Conditional human VEGF-mediated vascularization in chicken embryos using a novel temperature-inducible gene regulation (TIGR) system

Abstract

Advanced heterologous transcription control systems for adjusting desired transgene expression are essential for gene function assignments, drug discovery, manufacturing of difficult to produce protein pharmaceuticals and precise dosing of gene-based therapeutic interventions. Conversion of the Streptomyces albus heat shock response regulator (RheA) into an artificial eukaryotic transcription factor resulted in a vertebrate thermosensor (CTA; cold-inducible transactivator), which is able to adjust transcription initiation from chimeric target promoters (P(CTA)) in a low-temperature- inducible manner. Evaluation of the temperature-dependent CTA-P(CTA) interaction using a tailored ELISA-like cell-free assay correlated increased affinity of CTA for P(CTA) with temperature downshift. The temperature-inducible gene regulation (TIGR) system enabled tight repression in the chicken bursal B-cell line DT40 at 41 degrees C as well as precise titration of model product proteins up to maximum expression at or below 37 degrees C. Implantation of microencapsulated DT40 cells engineered for TIGR-controlled expression of the human vascular endothelial growth factor A (hVEGF121) provided low-temperature-induced VEGF-mediated vascularization in chicken embryos.

Figure 1

TIGR system. Top three expression units show the molecular details of TIGR. The cold-inducible transactivator (CTA) was constructed by fusing the S.albus heat shock response regulator rheA to the VP16 transactivation domain of herpes simplex virus (CTA, rheA-VP16). CTA was placed under control of the human elongation factor 1-α promoter, which mediates constitutive high level expression in chicken cells (pWW255, P_hEF1α-CTA-pA). The CTA-responsive target promoter P_CTA was assembled by cloning a single rheA-specific operator module (rheO) adjacent to a minimal version of the human cytomegalovirus immediate early promoter (P_hCMVmin) (P_CTA, rheO-P_hCMVmin). P_CTA was configured to drive either the cDNA encoding the human vascular endothelial growth factor A (hVEGF₁₂₁; pWW277, P_CTA-hVEGF₁₂₁-pA) or the SAMY reporter gene (pWW254, P_CTA-SAMY-pA). Selected sites for restriction endonucleases are indicated. Bottom two expression units illustrate TIGR-controlled transgene expression at different temperatures. At the permissive temperature of 37°C, CTA binds and transactivates P_CTA, which results in expression of the gene of interest. At 41°C, the P_CTA binding affinity of CTA is abolished and expression of the gene of interest remains silent.

Figure 2

ELISA-based quantification of the thermosensitivity of the CTA–P_CTA interaction. (A) Schematic representation for scoring the CTA affinity for P_CTA. A PCR-generated biotin (BT)-labeled P_CTA fragment is immobilized via streptavidin (ST) in a multi-well chamber. This set-up is incubated with the lysate of an E.coli CTA production strain (harboring pWW317, P_T7-CTA-pA) at different temperatures. After washing steps, the affinity of CTA for P_CTA is quantified by anti-VP16-based immunodetection visualized by an HRP-conjugated secondary antibody (targeted against the Fc region of the anti-VP16 antibody) which converts TMB into a color readout at 450 nm. (B) Quantitative ELISA-based cell-free analysis of CTA–P_CTA interaction at different temperatures. Whereas CTA showed lower affinity for its cognate rheO operator module at increasing temperatures (black bars), the interaction of the control configuration consisting of the macrolide-responsive transactivator (ET1) and its target sequence ETR was insensitive to temperature changes (white bars). Furthermore, the CTA binding capacity was specific for rheO as it did not interact with the ETR operator module at any of the temperatures (shaded bars).

Figure 3

TIGR-mediated temperature-dependent transgene expression switches in chicken DT40 cells. Triplicate DT40 cultures were co-transfected with the CTA expression construct pWW255 (P_hEF1α-CTA-pA) as well as the CTA-responsive SAMY expression vector pWW254 (P_CTA-SAMY-pA) and incubated at 37 and 41°C. At 48 h post-transfection, SAMY expression profiles were quantified in the supernatant of both cultures. DT40 control cells transfected with the constitutive SAMY expression vector pSS211 (P_hEF1α-SAMY-pA; S.Schlatter, unpublished) showed similar reporter gene expression profiles at both temperatures.

Figure 4

Temperature adjustability of the TIGR system. DT40 cells co-transfected with pWW255 (P_hEF1α-CTA-pA) and pWW254 (P_CTA-SAMY-pA) were incubated at varying temperatures, and SAMY production profiles were quantified at different time points post-transfection.

Figure 5

hVEGF₁₂₁-mediated vascularization in chicken embryos. Parallel grafting experiments were performed following supra-CAM application of microencapsulated DT40 cells engineered for TIGR-controlled hVEGF₁₂₁ and incubation at 41°C (a, c, e and g) and 37°C (b, d, f and h) for 2 days (until embryonic day 11). (a–d) Still video images of the growing CAM microvasculature at embryonic day 11 (application site indicated by asterisks) following intravenous injection of FITC–dextran (a and b overview; c and d insets of a and b at higher magnification). As shown in (b) and (d) in comparison with (a) and (c), only TIGR-mediated induction of hVEGF₁₂₁ at 37°C results in a massive angiogenic response exemplified by (i) the increased number of the feeding vessels; and (ii) their tortuous shape (arrows shown in d) and the formation of arterial venous shunts (arrowhead in d). Scanning electron micrographs confirm increased numbers of arterioles and veins as well as a densely packed capillary plexus in CAMs incubated at 37°C (f) when compared with an isogenic set-up grown at 41°C (e). Cross-sections through microcapsules and underlying CAM reveal a local angiogenic response at sites of application at 37°C (h) but not at 41°C (g). The wild-type CAM consisting of capillary plexus (arrows in g) in the subepithelial region is transformed by TIGR-controlled production of hVEGF₁₂₁ at permissive temperatures into a compact and stacked multitude of newly formed capillary vessels (arrows in h). The supplying vessels are indicated by asterisks. Size bars: (a) and (b) = 50 µm; (c), (d), (e) and (f) = 200 µm, (g) and (h) = 20 µm.