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Therapeutic use of site-directed endonucleases relies on safe and effective cellular delivery, preferentially resulting in short-term enzymatic activity. Based on the packaging of Gag/GagPol-fused heterologous proteins into VSV-G-pseudotyped lentivirus-derived particles, we have established lentiviral protein transduction for delivery of DNA transposases and custom-made endonucleases. Up to 24% of targeted CCR5 and AAVS1 alleles were disrupted in primary cells, including normal human dermal fibroblasts and primary keratinocytes, exposed to lentiviral particles loaded with zinc-finger nucleases (ZFNs). By exposing human 293 cells to ‘all-in-one’ integrase-defective lentiviral vectors (IDLVs) containing a complete gene repair kit consisting of ZFNs and viral RNA carrying the donor sequence for homology-directed repair, correction of genomic mutations was obtained in more than 8% of treated cells. As shown by confocal microscopy, ZFN proteins were abundant within transduced cells one hour after initial virus exposure, but were short-lived and gone after 24 hours. In accordance, under conditions supporting comparable CCR5 indel rates, disruption of the nearby CCR2 off-target site was reduced by lentiviral delivery of ZFNs targeting CCR5 relative to a conventional transfection-based approach. As biased and uncontrolled integration into genes remains a key challenge for gene therapies based on lentiviral vector technologies, we engineered ZFN-loaded IDLVs with the capacity to insert transgenes into the human CCR5 and AAVS1 loci by a homology-driven mechanism. Targeted gene integration into safe genomic loci was observed in human cell lines (85% of analyzed clones) and in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted transgene insertion into safe harbors was identified in all of 23 analyzed iPSC clones. Altogether, our findings generate a new platform for targeted genome engineering based on lentiviral delivery of complete gene repair or gene insertion kits.

The PiggyBac (PB) transposon system is a potent nonviral gene delivery tool with relevance in both gene therapy and cell reprogramming for production of induced pluripotent stem cells (iPSCs). Moreover, by taking advantage of the ability of the PB transposase to excise transposon-embedded gene cassettes from the genome without leaving footprints, the PB system is unique in facilitating seamless genome editing. The need for intracellular production of the transposase, however, raises concerns related to delivery and to cytotoxicity caused by sustained transposase expression and insertional mutagenesis. Furthermore, transposon re-integration may decrease the overall efficiency of the PB-mediated excision as well as increasing the risk of adverse secondary insertions. Based on our previous work, we present a new approach for lentivirus-based delivery of PB transposase. By fusing the hyperactive PB transposase, hyPBase, to the C-terminus of the GagPol polyprotein, we show robust incorporation and subsequent release of the transposase in matured lentiviral particles. Furthermore, in an effort to limit transposon re-integration, we engineered a hyPBase variant carrying three missense mutations. This novel hyPBase variant, hyPBaseExc+/Int−, demonstrates integration levels very close to background levels, thus limiting the risk of reintegration. Notably, the ability of hyPBaseExc+/Int− to excise transposons from plasmid-borne as well as from genomically integrated PB transposon cassettes is increased up to 6.3-fold relative to the original hyPBase transposase. By fusing the hyPBaseExc+/Int− to the C-terminus of GagPol, transposase protein can be efficiently delivered to cells by lentiviral protein transduction and, in our model system, performs seamless genomic excision of PB transposon cassettes in a copy number and dose-dependent manner, resulting in excision in up to 23.6% of virus-treated cells. Using a transposon containing the puro-deltaTK transgene cassette, we furthermore show that cells with successful excision can be enriched 17-fold by negative selection with FIAU. We believe that protein transduction of hyPBaseExc+/Int− may increase the applicability and safety of transposase-directed genomic excision in iPSCs and in hard-to-transfect cell types including hematopoietic cells.

Diffuse large B-cell lymphoma (DLBCL) is characterized by great genetic and clinical heterogeneity which complicates prognostic prediction and influences treatment efficacy. The most common regimen, R-CHOP, consists of a combination of anthracycline- and immuno-based drugs including Rituximab. It remains elusive how and to which extent genetic variability impacts the response and potential tolerance to R-CHOP. Hence, an improved understanding of mechanisms leading to drug tolerance in B-cells is crucial, and modelling by genetic intervention directly in B-cells is fundamental in such investigations. Lentivirus-based gene vectors are widely used gene vehicles, which in B-cells are an attractive alternative to potentially toxic transfection-based methodologies. Here, we investigate the use of VSV-G-pseudotyped lentiviral vectors in B-cells for exploring the impact of microRNAs on tolerance to Rituximab. Notably, we find that robust lentiviral transduction of cancerous B-cell lines markedly and specifically enhances the resistance of transduced germinal center B-cells (GCBs) to Rituximab. Although Rituximab works partially through complement-mediated cell lysis, increased tolerance is not achieved through effects of lentiviral transduction on cell death mediated by complement. Rather, reduced levels of PARP1 and persistent high levels of CD43 in Rituximab-treated GCBs demonstrate anti-apoptotic effects of lentiviral transduction that may interfere with the outcome and interpretation of Rituximab tolerance studies. Our findings stress that caution should be exercised exploiting lentiviral vectors in studies of tolerance to therapeutics in DLBCL. Importantly, however, we demonstrate the feasibility of using the lentiviral gene delivery platform in studies addressing the impact of specific microRNAs on Rituximab responsiveness.

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.

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.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor that plays a critical role in several diseases, including cancer, rheumatoid arthritis and diseases of the eye. Persistent regulation of VEGF by expression of small interfering RNAs targeting VEGF represents a potential future strategy for treatment of such diseases. As a step toward this goal, the present study combines the potency of VEGF-targeted miRNA mimics, produced from a miRNA cluster, with delivery by adeno-associated virus (AAV)-based vectors.Nine different engineered tri-cistronic miRNA clusters encoding anti-VEGF effectors were generated and tested in adult human retinal pigment epithelial (ARPE-19) cells using Renilla luciferase screening, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), western blotting and immunostaining analysis. In vivo efficacy was tested by the injection of scAAV2/8 vectors expressing the most effective miRNA cluster into murine hindlimb muscles, followed by quantitative RT-PCR.Plasmids containing anti-VEGF miRNA clusters showed efficient silencing of VEGF and demonstrated a combined gene silencing effect for miRNA clusters composed of multiple miRNA-mimicked RNA interference effectors. The most potent molecule, miR-5,10,7, resulted in a knockdown of VEGF by approximately 75%. Injection of scAAV2/8 vectors expressing miR-5,10,7 into murine hindlimb muscles, resulted in a 44% reduction of endogenous VEGF.We have developed miRNA clusters encoding anti-VEGF effectors and shown, in a mouse model, that VEGF is efficiently down-regulated by scAAV2/8-delivered miRNA clusters, allowing potent attenuation of VEGF. These findings may contribute to the development of gene therapy based on AAV-mediated delivery of miRNA clusters.

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.

Background 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. Methods 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. Results 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 mimick 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. Conclusions 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.

We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian species suitable for locus-directed transgene expression in cell cultures and, in addition, for transgene analyses in the very early embryonic stages.

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition.

The proinflammatory cytokine Tumor Necrosis Factor alpha (TNF-) is upregulated in inflammatory psoriatic skin. The increased level of TNF- protein is thought to cause keratinocyte hyperproliferation, leukocyte infiltration as well as growth and dilation of superficial blood vessels, which are all characteristics of human psoriasis skin. Blockade of TNF- function with specific inhibitors at the protein level has resulted in a rapid clinical improvement in psoriasis patients, demonstrating that TNF- inhibition offers a promising therapy of psoriasis. Whether TNF--encoding RNA is a valid therapeutic target, however, is still a matter of speculation, as recent findings have suggested that the level of TNF- is not increased in psoriatic skin. To test the hypothesis that TNF- in skin can be stably down-regulated by RNA interference (RNAi), we designed a panel of short hairpin RNAs (shRNAs) targeting human TNF-. Their efficiency in down-regulating TNF- protein expression was evaluated using a Renilla luciferase screening-assay based upon targeting of luc-TNF- fusion RNAs and a transient co-transfection assay. The three most potent shRNAs, which at most reduced the expression from target templates to 15% of the control samples treated with irrelevant shRNAs, were selected and cloned into lentiviral vectors. The lentiviral vectors expressing TNF- shRNAs were used to transduce HEK-293 cells and verify vector-derived knock-down of stable TNF- expression in vitro. The most efficient TNF--directed shRNA, which in cell lines reduced the amount of released TNF- more than 50% upon viral transduction, was selected for in vivo studies. In vivo studies were carried out in a xenograft mouse model in which human psoriatic plaques keratome skin biopsies were transplanted onto SCID mice. Initial studies using eGFP-encoding lentiviral vectors demonstrated efficient transduction of human psoriatic skin. Grafted psoriatic skin was exposed to viral vector-encoded TNF- shRNAs by a single intradermal injection of purified VSV-G-pseudotyped lentiviral vectors (150l containing 46.4 ng p24/l was injected at a single site). Biopsies were taken three weeks after injection. qPCR-based measurements of TNF- mRNA in skin treated with lentiviral vector-encoded TNF- shRNA demonstrated a 50% reduction in the level of TNF- mRNA. Most interestingly, the epidermal thickness of the human psoriatic plaques was reduced relative to mice treated with lentiviral vectors encoding an irrelevant shRNA. In conclusion, our results demonstrate that lentiviral vector-encoded TNF- shRNAs have the potential to down-regulate TNF- production both in vitro and in vivo. Phenotypic changes in shRNA-treated psoriatic skin suggest that TNF--encoding RNA is a valid therapeutic target in psoriasis treatment.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates approximately 20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2'-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.

RNA interference (RNAi) is an evolutionarily conserved cellular mechanism by which gene expression is suppressed by double-stranded RNA (dsRNA) in a sequence-specific manner. Enzymatically processed dsRNA molecules (siRNAs) target and facilitate cleavage of mRNA. By harnessing the RNAi machinery using synthetic or DNA-encoded RNA effectors, the siRNA technology offers new therapeutic strategies for genetic diseases. We outline the basic mechanisms of RNAi and present state-of-the-art examples of in vivo siRNA delivery towards development of RNAi-based individualized drugs.

Top of pageAbstract The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is one of the most efficient non-viral integrating systems described to date, all previous estimations of its integrative potential have relied on the use of antibiotic selection schemes. Based on previous studies demonstrating post-integrative gene silencing with other gene transfer systems (e.g. retroviruses), we investigated how genomic integration influenced transposon expression in human cells. To do this, we devised a novel strategy to monitor SB integration irrespective of transgene expression. We first constructed single plasmids (pT/RSV- YFP.CMV-SB, pT/EF1|[alpha]|-eGFP.CMV-SB, and pT/dmEF1|[alpha]|- dmGFP.CMV-SB) encoding a GFP variant-marked transposon and an externally-located transposase expression cassette and then transfected each of these plasmids into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS and then grown in the absence of antibiotic selection to generate a large number of clonal cell lines, each of which was screened via Southern blot analysis for transposon integrations. Results indicate that in the absence of selection, the Sleeping Beauty transposition system has a true integration efficiency of 41-52%, a value that is significantly higher than the 3-10% estimates previously generated using antibiotic selection-based approaches. In addition, we find that, irrespective of promoter usage or CpG content, many transposon copies undergo post-integrational gene silencing during a 12 week time period, with silencing in 13-18% of single-integration cell lines. Furthermore, upon continued passage of the pT/RSV-YFP.CMV-SB-derived lines, the frequency of transposon silencing had increased substantially from 18% to 71% after a 42 week time period. Studies utilizing the methylation- sensitive restriction enzyme McrBC indicate that DNA methylation contributes substantially to transgene repression in the vast majority of integrated transposon copies, a finding which was further substantiated through experimental application of the DNA methyltransferase inhibitor, 5-aza-2|[prime]|-deoxycytidine. Histone deacetylation was also found to play a role in transgene repression using the histone deacetylases inhibitor trichostatin A. Overall, these transposon clones are proving invaluable in helping unravel the molecular mechanism(s) governing transcriptional gene silencing, and may someday provide the insight needed to design an appropriate integrating vector system that is capable of maintaining long-term gene expression regardless of its integration site.

Retroviral particles contain a dimeric RNA genome, which serves as template for the generation of double-stranded DNA by reverse transcription. Transfer between RNA strands during DNA synthesis is governed by both sequence similarity between templates and structural features of the dimeric RNA. A kissing hairpin, believed to facilitate intermolecular recognition and dimer formation, was previously found to be a preferred site for recombination. To investigate if hairpin loop–loop-complementarity is the primary determinant for this recombination preference, we have devised a novel 5′ leader recombination assay based upon co-packaging of two wild-type or loop-modified murine leukemia virus vector RNAs. We found that insertion of an alternative palindromic loop in one of the two vectors disrupted site-directed template switching, whereas site-specificity was restored between vectors with complementary non-wild-type palindromes. By pairing vector RNAs that contained identical non-palindromic loop motifs and that were unlikely to interact by loop–loop kissing, we found no preference for recombination at the kissing hairpin site. Of vector pairs designed to interact through base pairing of non-palindromic loop motifs, we could in one case restore hairpin-directed template switching, in spite of the reduced sequence identity, whereas another pair failed to support hairpin- directed recombination. However, analyses of in vitro RNA dimerization of all studied vector combinations showed a good correlation between efficient dimer formation between loop-modified viral RNAs and in vivo cDNA transfer at the kissing hairpin. Our findings demonstrate that complementarity between wild-type or non-wild-type hairpin kissing loops is essential but not sufficient for site-specific 5′ leader recombination and lend further support to the hypothesis that a specific ‘kissing’ loop–loop interaction is guided by complementary sequences and maintained within the mature dimeric RNA of retroviruses.

Therapeutic gene transfer may constitute an alternative to current treatments. Despite promising results from animal studies common use of genes as drugs awaits improved vector efficiency and easier large-scale vector production. New developments in the field include high-capacity vectors with low immunogenicity, hybrid vectors, site-specific vector integration, RNA interference, and efficient non-viral gene transfer. In this article, we review the recent progress in somatic gene therapy with focus on vector development and current examples of clinical efficiency.

A major limitation of adenovirus-mediated gene therapy for inherited diseases is the instability of transgene expression in vivo, which originates at least in part from the loss of the linear, extrachromosomal vector genomes. Herein we describe the production of a gene-deleted adenovirus-transposon vector that stably maintains virus-encoded transgenes in vivo through integration into host cell chromosomes. This system utilizes a donor transposon vector that undergoes Flp-mediated recombination and excision of its therapeutic payload in the presence of the Flp and Sleeping Beauty recombinases. Systemic in vivo delivery of this system resulted in efficient generation of transposon circles and stable transposase-mediated integration in mouse liver. Somatic integration was sufficient to maintain therapeutic levels of human coagulation Factor IX for more than six months in mice undergoing extensive liver proliferation. These vectors combine the versatility of adenoviral vectors with the integration capabilities of a eukaryotic DNA transposon and should prove useful in the treatment of genetic diseases.

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1 b cells with respect to infection though the ecotropic receptor pathway.

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.

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.

Nonviral integration systems are widely used genetic tools in transgenesis and play increasingly important roles in strategies for therapeutic gene transfer. Methods to efficiently regulate the activity of transposases and site-specific recombinases have important implications for their spatiotemporal regulation in live transgenic animals as well as for studies of their applicability as safe vectors for genetic therapy. In this report, strategies for posttranslational induction of a variety of gene-inserting proteins are investigated. An engineered hormone-binding domain, derived from the human progesterone receptor, hPR891, and specifically recognized by the synthetic steroid mifepristone, is fused to the Sleeping Beauty, Frog Prince, piggyBac and Tol2 transposases as well as to the Flp and PhiC31 recombinases. By analyzing mifepristone-directed inducibility of gene insertion in cultured human cells, efficient posttranslational regulation of the Flp recombinase and the PhiC31 integrase is documented. In addition, fusion of the PhiC31 integrase with the ER(T2) modified estrogen receptor hormone-binding domain results in a protein, which is inducible by a factor of 22-fold and retains 75% of the activity of the wild-type protein. These inducible PhiC31 integrase systems are important new tools in transgenesis and in safety studies of the PhiC31 integrase for gene therapy applications.

DNA transposons, incorporated in nonviral or viral gene delivery systems, represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. In a nonviral gene therapy approach based on a single plasmid vector containing (i) a transgene-tagged Sleeping Beauty (SB) transposon and (ii) a SB-derived transposase we have previously demonstrated efficient transposition and therapeutic gene expression in mouse liver (Mikkelsen et al., Mol. Ther. 2003, 8:654). Although this vector technology holds great promise as a nonviral therapeutic tool, incorporation of the transposon integration machinery into viral vector systems may be required for some applications. We have previously shown that circular DNA serves as an efficient substrate for SB transposition, whereas linear DNA only supports limited transposition. Therefore, to create a gutless 'all-in-one' adenoviral vector containing all components required for transposition we have generated adenoviral vector constructs from which plasmid-like circular DNA carrying a transgene-marked transposon and a transposase expression cassette can be released by Flp-recombinase-based recombination. To reduce possible premature DNA circle formation during vector preparation, we fused Flpe with a modified human progesterone receptor (hPR) domain that facilitates specific binding to and activation by the synthetic steroid RU486. By co-transfections with a |[beta]|-gal reporter plasmid, the Flpe-hPR protein was found to retain 20 to 50% of the wildtype Flpe activity (depending on cell line) in the presence of RU486 and was induced 6- to 44-fold by addition of RU486. Next, by using plasmid vectors containing both (i) a neo-tagged transposon and (ii) a Flpe-activated transposase expression cassette we found that the level of transposition in Flpe-hPR-transfected HeLa cells was 5-fold higher than the background level in the presence of RU486 and only slightly reduced (1.4-fold) compared to cells transfected with wildtype Flpe plasmid. We have successfully produced 'all-in-one' adenoviral vectors carrying the transposase coding sequence, the Flpe-hPR recombinase gene, and a human factor IX-marked transposon and are currently studying RU486-induced generation of circular substrates for SB-mediated transposition in in vitro-transduced cells. In summary, this study demonstrates a new approach for drug-inducible SB transposition and its use in a novel integrating adenoviral vector system with potential applications in gene therapy approaches which require long-term transgene expression.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single plasmid (pT/RSV-YFP.CMV-SB) containing both a YFP-marked transposon and a separate transposase expression cassette (external to the transposon), then transfected the plasmid into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate approximately 100 clonal cell lines. After 8 weeks of growth (>50 cell divisions) 30% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy and FACS analyses. We have screened these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Results indicate approximately 50% of the total cell lines contain an integrated transposon sequence(s), of which roughly 20% show little or no YFP expression. Our early results, based upon Hpa II (a methylation sensitive restriction enzyme) cleavage of genomic DNA, suggested the lack of YFP expression was not due to DNA methylation but to some other regulatory system. However, more recent data (including the use of the methylation specific McrBC enzyme) indicates that some of the integrated transposons do in fact become methylated over time. Possibly related, most individual cell lines show heterogeneous YFP levels throughout the cell population. Using aphidicolin (a cell cycle inhibitor) we determined that the varied YFP expression is not cell cycle dependent. We are continuing to explore post-integrative gene expression/silencing using a variety of approaches including a detailed analysis of how CpG concentration within the transposon expression cassette affects long-term gene expression. Overall, our transposon clones are proving to be an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of transcriptional silencing of integrating gene transfer vectors.

The Sleeping Beauty (SB) transposon system mediates stable genomic integration by a cut-and-paste transposition process and has shown tremendous success as a therapeutic vehicle for in vivo gene delivery. Nevertheless, the overall rate of transposition with SB is still insufficient for many clinical applications. Herein, we generated 141 missense mutants for the SB transposase and screened each for altered gene transfer activity in human cells using a genetic assay. We show that despite equivalent steady-state levels of wild-type (WT) and mutant transposases in transfected cells, many mutations introduced into the N-terminal DNA-binding domain (n=10) and C-terminal catalytic core domain (n=6) resulted in 2- to 4-fold higher transpositional efficiencies compared to WT. In order to better understand the transposition reaction for potential future development, we compared the functional activities of these hyperactive recombinases to that of the WT transposase using a combination of PCR and EMSAs. Results of these studies identified a number of pathways that contribute to improved transposase activity, including an altered affinity of the transposase for transposon ends, transposase conformational changes, enhanced donor cleavage activity, and increased rates of strand transfer. Importantly, many of these hyperactive SB (HSB) mutations functioned synergistically and, when combined with a hyperactive transposon, elevated transposition by 14-fold compared to the first-generation transposon system. Moreover, although WT and mutant transposases were negatively regulated at high enzyme concentrations, the HSB variants supported ~10-fold higher transpositional activity at all experimental doses, suggesting that lower doses could be administered to further improve the safety profile for SB in vivo. In addition, the use of HSB instead of the WT transposase dramatically improved the integration frequency of larger-sized elements, including ones greater than 14 kb in length. These HSB mutants should greatly extend the carrying capacity for SB, thus overcoming one of this system's major limitations. Finally, we studied the long-term in vivo persistence of two transposon reporters, β-galactosidase and human coagulation factor IX (hFIX), in the livers of adult mice following a single administration of WT- or hyperactive transposase-expressing plasmids. Results of these analyses demonstrated that these mutated enzymes could also greatly enhance SB's gene transfer capabilities in vivo, supporting stable integration in an estimated 57% of transfected mouse hepatocytes, compared to ~8% for the WT. This level of transposition was sufficient to maintain ~7-fold higher serum hFIX levels (955 ng/ml ± 241 ng/ml) than achieved in SB-expressing animals (140 ng/ml ± 56 ng/ml) for a period of 4 months (length of study). This level of hFIX obtained with the HSB mutant is ~19% of normal human levels and is well within a curative range capable of converting a severely affected hemophilia B patient to one with a much milder phenotype. Therefore, our work demonstrates the potential for improved in vivo gene delivery through directed transposase evolution, and provides a number of important insights into Sleeping Beauty transposon biology that could be exploited for future development.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single autonomously integrating plasmid (pT/YFP-SB) containing a YFP-marked transposon and an external plasmid-encoded transposase expression cassette, and transfected it into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate a total of 183 clonal cell populations. Unexpectedly, after 6 weeks of growth (> 30 cell divisions) 28% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy. We have begun screening these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Interestingly, preliminary results indicate that up to 15% of the total cell lines contain non-expressing integrated transgenes and we are currently investigating whether this may be due to post-integrative gene silencing. Surprisingly, up to 40% of the expressing cell lines contain persisting (non-dividing) episomal plasmid, as determined by DpnI/Southern blot analyses. Consequently, we are investigating whether co-delivery of SB and transposon sequences can facilitate episomal persistence in mammalian cells, possibly through transposase-mediated nuclear retention. Also of note, many individual cell lines showed heterogeneous YFP levels throughout the cell population. Currently, we are testing whether expression from an integrated transposon varies with the cell cycle, as has been seen in other integrating systems. The generation of stably silenced transposon clones provides an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of Sleeping Beauty biology.

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies. Currently, the only efficient therapy is bone-marrow transplantation from a matched sibling donor. Therefore, FA is considered a good candidate disease for hematopoietic gene therapy. Our previous preclinical gene therapy experiments with Fancc knock-out mice have shown that wild-type hematopoietic stem cells (introduced by transplantation or generated by gene transfer) have a strong selective growth advantage in vivo. However, despite the successful modeling of FA gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for ex vivo manipulation and scarcity of HSCs in FA patients. Consequently, the development of new gene transfer methods that support stable integration of therapeutic transgenes in vivo are greatly desired. Here, we have utilized the Sleeping Beauty (SB) transposon system to facilitate stable, transposase-mediated gene transfer of the human FANCC cDNA in vivo from a plasmid-based system. The transposase and FANCC transposon plasmid constructs were co-transfected into bone-marrow cells of Fancc mutant mice by two different methods: 1) via electroporation into enriched hematopoietic cells; 2) via direct injection into the femur cavity. In each case, the presence of the transgene was followed in treated animals by semi-quantitative PCR. Although no transgene was detectable in peripheral blood DNA 2 weeks after injection, 2/4 mice injected into the femur cavity were PCR positive 4 weeks after injection, even in the absence of selective pressure. At this time, a single low dose of cyclophosphamide (CPA) was given to provide a selective advantage for FANCC corrected cells. Four weeks after CPA administration, all the experimental electroporated animals (N=2) and femur injected animals (N=8) were strongly PCR positive in their peripheral blood and have remained stably positive for at least 9 months. Furthermore, upon transplantation of bone marrow from these mice into wild-type lethally irradiated recipients, we observed that 81/110 (74%) of day 12 CFU-S colonies were transgene positive. Similarly, 64/110 (64%) of the 12-day CFU-S colonies were transgene positive from the femur-injected donors, thus confirming that transgene integration had occurred in bona fide stem cells. Collectively, these studies demonstrate that a) hematopoietic stem cells from Fancc mutant mice are proficient in taking up plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells is possible and c) the efficiency of stable gene transfer using these nonviral approaches compares favorably to that achieved by alternative viral-based methods. We are in the process of analyzing transposon host cellular DNA junctions to determine the mechanism of transgene persistence in each experimental group. The above experiments have been repeated independently with more animals: N=5 for electroporated and N=6 for femur injected. Details of all these results will be discussed.

Retroviruses encapsidate two copies of full-length viral RNA molecules linked together as a dimeric genome. RNA stem loop structures harboring palindromic (or “kissing”) loop sequences constitute important cis-elements for viral dimerization known as dimer initiation sites (DIS). In murine leukemia virus (MLV), a 10-mer and a 16-mer palindrome (DIS-1 and DIS-2, respectively) located in the viral leader region mediate dimerization in vitro and affect dimer stability of vector RNA in vivo. We have investigated the effect on viral replication of introducing deletions or nucleotide substitutions within these palindromes in a full-length MLV genome. Our results demonstrate that viruses modified at the dimer initiation site regions are viable and show wild-type levels of RNA encapsidation. One mutant lacking the DIS-1 palindrome was severely impaired and displayed an increased cellular ratio of spliced versus genomic RNA that most likely contributes to the inefficient replication. The implications for development of DIS-modified retrovirus-based vectors are discussed.

Sleeping Beauty (SB) DNA transposon-based vectors belong to a growing family of nonviral integrating vectors that represent attractive alternatives to conventional virus-based integrating gene vehicles. Because of concerns related to mutagenesis and/or activation of cellular genes by integrating vectors, much attention has been paid to integration site preferences and the ability of vectors to influence expression of neighboring genes. Here, we test the hypothesis that terminal repeats of transposons carry cis-acting regulatory sequences. In transient gene expression studies, we demonstrate that the inverted repeats of SB direct gene expression in HeLa cells to levels that are 3-fold higher than in promoter-deficient controls. Inverted repeats pointing toward the transposon center consistently facilitate the highest levels of activity in a number of cell lines. We show that transposon sequences flanking the inverted repeats of SB are required for positive effects on gene expression and, moreover, that these regions contain both stimulatory and inhibitory cis-acting elements. In the context of an integrated SB vector the regulatory activities of the transposon termini are sufficient to drive expression of selectable marker genes carried by the transposon, indicating that opposing transcriptional activities originating from the transposon termini may influence expression of its genetic cargo. Finally, detection of regulatory properties of the terminal repeats of the active Tc3 element from Caenorhabditis elegans leads to the suggestion that transcriptional activities of the inverted repeats are conserved among Tc1/mariner transposons in nature. Our data suggest that SB-based gene vectors may carry ancient properties of self-regulation with potential relevance for SB-directed therapeutic gene transfer.

For cellular and animal transgenesis, FLP- and Cre-recombinase gene capture systems are highly effective to provide stable integration of a donor plasmid carrying the transgene cassette of interest into an engineered genomic locus in a given cell line. However, in many protocols, the entire plasmid bacterial backbone is integrated along with the transgene cassette. Here, we present a very simple yet highly efficient method for excluding plasmid bacterial backbone integration. The transgene cassette, including a single FLP recognition target site, is specifically amplified by PCR, and the resulting DNA ligated into minicircles can serve as donor DNA in FLP-mediated recombination. Interestingly, the elimination of the bacterial backbone increased expression of the inserted transgene. The presented method is simple and efficient for generating transgene cassette insertions devoid of the bacterial backbone.

Gene vehicles derived from lentiviruses have become highly esteemed tools for gene transfer and genomic insertion in a wealth of cell types both in vivo and ex vivo. However, accumulating evidence of preferred insertion into actively transcribed genes, driven by biological properties of the parental human immunodeficiency virus type 1, has questioned the safety of this vector technology. As a consequence, integrase-defective lentiviral vectors [IDLVs], carrying an inactive integrase protein, have been developed and used with success for persistent in vivo gene transfer to quiescent or slowly dividing cells. We and others have shown that episomal DNA delivered by IDLVs may serve as a substrate for heterologous integration machineries, including recombinases and transposases, and homologous recombination triggered by nuclease-induced DNA damage. New vector systems that combine the best of lentiviral gene delivery and nonviral integration systems are under development. The first prototypes of such hybrid lentiviral vectors facilitate efficient gene transfer and show profiles of insertion that are not dictated by the biological constraints of the normal integration pathway and are, therefore, significantly different from the profile of conventional lentiviral vectors. The stage is set for further exploration of these vectors. In this review, we summarize the background and short history of hybrid IDLV-based vector systems and discuss their applicability in gene therapy and treatment of genetic disease.

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.