Science Editor

There's a lot of false leads, and a lot of heartbreak, in and behind the search for any cure. But X-linked severe combined immunodeficiency disorder, or X-SCID, is especially heartbreaking - both because of the disease itself, which leaves the "bubble boys" born without an immune system and vulnerable to death from any sort of infectious disease, no matter how mild, and because of the cruel twists of fate in the attempts to treat it.

In 2000, the successful treatment of babies suffering from X-SCID by gene therapy was hailed as gene therapy's first triumph. However, for some of those children the cure turned out to be as bad as the disease: To date, three of 14 patients have developed leukemia after the therapy. One of the children died. While not scrapping gene therapy trials altogether, the FDA has recommended that only patients for whom conventional therapy has failed be treated with gene therapy. (See BioWorld Today, May 1, 2000; Jan. 16, 2003; and March 7, 2005.)

The reasons for some of the children developing leukemia are not fully understood, but one apparent problem is that the corrective gene, which is randomly inserted into the host genome, was inserted in or near an oncogene.

In an advance online publication of Nature, scientists from Richmond, Calif.- based Sangamo BioSciences Inc. and the University of Texas Southwestern Medical Center reported on an alternative method for gene correction.

The paper is titled "Highly efficient endogenous human gene correction using designed zinc finger nucleases." Though the method is not yet ready for clinical use, in principle it avoids random insertion of genes into the genome, as well as the use of exogenous promoters, which are another potential headache for classical gene therapy.

"The critical distinction [between gene therapy and the method described in the paper] is that this is a way of actually repairing a gene, leaving nothing behind and having it still driven by its own promoter, with all of the normal feedback of normal gene regulation," Edward Lanphier, president and CEO of Sangamo, told BioWorld Today. "When you look at the cases that have received a lot of attention recently, such as the French cases, it's really the random integration of the exogenous promoter that has caused problems."

Gene Correction: First Break It, Then Fix It

In the paper, the scientists combined DNA binding proteins known as zinc finger proteins to target specific DNA sequences slated for repair. Those zinc finger proteins are attached to an enzyme that induces double-stranded DNA breaks.

If only one DNA strand is broken, it will be repaired using the complementary strand as a template. But double-stranded DNA breaks spur the cells' repair machinery to search the cell for homologous DNA to use as a template; in that case, that DNA comes from a plasmid bearing a functional copy of the gene. The undesirable possibility is that the repair is made using mutated DNA on the cell's own homologous chromosome or sister chromatid as a template, but Lanphier said that "since there is an excess of plasmid DNA present, mass action makes it more likely that the plasmid DNA will be used as a template." The plasmid itself ultimately is degraded by the cell's cleanup mechanisms.

In the studies reported in Nature, the scientists engineered zinc finger proteins to bind to sequences near an X-SCID-causing mutation in the IL2Rgamma gene. When K562 cells were treated with both the zinc finger nucleases and the corrected gene on a plasmid, nearly 20 percent of them acquired the corrected gene on at least one allele; 7 percent had the corrected version on both alleles. The correction was present a month after treatment. Primary CD4 cells showed the same changes, albeit at lower transduction levels than the K562 cell line. The scientists also showed that the corrected genes led to the production of functional gammaC mRNA and protein, which are produced by the IL2Rgamma gene.

Gene Disruption: If It Ain't Broke, Break It

The technology has two applications. As demonstrated in the Nature paper, it can be used for gene correction, in which a functional template is used to repair a gene.

But it also can be used to disrupt genes that contain workings detrimental to the organism. In fact, Sangamo's most advanced program applying the technology is in gene disruption, where the company is working on creating helper T cells with a disrupted CCR5 receptor. Individuals with that disruption have been shown to be resistant to HIV infection, and the company hopes such T cells would provide HIV patients with a reservoir of uninfectable T cells that would allow their immune system to continue functioning. Sangamo plans to enter the clinic with T cells bearing the disrupted CCR5 gene in 2006.