THE genetic equivalent of a tailor who uses molecular 鈥渇ingers鈥� to grab onto DNA, before snipping it apart and stitching in a new sequence could lead to safer gene therapies.
In principle, genetic engineering is simple, but inserting a new gene into the right place in an organism鈥檚 genome is fraught with difficulty. For example, in a gene therapy trial for X-SCID 鈥� or 鈥渂ubble-boy鈥� disease 鈥� inserting a gene in the wrong place triggered cancer in some of the recipients.
One approach for locating and snipping DNA strands involves 鈥渮inc fingers鈥� 鈥� proteins that bind to DNA and can be linked together to recognise extended stretches of DNA with very high specificity. Zinc fingers are usually attached to enzymes called nucleases, dubbed ZFNs, which cut both strands of DNA.
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However, the problem with this approach is that it relies on enzymes, the cell鈥檚 natural repair machinery to fix the break, and either insert a new gene or a mutation that knocks out the function of a gene. 鈥淚n some cells these enzymes work better than in others,鈥� says of the University of Glasgow, UK.
Instead, Carlos Barbas of the Scripps Research Institute in La Jolla, California, and his colleagues have taken viral enzymes called recombinases and attached these to zinc fingers, called ZFRs.
While a ZFN is essentially just a pair of scissors, the recombinase in a ZFR both cuts and mends the break without resorting to unreliable enzymes.
Unlike nucleases, recombinases cut a double-stranded piece of DNA and then wait around on the exposed ends. When the intended gene, which would have been inserted at the same time, comes along, the recombinase recognises it, and binds the DNA to the ends, repairing the break (see diagram). 鈥淭he advantage of a site-specific recombinase is that the enzyme does everything,鈥� says Stark, who is also investigating the potential of ZFRs in genetic engineering.
As proof of principle, Barbas鈥檚 team has taken human cells and inserted a gene that their ZFR would recognise. They then used the recombinase to insert the gene into the cells鈥� genome. The gene was inserted correctly in more than 98 per cent of cases, says Barbas, who presented his results earlier this week at the Strategies for Engineered Negligible Senescence meeting in Cambridge, UK.
Their results are promising, says of the University of Texas in Dallas. 鈥淚t will be interesting to see how efficiently they can [target genes] to a natural site in the human genome,鈥� he adds.
鈥淚f you could target specific sequences in a genome you could introduce genes at safe places鈥�
According to Philip Gregory of Sangamo BioSciences in Richmond, California, zinc finger-based gene editing holds significant promise. 鈥淓fficient and specific gene editing will be important in advancing the post genomic era of medicine and zinc-finger based reagents will play a prominent role,鈥� he says.
Sangamo is testing the ability of a ZFN to disrupt the expression of a key protein that HIV uses as a door handle to enter cells. He adds that it is too early to say whether the recombinase fingers will prove as useful as ZFNs.
