Activation of proto-oncogenes by disruption of chromosome neighborhoods
2016; 351 (6280): 1454-1458
Influenza A virus targets a cGAS-independent STING pathway that controls enveloped RNA viruses.
2016; 7: 10680
Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.
View details for DOI 10.1126/science.aad9024
View details for Web of Science ID 000372756200050
View details for PubMedID 26940867
Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.
2015; 33 (9): 985-989
Stimulator of interferon genes (STING) is known be involved in control of DNA viruses but has an unexplored role in control of RNA viruses. During infection with DNA viruses STING is activated downstream of cGAMP synthase (cGAS) to induce type I interferon. Here we identify a STING-dependent, cGAS-independent pathway important for full interferon production and antiviral control of enveloped RNA viruses, including influenza A virus (IAV). Further, IAV interacts with STING through its conserved hemagglutinin fusion peptide (FP). Interestingly, FP antagonizes interferon production induced by membrane fusion or IAV but not by cGAMP or DNA. Similar to the enveloped RNA viruses, membrane fusion stimulates interferon production in a STING-dependent but cGAS-independent manner. Abolishment of this pathway led to reduced interferon production and impaired control of enveloped RNA viruses. Thus, enveloped RNA viruses stimulate a cGAS-independent STING pathway, which is targeted by IAV.
View details for DOI 10.1038/ncomms10680
View details for PubMedID 26893169
Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases
CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
View details for DOI 10.1038/nbt.3290
View details for PubMedID 26121415
IFI16 senses DNA forms of the lentiviral replication cycle and controls HIV-1 replication
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (48): E4571-E4580
Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in 'all-in-one' lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.DOI: http://dx.doi.org/10.7554/eLife.01911.001.
View details for DOI 10.7554/eLife.01911
View details for Web of Science ID 000334923200001
View details for PubMedID 24843011
Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus
ARTHRITIS RESEARCH & THERAPY
Overexpression of microRNA-155 increases IL-21 mediated STAT3 signaling and IL-21 production in systemic lupus erythematosus.
Arthritis research & therapy
2015; 17: 154-?
Replication of lentiviruses generates different DNA forms, including RNA:DNA hybrids, ssDNA, and dsDNA. Nucleic acids stimulate innate immune responses, and pattern recognition receptors detecting dsDNA have been identified. However, sensors for ssDNA have not been reported, and the ability of RNA:DNA hybrids to stimulate innate immune responses is controversial. Using ssDNAs derived from HIV-1 proviral DNA, we report that this DNA form potently induces the expression of IFNs in primary human macrophages. This response was stimulated by stem regions in the DNA structure and was dependent on IFN-inducible protein 16 (IFI16), which bound immunostimulatory DNA directly and activated the stimulator of IFN genes -TANK-binding kinase 1 - IFN regulatory factors 3/7 (STING-TBK1-IRF3/7) pathway. Importantly, IFI16 colocalized and associated with lentiviral DNA in the cytoplasm in macrophages, and IFI16 knockdown in this cell type augmented lentiviral transduction and also HIV-1 replication. Thus, IFI16 is a sensor for DNA forms produced during the lentiviral replication cycle and regulates HIV-1 replication in macrophages.
View details for DOI 10.1073/pnas.1311669110
View details for Web of Science ID 000327390400010
View details for PubMedID 24154727
miRNA sponges: soaking up miRNAs for regulation of gene expression
WILEY INTERDISCIPLINARY REVIEWS-RNA
2014; 5 (3): 317-333
Interleukin (IL-)21 is a key cytokine in autoimmune diseases such as systemic lupus erythematosus (SLE) by its regulation of autoantibody production and inflammatory responses. The objective of this study is to investigate the signaling capacity of IL-21 in T and B cells and assess its possible regulation by microRNA (miR)-155 and its target gene suppressor of cytokine signaling 1 (SOCS1) in SLE.The signaling capacity of IL-21 was quantified by stimulating peripheral blood monocuclear cells (PBMCs) with IL-21 and measuring phosphorylation of STAT3 (pSTAT3) in CD4+ T cells, B cells, and NK cells. Induction of miR-155 by IL-21 was investigated by stimulating purified CD4+ T cells with IL-21 and measuring miR-155 expression levels. The functional role of miR-155 was assessed by overexpressing miR-155 in PBMCs from SLE patients and healthy controls (HCs) and measuring its effects on STAT3 and IL-21 production in CD4+ and CD8+ T cells.Induction of pSTAT3 in CD4+ T cells in response to IL-21 was significantly decreased in SLE patients compared to HCs (p < 0.0001). Further, expression levels of miR-155 were significantly decreased and SOCS1 correspondingly increased in CD4+ T cells from SLE patients. Finally, overexpression of miR-155 in CD4+ T cells increased STAT3 phosphorylation in response to IL-21 treatment (p < 0.01) and differentially increased IL-21 production in SLE patients compared to HCs (p < 0.01).We demonstrate that SLE patients have reduced IL-21 signaling capacity, decreased miR-155 levels, and increased SOCS1 levels compared to HCs. The reduced IL-21 signaling in SLE could be rescued by overexpression of miR-155, suggesting an important role for miR-155 in the reduced IL-21 signaling observed in SLE.
View details for DOI 10.1186/s13075-015-0660-z
View details for PubMedID 26055806
DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors
NUCLEIC ACIDS RESEARCH
2014; 42 (4)
MicroRNAs (miRNAs) are small regulatory RNAs that act in an entangled web of interactions with target mRNAs to shape the cellular protein landscape by post-transcriptional control of mRNA decay and translation. miRNAs are themselves subject to numerous regulatory mechanisms that adjust their prevalence and activity. Emerging evidence suggests that miRNAs are themselves targeted by regulatory RNA species, and the identification of several classes of noncoding RNA molecules carrying miRNA binding sites has added a new intricate dimension to miRNA regulation. Such miRNA 'sponges' bind miRNAs and competitively sequester them from their natural targets. Endogenous miRNA sponges, also termed competing endogenous RNAs (ceRNAs), act to buffer the activity of miRNAs on physiologically relevant targets. This class of sponges includes endogenously transcribed pseudogenes, long noncoding RNAs, and recently discovered circular RNAs and may act in large complex networks in conjunction with miRNAs to regulate the output of protein. With the growing demand of regulating miRNA activity for experimental purposes and potential future clinical use, naturally occurring miRNA sponges are providing inspiration for engineering of gene vector-encoded sponges as potent inhibitors of miRNA activity. Combined with potent and versatile vector technologies, expression of custom-designed sponges provides new means of managing miRNAs and soaking up miRNAs for therapeutic regulation of gene expression.
View details for DOI 10.1002/wrna.1213
View details for Web of Science ID 000334501800002
View details for PubMedID 24375960
Managing MicroRNAs with Vector-Encoded Decoy-Type Inhibitors
2013; 21 (8): 1478-1485
DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
View details for DOI 10.1093/nar/gkt1163
View details for Web of Science ID 000332381000007
View details for PubMedID 24270790
Inhibition of p53-Dependent, but Not p53-Independent, Cell Death by U19 Protein from Human Herpesvirus 6B
2013; 8 (3)
A rapidly growing understanding of the complex circuitry of microRNA (miRNA)-mediated gene regulation is attracting attention to miRNAs as new drug targets. Targeted miRNA suppression is achieved in a sequence-specific manner by antisense RNA "decoy" molecules. Such synthetic miRNA inhibitors have reached the clinic with remarkable pace and may soon appear as new therapeutic modalities in several diseases. Shortcomings, however, include high production costs, the requirement for repeated administration, and difficulty achieving tissue-specific delivery. With the many recent landmark achievements in clinical gene therapy, new and refined vector-encoded miRNA suppression technologies are attractive for many applications, not least as tools in innumerable daily studies of miRNA biology in laboratories worldwide. Here, we provide an overview of the strategies that have been used to adapt vector-encoded inhibitors for miRNA suppression and discuss advantages related to spatiotemporal and long-term miRNA attenuation. With the remarkable new discovery of miRNA management by naturally occurring circular RNAs, RNA circles generated by trans-splicing mechanisms may prove to be well-suited carriers of decoy-type miRNA inhibitors. The community will aspire to combine circles with high-affinity miRNA decoy methodologies, and such "vectorized" RNA circles may represent new solid ways to deliver miRNA inhibitors, perhaps even with therapeutic applications.
View details for DOI 10.1038/mt.2013.113
View details for Web of Science ID 000322578600006
View details for PubMedID 23752312
A lentiviral vector-based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin
2013; 22 (3): 178-183
Infection with human herpesvirus (HHV)-6B alters cell cycle progression and stabilizes tumor suppressor protein p53. In this study, we have analyzed the activity of p53 after stimulation with p53-dependent and -independent DNA damaging agents during HHV-6B infection. Microarray analysis, Western blotting and confocal microscopy demonstrated that HHV-6B-infected cells were resistant to p53-dependent arrest and cell death after γ irradiation in both permissive and non-permissive cell lines. In contrast, HHV-6B-infected cells died normally through p53-independet DNA damage induced by UV radiation. Moreover, we identified a viral protein involved in inhibition of p53 during HHV-6B-infection. The protein product from the U19 ORF was able to inhibit p53-dependent signaling following γ irradiation in a manner similar to that observed during infection. Similar to HHV-6B infection, overexpression of U19 failed to rescue the cells from p53-independent death induced by UV radiation. Hence, infection with HHV-6B specifically blocks DNA damage-induced cell death associated with p53 without inhibiting the p53-independent cell death response. This block in p53 function can in part be ascribed to the activities of the viral U19 protein.
View details for DOI 10.1371/journal.pone.0059223
View details for Web of Science ID 000317418500026
View details for PubMedID 23555634
Suppression of microRNAs by dual-targeting and clustered Tough Decoy inhibitors
2013; 10 (3): 406-414
Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.
View details for DOI 10.1111/exd.12091
View details for Web of Science ID 000316332000291
View details for PubMedID 23489420
Efficient Sleeping Beauty DNA Transposition From DNA Minicircles
MOLECULAR THERAPY-NUCLEIC ACIDS
MicroRNAs (miRNAs) are ubiquitous regulators of gene expression that contribute to almost any cellular process. Methods for managing of miRNA activity are attracting increasing attention in relation to diverse experimental and therapeutic applications. DNA-encoded miRNA inhibitors expressed from plasmid or virus-based vectors provide persistent miRNA suppression and options of tissue-directed micromanaging. In this report, we explore the potential of exploiting short, hairpin-shaped RNAs for simultaneous suppression of two or more miRNAs. Based on the "Tough Decoy" (TuD) design, we create dual-targeting hairpins carrying two miRNA recognition sites and demonstrate potent co-suppression of different pairs of unrelated miRNAs by a single DNA-encoded inhibitor RNA. In addition, enhanced miRNA suppression is achieved by expression of RNA polymerase II-transcribed inhibitors carrying clustered TuD hairpins with up to a total of eight miRNA recognition sites. Notably, by expressing clustered TuD inhibitors harboring a single recognition site for each of a total of six miRNAs, we document robust parallel suppression of multiple miRNAs by inhibitor RNA molecules encoded by a single expression cassette. These findings unveil a new potential of TuD-based miRNA inhibitors and pave the way for standardizing synchronized suppression of families or clusters of miRNAs.
View details for DOI 10.4161/rna.23543
View details for Web of Science ID 000316784400011
View details for PubMedID 23324610
Potent microRNA suppression by RNA Pol II-transcribed 'Tough Decoy' inhibitors
RNA-A PUBLICATION OF THE RNA SOCIETY
2013; 19 (2): 280-293
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.Molecular Therapy - Nucleic Acids (2013) 2, e74; doi:10.1038/mtna.2013.1; published online 26 February 2013.
View details for DOI 10.1038/mtna.2013.1
View details for Web of Science ID 000332460000005
View details for PubMedID 23443502
Regulation of pro-inflammatory cytokines TNF alpha and IL24 by microRNA-203 in primary keratinocytes
2012; 60 (3): 741-748
MicroRNAs (miRNAs) are key regulators of gene expression and modulators of diverse biological pathways. Analyses of miRNA function as well as therapeutic managing of miRNAs rely on cellular administration of miRNA inhibitors which may be achieved by the use of viral vehicles. This study explores the miRNA-suppressive capacity of inhibitors expressed intracellularly from lentivirus-derived gene vectors. Superior activity of two decoy-type inhibitors, a "Bulged Sponge" with eight miRNA recognition sites and a hairpin-shaped "Tough Decoy" containing two miRNA recognition sites, is demonstrated in a side-by-side comparison of seven types of miRNA inhibitors transcribed as short RNAs from an RNA Pol III promoter. We find that lentiviral vectors expressing Tough Decoy inhibitors are less vulnerable than Bulged Sponge-encoding vectors to targeting by the cognate miRNA and less prone, therefore, to reductions in transfer efficiency. Importantly, it is demonstrated that Tough Decoy inhibitors retain their miRNA suppression capacity in the context of longer RNA transcripts expressed from an RNA Pol II promoter. Such RNA Pol II-transcribed Tough Decoy inhibitors are new tools in managing of miRNAs and may have potential for temporal and spatial regulation of miRNA activity as well as for therapeutic targeting of miRNAs that are aberrantly expressed in human disease.
View details for DOI 10.1261/rna.034850.112
View details for Web of Science ID 000313680200014
View details for PubMedID 23249752
The Impact of cHS4 Insulators on DNA Transposon Vector Mobilization and Silencing in Retinal Pigment Epithelium Cells
2012; 7 (10)
Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFα and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFα and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.
View details for DOI 10.1016/j.cyto.2012.07.031
View details for Web of Science ID 000311248000023
View details for PubMedID 22917968
Lentiviral vectors for cutaneous RNA managing
2012; 21 (3): 162-170
DNA transposons have become important vectors for efficient non-viral integration of transgenes into genomic DNA. The Sleeping Beauty (SB), piggyBac (PB), and Tol2 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 Tol2 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.
View details for DOI 10.1371/journal.pone.0048421
View details for Web of Science ID 000310262500066
View details for PubMedID 23110238
Comparative Genomic Integration Profiling of Sleeping Beauty Transposons Mobilized With High Efficacy From Integrase-defective Lentiviral Vectors in Primary Human Cells
2011; 19 (8): 1499-1510
Post-transcriptional managing of RNA plays a key role in the intricate network of cellular pathways that regulate our genes. Numerous small RNA species have emerged as crucial regulators of RNA processing and translation. Among these, microRNAs (miRNAs) regulate protein synthesis through specific interactions with target RNAs and are believed to play a role in almost any cellular process and tissue. Skin is no exception, and miRNAs are intensively studied for their role in skin homoeostasis and as potential triggers of disease. For use in skin and many other tissues, therapeutic RNA managing by small RNA technologies is now widely explored. Despite the easy accessibility of skin, the natural barrier properties of skin have challenged genetic intervention studies, and unique tools for studying gene expression and the regulatory role of small RNAs, including miRNAs, in human skin are urgently needed. Human immunodeficiency virus (HIV)-derived lentiviral vectors (LVs) have been established as prominent carriers of foreign genetic cargo. In this review, we describe the use of HIV-derived LVs for efficient gene transfer to skin and establishment of long-term transgene expression in xenotransplanted skin. We outline the status of engineered LVs for delivery of small RNAs and their in vivo applicability for expression of genes and small RNA effectors including small hairpin RNAs, miRNAs and miRNA inhibitors. Current findings suggest that LVs may become key tools in experimental dermatology with particular significance for cutaneous RNA managing and in vivo genetic intervention.
View details for DOI 10.1111/j.1600-0625.2011.01436.x
View details for Web of Science ID 000300931700301
View details for PubMedID 22379961
A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues
It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.
View details for DOI 10.1038/mt.2011.47
View details for Web of Science ID 000293378500014
View details for PubMedID 21468003
Mobilization of DNA transposable elements from lentiviral vectors.
Mobile genetic elements
2011; 1 (2): 139-144
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.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.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.
View details for DOI 10.1186/1472-6750-11-33
View details for Web of Science ID 000289954400001
View details for PubMedID 21473770
Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin.
2011; 11: 5-?
With the Sleeping Beauty (SB) DNA transposon, a reconstructed Tc1/mariner element, as the driving force, DNA transposable elements have emerged as new gene delivery vectors with therapeutic potential. The bipartite transposon vector system consists of a transposon vector carrying the transgene and a source of the transposase that catalyzes transposon mobilization. The components of the system are typically residing on separate plasmids that are transfected into cells or tissues of interest. We have recently shown that SB vector technology can be successfully combined with lentiviral delivery. Hence, SB transposons are efficiently mobilized from HIV-based integrase-defective lentiviral vectors by the hyperactive SB100X transposase, leading to the genomic insertion of lentivirally delivered DNA in a reaction controlled by a nonviral integration machinery. This new technology combines the better of two vector worlds and leads to integration profiles that are significantly altered and potentially safer relative to conventional lentiviral vectors. In this short commentary, we discuss our recent findings and the road ahead for hybrid lentivirus-transposon vectors.
View details for DOI 10.4161/mge.1.2.17062
View details for PubMedID 22016863
Regulation of cytokines by small RNAs during skin inflammation
JOURNAL OF BIOMEDICAL SCIENCE
Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs.Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels.Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimic the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination.Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.
View details for DOI 10.1186/1471-5945-11-5
View details for PubMedID 21352568
Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.
View details for DOI 10.1186/1423-0127-17-53
View details for Web of Science ID 000280092200001
View details for PubMedID 20594301