So that the urine does not beat in the head

Researchers from the University of Pennsylvania have achieved a clinical improvement in the condition of mice with a genetic liver disease caused by hundreds of different mutations. The data obtained indicate the prospects of CRISPR for the treatment of patients with a rare urea cycle metabolism disorder caused by a deficiency of the ornithine transcarbamylase enzyme (ornithine transcarbamylase, OTC), as well as other hereditary diseases caused by various mutations of the same gene.

CRISPR was previously limited to correcting only one mutation and brought relief only in newborn mice. The new study used a double adeno-associated viral vector (AAV) to deliver CRISPR components that inserted a minigene into the genome to achieve stable expression of OTC in liver cells. The mutations were not corrected, the cells were simply given a new set of instructions. As a result, the researchers observed clinical efficacy in newborn mice, which was maintained in adulthood.

Violation of OTC synthesis occurs in almost one in 40,000 newborns, it can be caused by more than 300 different mutations of the OTC gene. Since these mutations are localized on the X chromosome, they usually manifest in men, whereas women are carriers. The urea cycle is a series of six enzyme reactions in the liver that help rid the body of ammonia. With a deficiency or absence of one of these enzymes, ammonia accumulates in the blood and enters the brain, causing damage to it and even leading to the death of the patient. To treat this disease, patients take medications for life that stimulate alternative ways of purification from nitrogen compounds, and in severe cases they need liver transplantation. Nevertheless, mortality is still quite high, so there is a need for newer and more effective methods of treatment.

To develop a genome editing tool for urea cycle metabolic disorders, the group created a new AAV double vector containing the RNA-controlled bacterial protein Cas9, which searches for and manipulates genes in cells. This AAV8 vector has a specific affinity for liver cells. The second AAV contained a fully functioning minigene expressing human OTC – a donor DNA-driven liver-specific promoter that, when injected into the blood, functions only in liver cells. First, Cas9 cuts the DNA in a certain area along the gene, and then a minigene is added to this gap.

Unlike other CRISPR subtypes, which remove or modify part of a normal gene, this method creates a new section of DNA. The researchers did not correct the mutations that prevent liver cells from producing OTC, they added a new minigene so that the cells could do it.

In mice treated with a double vector, 25% and 35% of OTC-expressing cells were detected in the liver after three and eight weeks, respectively, this is four and three times higher than in mice treated with a non-target vector. At both time points (three and eight weeks), most of the OTC-synthesizing cells were located in clusters scattered throughout the liver.

The researchers also observed a 60% decrease in ammonia levels in experimental mice compared to untreated mice who received a high-protein diet – this is a clinical sign indicating the effectiveness of therapy.

The next step, as the authors write, will be to conduct additional preclinical studies in order to find a safe DNA site for inserting a gene in human liver cells, and then testing a similar gene editing approach.

Article L.Wang et al. A mutation-independent CRISPR-Cas9–mediated gene targeting approach to treat a murine model of ornithine transcarbamylase deficiency is published in the journal Science Advances.

Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on Penn Medicine: CRISPR "Minigene" Approach Stops Genetic Liver Disease in Mice.