title: c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.
abstract: BACKGROUND
  The human neural stem cell line CTX0E03 was developed for the cell based treatment of chronic stroke disability. Derived from fetal cortical brain tissue, CTX0E03 is a clonal cell line that contains a single copy of the c-mycERTAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycERTAM enabled large-scale stable banking of the CTX0E03 cells. In this study, we investigated the fate of this transgene following growth arrest (EGF, bFGF and 4-OHT withdrawal) in vitro and following intracerebral implantation into a mid-cerebral artery occluded (MCAo) rat brain. In vitro, 4-weeks after removing growth factors and 4-OHT from the culture medium, c-mycERTAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycERTAM protein. To examine the transcription of the transgene in vivo, CTX0E03 cells (450,000) were implanted 4-weeks post MCAo lesion and analysed for human cell survival and c-mycERTAM transcription by qPCR and qRT-PCR, respectively.
  
  
  RESULTS
  The results show that CTX0E03 cells were present in all grafted animal brains ranging from 6.3% to 39.8% of the total cells injected. Prior to implantation, the CTX0E03 cell suspension contained 215.7 (SEM = 13.2) copies of the c-mycERTAM transcript per cell. After implantation the c-mycERTAM transcript copy number per CTX0E03 cell had reduced to 6.9 (SEM = 3.4) at 1-week and 7.7 (SEM = 2.5) at 4-weeks. Bisulfite genomic DNA sequencing of the in vivo samples confirmed c-mycERTAM silencing occurred through methylation of the transgene promoter sequence.
  
  
  CONCLUSIONS
  In conclusion the results confirm that CTX0E03 cells downregulated c-mycERTAM transgene expression both in vitro following EGF, bFGF and 4-OHT withdrawal and in vivo following implantation in MCAo rat brain. The silencing of the c-mycERTAM transgene in vivo provides an additional safety feature of CTX0E03 cells for potential clinical application.
authors.affiliation: ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com
authors.forename: Lara
authors.initials: L
authors.lastname: Stevanato
authors.forename: Randolph L
authors.initials: RL
authors.lastname: Corteling
authors.forename: Paul
authors.initials: P
authors.lastname: Stroemer
authors.forename: Andrew
authors.initials: A
authors.lastname: Hope
authors.forename: Julie
authors.initials: J
authors.lastname: Heward
authors.forename: Erik A
authors.initials: EA
authors.lastname: Miljan
authors.forename: John D
authors.initials: JD
authors.lastname: Sinden
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000098:blood supply / Q000637:transplantation*; D053686:Fetal Stem Cells / Q000166:cytology / Q000637:transplantation*; D020868:Gene Silencing; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics*; D008297:Male; D009474:Neurons / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D019076:Transgenes; D014183:Transplantation, Heterologous