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Scientists modify CRISPR to epigenetically treat diabetes, kidney disease, muscular dystrophy

The advanced Cas9-based gene activation system in Cas9 from the Belmonte laboratory improves skeletal muscle mass (above) and growth in size of the fiber (bottom) in a mouse treatment (right) compared to an independent control (left). The fluorescent microscopy images in the lower part show the purple staining of the glycoprotein laminin in the anterior tibial muscle fibers. Credit: Salk Institute

Salk scientists have created a new version of the CRISPR / Cas9 genome editing technology that allows them to activate genes without creating interruptions in DNA, avoiding a major obstacle to using gene editing technologies to treat human diseases.

Most CRISPR / Cas9 systems work by creating "double-stranded divisions" (DSB) in selected regions of the genome to be edited or deleted, but many researchers are opposed to creating such breaks in the DNA of living humans . As a proof of concept, the Salk group used its new approach to treat several diseases, including diabetes, acute kidney disease and muscular dystrophy, in mouse models.

"Although many studies have shown that CRISPR / Cas9 can be applied as a powerful tool for gene therapy, there are growing concerns regarding the unwanted mutations generated by double-strand breaks through this technology," he says. Juan Carlos Izpisua Belmonte, professor of Salk's Laboratory of Gene Expression and main author of the new document, published in Cell on December 7, 2017. "We were able to avoid that concern".

In the original CRISPR / Cas9 system, the Cas9 enzyme is coupled with guiding RNA that directs it to the correct place in the genome to create DSB. Recently, some researchers have begun using a "dead" form of Cas9 (dCas9), which can still point to specific places in the genome, but no longer cuts the DNA. In contrast, dCas9 has been coupled with transcriptional activation domains (molecular switches) that activate specific genes. But the resulting protein-dCas9 bound to the activating switches is too large and bulky to fit in the vehicle typically used to deliver these types of therapies to cells in living organisms, namely, adeno-associated viruses (AAV). The lack of an efficient delivery system makes it very difficult to use this tool in clinical applications.

The Izpisua Belmonte team combined Cas9 / dCas9 with a range of different activators to discover a combination that worked even when the proteins were not fused to one another. In other words, Cas9 or dCas9 was packed into an AAV, and the switches and guide RNAs were packed into another. They also optimized the guide RNAs to ensure that all the pieces end up in the desired place in the genome, and that the target gene is strongly activated.

"All components work together in the body to influence endogenous genes," says Hsin-Kai (Ken) Liao, a staff researcher at the Izpisua Belmonte laboratory and first author of the new paper. In this way, the technology operates epigenetically, which means that it influences gene activity without changing the DNA sequence.

To test the method, the researchers used mouse models of acute kidney injury, type 1 diabetes and one form of muscular dystrophy. In each case, they designed their CRISPR / Cas9 system to boost the expression of an endogenous gene that could reverse the symptoms of the disease. In the case of kidney disease, they activated two genes that were known to be involved in kidney function, and they observed not only an increase in the levels of the proteins associated with those genes, but also an improved renal function after an acute injury . For type 1 diabetes, they aimed to increase the activity of genes that could generate insulin-producing cells. Once again, the treatment worked, lowering blood glucose levels in a model of diabetes in mice. For muscular dystrophy, the researchers expressed genes that had previously been shown to reverse the symptoms of the disease, including a particularly large gene that can not be easily administered through gene therapies mediated by traditional viruses.

"We were very excited when we saw the results in mice," adds Fumiyuki Hatanaka, a research associate in the laboratory and co-first author of the article. "We can induce the activation of genes and at the same time see physiological changes."

The Izpisua Belmonte team is now working to improve the specificity of its system and apply it to more types of cells and organs to treat a wider range of human diseases, as well as to rejuvenate specific organs and reverse the aging process and conditions related to age, such as hearing loss and macular degeneration. More safety testing is needed before human trials, they say.

Explore more:
Researchers design an improved gene editing process for Duchenne muscular dystrophy

More information:
Cell Liao et al .: "In vivo Target Gene activation through trans-epigenetic modulation mediated by CRISPR / Cas9" www.cell.com/cell/fulltext/S0092- 8674 (17) 31247-3, DOI: 10.1016 / j.cell.2017.10.025

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Salk Institute

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