CRISPR-carrying nanoparticles can edit the genome

A research team led by the Massachusetts Institute of Technology (MIT) has developed nanoparticles that can deliver the CRISPR genome-editing system into mice, where it can be used to modify specific genes. By using nanoparticles to carry the CRISPR components, the team eliminated the need to use viruses for delivery.

Many scientists are trying to develop safe and efficient ways to deliver the components needed for CRISPR, which consists of a DNA-cutting enzyme called Cas9 and a short RNA that guides the enzyme to a specific area of the genome, directing Cas9 where to make its cut. In most cases, researchers rely on viruses to carry the gene for Cas9, as well as the RNA guide strand.

In 2014, MIT researchers Daniel Anderson, Hao Yin and their colleagues developed a nonviral delivery system in the first-ever demonstration of curing a disease (the liver disorder tyrosinemia) with CRISPR in an adult animal. However, this type of delivery requires a high-pressure injection, a method that can also cause some damage to the liver.

The researchers later showed that they could deliver the components without the high-pressure injection by packaging messenger RNA (mRNA) encoding Cas9 into a nanoparticle instead of a virus. Using this approach, in which the guide RNA was still delivered by a virus, the researchers were able to edit the target gene in about 6% of hepatocytes, which is enough to treat tyrosinemia.

While that delivery technique holds promise, in some situations it would be better to have a completely nonviral delivery system, according to Anderson. One consideration is that once a particular virus is used, the patient will develop antibodies to it, so it couldn’t be used again. Also, some patients have pre-existing antibodies to the viruses being tested as CRISPR delivery vehicles.

Now, the researchers have come up with a system that delivers both Cas9 and the RNA guide using nanoparticles, with no need for viruses. Anderson served as senior author on the study, while Yin was lead author. The results have been published in the journal Nature Biotechnology.

To deliver the guide RNAs, the researchers first had to chemically modify the RNA to protect it from enzymes in the body that would normally break it down before it could reach its destination. The scientists analysed the structure of the complex formed by Cas9 and the RNA guide, or sgRNA, to figure out which sections of the guide RNA strand could be chemically modified without interfering with the binding of the two molecules. Based on this analysis, they created and tested many possible combinations of modifications.

“We used the structure of the Cas9 and sgRNA complex as a guide and did tests to figure out we can modify as much as 70% of the guide RNA,” said Yin. “We could heavily modify it and not affect the binding of sgRNA and Cas9, and this enhanced modification really enhances activity.”

The researchers packaged these modified RNA guides (which they call enhanced sgRNA) into lipid nanoparticles, which they had previously used to deliver other types of RNA to the liver, and injected them into mice along with nanoparticles containing mRNA that encodes Cas9. They experimented with knocking out a few different genes expressed by hepatocytes, but focused most of their attention on the gene Pcsk9, which regulates cholesterol levels. Mutations in the human version of the gene are associated with a rare disorder called dominant familial hypercholesterolemia, and the FDA recently approved two antibody drugs that inhibit Pcsk9. However, these antibodies need to be taken regularly, and for the rest of the patient’s life, to provide therapy.

The researchers were able to eliminate the gene in more than 80% of liver cells — the best success rate ever achieved with CRISPR in adult animals — and the Pcsk9 protein was undetectable in the treated mice. The team also found a 35% drop in the total cholesterol levels of the mice.

“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,” said Anderson, with this editing having occurred following a single treatment.

The researchers are now working on identifying other liver diseases that might benefit from this approach, and advancing these approaches towards use in patients. As noted by Anderson, “I think having a fully synthetic nanoparticle that can specifically turn genes off could be a powerful tool not just for Pcsk9 but for other diseases as well.

“The liver is a really important organ and also is a source of disease for many people. 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.”

Image caption: In a new study, MIT researchers have developed nanoparticles that can deliver the CRISPR genome-editing system and specifically modify genes, eliminating the need to use viruses for delivery. Image courtesy of MIT News under CC BY-NC-ND 3.0