New CRISPR Technique Used to Integrate Minigene into Mouse Genome and Stop Liver Disease

New CRISPR Technique Used to Integrate Minigene into Mouse Genome and Stop Liver Disease

A new CRISPR method has been developed and used to insert a minigene into mouse DNA which prevented the development of a genetic liver disease and improved clinical symptoms.

The Penn Medicine researchers had previously used CRISPR to correct a single mutation which resulted in improved symptoms of liver disease in newborn mice, but was not effective in adult mice. The new study went a step further and delivered a minigene, rather than using a cut and paste approach to correct mutations. To get the minigene, Cas9 CRISPR enzyme, and guide RNA inside the cells, the researchers used a novel dual adeno-associated virus (AAV). The AAV has a special affinity for liver cells and was delivered along with a liver-specific promoter to ensure expression only inside liver cells.

One AAV was used to deliver the RNA-guided Cas9 enzyme used for cutting the DNA, and the second was used to deliver the minigene that contained the codon for expressing the enzyme ornithine transcarbamylase (OTC). The minigene was inserted and the DNA repaired using homology directed repair (HDR).

Using the new technique, there was a clinical benefit in newborn mice that lasted into adulthood. Treated mice displayed 25% and 35% of OTC-expressing cells in the liver at 3 and 8 weeks respectively, compared to mice that received the untargeted vector. Ammonia levels fell by 60% compared to untreated mice showing the liver cells were producing OTC.

OTC is one of six enzymes in the liver used to eliminate ammonia from the body. If ammonia is not eliminated it can accumulate in the blood and can cause brain damage. Patients with OTC disorder can take medications to stimulate other pathways for clearing ammonia but even with treatment the mortality rate is high. Ideally patients require a liver transplant, but there is a shortage of donors. Without the medications or a liver transplant OTC disorder is fatal.

The researchers hope to be able to develop their new technique for use in humans to correct genetic liver disease caused by mutations on the OTC gene. OTC disorder in humans affects around one in 40,000 individuals. OTC disorder can be caused by around 300 different mutations on the OTC gene. The minigene approach is therefore the ideal solution for treating OTC disorder in humans.

“The next step, through additional preclinical studies, is to find a safe harbor site on the gene in human liver cells and then to test a similar gene-editing approach,” said James Wilson, MD, Ph.D., a professor of Medicine and director of the Gene Therapy Program and the Orphan Disease Center at Penn Medicine.

You can read more about the study in the paper – A mutation-independent CRISPR-Cas9–mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency – which was recently published in the journal Science Advances. DOI: 10.1126/sciadv.aax5701

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