Using the CRISPR-Cas9 technique, researchers were able to remove the Proviral DNA of SIV, the equivalent of HIV in monkeys, from infected cells. They see this proof of concept as a potential definitive treatment for AIDS.
This year’s World AIDS theme is “Global Solidarity and Shared Responsibility,” which echoes another viral pandemic we’ve been living with for nearly a year, Covid-19. The WHO estimates that 33 million people worldwide are living with HIV (as of 2019) and that 68% of infected adults and 53% of infected children are on lifelong antiretroviral treatment.
When HIV (the human immunodeficiency virus) infects the body, it contains a genome of RNA in its envelope and capsule. But the virus also carries an enzyme called reverse transcriptase. This enzyme is able to retrotranscribe viral RNA into single-stranded proviral DNA. It also synthesizes the second strand of proviral DNA, and only in the double-stranded form can proviral DNA be integrated into the genome of the cell.
Thanks to an integrase, the proviral DNA is hidden between our own genes, safe from the immune system. It can remain there for years before reactivating and forming infectious virions again, which then invade other cells. HIV infects cells that express CD4 cell receptors. CD4+ T lymphocytes express it the most, but other cell populations, such as macrophages, dendritic cells, and microglial cells, also express it.
Treatment of retroviral infections
Current therapies target proteins essential for viral replication (reverse transcriptase, integrase, and protease) and keep their levels below the limit of detection. Thus, an HIV-positive person can live without infecting others. But the proviral DNA is always hidden in the cells and is eventually reactivated. AIDS, Acquired Immunodeficiency Syndrome, is the final stage of HIV infection in which the large number of CD4 T lymphocytes destroyed by the virus plunges the patient into a state of profound immunosuppression. Thus, a simple opportunistic disease can lead to death.
To eliminate it and get rid of it forever, genetic tools must be used. Currently, the most promising genetic engineering technique is CRISPR-Cas9, the development of which by two researchers was awarded the 2020 Nobel Prize in Chemistry.
Scientists at Temple University’s Lewis Katz School of Medicine in Philadelphia used CRISPR-Cas9 to remove proviral DNA of SIV, the equivalent of HIV that infects monkeys, and a proven model to study the human form of the infection. The results of these in vivo experiments are published in Nature Communications.
The genome of SIV removed from cells
To achieve SIV excision from rhesus monkey tissue, the scientists used a complex molecular biology gene editing construct. The construct included the CRISPR-Cas9 tool built specifically for SIV provirus DNA. This construct, which takes the form of a plasmid, is integrated into an adeno-associated virus (or AAV). AAVs are small, non-pathogenic DNA viruses used in gene therapy.
This platform was injected intravenously into three rhesus monkeys. Three weeks later, scientists collected blood and tissue from these monkeys after they died. The results obtained from these samples were compared to samples taken from a monkey that was not treated with the platform.
Specific excision of proviral SIV DNA by CRISPR-Cas9 was confirmed in the blood cells of the three treated monkeys, in contrast to the control monkeys. The efficiency of excision varied widely between the animals: 37%, 65%, and 92%.
The scientists also examined the bio-distribution of their platform in the monkeys’ organs. Using an improved PCR technique, they found evidence of the platform in the bone marrow, spinal cord, tonsils, brain, spleen, liver, lymph nodes, thymus, and heart. In short, the platform has spread throughout the body, including the brain, which is protected by the blood-brain barrier.
In several of these organs, including the spleen, lungs, and lymph nodes, the ability of the molecular scissors to remove proviral DNA without damaging neighboring genes was also demonstrated. These results provide a proof of concept for the use of CRISPR-Cas9 as a potential strategy for HIV treatment in an animal model.
“This is an important development in what we hope will be the end of HIV/AIDS,” Andrew MacLean, a researcher involved in this work, said in a news release. “The next step is to evaluate this treatment over a longer period of time to see if we can achieve the complete elimination of the virus or even free people from antiretroviral treatment. “The scientists hope to one day see their treatment in a human clinical trial after the Food and Drug Agency approves it.