Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair.
BACKGROUND
Single-stranded oligonucleotides (ssODN) are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time.


RESULTS
First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours.


CONCLUSIONS
Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes.
Engstrom
Julia U
JU
Department of Biological Sciences, University of Delaware, Delaware Biotechnology Institute, 15 Innovation Way, Newark, DE 19711, USA. juliaeng@udel.edu <juliaeng@udel.edu>
Kmiec
Eric B
EB
D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics*; D004261:DNA Replication / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D009841:Oligonucleotides / Q000627:therapeutic use*; D013936:Thymidine / Q000494:pharmacology; D013997:Time Factors
D009841:Oligonucleotides; D013936:Thymidine