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

BACKGROUND: Analogues 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. RESULTS: The 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. CONCLUSIONS: Our 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

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system

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

Abstract Background Analogues 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. Results The 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. Conclusions Our 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.</p

The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells

<div><p>DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The <em>Sleeping Beauty</em> (SB), <em>piggyBac</em> (PB), and <em>Tol2</em> transposable elements have distinct biological properties and currently represent the most promising transposon systems for animal transgenesis and gene therapy. A potential obstacle, however, for persistent function of integrating vectors is transcriptional repression of the element and its genetic cargo. In this study we analyze the insulating effect of the 1.2-kb 5′-HS4 chicken β-globin (cHS4) insulator element in the context of SB, PB, and <em>Tol2</em> transposon vectors. By examining transgene expression from genomically inserted transposon vectors encoding a marker gene driven by a silencing-prone promoter, we detect variable levels of transcriptional silencing for the three transposon systems in retinal pigment epithelium cells. Notably, the PB system seems less vulnerable to silencing. Incorporation of cHS4 insulator sequences into the transposon vectors results in 2.2-fold and 1.5-fold increased transgene expression levels for insulated SB and PB vectors, respectively, but an improved persistency of expression was not obtained for insulated transgenes. Colony formation assays and quantitative excision assays unveil enhanced SB transposition efficiencies by the inclusion of the cHS4 element, resulting in a significant increase in the stable transfection rate for insulated SB transposon vectors in human cell lines. Our findings reveal a positive impact of cHS4 insulator inclusion for SB and PB vectors in terms of increased transgene expression levels and improved SB stable transfection rates, but also the lack of a long-term protective effect of the cHS4 insulator against progressive transgene silencing in retinal pigment epithelium cells.</p> </div

Transposition of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Schematic representation of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP vectors. IR, inverted repeat; RSV, Rous sarcoma virus promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; puro, puromycin resistance gene; pA, polyadenylation site. (<b>B</b>) Stable transfection rates of SB, PB, and <i>Tol2</i> transposon vectors in ARPE-19 cells. 0.125 pmol of pSBT/RGIP, pPBT/RGIP, and pTol2T/RGIP plasmid were cotransfected together with 0.02 pmol pcDNA3.1D/V5.TOPO plasmid (empty vector) or 0.02 pmol helper plasmid expressing either <i>SB100X</i> transposase, <i>iPB</i> transposase, or <i>Tol2</i> transposase. The pcDNA3.1D/V5.TOPO plasmid was also included as non-specific DNA to ensure that the total amount of DNA was 1 µg in each transfection. After 8 days of selection, puromycin resistant colonies were stained and counted. Mean ± SEM values are shown (N = 3). P values listed above the brackets were obtained by student's t-tests. (<b>C</b>) Transposon copy number of stably transfected ARPE-19 clones. Genomic DNA from ARPE-19 cell clones carrying SBT/RGIP, PBT/RGIP, or Tol2T/RGIP transposons was purified and examined by Southern blot analysis to determine the transposon copy number. A representative Southern blot is shown.</p

Insulation of SB, PB, and Tol2 transposon vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones carrying insulated transposon vectors. Measurements were obtained as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048421#pone-0048421-g002" target="_blank">Figure 2a</a>. (<b>B</b>) Comparison of mean MFI levels for insulated and uninsulated clones. Stably transfected ARPE-19 cell clones were grown for 8 weeks in the absence of selection, and eGFP expression levels were measured by flow cytometry at day 0 and day 56 of passage. (<b>C</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 1-3 transposon insertions. (<b>D</b>) Comparison of mean MFI levels for insulated and uninsulated clones carrying 9 or more transposon insertions.</p

Silencing of SB, PB, and <i>Tol2</i> transposon-based vectors in ARPE-19 cells.

<p>(<b>A</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones. ARPE-19 cells were transfected with pSBT/RGIP, pPBT/RGIP, or pTol2T/RGIP together with a transposase expressing plasmid, and selected for puromycin resistance. Individual clones were expanded and then passaged for 8 weeks in the absence of selection. Their eGFP expression level was determined at day 0 and day 56 of passage, and their percentage of retained MFI was calculated. (<b>B</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 1–3 transposon insertions. (<b>C</b>) Percentage of retained median fluorescence intensity (MFI) for stably transfected ARPE-19 clones containing 9 or more transposon insertions. (<b>D</b>) Reactivation of eGFP expression by TSA treatment. A subset of silenced ARPE-19 cell clones was grown in the presence of the deacetylase inhibitor Trichostatin A (TSA). The clones were treated 24 hours before analysis of eGFP expression by flow cytometry.</p

Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells

The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector–containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5′-HS4 chicken β-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer