CRISPR ‘minigene’ used to prevent genetic liver disease in mice

In a proof-of-concept study, researchers have successfully used a new CRISPR gene-editing technique in mice to prevent a genetic liver disease known to be driven by hundreds of different mutations and improve clinical symptoms.

According to the scientists, from Penn Medicine, US, the findings suggest a promising CRISPR tool that could potentially treat patients with a rare metabolic urea-cycle disorder caused by a deficiency in the enzyme ornithine transcarbamylase (OTC), as well as other hereditary diseases triggered by different mutations on the same gene.

The CRISPR approach was developed from a previous one created at Penn Medicine which was limited to correcting only one mutation and benefitted new-born mice but not adults. This time, the technique, which employs a novel, dual adeno-associated virus (AAV) to deliver its components, inserted a ‘minigene’ into the genome to accomplish a sustained expression of OTC in liver cells. No mutations are corrected; instead, a new set of instructions are added to the cells. As a result, researchers observed a clinical benefit in new-born mice that was maintained in adulthood.

“Like most genetic diseases that present lethal effects in new-borns, early treatments that are effective for the long term are essential,” said Dr James Wilson, a professor of Medicine, and director of the Gene Therapy Program and the Orphan Disease Center at Penn. “Here, we moved a CRISPR approach forward to not only sustain expression of OTC in the cells but also broaden the tool’s abilities.”

Mechanism of the system

To develop a broadly applicable genome-editing tool, the team constructed a new, dual AAV vector containing an RNA-guided bacteria protein called Cas9. Known as AAV8, this Penn-developed vector specifically has an affinity for liver cells. The second AAV contained a fully functioning minigene expressing a codon-optimised human OTC, the donor DNA, driven by a liver-specific promoter to ensure it only expresses in liver cells when injected into the blood.

The first step the researchers took was to create a break in the DNA by Cas9 at the targeting site along the gene, which enables the addition of the minigene for homology directed repair (HDR).

“Unlike other CRISPR approaches that delete or modify a portion of the normal gene, this technique integrates a new portion,” said first author Dr Lili Wang, a research associate professor of Medicine. “We’re not trying to correct mutations that stop liver cells from producing OTC, we’re adding this new minigene so the cells can.”

Mice treated with the targeted vector showed 25 and 35 percent of OTC-expressing cells in the liver at three and eight weeks, respectively. This is four- and three-fold higher than the mice treated with the untargeted vector. At both three and eight weeks, most OTC-positive liver cells were located in clusters scattered throughout all portions of the liver in the targeted mice.

Researchers also observed a 60 percent reduction in ammonia levels in targeted mice compared to untreated mice fed on a high protein diet, a clinical sign that suggests the liver cells are producing OTC.

“With these successful animal studies, we’ve moved closer to a potential ‘broad spectrum’ gene-editing approach to treat patients with the OTC deficiency, irrespective of mutation and clinical state,” Wilson said. “The next step, through additional pre-clinical studies, is to find a safe harbour site on the gene in human liver cells and then to test a similar gene-editing approach.”

The results were published in Science Advances.