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    Gene therapy partially restores vision in rats

    Mutant (left) and treated retinas.© Keiichiro Suzuki
    A team made up of researchers spanning five countries has used gene-replacement therapy to partially restore vision in genetically blind rats. The new practice is the first successful gene editing tool that can replace damaged genes with fully functioning versions of DNA in adult cells, according to the Salk Institute study, which was published in Nature journal on Wednesday.
    To repair the rats’ genetic defect, the team, which included researchers from Saudi Arabia’s King Abdullah University of Science and Technology (KAUST), developed a new method which allowed them to introduce DNA at a targeted location in the non-dividing cells which make up most adult tissues and organs, such as neurons and muscles. The new approach builds on the genome-editing technique CRISPR-Cas9, which co-opts an immune mechanism from bacteria to enable scientists to direct a payload to a specified target location in the genome. In tandem with native DNA repair mechanisms, this has brought precise genome-editing within scientists’ reach.
    So far these technologies have primarily been used to knock out genes rather than insert DNA at a specific location. For example, in a study published this year, CRISPR-Cas9 was used to disable one of the malfunctioning genes in rats with the inherited degenerative eye disease, retinitis pigmentosa, preventing degradation of the rats’ retinas.
    A major shortcoming has been the restriction of these techniques to dividing cells. To overcome these limitations, the team developed a strategy called homology-independent targeted integration (HITI) which combines a customized nucleic acid cocktail with CRISPR-Cas9 to take advantage of a DNA repair mechanism that is active even in nondividing cells. The new technique was tested by using it to introduce DNA encoding a green fluorescent protein (GFP) downstream of a specific gene in cultured human neurons.
    “To our excitement, a couple of days later, we found many neurons with GFP! That was the first indication that HITI could work in non-dividing cells,” says Juan Carlos Izpisua Belmonte of the Salk Institute in California, the lead scientist of the project. “We immediately designed a series of experiments and applied HITI directly in vivo using rodent models.”
    The team then used different techniques to deliver the HITI cocktail into muscle, kidney and brain cells in live mice, including local and systemic injections of a viral vector called AAV carrying the HITI components. All of the experiments worked to some extent, though the efficiency varied depending on the tissue and delivery mode. “The development of more efficient gene delivery methods, which is a bottleneck for in vivo genome editing in general, will improve HITI efficiency in the near future” says Izpisua Belmonte.
    As a final test, the researchers used HITI to repair the defective Mertk gene in the eyes of three-week-old rats with retinitis pigmentosa, which causes blindness. They introduced a functional copy of the gene at the right genome location, thereby repairing the disease-causing mutation. The rats were able to detect light five weeks following the injection, and showed significant recovery of their visual functions.
    The ability to correct loss-of-function mutations in adult tissues holds tremendous therapeutic appeal, and HITI also has the potential to advance basic and translational research. However, other scientists have noted that the technique still needs refinement and its safety needs to be studied. Further research would need to be conducted before scientists can test HITI on humans, Salk Institute scientists said. But they were confident clinical trials would be underway after more extensive studies were conducted.

    Read more: Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature (2016)