The new delivery mechanism completely turned off a gene responsible for high cholesterol in mice.
This article is part of DNA/IDK, a semi-regular column exploring how genetic modification is shaping the future. Follow along here.
Since its was first harnessed by scientists in 2013, the natural gene-editing technology known as CRISPR has sparked a designer baby controversy, dreams of ending hereditary diseases, and fears of sophisticated biological terrorism. Yet for all of CRISPR’s peril and promise, figuring out a way to effectively deliver the system to the target DNA has remained a significant technical hurdle.
Usually, a CRISPR system—which consists of an enzyme called Cas9 that cuts out a portion of a target DNA strand, as well as a short strand of RNA that guides the enzyme to its target—catches a ride through the body in a virus. This is a less than ideal solution because patients receiving a CRISPR treatment can quickly develop, or may already possess, antibodies that would destroy it.
As detailed today in Nature Biotechnology, a team of researchers at MIT has created a highly effective, non-viral solution: a nanoparticle system that can deliver CRISPR to target genes. Moreover, the nanoparticle CRISPR-delivery system was able to completely turn off a gene responsible for high cholesterol level when administered to mice.
The group was led by MIT research scientist Hao Yin and associate professor of chemical engineering Daniel Anderson, both of whom have made ground-breaking discoveries in the science of gene editing in recent years.
In 2014, Yin, Anderson, and their colleagues at MIT became the first to cure a disease in an adult animal using CRISPR. In this case, it was a liver disease called tyrosinemia and the “patients” were adult mice. The problem, however, was this CRISPR method was dependent on viral delivery, as well as a high pressure injection, which could cause damage to the liver.
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Anderson and Yin further modified this approach in 2016 by encasing the Cas9 enzyme in a lipid nanoparticle. These are particles existing on the nanoscale that basically act like synthetic cells, protecting their contents with a wall of fatty material known as lipids.
The nanoparticles obviated the need for a high-pressure viral delivery system for Cas9, although the guiding RNA strand still required a virus for delivery. Deploying single strands of RNA is a difficult problem since our body associates them with viral infections. This can prompt the body to trigger certain immune responses that can result in the degradation of the RNA, rendering it useless.
One solution to this problem that has been explored in RNA-based medicines is to modify the RNA so that it doesn’t trigger an immune response. Things get a bit tricker when modifying RNA for CRISPR, since the modified RNA still needs to be able to interface with the Cas9 enzyme.
In their new research, Anderson and Yin worked on modifying the guide RNA so that it wouldn’t trigger the body’s immune system as it worked its way toward its target in a nanoparticle, while still being able to link up with the Cas9 enzyme. As they found in their research, they could modify up to 70 percent of the RNA before it was unable to bind to the Cas9 enzyme as a CRISPR system.
Importantly, the ability to use chemically modified RNA in the CRISPR system meant that a virus was no longer needed as a host for the guide RNA. It could instead be encased in a synthetic nanoparticle.
The guide RNA is programmed to seek out specific genes in the liver by reversing the genetic code found in a target strand of DNA. Once it locates this target DNA sequence, the Cas9 enzyme effectively splits the double helix to allow the guide RNA to bind to the DNA. If it’s a good match, this portion of the helix is excised from DNA strand. Usually, the DNA strand tries to repair itself, at which point the CRISPR system does its thing again. This process is repeated until the DNA no longer repairs itself, at which point the targeted gene has effectively been turned off.
In their new research, Anderson and his colleagues at MIT looked at a handful of different genes for their CRISPR system, but they mostly focused on PCSK9, a gene that produces a protein responsible for regulating cholesterol levels.
This gene is also responsible for hypercholesterolemia, or high cholesterol, which a genetic condition that affects about 1 in 500 people. High cholesterol significantly increases the risk of strokes and heart disease, and while a new suite of drugs targeting PCSK9 have proven to be effective, these drugs must be taken daily for the rest of a patient’s life.
“PCSK9 is an exciting and clinically relevant target for the treatment of hypercholesterolemia as there are some people who genetically get these incredibly high levels of cholesterol,” Anderson told me on the phone. “We reasoned that it may be possible to permanently inactivate this gene using a nanoparticle and that might provide a lifetime of therapy for patients.”
When Anderson and Yin injected these CRISPR-carrying nanoparticles into the livers of mice, the CRISPR system was able to eliminate the PCSK9 gene in 80 percent of their liver cells. This resulted in a 35 percent drop in cholesterol in treated mice. According to Anderson, this same technique may be used to treat other liver diseases, and may be slightly modified for use treating diseases in other body tissues.
It’s uncertain how long it will be before nanoparticle CRISPR treatments like the one pioneered by Anderson and Yin see widespread clinical use in humans.
For now, the nanoparticle technology will likely remain confined to non-human experiments, if for no other reason than CRISPR is still quite error prone. Although scientists are also exploring alternative gene-editing technologies that are more accurate than CRISPR, Anderson believes this new nanoparticle delivery system marks a significant step toward the medical adoption of CRISPR in humans.
“Nanoparticles like these have real potential to treat people with diseases,” Anderson said. “My hope is that these types of formulations really do end up helping people, that’s what really motivates us.”
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