1. Viral vectors were used to deliver the correct sequence for the ornithine transcarbamylase (OTC) gene, the guide sequence to locate OTC, and the gene for the Cas9 editing enzyme.
2. OTC-deficient newborn mice treated with the vectors showed restored OTC activity, decreased levels of toxic ammonia, as well as enhanced survival.
Evidence Rating Level: 2 (Good)
Study Rundown: CRISPR-Cas9 is a genome editing system with the capability to correct fatal genetic conditions caused by single point mutations. This study attempted to correct a metabolic liver condition characterized by hyperammonemia, caused by a hereditary mutation in OTC. Newborn OTC-deficient mice were administered viral vectors containing the Cas9 gene, the correct OTC gene sequence, and a single guide RNA sequence to guide Cas9 to the correct location. This was ultimately successful, leading to an increase in liver cell OTC expression and a significant reduction in ammonia levels of treated mice. As the liver continued to develop in the newborns, Cas9 expression decreased, preventing further unwanted genomic changes. The researchers then tried OTC restoration in adult mice. These attempts were unsuccessful, with the adult mice incurring more undesirable mutations and less successful gene correction. None of the treated adult animals survived due to the severe toxicity of these mutations.
Safety is a large issue with the CRISPR-Cas9 system. In order for the technology to be used effectively as a treatment, it needs to be ensured that patients would not acquire unexpected and potentially fatal mutations. In addition, the ethical dilemmas regarding genome editing must be considered if this potential treatment does prove to have efficacy in humans. Despite these complications, this technique could impact newborns with fatal OTC deficiencies and potentially other genetic conditions.
In-Depth [animal study]: Previous studies have shown that the adeno-associated virus 8 vector specifically targets the liver. Therefore, it was used to deliver the Cas9 gene, the correct version of the OTC gene, and the single guide RNA to make sure that editing occurred in the correct location.
Newborn pups with the OTC deficiency were given an intravenous injection of the vectors on their second day of life. Using immunohistochemistry to stain for OTC, there was a 100-fold increase in OTC-expressing cells in treated animals compared to controls (p<0.01). There was also a 20% and 16% increase in OTC activity after 3 and 8 weeks, respectively (p<0.01 compared to controls). Since the Cas9 enzyme can be toxic, its persisting levels following injection were measured using RT-qPCR. Enzyme expression levels decreased 43-fold over 7 weeks (p<0.01). There was also a 25-fold reduction in Cas9 DNA, showing that the vector genomes were eliminated in the proliferating hepatocytes. Finally, a high protein diet was used to induce hyperammonemia. Treated animals showed a 40% reduction in ammonia levels compared to untreated mice (p<0.01). While 30% of untreated newborn pups developed fatal hyperammonemia, all of the treated mice survived and no liver pathology or toxicity was found in histological analyses.
The researchers then attempted to correct the OTC deficiency in adult mice. Undesirable mutations were seen 42-44.6% of the time, while successful gene correction occurred only around 1% of the time, showing the treatment to be ineffective in adult mice. In addition, by 5 weeks following treatment administration, all of the animals were euthanized due to sickness.
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