Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Abstract

Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Gö 6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations. Gene Therapy (2005) Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, 3-6 triple helixforming oligonucleotides containing a reactive group, 7-9 bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown. 6 The central problem in studying this phenomenon are low rates of in vivo sequence correction rates in cell culture. Generally, observed sequence alteration rates on in vivo chromosomal templates in selected cell lines are in the range of 0.1%, 6 while correction rates in untransformed primary cells are substantially lower. [13][14][15] In the absence of a suitable selection system, such low rates allow only limited functional analysis, although Yoon and co-workers have recently suggested a selection system based on simultaneous targeting of two loci. 16 A further problem is the variety of model systems and assays that were used to measure targeted sequence correction which lead to confusing and often conflicting results. 6 Despite these obstacles, several consistent observations have been made: targeted sequence correction depends centrally on transcription, and ssODN that are complementary to the nontranscribed strand show in most cases substantially higher correction rates than ssODN complementary to the transcribed strand in both episomal and chromosomal assays. 17,18 The involvement of transcription-coupled repair has therefore been suggested. Furthermore, the absence of MSH2, ...

Progress and prospects: oligonucleotide-directed gene modification in mouse embryonic stem cells: a route to therapeutic application

“…19 Over the past few years, it has become evident that DNA replication is the predominant cellular process underlying ssODN-mediated gene targeting in bacteria, [20][21][22] yeast 23,24 and various mammalian cell lines including ESCs. 16,25,26 According to this model, the ssODN anneals to its chromosomal complement when this becomes exposed as singlestranded DNA within the context of a replication fork. Upon annealing, the ssODN can serve as a primer for DNA synthesis to form a 'pseudo' Okazaki fragment, resulting in physical incorporation of the ssODN into the nascent DNA strand.…”

“…Increasing the number of cells that progressed through S phase or slowing down replication fork progression during ssODN exposure resulted in elevated targeting frequencies in various mammalian cell lines. [25][26][27] The discontinuous nature of lagging strand DNA synthesis may expose more ssDNA regions and thus facilitate ssODN annealing. Indeed, studies in E. coli demonstrated that ssODNs corresponding in sequence to the lagging strand consistently performed better, irrespective of the direction of transcription, thereby establishing a direct link between ssODN polarity and the direction of replication through the target locus.…”

“…However, besides improving the bioavailability of ssODNs, chemical modifications also have deleterious side effects. Olsen et al 25 demonstrated that the majority of cells that were initially corrected by PTO-modified ssODNs arrested in the G 2 phase of the cell cycle and were rapidly diluted by the proliferating uncorrected cells in the population. This G 2 arrest was due to the presence of unrepaired genomic double-stranded DNA breaks, leading to the activation of the ATM/ATR pathway and phosphorylation of histone H2AX.…”

Effective oligonucleotide-mediated gene disruption in ES cells lacking the mismatch repair protein MSH3

“…3,9,10 Also in mammalian cells, a modestly increased efficiency of lagging strand over leading strand oligonucleotides was observed and the efficacy of oligonucleotide-mediated gene modification was dependent on the phase of the cell cycle, the highest frequencies being found when oligonucleotides were delivered to cells immediately after release of a G 1 /S block. 11,12 Furthermore, the efficacy could be improved by slowing down progression of replication forks again suggesting that also in mammalian cells at least one mechanism of oligonucleotide-mediated gene modification involves replication of DNA. 13,14 We have previously identified another parameter affecting the frequency of oligonucleotide-mediated gene modification: DNA mismatch repair (MMR).…”

Site-specific gene modification by oligodeoxynucleotides in mouse bone marrow-derived mesenchymal stem cells

“…34,35 It is interesting to note that not all corrected cells were able to survive and in fact, the only one-third of EGFP + sorted cell produced viable clones. Recently, Olsen et al 49 found that the majority of corrected CHO-K1 cells were arrested at G 2 /M stage and only 2% of the initially EGFPcorrected cells made viable colonies. The authors concluded that targeted sequence alteration by ODN by itself is somehow arresting mitosis of the corrected cells, suggesting difficulties in expansion of corrected cells.…”