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, ...

We show that, on a chromosomal level, the orientation of the replication fork towards the targeted locus is not central in the strand bias of ssODN-based targeted sequence correction. We demonstrate the importance of accessible ssODN ends for sequence alteration. Finally, we provide evidence for the involvement of DNA synthesis in the process.

Overactivation of the WNT/β-CATENIN signaling axis is a common denominator in colorectal cancer. Currently, there is no available WNT inhibitor in clinical practice. Although TANKYRASE (TNKS) inhibitors have been proposed as promising candidates, there are many colorectal cancer models that do not respond positively to TNKS inhibition and Therefore, a combinatorial therapeutic approach combining a TNKS inhibitor (G007-LK) with PI3K (BKM120) and EGFR (erlotinib) inhibitors in colorectal cancer was investigated. The data demonstrate that TNKS inhibition enhances the effect of PI3K and EGFR inhibition in the TNKS inhibitor-sensitive COLO320DM, and in the nonsensitive HCT-15 cell line. In both cell lines, combined TNKS/PI3K/EGFR inhibition is more effective at reducing growth than a dual TNKS/MEK inhibition. TNKS/PI3K/EGFR inhibition affected in a context-dependent manner components of the WNT/β-CATENIN, AKT/mTOR, EGFR, and RAS signaling pathways. TNKS/PI3K/EGFR inhibition also efficiently reduced growth of both COLO320DM and HCT-15 tumor xenografts At the highest doses, tumor xenograft growth was halted without affecting the body weight of the tested animals. Combining TNKS inhibitors with PI3K and EGFR inhibition may expand the therapeutic arsenal against colorectal cancers. .

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