The CRISPR-Cas9 tool is emerging as a powerful gene therapy tool, showing promise in fighting diseases like cancer and HIV. Now, a team from Duke University has used the technique to switch off certain genes in mice to reduce cholesterol levels, marking the first time CRISPR has been used to silence genes – without making edits – in adult animals.

Normally, CRISPR-Cas9 works its magic by making precise cut-and-paste edits to specific sections of DNA, allowing scientists to correct mutations that cause disease, even snipping them out of the genome at the embryo stage. But a recent study showed that chopping and changing a living organism's DNA could have harmful side effects throughout the body, and although that was later officially withdrawn, questions of safety still need to be raised at this early stage of the technology.

Rather than hacking away with genetic scissors, recent work has instead used CRISPR to temporarily turn certain genes on and off through epigenetic modulation. In that vein, the new study developed a CRISPR-Cas9 repressor system that can silence a gene called Pcsk9, which regulates cholesterol levels. The Duke researchers managed to package this system and deliver it into the livers of adult mice.

"We previously used these same types of tools to turn genes on and off in cultured cells, and we wanted to see if we could also deliver them to animal models with an approach that is relevant for gene therapy," says Charles Gersbach, lead researcher on the study. "We wanted to change the genes in a way that would have a therapeutic outcome, and Pcsk9 is a useful proof-of-concept given its role regulating cholesterol levels, which in turn affect health issues like heart disease."

The researchers started with a Cas9 enzyme taken from the bacteria species Staphylococcus aureus, and to keep it from making cuts to the target DNA, they created a "dead" version dubbed dCas9. This was bundled with a KRAB protein that silences gene expression, and the combination was then packaged inside adeno-associated viral (AAV) vectors – viruses that are engineered to carry the active ingredients to the right DNA target.

In their tests, the Duke researchers delivered the system to adult mice, where it activated in their livers. Compared to a control group that had simple saline injections, the Pcsk9 genes in the test mice were successfully repressed and the animals' cholesterol levels dropped as a result. Better yet, the effects of a single treatment lasted six months.

The experiment wasn't without its problems, though. The researchers reported seeing an immune response to the treatment in the form of elevated levels of certain liver enzymes, and although these stayed below critical levels, the team says multiple injections might tip them over the edge.

"One of the interesting things we found looked like an immune response against the Cas9 protein," says Pratiksha Thakore, an author of the study. "Following injection, we saw that levels of our target gene, Pcsk9, were reduced, but we also observed increases in expression of many immune cell genes, which indicates that immune cells were infiltrating the liver after we delivered Cas9 to the mice. Gaining a better understanding of this immune response and how to modulate it will be important for using Cas9 technologies for therapies."

The key may lie in the choice of delivery system. Since AAV vectors are viruses, the immune system can respond violently to fight them off. Interestingly, a recent similar study from MIT managed to avoid this problem by using a non-viral delivery system, instead packaging the Cas9 enzyme into nanoparticles. That study, however, still snipped the target DNA instead of switching the genes off.

The Duke researchers plan to continue their investigation, which could one day lead to less invasive gene therapy techniques for various diseases.

"There are still lots of things for us to explore with this approach," says Thakore. "CRISPR-Cas9 tools have worked so well in cell culture models that it's exciting to apply them more in vivo, especially when we're examining important therapeutic targets and using delivery vehicles that would be relevant to treating human diseases."

The research was published in the journal Nature Communications.

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