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-mycER TAM transgene delivered by retroviral infection. Under the conditional regulation by 4-hydroxytamoxifen (4-OHT), c-mycER TAM 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-mycER TAM transgene transcription is reduced by ~75%. Furthermore, immunocytochemistry and western blotting demonstrated a concurrent decrease in the c-MycER TAM 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-mycER TAM transcription by qPCR and qRT-PCR, respectively.

Neural Stem Cell-based Gene Therapy for Brain Tumors

“…In a recent study in immortal human NSC line, silencing of c-myc expression in vivo was demonstrated and this finding is consistent with the absence of tumorigenesis [125]. In the samples prepared from grafts of human NSCs (containing a single copy of the c-myc transgene delivered by retroviral infection) in ischemia rodent brain, 62 to 100% of the transgene sites were methylated [125]. Direct modification of the DNA by methylation is an epigenetic mechanism to downregulate or silence gene expression.…”

“…Although tumor formation has never been observed with human NSCs in our numerous pre-clinical animal studies, it is important in determining the inherent risk of using immortal human NSCs for clinical applications. In a recent study in immortal human NSC line, silencing of c-myc expression in vivo was demonstrated and this finding is consistent with the absence of tumorigenesis [125]. In the samples prepared from grafts of human NSCs (containing a single copy of the c-myc transgene delivered by retroviral infection) in ischemia rodent brain, 62 to 100% of the transgene sites were methylated [125].…”

Fluorescence-based alternative splicing reporters for the study of epithelial plasticity in vivo

“…The fact that pRint animals also display variable expression of DsRed suggests that transcription of this open reading frame may be differentially silenced within a single cell type. Along these lines, others have observed downregulation of the CMV promoter in vivo (Brooks et al 2004;Stevanato et al 2009); however, these effects are often observed during nontargeted insertions of transgenes. To mitigate any potential silencing of the CMV promoter, we chose to insert the fluorescence reporters into the ROSA26 locus, which is not susceptible to transcriptional shutdown due to chromatin remodeling (Chen et al 2011).…”

Stem cells in stroke management

“…104 Once removed from culture with 4-OH tamoxifen, cells differentiate and animal studies indicate no reactivation of the gene after cell transplantation. 105 In rodent focal ischaemia models, CTX0E03 cells implanted in the striatum differentiate into neuronal tissue, 22 and improve behavioural deficits in a dose-dependent manner when administered 4 weeks after the stroke. An ongoing clinical trial is designed as a safety study that will involve ascending doses being implanted into the putamen in neurologically stable patients 6-24 months after an ischaemic stroke involving the basal ganglia (www.clinicaltrials.gov).…”