HIV/AIDS
Using the CRISPR/Cas9 gene editing technique, scientists at Temple University have eliminated HIV-1 DNA from T cell genomes in the lab experiments, a move that would prevent reinfection after the cells were re-exposed to the virus.
New research is paving the way to an eventual cure for patients infected with HIV, the virus that causes AIDS. In a study published in the Nature journal, Scientific Reports, researchers have shown that they can both effectively and safely eliminate the virus from the DNA of human cells grown in culture.
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Using the CRISPR/Cas9 gene editing technique, the scientists from Temple University eliminated HIV-1 DNA from T cell genomes in lab experiments, and prevented reinfection after the cells were re-exposed to the virus.The senior investigator on the new study, Kamel Khalili, says “Antiretroviral drugs are very good at controlling HIV infection. But patients on antiretroviral therapy who stop taking the drugs suffer a rapid rebound in HIV replication.” The presence of numerous copies of HIV weakens the immune system and eventually causes acquired immune deficiency syndrome, or AIDS.
Curing HIV/AIDS – which has claimed the lives of more than 25 million people since it was first discovered in the 1980s – is the ultimate goal in HIV research. Eliminating the virus after it has become integrated into CD4+ T-cells, the cells primarily infected with HIV, has proven difficult. Recent attempts have focused on intentionally reactivating HIV, aiming to stimulate a robust immune response capable of eradicating the virus from infected cells. However, to date, none of these “shock and kill” approaches has been successful.
Khalili and colleagues decided to try a different approach, specifically targeting HIV-1 proviral DNA (the integrated viral genome) using uniquely tailored gene editing technology. Their system includes a guide RNA that specifically locates HIV-1 DNA in the T-cell genome, and a nuclease enzyme, which cuts the strands of T-cell DNA. Once the nuclease has edited out the HIV-1 DNA sequence, the loose ends of the genome are reunited by the cell's own DNA repair machinery.
"These experiments had not been performed previously to this extent but the questions they address are critical, and the results allow us to move ahead with this technology."
In previous work, Dr. Khalili's team had demonstrated the ability of their technology to snip out HIV-1 DNA from human cell lines. In their latest study, however, they concentrated on latently and productively infected CD4+ T cells to show not only that the technology eliminates the virus from cells but also that its persistent presence in HIV-1-eradicated cells actually protects them against reinfection. More importantly, they carried their work over to ex vivo experiments, in which T-cells from patients infected with HIV were grown in cell culture, showing that treatment with the gene editing system can suppress viral replication and dramatically reduce viral load in patient cells.In another major component of the study, Dr. Khalili's team addressed questions about off-target effects and toxicity. Using an approach known as ultra-deep whole-genome sequencing, which is considered the gold standard for genomic assessment, the researchers analyzed the genomes of HIV-1-eradicated cells for mutations in genes outside the region targeted by the guide RNA. Their analyses ruled out off-target effects on genes, including potential collateral effects on cellular gene expression. Studies of cell viability and proliferation showed that HIV-1-eradicated cells were growing and functioning normally.
“The findings are important on multiple levels,” Dr. Khalili said. “They demonstrate the effectiveness of our gene editing system in eliminating HIV from the DNA of CD4 T-cells and, by introducing mutations into the viral genome, permanently inactivating its replication. Further, they show that the system can protect cells from reinfection and that the technology is safe for the cells, with no toxic effects.”
“These experiments had not been performed previously to this extent,” he added. “But the questions they address are critical, and the results allow us to move ahead with this technology.”
More work is needed to adapt the techniques into human treatments, but the promise is there, and according to many associated in the field, the work is only a sign of things to come in terms of CRISPR breakthroughs.
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