A single treatment with CRISPR/Cas9 has reduced cholesterol levels in mice for six months. The treatment was used to switch off a gene responsible for regulating cholesterol levels. While CRISPR/Cas9 has been used in various studies using cultured cells, this is the first time that targeted therapeutic gene silencing has been used successfully in adult animal models.
The study serves as a proof-of-concept that CRISPR/Cas9 could potentially be used to reduce cholesterol levels in patients suffering from cardiovascular disease.
The gene in question is Pcsk9. Drugs have already been developed to reduce Pcsk9 activity to lower cholesterol levels in patients with cardiovascular disease, this approach would prevent Pcsk9 proteins from being synthesized.
The researchers from Duke University used adeno-associated viral (AAV) vectors to deliver Cas9 to the mouse liver, but since the standard Cas9 enzyme from Streptococcus pyogenes was too large, the researchers used the smaller Cas9 from Staphylococcus aureus.
The Cas9 part of CRISPR/Cas9 is responsible for making the cut to the double stranded DNA, although the researchers did not want to remove the gene, only suppress activity. Instead they deactivated the cutting enzyme. The Cas9 enzyme still bound to the appropriate gene, but no cut was made to the DNA. Instead they combined the enzyme with a KRAB protein that silenced gene expression.
While the experiment was a success and cholesterol levels were reduced and maintained at a low level, the researchers did note that liver enzymes were released into the blood following treatment with Cas9 suggesting there was an immune response to the Cas9 enzyme. They enzymes were only released at a low level and were mitigated over time with no intervention.
Due to the low levels of enzymes released, this would not necessary be a problem, but for the treatment to be used in humans for long term reduction of cholesterol levels, multiple injections of Cas9 would be required. Further research is therefore required to investigate the immune response.
“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,” said Pratiksha Thakore, lead researcher for the study.
The results of the study can be found in the paper RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors, which was recently published in the journal Nature Communications.