Tests in animal models by researchers at the Lewis Katz School of Medicine at Temple University, and at the University of Nebraska Medical Center (UNMC) have shown how gene editing two targets, HIV-1 and CCR5—the co-receptor that helps the virus get into cells—can effectively eliminate HIV infection. The team used CRISPR-based technology to inactivate CCR5 and excise HIV-1 DNA fragments from infected cells, in humanized mice (hu-mice) that were also treated using long-acting antiretroviral drugs. The work is, the investigators stated, the first to combine a dual gene editing strategy with antiretroviral drugs to cure animals of HIV-1.

“The idea to bring together the excision of HIV-1 DNA with inactivation of CCR5 using gene-editing technology builds on observations from reported cures in human HIV patients,” explained Kamel Khalili, PhD, Laura H. Carnell Professor and Chair of the Department of Microbiology, Immunology, and Inflammation, Director of the Center for Neurovirology and Gene Editing, and Director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine. “In the few instances of HIV cures in humans, the patients underwent bone marrow transplantation for leukemia, and the donor cells that were used carried inactivating CCR5 mutations.”

Added Howard E. Gendelman, MD, Professor and Chair of the Department of Pharmacology and Experiential Neuroscience at UNMC “Curing HIV is the big picture. Through our ongoing collaboration, Temple and UNMC have carried out meaningful research that could ultimately impact the lives of many people.”

Reporting on their work in PNASCRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretrovial drug-suppressed virus-infected humanized mice,” the researchers stated, “Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments.”

Current HIV-1 treatment includes both antiretroviral therapy (ART) and broadly neutralizing antibodies, the authors noted. Such treatments can reduce, but not eliminate infectious virus. HIV integrates its DNA into the genome of host cells, and can lie dormant in tissue reservoirs for long periods of time, out of reach of antiretroviral drugs. As a consequence, when ART is stopped, HIV replication renews, giving rise to AIDS. “HIV-1 establishes latency in CD4+ T cells and mononuclear phagocytes (monocytes, macrophages, and dendritic cells), which represents the primary barrier for cure,” the team noted.

Functional cure of HIV-1 infection has been documented in three cases, however, and in each case, treatment of acute myeloid leukemia was performed with allogenic hematopoietic stem-cell transplants containing heterozygous or homozygous mutations in the HIV-1 chemokine C-C chemokine receptor type five (CCR5 Δ32) gene. “HIV-1 cure included attenuation of spreading viral infection, presence of virus-infected cells and tissue reservoirs, and absence of latent integrated proviral DNA,” the investigators explained.

Given the curative results in these patients, the team hypothesized that the current barrier to eliminating infection might be overcome using CRISPR-based gene editing. The newly reported study’s senior investigators, Khalili and Gendelman, have been long-time collaborators, and in previous work, the Khalili and Gendelman teams showed that HIV can be edited out from the genomes of live, humanized HIV-infected mice, leading to a cure in some animals.

For that research, Khalili and co-investigator, Rafal Kaminski, PhD, assistant professor at the Center for Neurovirology and Gene Editing at the Katz School of Medicine, combined their expertise in CRISPR gene-editing technology for targeting HIV-1 with a therapeutic strategy known as long-acting slow-effective release (LASER) antiretroviral therapy (ART), which was co-developed by Gendelman and Benson Edagwa, PhD, Assistant Professor of Pharmacology at UNMC. LASER ART holds HIV replication at low levels for long periods of time, decreasing the frequency of ART administration.

However despite being able to eliminate HIV in LASER-ART mice, the researchers found that HIV could eventually re-emerge from tissue reservoirs and cause rebound infection. This effect is similar to rebound infection in human patients who have been taking ART but suddenly stop or experience a disruption in treatment. To prevent rebound infection, Khalili and colleagues worked on next-generation CRISPR technology for HIV excision, developing a new, dual system aimed at permanently eliminating HIV from the animal model. “From success stories of human HIV patients who have undergone bone marrow transplantation for leukemia and been cured of HIV, our hypothesis was that the loss of the virus’s receptor, CCR5, is important to permanently eliminating HIV infection,” Khalili explained.

To achieve this the researchers developed a simple and more practical procedure for the inactivation of CCR5 that includes an IV inoculation of the CRISPR gene editing molecule. “The use of two CRISPR formulations administered in a sequential manner was designed to block “infection spread” present at low levels during ART,” the investigators explained further. “This was followed by clearance of residual integrated HIV-1 DNA. The strategy was designed to affect viral elimination in HIV-1-infected mice.”

Experiments in humanized LASER-ART mice carried out by Gendelman’s team showed that the constructs developed at Temple, when administered together, resulted in viral suppression, restoration of human T-cells, and elimination of replicating HIV-1 in 58% of infected animals. Their findings supported the notion that CCR5 plays a key role in facilitating HIV infection. “CRISPR gene editing by targeting host CCR5 and HIV-1 LTR-Gag while controlling viral replication by antiretroviral drugs can lead to HIV-1 elimination in tissue reservoirs of infected animals,” the team concluded. “Evidence was provided by the absence of viral rebound after 11 wk following ART cessation.” The researchers used sensitive PCR and viral rescue assays to confirm their findings.

Noting limitations of their work, the team also stated, “To achieve 100% HIV elimination, the CRISPR cargo carrying the excision agent needs to reach all the HIV-1 latently infected cells and compartments.” Nevertheless, they wrote, “In conclusion, despite the noted limitations, the sequential administration of combinations of two CRISPR-mediated antiretroviral strategies with ART achieved effective HIV-1 elimination and has potential for translation to the clinic … We demonstrate that further improvements in LASER ART and CRISPR for combinatorial editing of viral and strategically important cellular genes such as CCR5 in hu-mice may achieve and serve as a proof-of-principle for further investigation toward clinical trials.”

Gendelman added, “We are true partners, and what we achieved here is really spectacular. Khalili’s team generated the essential gene-editing constructs, and we then applied those constructs in our LASER-ART mouse model at Nebraska, figuring out when to administer gene-editing therapy and carrying out analyses to maximize HIV-1 excision, CCR5 inactivation, and suppression of viral growth.”

The Temple team anticipates that they could soon start evaluating the dual gene-editing strategy in non-human primates. To do so, Khalili will collaborate with their co-author Tricia H. Burdo, PhD, Professor and Vice Chair in the Department of Microbiology, Immunology, and Inflammation at the Katz School of Medicine, a known expert in the use of non-human primate models for studying HIV-1. Burdo and her team are interested in understanding the involvement of CCR5 in SIV-infected primates and the Burdo laboratory previously played a key part in research demonstrating the effectiveness and safety of CRISPR-based technology in removing HIV DNA from primate cells.

The new dual CRISPR gene-editing strategy holds exceptional promise for treating HIV in humans, the researchers believe. “It is a simple and relatively inexpensive approach,” Khalili noted. “The type of bone marrow transplant that has brought about cures in humans is reserved for patients who also have leukemia. It requires multiple rounds of radiation and is not applicable in resource-limited regions, where HIV infection tends to be most common.”

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