Much of the enthusiasm around gene-editing techniques, particularly the CRISPR-Cas9 technology, centers on the ability to insert or remove genes or to repair disease-causing mutations. A major concern of the CRISPR-Cas9 approach, in which the double-stranded DNA molecule is cut, is how the cell responds to that cut and how it is repaired. With some frequency, this technique leaves new mutations in its wake with uncertain side effects.
In a paper appearing in the journal Cell on December 7, scientists at the Salk Institute report a modified CRISPR-Cas9 technique that alters the activity, rather than the underlying sequence, of disease-associated genes. The researchers demonstrate that this technique can be used in mice to treat several different diseases.
"Cutting DNA opens the door to introducing new mutations," says senior author Juan Carlos Izpisua Belmonte of the Salk Institute for Biological Studies whose laboratory developed the new technique. "That is something that is going to stay with us with CRISPR or any other tool we develop that cuts DNA. It is a major bottleneck in the field of genetics - the possibility that the cell, after the DNA is cut, may introduce harmful mistakes."
That fact guided every experiment in the Belmonte lab as they developed the technique using a modified CRISPR-Cas9 system that does not cut the DNA. Their findings are the first to provide evidence that one can alter the phenotype of an animal with a epigenetic editing technology, preserving DNA integrity.
The principal idea behind the Salk technique is the use of two adeno-associated viruses (AAVs) as the machinery to introduce their genetic manipulation machinery to cells in post-natal mice. The researchers inserted the gene for the Cas9 enzyme into one AAV virus. They used another AAV virus to introduce a short single guide RNA (sgRNA), which specifies the precise location in the mouse genome where Cas9 will bind, and a transcriptional activator. The shorter sgRNA is only 14 or 15 nucleotides compared with the standard 20 nucleotides used in most CRISPR-Cas9 techniques, and this prevents Cas9 from cutting the DNA.
"Basically, we used the modified guide RNA to bring a transcriptional activator to work together with the Cas9 and delivered that complex to the region of the genome we were interested in," says co-first author Hsin-Kai Liao of the Belmonte laboratory.
The complex sits in the region of DNA of interest and promotes expression of a gene of interest. Similar techniques could be used to activate virtually any gene or genetic pathway without the risk of introducing potentially harmful mutations.
"We wanted to change the cell fate with therapeutic efficiency without a DNA cut," co-first author Fumiyuki Hatanaka explains.
Strikingly, the team demonstrated disease reversal in several disease models in mice. In a mouse model of acute kidney disease, they showed that the technique activated previously damaged or silenced genes to restore normal kidney function. They were also able to induce some liver cells to differentiate into pancreatic ?-like cells, which produce insulin, to partially rescue a mouse model of type 1 diabetes.
The team also showed that they could recover muscle growth and function in mouse models of muscular dystrophy, a disease with a known gene mutation. Instead of trying to correct the mutated gene, the researchers increased the expression of genes in the same pathway as the mutated gene, over-riding the effect of the damaged gene. "We are not fixing the gene; the mutation is still there," says Belmonte, "Instead, we are working on the epigenome and the mice recover the expression of other genes in the same pathway. That is enough to recover the muscle function of these mutant mice."
Preliminary data suggest that the technique is safe and does not produce unwanted genetic mutations. However, the researchers are pursuing further studies to ensure safety, practicality, and efficiency before considering bringing it to a clinical environment.
Belmonte sees this technology as a way of potentially treating neurological disorders such as Alzheimer's and Parkinson's diseases. Just as the technique restored kidney, muscle, and insulin-producing function in the mouse models, he sees a future for rejuvenating neuronal populations, maybe even one day in human patients.
The work was supported by the Moxie Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the G. Harold and Leila Y. Mathers Charitable Foundation, Fundacion Dr. Pedro Guillen, AFE, UCAM, and NIH.
Cell, Liao et al.: "In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-Epigenetic Modulation" http://www.cell.com/cell/fulltext/S0092-8674(17)31247-3
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