As part of an innovative research, scientists from MIT have created nanoparticles with the ability to deliver the CRISPR genome-editing system for particularly modifying the genes in mice. The researchers used the nanoparticles to deliver the CRISPR components, thereby avoiding the need for using viruses for carrying the components.
By applying the innovative delivery methods, the team was successful in eliminating specific genes in nearly 80% of liver cells, which is the highest success rate accomplished ever by using CRISPR in full-grown 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,” stated Daniel Anderson, a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science (IMES), who is an Associate Professor in MIT’s Department of Chemical Engineering.
One gene targeted in this research, known as Pcsk9, is responsible for regulating cholesterol levels. Mutations caused in the human version of this gene lead to a rare disorder known as dominant familial hypercholesterolemia. Recently, the FDA approved two antibody drugs with the ability to curb the Pcsk9 gene. The downside is that in order to provide therapy, these antibodies have to be regularly taken for the rest of the patient’s life. The MIT researchers stated that the innovative nanoparticles have the ability to permanently modify the gene in just a single treatment, and that the method looks promising for the treatment of various other liver disorders.
The senior author of the study, which was reported in a paper published in the Nature Biotechnology journal on November 13th, 2017, is Anderson. Hao Yin, a research scientist from the Koch Institute, is the lead author of the paper. David H. Koch Institute Professor Robert Langer of MIT, Professors Victor Koteliansky and Timofei Zatsepin from the Skolkovo Institute of Science and Technology, and Professor Wen Xue from the University of Massachusetts Medical School are the other authors of the study.
Globally, a number of researchers have been attempting to devise effective and safe techniques for delivering the components required for CRISPR, which includes Cas9 (a DNA-cutting enzyme) and a short RNA for guiding the enzyme to a targeted area of the genome, instructing Cas9 on the area to be cut.
In most cases, scientists use viruses to deliver the gene for Cas9, and also the RNA guide strand. In the year 2014, Anderson, Yin, and their collaborators created a nonviral delivery system in the very first demonstration of treating a disease, namely a liver disorder called tyrosinemia, by administering CRISPR in a full-grown animal. This mode of delivery, however, mandates a high-pressure injection, a technique that can even damage the liver.
After some time, the scientists demonstrated that they can deliver the components without adopting the high-pressure injection by wrapping messenger RNA (mRNA) encoding Cas9 with a nanoparticle in the place of a virus. By adopting this technique, where the guide RNA was still carried by a virus, the team was able to modify the target gene in nearly 6% of hepatocytes, a percentage adequate to cure tyrosinemia.
According to Anderson, although this delivery method looks promising, under certain conditions, it might be better to use an entirely nonviral delivery system. One factor is that when a specific virus is used, the patient develops antibodies to the virus. Hence it cannot be used again. Moreover, the antibodies to the viruses, tested for CRISPR delivery, pre-exist in certain patients.
In the new paper published in Nature Biotechnology, the team has reported about a system with the ability to deliver the Cas9 as well as the RNA guide by using nanoparticles, thereby eliminating the need for using viruses. In order to deliver the guide RNAs, the team had to initially edit the RNA chemically to safeguard it from enzymes present in the body that might usually disintegrate it before attaining its target location.
The researchers investigated the structure of the complex formed by the RNA guide and Cas9 (i.e. sgRNA) to find out the sections of the guide RNA strand that could be chemically edited without hampering the binding of the two molecules. Using this analysis, the team developed and tested various probable 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 percent of the guide RNA. We could heavily modify it and not affect the binding of sgRNA and Cas9, and this enhanced modification really enhances activity.
Hao Yin, Research Scientist, The Koch Institute and the Lead Author of the paper
Reprogramming the Liver
The team wrapped these altered RNA guides, or the enhanced sgRNA, into lipid nanoparticles that were earlier used by them to deliver other kinds of RNA to the liver. Then, they injected the RNA guides into mice along with the nanoparticles including mRNA that encodes Cas9.
They conducted experiments by inhibiting a few disparate genes expressed by hepatocytes. However, their main focus was on the cholesterol-regulating Pcsk9 gene. They were successful in inhibiting this gene in over 80% of liver cells, and the Pcsk9 protein could not be detected in these mice. The team also discovered a 35% dip in the total cholesterol levels in the treated mice.
At present, the team is attempting to identify other liver diseases that can be cured by using this technique, and to develop these methods for use in patients.
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.
Daniel Anderson, Department of Chemical Engineering, MIT
“We are very excited to see this new application of nanotechnology open new avenues for gene editing,” added Langer.
The National Institutes of Health (NIH), the Russian Scientific Fund, the Skoltech Center, and the Koch Institute Support (core) Grant from the National Cancer Institute funded the study.