CRISPR and LASER ART Eliminate HIV from Mice

×

Subscribe Us

By subscription, you consent to allow Ubigene Biosciences to store and process the information provided above to deliver the latest news, research spotlight, and promotions. You can unsubscribe from these communications at anytime.

Location:Home > About Us > Blogs >

CRISPR and LASER ART Eliminate HIV from Mice

A cure for HIV has been one of the biggest challenges facing the scientific and medical communities for decades. The virus has managed to successfully evade almost every technique that has been thrown up against it. Not only can HIV mutate frequently—making new variants quickly and constantly, it can also achieve latency and sequester itself. Although antiretroviral therapy has turned HIV into a chronic disease, curing a person of the infection has remained the holy grail of retrovirologists.


Today, a paper in Nature Communications, titled “Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice” reports new work from a collaborative effort showing that a combination of long-acting slow-effective release antiviral therapy (LASER) and CRISPR-Cas9 successfully cleared HIV from infected humanized mice.


Howard Gendelman, MD, professor of internal medicine and infectious diseases at the University of Nebraska Medical Center and senior author on the paper, does not withhold his excitement over the result, which may the reason for his hyperbole. He tells GEN that the conclusion is “almost unbelievable, but, it’s true” an idea, he adds, that “has been science fiction up until now.” He notes that “for the first time in the world” they have shown total elimination of HIV infection from a model with an established infection and, even though there are caveats, “there is a real possibility that an HIV cure can be realized.”


The team used a technique developed by co-author Kamel Khalili, PhD, professor in the department of neuroscience at the School of Medicine at Temple University, that uses the CRISPR-Cas9 system to remove the integrated HIV DNA from genomes. They combined the genome editing technique with the LASER ART, a technique developed by Gendelman’s lab that targets viral sanctuaries by packaging the ART drugs into nanocrystals. LASER ART distributes the drugs to areas of the body where HIV harbors and releases them slowly over time. Testing the combination of these two methods together in mice led the authors to conclude that permanent elimination of HIV is possible.


Pumping the HIV cure brakes



"This is a step. But, it’s a small step” notes John Coffin, PhD, professor at Tufts University School of Medicine, that is “not going to generate huge excitement in the field.” It’s a very interesting start, he notes, but “more work is clearly needed to understand what is happening in this model, before proceeding toward the development of studies in other species, and eventually humans.”


Although there are interesting aspects to the work, notes Coffin, a result in only 2 mice out of 7 requires follow up. The small numbers, he asserts, without reproduction, could be nothing more than an accident or a statistical fluke.



John Coffin, PhD, Tufts University School of Medicine



One experiment in particular, that Coffin would like to have been designed differently, is the DNA analysis of the mice. He notes that without having analyzed the DNA before and immediately the CRISPR addition, it is unclear whether or not the two mice that were successes had a significantly lower viral load than the other mice, even before the CRISPR treatment. Because it was not measured, the experiment doesn’t reveal the effect of the CRISPR. It’s possible, he adds, that the effect seen in those two mice had nothing to do with the CRISPR and was due entirely to the ART. And, because the mice were harvested just five weeks after the CRISPR treatment, there is no way to know if the virus may have rebounded or if resistant populations might pop up, given more time.


LASER ART and CRISPR – stronger together  


The authors conclude that both LASER ART and CRISPR are required for the elimination. And, although we’ve known for decades that ART cannot cure HIV on its own, why not CRISPR alone? In this paper, results showed 60% to 80% efficiency of viral DNA excision by CRISPR which, Coffin notes, is not even close to enough.


Gendelman explains that “It’s kind of like being in a beach and trying to find the right shell—you might want a certain color or shape.” When HIV replicates, he says, there are “billions and trillions” of particles so you’re asking CRISPR to excise every single DNA provirus in this morass. He adds, “it would be inconceivable that it would be efficient enough to destroy every DNA molecule…. If one infectious particle remains, it will grow and replicate. You have to destroy every single one in the body.” So, the ART reduced the viable targets. If you are inhibiting viral replication, he explains, you reduce the amount of HIV DNA in the host—in the cells and in the body, and that allows the CRISPR to be more effective. “It’s a numbers game,” Gendelman notes.


But, efficiency is not the only problem in the relationship between CRISPR and HIV. The sequence specificity of the approach a double-edged sword, notes Coffin. On the one hand, it minimizes off-target effects. But, as Coffin explains, it also sets the stage for rapid selection of resistance. In a virus that mutates as rapidly as HIV, the changes could quickly render CRISPR useless. Lastly, the mice were infected with a clonal virus with CRISPR delivered shortly after the infection, leaving little opportunity to generate a diverse population. However, this is not analogous to human patients as most patients do not report for treatment so soon after an infection. An effective treatment for humans would have to be designed to treat diverse viral populations with lots of mutations.


The path from mice to humans is long and winding (with lots of ditches to fall into)


Gendelman asserts that their mouse model is one of the reasons that this work has translational value, because it is a “truly humanized mouse.” Prasanta Dash, PhD, an instructor in the Gendelman lab and first author on the paper, agrees that the mouse model is "more advantageous than other existing mouse models." Developed by the Gendelman group more than a decade ago, the mouse has been engineered to produce human T cells susceptible to HIV infection, allowing the team to use experiments with long-term viral infection and ART-induced latency. Because HIV only infects human cells, “the cells, the distribution, the pathobiology, destruction of lymphocytes, is all replicated” according to Gendelman.


Even with the strong mouse model, and the excitement surrounding the result, the road to elimination of HIV in humans is long. First, notes Dash, they need to understand why many of the animals were not cured. Second, they have to be able to deliver CRISPR into all of the compartments in the body, which is not trivial in a human. Humans are complicated, with many cells and tissues. Gendelman notes that his lab is working on a homing device—a CRISPR system that will go after the places in the body where the virus is located.


Fyodor Urnov, deputy director, Altius Institute for Biomedical Sciences, explains that there are even more potholes in the road. "The developments of the past decade,” since the first gene editing trial for HIV initiated in 2009, notes Urnov, “have been the establishment of clear regulatory guidelines on the path to using an approach of this type for first-in-human clinical trials." He explains that these involve engineering nucleases of clinical-grade potency and specificity, and de-risking them using FDA-established preclinical models of efficacy and safety.


"A particular issue of regulatory concern with an approach of this type," asserts Urnov, “would be the immunogenicity of both the virus used to deliver Cas9, and of Cas9 itself.” Finally, he explains, there is the known challenge of manufacturing the therapeutic (in this case, an AAV harboring Cas9 and the two gRNAs) to GMP standards—a known nontrivial challenge in the field.


"If one assumes that a biotechnology company or a well-funded and motivated academic research group chooses to pursue an approach of this type, a clinical trial is ~3 years away in a best-case scenario (likely more),” asserts Urnov.



Contact us