Huntington's disease (HD) is a neurodegenerative disorder that follows an autosomal-dominant inheritance pattern. The pathogenesis of the disease depends on the degree of expansion of triplet (CAG) repeats located in the first exon on the gene. An expanded polyglutamine tract within the protein huntingtin (Htt) enables a gain-of-function phenotype that is often exhibited by a dysfunctional oligomerization process and the formation of protein aggregates. How this process leads to neurodegeneration remains undefined. We report that expression of a Htt-fragment containing an expanded glutamine tract induces DNA damage and activates the DNA damage response pathway. Both single-strand and double-strand breaks are observed as the mutant protein accumulates in the cell; these breaks precede the appearance of detectable protein aggregates containing mutant Htt. We also observe activation of H2AX, ATM, and p53 in cells expressing mutant Htt, a predictable response in cells containing chromosomal breakage. Expression of wild-type Htt does not affect the integrity of DNA, nor does it activate the same pathway. Furthermore, DNA damage and activated H2AX are present in HD transgenic mice before the formation of mutant Htt aggregates and HD pathogenesis. Taken together, our data suggest that the expression of mutant Htt causes an accumulation of DNA breaks that activates the DNA damage response pathway, a process that can disable cell function. Because these events can lead to apoptosis, it is possible that the DNA damage response pathway activated by single- and double-strand breaks that we found contributes to neurodegeneration.

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.

The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models. Gene Therapy (2005) 12, 639-646.