A gene-editing process that corrects mutations without weaving foreign genetic material into the chromosome has been demonstrated in diseased human cells for the first time. It could provide a less risky and more efficient alternative to gene therapy, which has resulted in leukaemia in some patients.
A team led by scientists at Sangamo Biosciences in Richmond, California, US, say they have corrected the single gene mutation that causes the fatal X-chromosome-linked severe combined immune deficiency (X-SCID) – or “bubble boy” disease – in human T-cells. They treated the cells in test tubes with the company’s proprietary type of “zinc finger nucleases” (ZFNs) and have published their results in Nature.
ZFNs are proteins made up of “fingers” of around 30 amino acids and stabilised by a zinc atom. Each finger binds to a specific combination of DNA bases and is attached to a DNA-cutting enzyme called a nuclease.
By using different combinations of amino acids, they can be designed to latch on to DNA at exactly the place where the mutated gene lies and cut it. This triggers the body’s natural repair process, called homologous recombination, which corrects the gene where the DNA was cut, The researchers provided the cells with a copy of the correct gene as a template.
Cancer-suppression disrupted
Boys with X-SCID have a faulty gene on their X chromosome which renders their immune systems highly deficient. In 2000 Alain Fischer at the Necker Hospital in Paris, France, treated 10 boys with X-SCID using a mouse retrovirus to add a healthy copy of the gene.
Although the treatment worked, at least two of the boys developed leukaemia as a result and one died. This was because the position of insertion of the new gene cannot be controlled and in these cases ended up being inserted near another gene called Lmo2. This helps control cell growth and can contribute to cancer if turned on at the wrong time.
In contrast, ZFNs are highly specific. “ZFN-induced gene targeting places the normal gene at its normal chromosomal location, where it should have no untoward genetic consequences,” explains Dana Carroll, a biochemist at the University of Utah in Salt Lake City, US, who has used ZFN to correct genes in fruit flies.
But Carroll warns that side effects cannot be ruled out: “There is still the possibility that some alternative reactions may occur.” For example, some cancers are thought to be caused by chromosomes that are broken and then stuck back together incorrectly.
Therapeutically viable
Matthew Porteus at the University of Texas Southwestern Medical Center in Dallas, US, an author of the Nature paper, established in 2003 that ZFNs can stimulate homologous recombination in human cells. But he corrected “model” genes, rather than disease-causing ones. Furthermore, both Porteus and Carroll only managed to correct a few per cent of treated cells.
In the latest work, the gene was corrected in 18% of the cells treated, enough to finally make the method therapeutically viable. “The Sangamo group has achieved truly remarkable efficiencies,” says Carroll.
The researchers say the advance was achieved thanks to the specificity of a pair of two-fingered ZFNs, which bind to six base pairs each, to home in precisely on the target. “They used more complex ZFN combinations than had previously been used,” says Carroll. “This is a very satisfying demonstration of the power of the basic ZFN technology.”
Sangamo’s primary aim is to take blood from patients, correct the genetic errors and then infuse it back into them. As well as X-SCID, it says it will be targeting other genetic diseases caused by single gene mutations, including sickle cell anaemia and beta thalassemia. And it suggests that immune cells could perhaps be altered to prevent infection with HIV.
Journal reference: Nature (DOI: 10.1038/nature03556)
