The virus that causes AIDS is an efficient and crafty retrovirus. Once HIV inserts its DNA into the genome of its host cells, it has a long incubation period, and can remain dormant and hidden for years. And while physicians can mix a cocktail from a variety of antiretroviral drugs to keep it in check, the virus can reactivate if treatment is stopped.
In an attempt to render latent HIV completely harmless, UMass Medical School researchers are using CRISPR/Cas9, a powerful gene editing tool, to develop a novel technology that can potentially cut the DNA of the latent virus out of an infected cell.
“On the simplest level, we’re employing a very precise pair of scissors to go in and clip out all, or part of, the HIV genome and reattach the severed ends of the human genome,” said principal co-investigator Scot Wolfe, PhD, associate professor of molecular, cell & cancer biology. “If we could do that, the hope is that this would be a step on the road to getting a functional cure for HIV.”
CRISPR is a component of the immune system found in normal bacteria. In its natural state, it protects bacteria from viral invasion. Since its discovery, researchers have been seeking ways to program this system to quickly and selectively edit specific genetic sequences for study.
For all its versatility, applications for the CRISPR system remain confined to the lab. Despite recent advances showing that CRISPR/Cas9 can edit HIV from an infected cell in culture, this technique remains too imprecise to be used clinically because of its tendency to cut into random regions of the genome, producing deleterious, off-target effects.
To improve the fidelity and precision of the CRISPR/Cas9 gene editing system for this project, Dr. Wolfe has proposed fusing it with an additional domain that improves its specificity. This would conceivably allow the CRISPR system to edit out only the HIV DNA without the potential for stray cuts in the human genome.
The other hurdle to using current CRISPR/Cas9 technology against HIV is that while researchers have some notions where the virus might be hiding, they still don’t know how to find the virus in latently infected cells.
“Cells that are infected with HIV are permanent carriers of the viral genome. They are a kind of time bomb that can be reactive at any time if a patient stops taking their antiretroviral treatment,” said principal co-investigator Jeremy Luban, MD, the David J. Freelander Professor in AIDS Research and professor of molecular medicine. “In order to attack the virus in its latent state, we really need to understand where the virus lives and what it needs to survive.”
Dr. Luban and Wolfe will use a combination of innovative technologies to describe and model HIV DNA integrated into the genome of reservoir cells, also known as provirus. Characterizing the genomic landscape of these latently infected cells will allow the researchers to identify vulnerable and accessible genetic sequences that can be potentially cut out of the HIV virus to make it permanently inactive.
“Many scientists are looking for tools that will activate the virus so it will be visible to the immune system or drugs. We’ve chosen a different approach that looks to isolate and excise the provirus directly from resting cells,” said Luban.
With a model of the latently infected cells’ genome from which to work, Wolfe hopes to use his precise gene editing tool to excise the latent virus from cells. Part of the project will be to assess whether the precision of the system has improved enough to allow for selected removal of the HIV genome in humanized mouse models and cells from infected patients without causing collateral damage to the human genome.
“The underlying premise of this project that Scot has pushed forward using new technologies that he has developed, is to genetically engineer a system that can potentially remove the HIV genome from infected cells,” said Luban. “The hope is that one might develop the tools to deliver these agents to cells of the human immune system and actually eliminate the virus from where it is hiding.”
Joining Luban and Wolfe on the five-year, $4.6 million National Institute of Allergy and Infectious Diseases funded project are Dale Greiner, PhD, the Dr. Eileen L. Berman and Stanley I. Berman Foundation Chair in Biomedical Research and professor of molecular medicine; Oliver J. Rando, MD, PhD, professor of biochemistry & molecular pharmacology; Job Dekker, PhD, professor of biochemistry & molecular pharmacology; and Manuel Garber, PhD, associate professor of molecular medicine. Each will lend their respective expertise in developing humanized mouse models; mapping chromatin structure; modeling 3D chromosome organization; and computational biology. Additionally, Katherine Luzuriaga, MD, professor of molecular medicine, pediatrics and medicine, and Thomas C Greenough, MD, assistant professor of medicine, will provide clinical expertise on the project.
“We’ve assembled a team of researchers here at UMass Medical School with the goal of better understanding the intricate structure of the latent HIV virus when integrated into immune cells because we believe that will allow us to better target it with CRISPR for gene editing,” Wolfe explained.