Transfer of drug resistance genes to the hematopoietic system has been advocated for myeloprotection during anti-cancer chemotherapy, however, for malignancies manifestated in blood or bone marrow compartments this strategy carries the risk of inadvertent transduction of tumor cells. Hematopoietic differentiation of induced pluripotent stem cells (iPSCs) has the potential to overcome this problem, and we here have investigated this concept in the context of Cytidine Deaminase (CDD)-mediated myeloprotection. However, epigenetic silencing of transgenic promoter/enhancer elements during differentiation is a major drawback when using genetically modified iPSCs. Therefore, we have transduced iPSCs with lentiviral constructs overexpressing CDD from different constitutive promoter/enhancer elements and have investigated the effects of the ubiquitous chromatin opening element (UCOE) on transgene stability and CDD-mediated drug resistance in hematopoietically differentiated and naïve iPSCs.
Materials/Methods: Murine iPSCs were transduced with 3rd-generation self-inactivating (SIN) lentiviral constructs overexpressing CDD and an IRES-coupled dTomato reporter from a truncated elongation factor 1α (EFS) or spleen focus-forming (SFFV) promoter/enhancer. Optionally, the UCOE site was cloned upstream of the respective promoter/enhancer. Transgene expression, Ara-C resistance and selection potential were investigated for naïve iPSCs and after differentiation along the hematopoietic lineage (d 0–8). Ara-C resistance was analyzed for colony-forming cells (d8-16). Expression of transgenic dTomato and CDD was measured by FACS, western blot and qRT-PCR. Bisulfite sequencing was performed to assess promoter methylation for the different lentiviral constructs.
Our studies demonstrated efficient transduction and stable EFS-driven CDD expression in undifferentiated iPSCs irrespective of the UCOE site. In contrast, SFFV-driven CDD expression was rapidly silenced. Although transgene expression levels were higher in UCOE.EFS.CDD- versus EFS.CDD-iPSCs (MFI: 89.5 vs 39.0), both, EFS.CDD- and UCOU.EFS.CDD-iPSCs were significantly protected against exposure to Ara-C (2000 nM/ 48 h) and were efficiently selected by continuous exposure to 2000 nM Ara-C (increase of dTomato+ cells from 5–8 % to 75–98 % within 16 days). No influence of CDD expression on iPSC morphology, growth characteristics, and expression of the pluripotency markers Oct4, Sox2, or Nanog was noted. Upon hematopoietic differentiation profound transgene silencing was observed in EFS.CDD-iPSCs. Silencing occurred during the first days of differentiation. Only 5–9% dTomato+ cells were observed on days 4 or 8, and reduced CDD expression levels were detected on days 4, 8, and 16 by Western blot and qRT-PCR analysis. Nevertheless, hematopoietic colony-forming units displayed significant resistance to Ara-C when compared to non-transduced controls. In contrast, UCOE.EFS.CDD-iPSCs only showed minor degrees of differentiation-induced transgene silencing with approx. 50% of the cells still expressing the dTomato transgene on day 8. Moreover, when subjected to clonogenic assays in the presence of 1000nM Ara-C, UCOE.EFS.CDD- in comparison to EFS.CDD-transduced cells exhibited significantly increased drug resistance (colony survival: 74±15 vs. 48±7%, p<0.05, n=3). In addition, bisulfite sequencing demonstrated significantly reduced CpG methylation in UCOE.EFS.CDD transduced cells upon hematopoietic differentiation.
Our data suggest that Ara-C resistance in the hematopoietic system can be achieved by hematopoietic differentiation of CDD-overexpressing iPSCs. While EFS and SFFV promoter/enhancer elements upon hematopoietic differentiation are prone to epigenetic silencing, this may be overcome by the use of a UCOE element, which stabilizes transgene expression during hematopoietic differentiation and significantly reduces CpG methylation in regulatory elements of the provirus. Thus, our UCOE.EFS.CDD vector allows for long-term transgene expression in hematopoietic progeny of iPSCs, and hematopoietic differentiation of gene modified, patient-derived iPSCs may be suitable to increase the safety of drug resistance gene therapy in malignant diseases with manifestation in the hematopoietic system.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.