0912 GMT November 21, 2019
Duke University engineers, using the CRISPR/Cas9 technique, initially reduced blood cholesterol levels and repressed the gene for six months after one treatment. Their findings were published this week in the journal Nature Communications, upi.com reported.
This is the first time researchers have delivered CRISPR/Cas9 repressors for targeted therapeutic gene silencing in adult animal models, according to Duke engineers.
CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, combines a scissor-like protein called Cas9 and a guide molecule that seeks a precise site in the genome. Then, Cas9 snips the DNA, which disables the targeted gene.
The CRISPR/Cas9 system not only locates and cuts specific sequences of DNA, it can also turn on or off the expression of targeted genes without permanent changes to the DNA coding sequence.
"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," Charles Gersbach, associate professor of biomedical engineering at Duke University, said in a press release.
"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."
In their tests, they silenced Pcsk9, a gene that regulates cholesterol levels.
Several drugs to treat high cholesterol and cardiovascular disease block the activity, but they don't prevent Pcsk9 from being made.
In adult mice 6 to 8 weeks old, the researchers used adeno-associated viral vectors, which are small viruses that have been engineered to target tissues in human gene therapy clinical trials.
Because of the vector's small cargo limit, they used a common Cas9 enzyme from Streptococcus pyogenes. They also created a ‘dead’ version of the enzyme, dCas9, that binds to but does not cut the DNA sequence.
In the mice, the researchers reduced Pcsk9 and cholesterol levels.
But they noted liver enzymes were released into the blood of treatments that included Cas9. Although the levels are below a critical threshold and normalized over time, the researchers said it raises questions about the efficacy of multiple injections.
"Gaining a better understanding of this immune response and how to modulate it will be important for using Cas9 technologies for therapies," said Pratiksha Thakore, the PhD student who led the work in the lab.
Researchers are learning how the immune system of living organisms responds to delivery of CRISPR/Cas9 system. The immune system may recognize it as a foreign protein from an invading organism and mount a response because the Cas9 enzyme is derived from bacteria.
In addition, potential patients for the therapy may already include immune responses against these systems because the enzymes come from common bacteria.
"The field is just starting to look at this, and it's clear that immune response is an important issue," Gersbach said. "Although we did see an immune response in the mice when we administered Cas9, the levels of liver enzymes in the serum seemed to mitigate over time without any intervention, and the effect of Pcsk9 repression was sustained regardless."
The researchers hope to gather more information to better understand the immune response against Cas9.
"There are still lots of things for us to explore with this approach," Thakore said.
"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."