Team:Freiburg/Demonstrate
Proof of Concept
The CARTELTM AND gate is a genetic circuit which we have designed to be integrated in T cells, for a controlled expression of one output, the chimeric antigen receptor, in response to two inputs that are only found in a tumor microenvironment. The AND gate is constructed of two promoters interconnected through a protein, (HIF1A), to integrate two inputs into one output. Two alternative AND Gates were designed as follows: high VEGF concentrations and hypoxic conditions or alternatively low pH and hypoxic conditions.
As a proof of concept we characterized all the promoters separately in mammalian cell lines.
We therefore generated stable cell lines, containing the promoters for the inputs driving a reporter protein. Single and multiple enhancer elements for the inputs: pH, VEGF and hypoxia were designed and integrated in Jurkat and HEK293T cells.
We could show an induced activity of the hypoxia response element promoter and the cAMP response element promoter in Jurkat cells (Fig. 1). Flow cytometry of Jurkat 4xHRE-pTal:eCFP cells showed an increase of eCFP positive cells with rising concentration of CoCl2 (Fig. 1a). In addition, we could show an induced activity of the cAMP response element promoter in Jurkat 4xCRE-pTal:eCFP cells treated with forskolin and IBMX (Fig. 1b). With this results, we can demonstrate that our constructs 4xHRE-pTal:eCFP and 4xCRE-pTal:eCFP are functional and responsive to hypoxic conditions and low pH, respectively.
Figure 1: Detection of hypoxia response element and cAMP response element promoter activity by flow cytometry. a) Jurkat cells stably expressing 4xHRE-pTal:eCFP were incubated 24 h with indicated concentrations of CoCl2 to mimic hypoxic conditions. A significant increase in eCFP positive cells was observed for 80 µM CoCl2. b) Jurkat cells stably expressing 4xCRE-pTal:eCFP were incubated 24 h in pH adjusted RPMI 1640 and induced with forskolin (10 µM) and IBMX (10 µM). The highest amount of eCFP positive cells was observed for pH 6.5. Data points are mean values of triplicates, error bars represent the standard deviation. Significant differences were determined by using one-tailed student’s t-test (Excel 2017) followed by Bonferroni-Holm correction (a) and by using ANOVA (b); * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant and decreasing differences are not marked.
The CARTELTM AND gate requires the knockdown or knockout of the endogenous gene coding for hypoxia-inducible factor 1 alpha (HIF1A) to ensure exclusive control over HIF1A by the introduced AND gate. We generated stable Jurkat and HEK293T cell lines in which HIF1A was efficiently knocked down. In addition, we could show that transient expression of HA tagged HIF1A in knockdown cells behaves as the endogenous HIF1A, without being affected by the siRNA used to generate the knockdown (Fig. 2).
Figure 2: Transient introduction of artificial HIF1A in HIF1A shRNA1 knockdown HEK293T cell line.
Induction of the overexpressed HIF1A-HA can be observed in the last lane. Endogenous and artificial HIF1A were detected with HIF1A antibody; reintroduced HIF1A was detected via an HA-tag; GAPDH served as a loading control. Wild type (WT) and knockdown (HIF1A shRNA1) cells were transfected with CMV:HIF1A-HA one day prior to induction with CoCl2 to simulate hypoxia. Cells were harvested 24 hours after induction. Immunoblot assay was performed using antibodies against HIF1A, HA-tag and GAPDH.
As an additional safety feature for the CARTELTM T cells we generated stable Jurkat T cell lines with the kill switch gene thymidine kinase. In the event of uncontrolled actions by the T cells, they can be eliminated by the administration of the drug ganciclovir. It is only affecting cells containing the kill switch, unmodified cells remain intact. To evaluate the efficiency of our kill switch we performed an apoptosis assay with ganciclovir (Fig. 3, 4). Both cell lines expressing HSV-TK show significantly reduced survival rates compared to wild type cells following treatment with ganciclovir. We were able to show that we can add an additional level of safety to our project design by introducing a functional kill switch.
Figure 3: Survival curve of Jurkat cells treated with ganciclovir. Depicted is the survival rate in percent after application of ganciclovir. The assay was done for 120 h by counting living cells of wild type (WT) and stable cell lines containing the suicide gene HSV-TK (SG). All experiments were performed in triplicates, data represents mean ± SD.
Figure 4: Survival curve of HEK293T cells treated with ganciclovir. Depicted is the survival rate in percent after application of ganciclovir. The assay was done for 72 h by counting living cells of wild type (WT) and stable cell lines containing the suicide gene HSV-TK (SG). All experiments were performed in triplicates, data represents mean ± SD.
This summer we could demonstrate that all parts of the CARTELTM AND gate are separately functional. After optimization and improvement of individual parts the system needs to be tested in primary T cells and may then proceed to pre-clinical studies with animal models. To learn more about possible improvements and future applications visit our outlook page!
Results
Applied Design
