It started like any other day. Dr. Hao Yin walked into the lab at MIT, ready to check on his transgenic mice. He had no idea he was about to make history.

Yin’s mice harbored a single mutated gene that gave them a terrible liver disease. Left untreated, the deteriorating liver fails to process nutrients, and the mice eventually whittle down to skin and bones.

Yin was testing an audacious new gene therapy, first discovered a year prior in bacteria. What he saw stopped him stone cold: while the control mice had shed one-fifth of their normal body weight, their treated counterparts remained plump and healthy.

The “miracle” treatment in question? CRISPR.

Yin and colleagues were the first to definitively show that CRISPR could be used in living animals to correct genetic mutations and treat disease. That was in 2014.

A scientific firestorm followed: by 2016, CRISPR-enhanced immune warriors were already given to a cancer patient to battle his rogue cells. A year later, the technique was used to edit human embryos, sparking debates about designer babies. Roughly 20 CRISPR clinical trials are gearing up for action. Momentum keeps building.

Yet CRISPR has a dirty secret: there’s really no perfect way to deliver the “molecular scissors” safely into cells. Most methods currently rely on viruses: the DNA that encodes the CRISPR machinery is spliced into a “viral vector” then injected into the troubled tissue.

That’s all well and good for diseases that affect blood and muscle. But for destinations buried deep within the body, delivery becomes a serious issue.

Yin’s workaround wasn’t ideal: he shot two milliliters of solution containing the CRISPR machinery into the mice’s veins using incredibly high pressure. The human equivalent? Your entire blood volume, injected in just five minutes.

Unsurprisingly, the method can sometimes cause tissue damage. “We’re working on safer and more efficient delivery methods,” said the study’s lead author, Dr. Daniel Anderson, at the time.

This week his team finally delivered. They created a nanoparticle system that envelops the CRISPR components in a protective sphere. A skin prick sends the treatment on its way to the liver—no blasting or virus required.

In mice with genetically high cholesterol, the nanoparticles successfully turned off the faulty gene in over 80 percent of liver cells, bringing their blood lipid back to normal levels.

That’s the highest success rate ever achieved with CRISPR in adult animals.

“What’s really exciting here is that we’ve shown you can make a nanoparticle that can be used to permanently and specifically edit the DNA in the liver of an adult animal,” says Anderson.

“If you can reprogram the DNA of your liver while you’re still using it, we think there are many diseases that could be addressed.”

Viral Trouble

Gene therapy has always had a love-hate relationship with viruses, and CRISPR is no different.

Thanks to their infectious nature, viruses are the perfect hosts for tunneling all sorts of genetic material into the body. Both components of CRISPR—a DNA-cutting protein called Cas9 and a short RNA sequence that guides the scissors to the target site on the genome—can be encoded as DNA and stuffed into a viral backbone.

But viruses are also trouble. Scientists always remove the disease-causing genes in these living shuttles before they’re deployed. Sometimes, however, viruses can still trigger an immune attack, destroying their precious cargo as collateral damage. What’s more, an overactive immune system causes all sorts of flu-like symptoms. In severe cases, it kills.

Scientists have long sought an alternative delivery solution. To Yin and Anderson, lipid nanoparticles may be the answer. These tiny, non-toxic molecules wrap around cargo in a fatty bubble, protecting their contents much like a cell’s membrane.

Back in 2014, the team made waves for the first proof-of-concept that such a system could work in a living animal. The seminal study cured a severe genetic liver disease after just one shot—but unfortunately, a gigantic, bulldozing shot.

Not willing to trade one problem for another, the team next worked to figure out a gentler alternative to their system.

Two years later, they published an updated version. Here, the Cas9 protein was encased inside nanoparticles and injected into the vein much like you would for a flu vaccine. However, the guide RNA sequence was still delivered by a virus. Another bummer: the method only targeted six percent of liver cells.

The guide RNA could be to blame, the authors hypothesized. The mice could have quickly generated antibodies to the virus carrying the RNA, or they may also already have had other antibodies that cross-reacted with the virus.

Reformed RNA

The ultimate solution is to enwrap both CRISPR components in nanoparticles. But without viruses to shield it from enzymes in the blood, a naked guide RNA quickly breaks down—long before it reaches its intended target.

The team took inspiration from RNA-based therapeutics, a field that explores ways to use RNA as drugs. As it happens, RNA molecules have several “hot spots” that enzymes latch onto. Blocking these spots with chemical alterations protects the RNA against getting chopped up. Similarly, hotspots that trigger the immune system into action could also be tamed by chemical modification.

However, because the guide RNA needs to be able to bind to Cas9 even after modification, the team had to throw out the usual hotspot playbook and start from scratch.

Analyzing the structure of the Cas9 guide RNA complex, they identified portions on the guide RNA that are absolutely needed for the binding process—the “no touch” zone.

They then figured out which sections could be chemically modified, and tested many possible combinations of the modifications in cells.

“We can modify as much as 70 percent of the guide RNA,” says Yin. “This enhanced modification really enhances activity.”

Doctored Livers

The team targeted the liver gene PCSK9, which produces a protein that regulates blood cholesterol. Mutations in this gene can cause dangerously high cholesterol levels, raising the risk of stroke and heart attacks.

Although the FDA has approved two antibody therapies that block the gene’s activity, those treatments are only like bandages on a septic wound—because they don’t treat the cause, they need to be taken regularly for life to keep the disease at bay.

“PCSK9 is an exciting and clinically relevant target,” says Anderson, because inactivating the gene could provide “a lifetime of therapy for patients.”

The team packaged the upgraded guide RNA into nanoparticles and injected them into mice along with the Cas9-containing ones.

Just five days later, the CRISPR-loaded nanoparticles had inhibited over 80 percent of the gene in liver cells. The gene’s product, the PCSK9 protein, was completely undetectable in the blood.

The treated mice also experienced a hefty 35 percent drop in total cholesterol levels. Experiments targeting other liver genes resulted in similarly high efficiencies.

Importantly, while the treatment did cause a small spike in one specific type of immune molecule called a cytokine, the animals did not show signs of mounting an all-out immune attack. The results were published in Nature Biotechnology.

Anderson is confident that the same strategy could be used for other liver diseases, which his team is now actively exploring.

Like other novel academic concoctions, it’s hard to say when these nanoparticles could be tested in humans. And delivery systems aren’t the only roadblock in gene therapy. For one, CRISPR still isn’t specific enough. For another, it doesn’t always work as intended.

But scientists are readily exploring CRISPR upgrades, which would also benefit from Anderson’s system. Compared to viruses, nanoparticles are much easier to scale up and customize, which may help reduce the sky-high cost of gene therapy.

We believe that the new system opens up the door to a range of new therapeutic and industrial applications, the authors conclude. “We are very excited.”

Image Credit: MIT News