A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues

Abstract

BackgroundAnalogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker.ResultsThe three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use.ConclusionsOur findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.

Adeno‐associated virus‐delivered polycistronic microRNA‐clusters for knockdown of vascular endothelial growth factor in vivo

“…Approximately 200 000 HEK‐293 cells were seeded in six‐well plates and cultured as described above. The next day, cells were co‐transfected with pT2/CMV‐VEGF SV40‐Neo and pCMV‐SB100X , using 1 µg of each vector and FuGENE®6 (Roche) . Twenty‐four hours post‐transfection, the cells were diluted (1:10 and 1:100) and transferred to p10 Cellstar tissue culture dishes (Greiner, In Vitro AS, Fredensborg, Denmark) and incubated for 72 h. Then, media was replaced with fresh medium containing 1.5 mg/ml Neomycin (G418; Roche).…”

A lentiviral vector‐based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin

“…In cells that were cotransduced with LV/ Gal4hVDR and LV/UAS-TK-Fluc, the luminescence increased 45-fold (P < 0.001) relative to the background level upon exposure to EB1213. Importantly, we have previously shown that the sensor readout is unaffected by the presence of unligated Gal4hVDR (20). Hence, these results demonstrate tight inducibility of the dual-LV sensor system and suggest that drug-dependent transactivation of Fluc expression is achieved by a 'double-hit' of the target cells with LVs carrying the essentials of the genetic sensor.…”

“…Notably, longer-lasting effects of vitamin D3 analogues, as shown here for EB1213, may help to reduce dosages or the number of administrations. Indeed, EB1213 exhibits anti-proliferative and differentiation-promoting properties at lower concentrations than other vitamin D3 analogues including calcipotriol (20,38,43,45). In summary, our findings underline the potential of an in vivo sensor system as a quantitative screening methodology in the pursuit of novel, therapeutically attractive vitamin D3 analogues.…”

“…Crucial to the development of novel, therapeutically attractive vitamin D3 analogues is a detailed understanding of drug uptake, biodistribution, and biochemical behaviour. We have previously described an SB DNA transposon-based, integratable vitamin D3-sensitive reporter system for functional screening of vitamin D3 analogues in vitro (20). Here, we describe a further development of the sensor concept, which allows introduction of the sensor in xenotransplanted skin by codelivery of LVs encoding components of the sensor.…”

Bio-applications Derived from Site-Directed Genome Modification Technologies

“…Recently, Staunstrup et al ( 2011 ) used a genomically integratable transposon system for the assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues (Staunstrup et al 2011 ) . A tri-cistronic Sleeping beauty transposon was cloned from which three different proteins are encoded: a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter and a resistance marker allowing clonal selection.…”