Two teams of researchers have successfully used CRISPR to correct genetic abnormalities that cause the inherited diseases phenylketonuria (PKU) and hereditary tyrosinemia type 1 (HT1). In both studies the researchers used base editing, rather than making double stranded DNA cuts with is believed to be safer.
The PKU study was conducted by researchers at ETH Zurich in Switzerland. PKU is a metabolic disorder caused by a mutation in the gene responsible for producing a liver enzyme called phenylalanine hydroxylase. Phenylalanine hydroxylase allows the amino acid phenylalanine to be metabolized. Without this enzyme, phenylalanine levels in the blood and brain increase which can cause seizures, atopic dermatitis, intellectual disability, mental health disorders and behavioral problems.
The researchers used the CRISPR-Cas9 gene editing tool but modified it to include an additional enzyme called cytidine deaminase. This enzyme corrected a DNA base pair to repair the mutation and ensure phenylalanine hydroxylase was synthesized. The team reports that up to 63% of copies of the mutated gene were corrected in the livers of adult mice, which was sufficient to reduce phenylalanine levels to near normal levels and eliminated symptoms of the disease.
The researchers note that the standard CRISPR-Cas9 method is used to make cuts to double stranded DNA at specific sites, with the cells’ homology-directed repair (HDR) mechanisms used to repair the damage. However, the technique is not very effective in slowly dividing cells. Further, making double-stranded cuts increases the chance of errors being made when the NA is repaired.
Base-editing on the other hand allows genes to be edited without making breaks to double stranded DNA and does not use HDR. The cytidine deaminase enzyme is bound to a deactivated cas9 enzyme which is used to covert a C-G pair to a T-A pair. The team showed that this method allowed the correction of the disease-causing mutation in nondividing hepatocytes at rates that were sufficient to cure phenylketonuria.
The team is now seeking funding to carry out trials in pigs, which more closely match humans. Further research is also necessary to investigate possible side effects such as the creation of cancer-causing mutations.
The second study was conducted by scientists at the Children’s Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania. In this study the gene editing occurred in utero and sought to prevent hereditary tyrosinemia type 1, a disease caused by the inability to effectively break down the amino acid tyrosine. As tyrosine levels builds they can cause liver and kidney damage, lead to the development of liver cancer or can cause liver failure. Untreated the disease is usually fatal.
This study also paired CRISPR-Cas9 with a base editor (base editor 3). In this trial, the team converted a single base but rather than targeting the mutation that leads to the development of HT1, they edited a base in a different gene – HPD. The genetic mutation remained, but the change made by the researchers prevented the build-up of toxic products from the breakdown of tyrosine, as occurs in individuals with the condition. Mice that didn’t have the edit were treated with the drug nitisinone, which is used to manage the condition in humans. The mice on nitisinone were not as healthy as those that had the BE3 edit performed.
The researchers report that following treatment, mice survived for up to three months with improved liver function and there were no off-target effects. Further research is needed to determine safety of the treatment before clinical trials can be considered.
The PKU study is detailed in the paper – Treatment of a metabolic liver disease by in vivo genome base editing in adult mice – which was published in Nature Medicine volume 24, pages1519–1525 (2018). The HT1 study is detailed in the paper – In utero CRISPR-mediated therapeutic editing of metabolic genes – which was also published in Nature Medicine volume 24, pages1513–1518 (2018).