Summary: Nanotechnology meets gene editing. MIT researchers use nanoparticles instead of viruses to deliver the CRISPR gene editing system. This article first appeared on LongevityFacts. Author: Brady Hartman]
In a new study, MIT scientists have developed nanoparticles that deliver the CRISPR gene editing system, eliminating the need to use viruses for delivery.
Using the new delivery technique, the gene editors were able to cut out genes in about 80 percent of liver cells, the best success rate ever achieved with CRISPR in adult animals. Speaking about the success of the project, Daniel Anderson, senior author of the study and an associate professor in MIT’s Department of Chemical Engineering, said
“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.”
One of the genes targeted in this study, called Pcsk9, regulates cholesterol levels. Mutations in the human version of the gene are associated with a rare disorder called dominant familial hypercholesterolemia, a condition that causes high cholesterol. The FDA recently approved two antibody drugs that inhibit Pcsk9. However, these drugs need to be taken regularly, and for the rest of the patient’s life. According to the MIT team, the new nanoparticle-based technique permanently edits the gene with a single treatment. Moreover, the method offers promise for treating other liver disorders.
The study was published in the Nov. 13 issue of Nature Biotechnology. The paper’s lead author is MIT research scientist Hao Yin. MIT’s Professor Robert Langer is also among the authors.
Treating Disease with CRISPR
Genetic engineers are trying to develop safe and efficient ways of delivering the components needed for CRISPR. The current CRISPR-Cas9 system, nicknamed CRISPR 1.0, consists of a DNA-cutting enzyme called Cas9 and a short RNA fragment that directs the Cas9 enzyme to a specific area of the genome, guiding it where to make its cut.
In most cases, gene editors rely on viruses to carry the gene for Cas9, as well as the RNA guide fragment. In 2014, Anderson, Yin, and colleagues developed a non-viral delivery system in the first-ever demonstration of curing a disease with CRISPR in an adult animal. The researchers successfully treated the liver disorder tyrosinemia. However, the non-viral delivery system requires a high-pressure injection, which can also cause some damage to the liver.
Later, the scientists showed 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 method, in which a virus still delivered the guide RNA, the research team was able to edit the target gene in about 6 percent of liver cells called hepatocytes, which is sufficient to treat tyrosinemia.
Anderson says while that delivery technique holds promise, in some situations, it would be better to have a completely non-viral delivery system. One consideration is that once a particular virus has been used, the patient will develop antibodies to it, so it cannot be reused. Moreover, some patients have pre-existing antibodies to the viruses being tested as CRISPR delivery vehicles.
In the new Nature Biotechnology paper, the scientists came up with a system that delivers both Cas9 and the RNA guide using nanoparticles, eliminating the need for viruses. To deliver the guide RNAs, the researchers first had to chemically modify the RNA to protect it from enzymes that would typically break it down before it could reach its destination in the body.
The gene editors analyzed 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, the researchers created and tested many possible combinations. As Yin says,
“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 percent of the guide RNA,” adding. “We could heavily modify it and not affect the binding of sgRNA and Cas9, and this enhanced modification really enhances activity.”
Using CRISPR to Reprogram the Liver
The researchers packaged the modified RNA guides, called enhanced sgRNA, into lipid nanoparticles, which they had previously used to deliver other types of RNA to the liver. They then injected them into mice along with nanoparticles containing mRNA that encodes Cas9.
The researchers experimented with knocking out a few different genes expressed by hepatocytes, but focused most of their attention on the cholesterol-regulating Pcsk9 gene. The scientists were able to eliminate this gene in more than 80 percent of liver cells, and the Pcsk9 protein was undetectable in these mice. The researchers also found a 35 percent drop in the total cholesterol levels of the treated mice.
The scientists are now working on identifying other liver diseases that might benefit from this approach using nanoparticles and translating these techniques for use in patients. Anderson says,
“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,” adding. “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.”
MIT’s Professor Robert Langer pointed out the importance of the novel technology, remarking,
“We are very excited to see this new application of nanotechnology open new avenues for gene editing,”
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Cover photo provided by MIT News.
Massachusetts Institute of Technology. “CRISPR-carrying nanoparticles edit the genome: New delivery system deletes disease-causing genes and reduces cholesterol.” ScienceDaily. ScienceDaily, 13 November 2017. Link.
Hao Yin, Chun-Qing Song, Sneha Suresh, Qiongqiong Wu, Stephen Walsh, Luke Hyunsik Rhym, Esther Mintzer, Mehmet Fatih Bolukbasi, Lihua Julie Zhu, Kevin Kauffman, Haiwei Mou, Alicia Oberholzer, Junmei Ding, Suet-Yan Kwan, Roman L Bogorad, Timofei Zatsepin, Victor Koteliansky, Scot A Wolfe, Wen Xue, Robert Langer, Daniel G Anderson. “Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing.” Nature Biotechnology, 2017; DOI: 10.1038/nbt.4005. Link.
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