Science in Society Archive

Safe Gene Therapy At Last?

Gene defect corrected without inserting foreign DNA. Dr. Mae-Wan Ho investigates

A research team in a company in Richmond, California, claims to have corrected the gene mutation associated with the fatal X-linked severe combined immune deficiency (X-SCID) in human cells [1] without insertion foreign DNA into their genomes, and published their results online in the journal Nature 2 June [2]. This raises hope of a safer form of gene therapy after three infants in Paris with X-SCID, who received gene therapy through their own bone marrow cells - isolated, genetically modified in the laboratory and injected back into the patient - came down with leukaemia (“Gene therapy woes”, SiS 26).

In the latest experiments, the human cells were treated with the company’s patented “zinc-finger nucleases” (ZFNs). ZFNs are proteins made up of “fingers” of about 30 amino acids, stabilized by a zinc atom. Each finger binds to a specific combination of DNA bases and is attached to nuclease, a DNA cutting enzyme. By using different combinations of amino acids, they can be designed to bind to DNA at the exact site where the gene is mutated to cut it out. This triggers the cell’s repair mechanism, which corrects the gene using a copy of the correct gene sequence provided in a plasmid, in a process of homologous recombination, in which the replacement depends on similarity in DNA sequence between the replacement and the resident copy of the gene.

Infants with X-SCID have a mutated gene on their X-chromosome that makes their immune system unable to function. More than 10 infants in the Necker Hospital in Paris, France had been treated with conventional gene replacement therapy since 2000 using a retrovirus as the vector (gene carrier) to insert the correct gene sequence into their bone-marrow cells. But the retroviral vector carrying the correct gene sequence cannot be targeted, so it ends up inserting in wrong places in the genome. To-date, three infants have developed leukaemia because the retroviral vector inserted near an oncogene (cancer-related gene), causing it to over-express, and the cell to multiple out of control. One of the infants has died earlier this year.

The ZFNs are highly specific. Each finger recognizes 3-4 base pairs of DNA via a single alpha-helix formed by the finger, and several fingers can be linked in tandem to recognize a broad spectrum of DNA sequences with high specificity. Earlier work from another laboratory [3] has shown that a zinc finger can be linked to a non-specific DNA- cutting domain of a DNA-cutting enzyme to produce the ZFN, which then cuts specifically at the zinc finger recognition site. An important feature is that two ZFNs bind to the same gene, in a precise orientation and spacing relative to each other, to create a double-strand break in the DNA, which then triggers the repair mechanism.

Mathew Proteus at the University of Texas Southwestern Medical Center, Dallas, Texas, a co-author of the Nature paper, had earlier used the technique to correct a marker gene in human cells. But he only managed to correct a few percent of the cells.

In the latest paper, they succeeded in modifying 18 percent of the cells without the need to select for them with selectable markers such as antibiotic resistance or fluorescent proteins. The advance was due to a more elaborate combination of zinc fingers than used previously, which are optimised for binding and cutting. A pair of four-fingered ZFNs, each binding to 12 base pairs (24 in all), home in precisely on the target between the pair of ZFNs, a mutation hotspot in the X-SCID gene, and replacing it with the correct copy.

In one experiment, they isolated single clones of cells after giving them the ZFNs and the correct copy of the gene, and found that 13.2% of the clones had converted one of the two X-chromosomes, while 6.6% had both X chromosomes corrected.

The researchers did other experiments confirming the findings, and demonstrated that corresponding changes occurred in levels of mRNA and protein expressed from the corrected gene.

The corrected gene sequence appeared to be stable for at least one month afterwards, and analysis showed there was no gross mis-integration of extra DNA or rearrangement or scrambling at the site of correction.

The company’s aim is to take blood from patients, correct the genetic defect in the blood cells and then infuse the cells back into the patients. Besides X-SCID, other ‘single gene’ diseases such as sickle cell anaemia or beta-thalassemia can also be treated, and perhaps immune cells could also be altered to prevent infection with HIV.

Dana Carroll, a biochemist at University of Utah, Salt Lake City, who has used ZFN to correct genes in fruit flies, said, ”FN-induced gene targeting places the normal gene at its normal chromosomal location, where it should have no untoward genetic consequences.” But he warned that side-effects cannot be excluded.

Is it safer?

The results look quite impressive, and as pointed out in the Nature article, “the ‘hit and run’ mechanism of ZFN action uncouples the therapeutically beneficial changes made to the genome from any need to integrate exogenous DNA, while still generating a permanently modified cell.”

This new technique thus avoids all the hazards associated with the viral vector and foreign gene constructs with aggressive promoter to force the cells to express the foreign gene, and also appears to be specific: the PCRs and Southern blots (which probe for the corrected gene sequences) all look quite clean. Further tests that could have been performed are genomic and expressed sequence microarrays, and protein gels, to see if other genes have also been corrected and/or changes in the pattern of RNA and protein expressed have occurred. It was microarray analysis that first alerted the gene therapy community to the problems of the ‘precision’ gene therapy of RNA interference hailed as 2002’s “breakthrough of the year” (“Controversy over gene therapy ‘breakthrough’”, SiS 26); although microarray analyses themselves are of questionable reliability (“Biotech wonder tool in disarray”, SiS 26).

It would also be important to show that the corrected protein does not cause side effects, such as immune rejection in patients whose bodies may treat the protein as ‘foreign’ [4].

Article first published 29/07/05


  1. “‘Gene-editing’ technique cuts out diseased DNA” Celeste Biever, 4 April 2005,
  2. Urnov FD, Miller JC, Lee Y-L, Beausejour CM, Rock JM, Augustus S, Jamieson AC, Porteus MH, Gregory PH and Holmes MC. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 2005, 435, 646-50. (Published online 3 April 2005).
  3. Kim YG, Cha J and Chandrasegaran S. Hybrid restriction enzyme: zinc finger fusions to Fok 1 cleavage domain. Proc Natl Acad Sci USA 1995, 93, 1156-60.
  4. Ho MW. Living with the Fluid Genome, pp. 136-7, TWN, Penang and ISIS, London, 2003.

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