immune cells called CD4 T cells in the colon (left) of an SIV-infected animal that received antibody infusions along with antiviral treatment. Fewer immune cells were found in the small intestine (right), while the liver (lower left) shows a high level of non-specific signal (orange).
Credit: Byrareddy et al., Science (2016).

The surprising results of an animal study are raising hopes for a far simpler treatment regimen for people infected with the AIDS-causing human immunodeficiency virus (HIV). Currently, HIV-infected individuals can live a near normal life span if, every day, they take a complex combination of drugs called antiretroviral therapy (ART). The bad news is if they stop ART, the small amounts of HIV that still lurk in their bodies can bounce back and infect key immune cells, called CD4 T cells, resulting in life-threatening suppression of their immune systems.

Now, a study of rhesus macaques infected with a close relative of HIV, the simian immunodeficiency virus (SIV), suggests there might be a new therapeutic option that works by a mechanism that has researchers both excited and baffled [1]. By teaming ART with a designer antibody used to treat people with severe bowel disease, NIH-funded researchers report that they have been able to keep SIV in check in macaques for at least two years after ART is stopped. More research is needed to figure out exactly how the new strategy works, and whether it would also work for humans infected with HIV. However, the findings suggest there may be a way to achieve lasting remission from HIV without the risks, costs, and inconvenience associated with a daily regimen of drugs.


Snapshots of Life: Imperfect but Beautiful Intruder

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RSV Particle

Credit: Boon Chong Goh, Beckman Institute, University of Illinois at Urbana-Champaign

The striking image you see above is an example of what can happen when scientists combine something old with something new. In this case, a researcher took the Rous sarcoma virus (RSV)—a virus that’s been studied for more than century because of its ability to cause cancer in chickens and the insights it provided on human oncogenes [1, 2]—and used modern computational tools to generate a model of its atomic structure.

Here you see an immature RSV particle that’s just budded from an infected chicken cell and entered the avian bloodstream. A lattice of proteins (red) held together by short peptides (green) cover the outer shell of the immature virus, shielding other proteins (blue) that make up an inner shell.


AIDS Vaccine Research: Better By Design?

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OD-GT8 60mer

Caption: eOD-GT8 60mer nanoparticle based on the engineered protein eOD-GT8. Yellow shows where eOD-GT8 binds antibodies; white is the protein surface outside the binding site; light blue indicates the sugars attached to the protein; dark blue is the nanoparticle core to which eOD-GT8 has been fused.
Credit: Sergey Menis and William Schief, The Scripps Research Institute

A while ago, I highlighted a promising new approach for designing a vaccine against the human immunodeficiency virus (HIV), the cause of AIDS. This strategy would “take the immune system to school” and teach it a series of lessons using several vaccine injections—each consisting of a different HIV proteins designed to push the immune system, step by step, toward the production of protective antibodies capable of fending off virtually all HIV strains. But a big unanswered question was whether most people actually possess the specific type of precursor immune cells that that can be taught to produce antibodies that kill HIV.

Now, we may have the answer [1]. In a study published in the journal Science, a research team, partly supported by NIH, found that the majority of people do indeed have these precursor cells. While the total number of these cells in each person may be low, this may be all that’s needed for the immune system to recognize a vaccine. Based in part on these findings, researchers plan to launch a Phase 1 clinical trial in human volunteers to see if their latest engineered protein can find these precursor cells and begin coaxing them through the complicated process of producing protective antibodies.


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