Nitric Oxide Signaling

Supplementary MaterialsDocument S1

Supplementary MaterialsDocument S1. generation of a modified hESC line harboring two suicide gene cassettes, whose expression results in cell death in the presence of specific pro-drugs. We show the efficacy of this system at enriching for cells and eliminating tumorigenic ones both and sites, is eliminated upon expression of Cre by the human insulin promoter (Kuhn and Torres, 2002). Therefore, insulin-expressing cells are rendered insensitive to CB1954. CNX-1351 HSV-TK is driven by the telomerase promoter, which is active only in undifferentiated cell types (Albanell et?al., 1999). This makes proliferating cells sensitive to GCV. Thus, our method provides a double fail-safe control such that (1) only insulin+, non-proliferating cells survive selection; (2) cells that may de-differentiate after transplantation (Fujikawa et?al., 2005) (and in which NTR was lost with the onset of insulin expression) may still be selectively killed by GCV, leaving the rest of the graft intact; and (3) undifferentiated cells are sensitive to two pro-drugs, making it less likely for tumorigenic cells to survive in case one single drug was insufficient to destroy 100% of them, or if they became resistant to one pro-drug due to spontaneous mutations of the relevant suicide gene (Kotini et?al., 2016). No other method reported thus far offers the same degree of safety and specificity, as conventional suicide gene-based strategies bring about the destruction of the entire graft or do not enrich for the cells of therapeutic interest. Our results offer proof-of-principle of this approach and open the door to the subsequent targeting of these constructs to specific safe harbor locations within the genome of clinical-grade hESCs. Results Suicide Cassette Construction DNA was synthesized by GenScript (Piscataway, NJ). Owing to the size of CNX-1351 both suicide cassettes, we generated two constructs that could be independently transfected. Figure?1A shows the composition of constructs A (sites flanking a region that is excised by Cre (Nagy, 2000). (2) Nitroreductase (NTR, T41L/N71S mutant). NTR is a flavoenzyme homodimer with flavin mononucleotide (FMN) cofactors, encoded by the gene (Searle et?al., 2004). CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] is reduced by the FMN to a 4-hydroxylamino derivative, which becomes a cytotoxic DNA crosslinking agent (Grove et?al., 1999). Since virus-mediated expression of NTR in tumor cells sensitizes them to CB1954, this strategy has been tested clinically for several types of cancer (Searle et?al., 2004, Williams et?al., 2015). The double mutant T41L/N71S sensitizes cells ERCC3 to CB1954 concentrations up to 15-fold lower than the native enzyme (Jaberipour et?al., 2010). In our construct, the T41L/N71S NTR gene is driven by the CMV promoter. This plasmid is selectable in neomycin/G418. Upon Cre expression, both NTR and neomycin resistance cassettes are eliminated (Figure?1A). Open in a separate window Figure?1 Genetically Modified Cells Are Sensitive to the Pro-drugs Ganciclovir (GCV) and CB1954 (A) The structure of construct A comprises: (1) a constitutive cytomegalovirus promoter-enhancer hybrid (CMV)-driven codon optimized (co) nitroreductase gene (NTR); (2) a neomycin resistance gene (NeoR); and (3) sites flanking the above two cassettes in their entirety. Construct B consists of: (1) a human telomerase reverse transcriptase promoter (hTERT)-driven codon optimized (co) herpes simplex virus thymidine kinase S39 mutant gene (HSV-TK/s39); (2) a human insulin promoter (hIP)-driven codon optimized Cre-recombinase gene (Cre); and (3) a puromycin resistance cassette. When the insulin promoter is active, Cre recombinase is produced, and the main elements of construct A (including the NTR cassette) are excised out. As shown in CNX-1351 the table, insulin+ cells (INS+) resulting from the differentiation of cells are therefore resistant (R) to GCV (since hTERT is not expressed in differentiated cells) and CB1954 (owing to the Cre-mediated excision). In contrast, HSV-TK/s39 and NTR are expressed in undifferentiated cells, which makes them sensitive (S) to both GCV and CB1954. Finally, cells differentiated into non-insulin+ cells are resistant to GCV but sensitive to CB1954, since the NTR cassette remains intact. (B and C) (B) Expression of NTR and HSV-TK genes in modified hESCs as determined by qRT-PCR versus -actin/18S. Asterisks denote statistical significance: ??p? 0.01, ???p? 0.001. (C) Photomicrographs of modified hESCs (and H1 hESCs express the pluripotency markers OCT4 (green), SSEA4 (red), SOX2 (green), TRA-1-60R (red), SSEA4 (green), and NANOG (red). DAPI (blue) is used as nuclear counterstaining. Insets show.