A major goal of molecular medicine is to replace or repair a dysfunctional gene in the context of its natural position in the chromosome. A number of avenues of investigation are being taken including those that utilize short oligonucleotides to direct the single base changes. Triplex-helix forming oligonucleotides conjugated to reactive chemical groups and those constructed as bifunctional oligonucleotides continue to show promise in model systems [1-4]. Most of the effort however, centers on the use of modified or unmodified single-stranded DNA oligonucleotides (ssODNs) ranging in length from 25 to 75 bases. The original concept for using ssODNs came from studies in yeast in the 1980s that were pioneered by Sherman and colleagues [see [5]] and adapted to episomal targets in mammalian cells by Campbell et al. 1989. Gamper et al. [6,7] and Liu et al. [8] then re-established single-stranded vectors as potential agents for the alteration of chromosomal sequences in eukaryotes.
The mechanism by which oligonucleotides-directed gene repair takes place is still being elucidated. One lab has proposed that transcription-coupled repair alone can account for base exchange or correction [9]. But, the majority of workers believe that DNA replication and DNA repair activities comprise the major pathway of gene repair. This concept was originally proposed by Brachman and Kmiec [10], subsequently extended by Parekh-Olmedo et al. [11], and now has been supported by data generated by numerous laboratories [12-17]. This model suggests that actively replicating genomes are more amenable to the hybridization of the ssODNs at the target site because the chromatin is in a more open configuration and the target sequence more accessible. The use of agents such as hydroxyurea [18], ddC [19], or thymidine [14,20,21] that retard the process of DNA replication enable higher levels of gene repair activity by expanding the window of time within which the ssODN can locate its target site.
The role of replication in the gene repair reaction, however, has recently been met with a paradoxical series of events. The initialization of the gene repair reaction results in a plethora of problems for the cell and its metabolism because an ATM-dependent DNA damage response pathway is activated by the transfer of the ssODN [22], confirming earlier observations by Liu [see [23]]. Activated ATM signals the induction of the checkpoint regulator proteins Chk1 and Chk2, which in turn direct cell cycle arrest predominantly in corrected cells. This result provided an explanation for the important observations of Olsen et al. [12,13] that the percentage of corrected cells in a population of treated cells decreases as a function of time, presumably because the uncorrected population continues to proliferate while the corrected cells are arrested. If this phenomenon is applicable to primary cells, then the use of gene repair for treating inherited diseases will suffer a severe setback. The goal of this work was to devise an experimental protocol to avert the dilution of corrected cells in a population of treated cells.
As mentioned, the addition of thymidine to cells being targeted for gene repair enhances the frequency with which single base exchange takes place [14,18,21] by synchronizing cells at the G1/S border (referred to as a thymidine block) [24] followed by their release into S phase en masse, which provides an increased number of accessible targets for repair. In addition to the increase level of gene repair observed after a thymidine treatment, we show that the agent is able to stabilize the correction frequency over several days, perhaps by inhibiting the replication of the entire cell population and preventing a dilution effect on the corrected cells. We also reveal a novel observation that long-term exposure to thymidine results in the reversion of DLD-1 cells into cellular senescence and that this can be avoided by the removal of the drug prior to this point. Based on this work and earlier observations, thymidine is an attractive reagent for downstream clinical applications due to its ability to elevate and maintain substantial levels of gene repair without inducing severe toxicity in vitro.
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