Genetic mutations are the root cause of many neurodegenerative disorders, including familial Alzheimer and Parkinson diseases. Because most of these mutations result in a gain of function, traditional gene therapy, where a good copy of the gene is introduced into the affected tissue, is of little benefit. What would be beneficial would be to remove or correct the damaged DNA. While this might sound like a pipe dream, in the April 3 edition of Nature online, researchers from Sangamo BioSciences, Inc., Richmond, California, described a novel approach that allowed them to correct mutations in T cells that cause X-linked severe combined immune deficiency (SCID) in humans. Their results raise the possibility that the technique, a modified version of homologous recombination, could be used to treat human diseases, including those affecting neurons.

In SCID, mutations in the interleukin 2 receptorγ (IL2Rγ) gene prevent normal T cell function. To correct the mutations, principal author Michael Holmes and colleagues designed a nuclease fused to a zinc-finger DNA binding protein to introduce a double strand break precisely at the spot to be repaired. T cells then repair that break by homologous recombination using exogenously supplied DNA carrying the correct IL2Rγ sequence. Previous work in model systems has shown that this kind of break-stimulated repair occurs at much higher efficiency than does the traditional method of homologous recombination used to generate “knock-in” or “knockout” mice, and does not require drug selection of modified cells, both advantages for therapeutic applications (Porteus and Baltimore, 2003; Bibicova et al., 2003).

To maximize the efficiency of repair, Holmes and colleagues generated two optimized zinc-finger-nuclease fusions by selecting the best DNA binders from a library of zinc-finger motifs, and then introduced additional amino acid changes to further enhance binding and recognition of the IL2Rγ gene sequence. Using these optimized zinc-finger nucleases, they measured the efficiency of gene conversion by introducing a silent mutation into the IL2Rγ gene in K562 human leukemia cells. Alteration of one gene copy occurred in a surprising 18 percent of cells, with 7 percent showing the change on both chromosomes. The gene conversion was stable after one month in culture, and so appeared permanent. The researchers then went on to introduce a SCID mutation into the same IL2Rγ gene, and showed they lost expression of the receptor. When they used the optimized zinc-finger nuclease and donor DNA to reverse the mutation, the repaired cells began to a make the correct mRNA and protein from the gene once again. The technique was useful not only for transformed cells in culture: Human primary T cells also showed gene conversion in 5 percent of the cells.

Coauthor Matthew Porteus of the University of Texas Southwestern Medical Center in Dallas says the technique should be applicable to any cell type, even non-dividing cells such as neurons and glia. “We haven’t done experiments to prove that, but if I had to take a guess, I’d say it would work. The recombination machinery is universal, and some of our experiments were done in cells that were arrested.” But the most powerful use of the recombination technique would be to apply it to human stem cells, according to Porteus. “The approach is to think about first trying to correct neuronal stem cells and then using those cells to treat disease.”—Pat McCaffrey


  1. I think that the general approach is a real tour de force, and could usher in a potential wave of corrective gene therapy, particularly for developmental disorders that can be identified in utero, or for disorders that affect the immune system, where immune cells could be removed, corrected, and reinserted. The relevance to neurodegenerative disease, though, is less clear for several reasons. One problem is that the efficiency of correction is still too low to correct a majority of neurons, although at 7 percent, it is orders of magnitude higher than conventional repair methods. Another problem for neurodegenerative disease is delivery. Even if the efficiency of correction were improved, the efficacy of viral delivery remains low and spatially restricted. Thus, the method faces many challenging hurdles before it could be applied to human neurodegenerative disease.

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Paper Citations

  1. . Chimeric nucleases stimulate gene targeting in human cells. Science. 2003 May 2;300(5620):763. PubMed.
  2. . Enhancing gene targeting with designed zinc finger nucleases. Science. 2003 May 2;300(5620):764. PubMed.

Further Reading

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Primary Papers

  1. . Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005 Jun 2;435(7042):646-51. PubMed.