Researchers have shown that by using a combination of genome editing and long-acting slow-effective release antiviral therapy (LASER ART), they were able to eradicate HIV reservoirs and cure nearly 40% of HIV-infected mice.

The report is not the first example of an HIV cure – two individuals, the Berlin patient and the London patient, have been cured of HIV through bone marrow transplants from donors that lacked the CCR5 co-receptor, making their T cells resistant to HIV infection. (See BioWorld, March 6, 2019.)

But both those cures were a collateral benefit of a high-risk procedure that was undertaken because the patients had leukemia.

Roughly 20% to 30% of bone marrow transplantees die within a year of receiving their transplant, making the procedure a complete nonstarter for an otherwise healthy HIV-positive individual whose infection can be controlled by a daily pill. (See BioWorld, March 6, 2019.)

In contrast, the new findings, which were published in the July 2, 2019, issue of Nature Communications, used methods for achieving their eradication cure that could be broadly useful for infected individuals.

LASER ART is a modified version of the standard of care of HIV-infected individuals, and gene editing – though using TALEN, not CRISPR, and targeting CCR5 rather than proviral DNA – is also in clinical trials for HIV. (See BioWorld, March 6, 2014.)

Co-corresponding author Kamel Khalili attributed his team's success to their approach to the problem.

In HIV infection, "the virus becomes part of the host genome, so we look at the disease not as an infectious disease... but as a genetic disease," he told BioWorld. "There is a bad gene, whose name is proviral DNA."

Khalili is Laura H. Carnell Professor and chair of the Department of Neuroscience, director of the Center for Neurovirology, and director of the Comprehensive NeuroAIDS Center at the Lewis Katz School of Medicine at Temple University.

Considering HIV through the lens of genetic disease led Khalili, co-corresponding author Howard Gendelman and their colleagues to apply the emerging toolkit of genetic diseases to the infection.

"About six years ago, we adapted a gene editing strategy – CRISPR technology – and developed methods that could completely excise proviral DNA... without any damage to cellular genes," Khalili said.

One of the major challenges to eliminating the HIV reservoir is that, anatomically speaking, it is not one reservoir but something more akin to dozens of wells. Though much of the latent virus is found in blood, lymph nodes and the gut, smaller reservoirs have been demonstrated at numerous other sites, including the brain.

As a result, "we knew we had to utilize a method that could spread" throughout the body, Khalili said, leading the team to develop a viral vector to do just that.

Using an AAV9-based delivery system, "any place the viral DNA is present can be targeted," he said.

The team combined the CRISPR technology with an antiretroviral therapy (ART) delivery strategy they called long-acting slow-effective release, or LASER, ART, which the team described as "highly hydrophobic lipophilic viral reservoir penetrating antiretroviral prodrugs" dolutegravir, lamivudine and abacavir in their paper. LASER ART can establish drug depots in macrophages that release their payloads over a course of weeks.

There are several methods, most prominently broadly neutralizing antibodies, that have been effective at delaying viral rebound for some time after treatment is stopped. "The real test is whether or not the virus can come back later on," Khalili said.

Along with the laboratory co-corresponding author Howard Gendelman, who is the Margaret R. Larson Professor of infectious diseases and internal medicine, chair of the Department of Pharmacology and Experimental Neuroscience and director of the Center for Neurodegenerative Diseases at the University of Nebraska Medical Center, Khalili and his colleagues looked for replication-competent virus in every nook and cranny of HIV-infected mice that had been treated with the combination of LASER ART and AAV9-delivered CRISPR targeting integrated provirus.

"We used a broad range of genetic and physiological assays," Khalili said. Those assays included adoptive lymph node transfer from treated into immunosuppressed mice, and whole-genome sequencing of potential reservoirs. "And all the studies showed that there is no viable virus."

The team is currently testing their approach in primates. When using mice, "a lot of success happens in the lab that cannot be translated," Khalili acknowledged.

So far, he said, the team has shown that in primates, too, CRISPR directed at the nonhuman primate version of HIV can cleave the virus in multiple tissues, including brain tissue, which presents a challenge for any and all drug development efforts due to the blood-brain barrier.

"We don't know at present time whether the cleavage can lead to cure," he said. "That experiment is underway."

But what his team's work demonstrates, he said, is that "HIV is a curable disease, and you can develop methods to cure it. But not with the old methods that we used. We need methods that make sense for this kind of disease."

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