22nd July 2014

HIV eliminated from human cell cultures

For the first time, researchers have demonstrated proof-of-concept that the HIV virus can be eliminated from the DNA of human cell cultures. Although years away from clinical application, this breakthrough has been described as an important step forward in the search for a cure.

The HIV-1 virus has proved to be tenacious – inserting its genome permanently into victims' DNA, forcing patients to take a lifelong drug regimen to control the virus and prevent a fresh attack. Now, a team of Temple University School of Medicine researchers has designed a way to "snip out" the integrated HIV-1 genes for good.

"This is one important step on the path toward a permanent cure for AIDS," says Kamel Khalili, PhD. He and colleague, Wenhui Hu, led the work which marks the first successful attempt to eliminate latent HIV-1 virus from human cells. "It's an exciting discovery – but it's not yet ready to go into the clinic. It's a proof-of-concept that we're moving in the right direction," added Dr. Khalili.

In a study published yesterday by the Proceedings of the National Academy of Sciences (PNAS), Dr. Khalili and colleagues detail how they created molecular tools to delete the HIV-1 proviral DNA. When deployed, a combination of DNA-snipping enzyme called a nuclease and targeting strand of RNA called a guide RNA (gRNA) hunt down the viral genome and excise the HIV-1 DNA. From there, the cell's own gene repair machinery takes over – soldering the loose ends of the genome back together – resulting in virus-free cells.

"Since HIV-1 is never cleared by the immune system, removal of the virus is required in order to cure the disease," said Khalili, whose work focuses on the neuropathogenesis of viral infections. The same technique could theoretically be used against a variety of viruses, he said. The research shows that these molecular tools also hold promise as a therapeutic vaccine; cells armed with the nuclease-RNA combination proved impervious to HIV infection.

Worldwide, over 35 million people have HIV, including more than 1 million in the United States. Every year, another 50,000 Americans contract the virus, according to the U.S. Centers for Disease Control and Prevention.

Although highly active antiretroviral therapy (HAART) has controlled HIV-1 for infected people in the developed world for the last 15 years, the virus can rage again with any interruption in treatment. Even when HIV-1 replication is well controlled with HAART, the lingering HIV-1 presence has longer-term health consequences. "The low level replication of HIV-1 makes patients more likely to suffer from diseases usually associated with aging," Khalili said. These include cardiomyopathy – a weakening of the heart muscle – bone disease, kidney disease, and neurocognitive disorders. "These problems are often exacerbated by the toxic drugs that must be taken to control the virus," he added.

His team based the two-part HIV-1 editor on a system that evolved as a bacterial defence mechanism to protect against infection, Khalili said. His lab engineered a 20-nucleotide strand of guide RNA to target the HIV-1 DNA and paired it with Cas9 (to induce strand breaks in DNA). The gRNA targets the control region of the gene called the long terminal repeat (LTR). LTRs are present on both ends of the HIV-1 genome. By targeting both LTRs, the Cas9 snips out the 9,709-nucleotides that comprise the HIV-1 genome. To avoid any risk of the gRNA accidentally binding with part of the patient's genome, the researchers selected nucleotide sequences that do not appear in any coding sequences of human DNA, thereby avoiding off-target effects and subsequent cellular DNA damage.

The editing process was successful in a number of cell types that can harbour HIV-1 – including microglia and macrophages, as well as in T-lymphocytes. "T-cells and monocytic cells are the main cell types infected by HIV-1, so they are the most important targets for this technology," Dr. Khalili said.

The HIV-1 eradication approach faces several significant challenges before the technique is ready for patients, Dr. Khalili said. The researchers must devise a method to deliver the therapeutic agent to every single infected cell. Finally, because HIV-1 is prone to mutations, treatment may need to be individualised for each patient's unique viral sequences.

"We are working on a number of strategies so we can take the construct into preclinical studies," Dr. Khalili said. "We want to eradicate every single copy of HIV-1 from the patient. That will cure AIDS. I think this technology is the way we can do it."

Last week, a report by the United Nations claimed that AIDS could be brought under control by 2030.

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