c-MycERTAM transgene silencing in a genetically modified human neural stem cell line implanted into MCAo rodent brain.
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.
Stevanato
Lara
L
ReNeuron Limited, Surrey Research Park, 10 Nugent Road, Guildford, Surrey, GU2 7AF, UK. lara-stevanato@reneuron.com
Corteling
Randolph L
RL
Stroemer
Paul
P
Hope
Andrew
A
Heward
Julie
J
Miljan
Erik A
EA
Sinden
John D
JD
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