News spread rapidly in July, 2019 that researchers had eliminated human immunodeficiency virus (HIV) from the genomes of animals for the first time. The accomplishment originated in the laboratories of Howard Gendelman from the University of Nebraska Medical Center (UNMC) and Kamel Khalili from Temple University, Pennsylvania.
Khalili and Gendelman combined forces to use a long-acting therapy to reduce the HIV viral load, followed by gene-editing to remove HIV from infected cells. With this approach, they hoped to come up with a better treatment for those suffering from the viral infection. Even so, Gendelman viewed his data skeptically when initial results showed that some of his HIV-infected humanized mice were no longer infected. “So we repeated the experiments,” he recalled. “After we saw it two or three times, we started incrementally believing that we had stumbled on something that might be important.”
Gendelman and Khalili are among the first researchers using CRISPR-Cas9 to approach HIV. Their efforts focused on removing integrated HIV viral genomes from the cells of animals. Just two months after a paper describing their work was released, another prominent team in the CRISPR-HIV space also announced their results. Hongkui Deng and his team from Peking University in Beijing, along with Hu Chen from the 307 Hospital in Beijing, approached HIV in a different way. They used CRISPR-Cas9 to engineer human stem cells to mimic natural immunity to the virus.
Humanizing HIV treatment
Because HIV integrates into a host cell’s genome, Khalili and Gendelman approached the problem by viewing the virus as if it were a genetic disorder. “We’re cutting it out,” Gendelman said. “If we are overzealous and deliver a tremendous amount of the excision vectors — in this case we’re using an adeno-associated viral vector — we can cut out HIV to the point that the disease can be eliminated. The disease can be cured.”
In collaboration with his UNMC colleague Benson Edagwa, Gendelman’s team developed a therapeutic strategy known as long-acting slow-effective release antiretroviral therapy (LASER ART). This approach relies on antiretroviral nanocrystals to keep HIV replication at low levels for a longer period of time than traditional ART therapy. LASER ART targets viral sanctuaries, decreasing the need for typical ART administration. Separately, Khalili’s team developed a novel CRISPR-Cas9 therapy for directly removing HIV DNA in vivo from cells where the viral genome had integrated, which worked after LASER ART had reduced the amount of HIV.
Neither Gendelman’s LASER ART nor Khalili’s gene-therapy independently eliminated HIV from mice. But together, the technologies eliminated HIV infection in more than one third of treated mice(1). The results show “simple proof-of-concept” that HIV can be eliminated using the dual therapy, Gendelman said.
The goal, of course, is to eventually bring this treatment into the clinic. But the team has a long road ahead before that becomes a reality. What was possible in a humanized mouse (a mouse carrying human bone marrow to imitate the human immune system) treated with an abundance of a vector delivery system may not translate to humans. Researchers will have to conduct extensive testing to determine potential toxicity, develop a delivery system that meets the needs of the human body, and balance the volume of the delivery system with the necessary level of HIV excision.
When HIV is present in the body in a latent state, it usually does not produce symptoms, but it can reactivate at any time. Scientists have to eradicate nearly all — if not all — of the latent virus for genetic HIV therapy to be effective. CRISPR treatment has to be very sensitive and very specific. Khalili’s team designed a CRISPR system that targeted every cell in the body — the majority of which were not infected. This may not be suitable for humans because of the sheer volume of therapy that would have to be administered.
The team will also need to explore the possibility of off-target effects. “Even though we didn’t see off-target effects in the mice, it doesn’t mean that we would not, or could not see that in a human. It’s still possible,” Gendelman explained.
Reducing the burden, honing the delivery
To solve these problems, Gendelman’s team plans to alter the ART delivery scheme; delivering the drugs more effectively across cell membranes will reduce latent virus and HIV replication capacity. His team has already gained traction with “next generation LASER ART.” Meanwhile, Khalili’s team is further developing the CRISPR-Cas treatment to target only cells that contain latent virus, with a focus on systems that can target common receptors on CD4+ cells. The team is also looking at new CRISPR delivery systems with greater carrying capacities.
Their next step is to look beyond obvious targets, such as common HIV receptors, to find targets for CRISPR that interfere with the latent HIV reservoir, such as co-transcriptional factors and regulatory factors. CRISPR-Cas doesn’t necessarily have to work only on HIV excision — it could work on targets that enhance, extend, or sustain proviral DNA content to excise genes in the host cell that affect that latent HIV reservoir. “Instead of going after the first violin, which is the major part of this symphony, you go after the horn, or the percussion, or other parts of the orchestra that are affecting the latent virus,” Gendelman said.
The team will also need to explore the possibility of off-target effects. “Even though we didn’t see off-target effects in the mice, it doesn’t mean that we would not, or could not see that in a human. It’s still possible,” Gendelman explained.
Reducing the burden, honing the delivery
To solve these problems, Gendelman’s team plans to alter the ART delivery scheme; delivering the drugs more effectively across cell membranes will reduce latent virus and HIV replication capacity. His team has already gained traction with “next generation LASER ART.” Meanwhile, Khalili’s team is further developing the CRISPR-Cas treatment to target only cells that contain latent virus, with a focus on systems that can target common receptors on CD4+ cells. The team is also looking at new CRISPR delivery systems with greater carrying capacities.
Their next step is to look beyond obvious targets, such as common HIV receptors, to find targets for CRISPR that interfere with the latent HIV reservoir, such as co-transcriptional factors and regulatory factors. CRISPR-Cas doesn’t necessarily have to work only on HIV excision — it could work on targets that enhance, extend, or sustain proviral DNA content to excise genes in the host cell that affect that latent HIV reservoir. “Instead of going after the first violin, which is the major part of this symphony, you go after the horn, or the percussion, or other parts of the orchestra that are affecting the latent virus,” Gendelman said.
A different slant
While Gendelman and Khalili’s research focuses on direct administration of CRISPR to a patient, Deng’s research concentrates on treating stem cells with CRISPR-Cas9 to mimic natural immunity to the virus. Deng’s research was inspired by the “Berlin patient,” a man suffering from both HIV and a lethal blood cancer. The man received a bone-marrow transplant in 2007 to treat cancer; this treatment also cured the HIV infection, rendering him the first in the world to be cured of HIV(2). On a whim, the Berlin patient’s treating doctor looked for a donor carrying two copies of CCR5 with a delta 32 mutation. Certain strains of HIV use the CCR5 receptor to gain entry to white blood cells, so people carrying the mutated CCR5 receptor are nearly immune to HIV. Deng was a member of the team that discovered this receptor in 1996(3).
In 2011, Hu Chen from the 307 Hospital in Beijing contacted Deng. Chen had spent decades researching hematopoietic stem cell (HSC) therapy for treating leukemia, and hoped that Deng would collaborate with him to modify HSC therapy to treat HIV. Deng agreed to team up.
Initially, Deng used a TALEN-based gene editing approach to try to insert mutated CCR5 into HSC genomes, but when CRISPR became popular a few years later, he switched tactics. Even with CRISPR’s greater efficiency, HSC genomes are notoriously difficult to work with. The team spent five years improving the efficiency before publishing their first study in 2017. In that paper, the team described a process for inserting mutated CCR5 into human HSCs to mimic natural immunity to the virus. When transplanted into mice, these modified HSC ablated HIV infection4.
In Deng’s latest research, the team transplanted these same modified stem cells into a man with HIV and acute lymphocytic leukemia. The altered stem cells survived in the man’s body for more than a year without causing detectable side effects. Ultimately, however, the number of cells was insufficient to significantly reduce the amount of HIV circulating in the man’s blood(5).
“It’s encouraging because [CRISPR-edited] stem cells have been in the HIV patient for more than two years now,” Deng said. “And it also suggests that we need to improve our editing efficiency, which is what we are currently focused on. We need our treatment for HIV to reproduce like in the Berlin patient case. We need to get better editing efficiency: ideally 100 percent efficiency.”
A work in progress
Gendelman and Deng acknowledge that there is still a great deal of research to be done before clinicians can combat HIV using CRISPR. Many groups are working on generating human HSCs via induced pluripotent stem cell technology, which may improve editing efficiency. “The gene editing efficiency of pluripotent stem cells is very high, so you can do very precise gene editing,” Deng said. However, how to go about this is a challenge.
For directly delivering CRISPR to patients, “It’s not until [LASER ART-CRISPR HIV therapy] is reproduced and affirmed by others, and when we extend from a couple of animals to a large number, to the majority, and move this to large animals, that we can really claim success,” Gendelman said. “I look at this as a process, not a Eureka. I hope that makes sense, but healthy skepticism is probably a good part of being a good scientist.”
1. P.K. Dash et al., “Sequential LASER ART and CRISPR treatments eliminate HIV-1 in a subset of infected humanized mice,” Nat Commun, 10:2753-2772, 2019.
2. M.D. Gero Hütter et al., “Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation,” N Engl J Med, 360:692-698, 2009.
3. H. Deng et al., “Identification of a major co-receptor for primary isolates of HIV-1,” Nature, 381(6584):661-666, 1996.
4. L. Xu et al., “CRISPR/Cas9-mediated CCR5 ablation in human hematopoietic stem/progenitor cells confers HIV-1 resistance in vivo,” Mol Ther, 25(8):1782-1789, 2017.
5. L. Xu et al., “CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia,” N Engl J Med, 381:1240-1247, 2019.
