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Swiss scientists have used improved CRISPR-Cas gene editing technology to correct the mutation in the gene responsible for the phenylketonuria (PKU) -related metabolic disorder in mice and restore blood levels to normal levels. amino acid phenylalanine. Reporting on technology in Medicine of nature, the researchers say their findings offer "compelling evidence" that the method can be used to reverse the disease. "This approach has great potential for application in humans," says Gerald Schwank, Ph.D., professor at the ETH Zurich.
In the reported studies, the new technology has allowed mRNA correction rates of up to 63%, which, according to the authors, suggests "an applicability to a large number of genetic diseases". Their published article called "Treatment of a Metabolic Liver Disease by In Vivo Genome". basic montage in adult mice. "
Phenylketonuria is an autosomal recessive disorder that causes a deficiency of the liver enzyme phenylalanine hydroxylase (PAH), which normally metabolizes the amino acid phenylalanine. People with PKU inherit a defective copy of each parent's Pah gene. This disorder means that they need a special diet to prevent the accumulation of phenylalanine in the body. Untreated infants may suffer from severe delay, microcephaly and convulsions. This is why some countries are routinely screening for PKU in newborns.
The EPF team has developed a method of treating the disease that uses gene editing to correct the gene mutation. The most widely used systems for in vivo The genome edition is based on the CRISPR Cas9 gene editing enzyme to introduce site-specific double-stranded DNA (dsDNA) breaks at target sites of the chromosome, which can then be repaired by repair mechanisms directed by the homology of the cell. However, the authors explain, this approach does not work well in very slow-dividing cells and tissues. "HDR in undivided cells is very inefficient …" they write. "The therapeutic application of CRISPR-associated nucleases to target genetic diseases in slow-proliferating tissues is therefore limited to a small group of disorders for which the inactivation of a gene is sufficient or the correction Precise mutation confers a selective growth advantage to the modified cell. . "
On the other hand, the basic editing strategy allows the editing of the genome without formation of ADsd breaks and without the use of HDR. Instead, base editors comprising a deaminase enzyme fused to a catalytically dead Cas9 enzyme (dCas9) can convert a C-G base pair to a T-A base pair, or vice versa. "It's important to note that these deaminases are specific to a single strand," note the authors. "We found that base editors allowed accurate correction of disease-causing mutations in non-dividing hepatocytes at a rate sufficient to cure the phenotype of a disease."
To test their hypothesis, the researchers developed a base editing technology associated with CRISPR-Cas to correct the homozygous Pah mutation in a mouse model of PKU. The system, packaged in adeno-associated viral vectors (AAV), effectively comprises the enzyme citidine deaminase fused to the dCas9 enzyme. The construct binds to the locus of the gene containing the Pah mutation and opens the strands of DNA. The deaminase enzyme then converts the incorrect C-G base pair of the Pah gene into the correct T-A base pair.
The team first tested the effectiveness of its basic editing approach on a cell line of hepatocyte reporter cells in culture, then on primary liver cells, before moving on to in vivo evaluation of the gene correction system in adult mice that received AAV constructs via tail vein injections.
The treatment allowed the abnormally high levels of phenylalanine in the blood of the animals to recover 20 times their normal physiological level after six weeks. Encouragingly, blood levels of the amino acid in the blood after treatment then remained normal for the full 26 weeks of the survey. The delayed-growth animals also gained weight after the gene modification therapy, and their paler, hypopigmented fur also took a more or less dark tint to about the same color as that of the control animals. normal, a few weeks after treatment.
Research has shown that up to 60% of all copies of the defective gene in mouse liver have been corrected, as well as Pah mRNA correction rates of up to 63%. after 14 weeks. "The restoration of PAH enzyme activity (1.7-22.8% of wild-type enzyme activity) was confirmed in whole liver lysates and correlated with the correction rates of the liver." MRNA and genomic DNA, "they write. There was also no evidence of untargeted effects or significant damage to the DNA. "… We have found no indication of excessive damage to DNA or cell proliferation after prolonged exposure to low levels of basic editors," the team said. "Our data suggest that base editors associated with highly specific guide RNAs have a low risk of generating untargeted mutations, even when they are expressed over longer time periods."
Previous methods of genome editing have been much less successful at correcting target mutations in live animals, and the rate of gene correction has been, at best, a few percent, Dr. Schwank comments. "We have achieved publishing rates that are several times higher – no one has succeeded so far."
"The use of a basic editor has been key to our success," says the study's lead author, Lukas Villiger, Ph.D. student at ETH. Before using basic editing technology, ETH researchers were working with traditional CRISPR / Cas approaches, but in 2016, they adopted the basic editing approach described for the first time. times by its Massachusetts Institute of Technology (MIT) developers. However, as Villiger acknowledges, "even with the new base editors, the path did not follow a straight line – we had to tinker a bit." The researchers say the biggest surprise was that the new system was revealed much more effective than the traditional CRISPR / Cas toolbox.
The EPF team is currently seeking funds to conduct additional preclinical tests on other animals, including pigs. They argue that follow-up studies will also be needed to determine if the AAV vector is likely to cause adverse effects and to further verify the presence of non-targeted mutations. "The human liver is made up of billions of cells," notes Dr. Schwank. "In none of them do we want to induce mutations that could cause cancer.
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