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Background: The etanercept biosimilar SB4 is a TNF inhibitor authorised for use as a targeted Biological Disease-Modifying Anti-Rheumatic Drug (bDMARD). Various administration devices have been developed for subcutaneous self-injection of bDMARDs. Objective: This study surveyed patient satisfaction with their experience of using the SB4 pre-filled pen device. Methods: This non-interventional, cross-sectional, multi-centre study enrolled adult rheumatoid arthritis and spondyloarthropathy patients who had been treated for at least three months with the SB4 pre-filled pen. Based on a standardized questionnaire, patients rated general satisfaction, handling, user-friendliness, physical characteristics, and training material received. A total of 492 eligible patients completed questionnaires at 43 centres across Germany between August 2017 and June 2018. Data were analysed descriptively. Pre-defined subgroup analyses by previous therapy and by indication were performed. Results: Overall, 87% (95% CI 83% - 90%) of patients reported being ‘satisfied’ or ‘very satisfied’ with the pen. 89% of patients reported that the pen was ‘simple’ or ‘very simple’ to use. Most patients (87%) self-injected. 93% of patients who received training on the use of the pen were ‘satisfied’ or ‘very satisfied’ with the training provided. In this cross-sectional study, 12 patients reported an Adverse Event (AE) and one patient reported a treatment-related AE (nausea). Conclusion: The results demonstrated a high level of general satisfaction among patients using the SB4 pre-filled pen as well as satisfaction with ease of use for patients who were either naïve to bDMARDs or who had switched to SB4 from other bDMARDs.

Reduced expression and disrupted corticostriatal transportation of brain-derived neurotrophic factor (BDNF) is proposed to contribute to the selective vulnerability of medium spiny striatal projection neurons (MSNs) in Huntington's disease (HD). We have previously demonstrated that BDNF overexpression in the quinolinic acid lesioned rat striatum attenuates motor impairment and reduces the extent of MSN cell loss. To further investigate the potential therapeutic properties of BDNF for HD, the current study examines the effect of bilateral AAV1/2-mediated BDNF expression in the striatum of a transgenic rat model of HD. Transfer of the BDNF gene to striatal neurons using an AAV1/2 serotype vector enhanced BDNF protein levels in the striatum. Bilateral BDNF expression attenuated the impairment of both motor and cognitive function when compared with AAV1/2-vehicle- or YFP-treated transgenic HD rats. Interestingly, a gender effect was apparent with female transgenic HD rats exhibiting less functional impairment than males. Quantification of NeuN and DARRP32 immunoreactivity and striatal volume revealed limited disease phenotype between wild type and transgenic HD animals. However, AAV1/2-BDNF-treated transgenic HD rats showed evidence of greater striatal volume and increased NeuN+ cell numbers compared with wild-type vehicle- and AAV1/2-vehicle- or YFP-treated transgenic HD rats. We propose BDNF holds considerable therapeutic potential for alleviating behavioral dysfunction and neuronal degeneration in HD, with further work required to examine the role of BDNF-TrkB signaling and the preservation of axonal and synaptic function.

Inflammation-induced chemoattraction plays a major role in adult subventricular zone (SVZ)-derived precursor cell migration following neural cell loss, in particular through the release of chemokines by activated microglia and macrophages. We previously demonstrated that monocyte chemotactic protein-1 (MCP-1) (chemokine (c-c motif) ligand (CCL)2), macrophage inflammatory protein-1α (MIP-1α) (CCL3) and growth regulatory protein-α (GRO-α) (chemokine (c-x-c motif) ligand (CXCL)1) are up-regulated following neural cell loss in the adult striatum and act as potent chemoattractants for SVZ-derived precursor cells in vitro. Based on these observations, the current study aimed to examine the individual effect of MCP-1, MIP-1α and GRO-α on the migration of adult SVZ-derived neural precursor cells in vivo. To address this without the confounding effects of injury-induced chemotactic cues, adeno-associated viral (AAV)2-mediated in vivo gene transfer was used to ectopically express either MCP-1, MIP-1α or GRO-α, or the control red fluorescent protein (RFP) in the normal adult rat striatum. The extent of doublecortin (Dcx)-positive cell recruitment from the SVZ into the striatal parenchyma was then determined at 4 and 8weeks following AAV2 injection. Ectopic expression either of MCP-1 or MIP-1α in the normal adult rat brain significantly increased the number of Dcx-positive cells and the extent of their migration into the striatum at both 4 and 8weeks after vector injection but did not promote either precursor cell proliferation or neural differentiation. In contrast, while over-expression of GRO-α 4weeks after vector injection induced a significant increase in Dcx-positive cell migration compared to control, this effect was reduced to control levels by 8weeks post injection. Further, direct comparison between MCP-1, MIP-1α and GRO-α at both 4 and 8weeks post vector injection indicated that GRO-α may have a reduced effect in inducing Dcx-positive cell migration when compared to MCP-1. Combined, these results confirm that over-expression of the chemokines MCP-1, MIP-1α and GRO-α can override cues directing precursor cell migration along the rostral migratory stream (RMS) and provides a mechanism by which neural precursor cell migration can be redirected into a non-neurogenic region. Differences in the migratory effect observed between individual chemokine may be due to ligand-binding affinity and/or receptor expression on SVZ-derived precursor cells.

Recent studies have reported direct reprogramming of human fibroblasts to mature neurons by the introduction of defined neural genes. This technology has potential use in the areas of neurological disease modeling and drug development. However, use of induced neurons for large-scale drug screening and cell-based replacement strategies is limited due to their inability to expand once reprogrammed. We propose it would be more desirable to induce expandable neural precursor cells directly from human fibroblasts. To date several pluripotent and neural transcription factors have been shown to be capable of converting mouse fibroblasts to neural stem/precursor-like cells when delivered by viral vectors. Here we extend these findings and demonstrate that transient ectopic insertion of the transcription factors SOX2 and PAX6 to adult human fibroblasts through use of non-viral plasmid transfection or protein transduction allows the generation of induced neural precursor (iNP) colonies expressing a range of neural stem and pro-neural genes. Upon differentiation, iNP cells give rise to neurons exhibiting typical neuronal morphologies and expressing multiple neuronal markers including tyrosine hydroxylase and GAD65/67. Importantly, iNP-derived neurons demonstrate electrophysiological properties of functionally mature neurons with the capacity to generate action potentials. In addition, iNP cells are capable of differentiating into glial fibrillary acidic protein (GFAP)-expressing astrocytes. This study represents a novel virus-free approach for direct reprogramming of human fibroblasts to a neural precursor fate.

We have previously demonstrated a role for the chemokines MCP-1, MIP-1α and GRO-α in directing subventricular zone (SVZ)-derived neural precursor cell migration towards the site of cell death in the adult rodent brain. However the influence of chemokines such as MCP-1, MIP-1α and GRO-α on the differentiation of adult neural precursor cells has not previously been investigated. Further, as the majority of neurological disorders and injuries occur during ageing, it is important to investigate the effect of chemokines on adult neural precursor cell cultures obtained from the ageing brain. This study therefore examined the effect of MCP-1, MIP-1α and GRO-α on SVZ-derived neural precursor cell differentiation in vitro, and assessed whether precursor cells from the middle-aged rat brain (13 months old) follow the same migratory and differential profile as neural precursor cells obtained from the young adult rat brain (2 months old). We observed that each of the chemokines examined generated differing effects in regards to neuronal or glial differentiation. Further, both MIP-1α and GRO-α increased total cell number, suggesting an effect on precursor cell proliferation and/or survival. In agreement with cultures obtained from young adult brains, SVZ-derived neural precursor cells cultured from the middle-aged brain exhibited chemotactic migration in response to a concentration gradient. These results indicate that the chemokines MCP-1, MIP-1α and GRO-α can influence both the migration and fate choice of SVZ-derived neural precursor cells, as well as promoting cell viability. While a response to each of these chemokines is maintained in the middle-aged brain, a distinct age-related alteration in differential fate can be identified.

Udgivelsesdato: 2010-Jan-11 Oligo(ethylene glycol) methyl ether methacrylates (OEGMA) of various chain lengths (i.e., 9, 23, or 45 EG units) and N,N-dimethylaminoethyl methacrylate (DMAEMA) were copolymerized by atom transfer radical polymerization (ATRP), yielding well-defined P(DMAEMA-co-OEGMA) copolymers with increasing OEGMA molar fractions (F(OEGMA)) but a comparable degree of polymerization (DP approximately 120). Increase of both F(OEGMA) and OEGMA chain lengths correlated inversely with gene vector size, morphology, and zeta potential. P(DMAEMA-co-OEGMA) copolymers prevented gene vector aggregation at high plasmid DNA (pDNA) concentrations in isotonic solution and did not induce cytotoxicity even at high concentrations. Transfection efficiency of the most efficient P(DMAEMA-co-OEGMA) copolymers was found to be >10-fold lower compared with branched polyethylenimine (PEI) 25 kDa. Although OEGMA copolymerization largely reduced gene vector binding with the cell surface, cellular internalization of the bound complexes was less affected. These observations suggest that inefficient endolysosomal escape limits transfection efficiency of P(DMAEMA-co-OEGMA) copolymer gene vectors. Despite this observation, optimized p(DMAEMA-co-OEGMA) gene vectors remained stable under conditions for in vivo application leading to 7-fold greater gene expression in the lungs compared with PEI. Tailor-made P(DMAEMA-co-OEGMA) copolymers are promising nonviral gene transfer agents that fulfill the requirements for successful in vivo gene delivery.

Coupling of targeting ligands to polyethylenimine (PEI) has been previously used to improve transfection efficiency of PEI gene vectors. Here, we show that the β 2 -adrenoceptor (β 2 -AR) agonist, clenbuterol (Clen), can be used to improve gene transfer efficiency of PEI gene vectors on alveolar epithelial cells in vitro and in the lungs of mice in vivo . Clenbuterol conjugated to fluorescein-labeled bovine serum albumin resulted in clenbuterol-specific cellular uptake predominantly into alveolar but not bronchial epithelial cells. Clen- g -PEI (4/1) conjugates were combined with increasing molar ratios of PEI for transfection. At optimized PEI- g -Clen/PEI composition, transfection efficiency on alveolar epithelial cells was up to 14-fold higher than for unmodified PEI and could be inhibited by an excess of free clenbuterol. No increase of transfection efficiency was observed on bronchial epithelial cells. Increasing the PEI- g -Clen/PEI molar ratio resulted in an increase of gene vector size, decrease of the zeta potential and cytotoxicity. Aerosol delivery of optimized PEI- g -Clen/PEI (1/5) gene vectors resulted in a significant 3-fold increase of gene expression in the lungs of mice compared with unmodified PEI gene vectors. We suggest that coupling of β 2 -adrenoceptor ligands to nonviral gene vectors represents a promising approach to improve gene delivery to the lungs.

Genomic integration by the Streptomyces bacteriophage ϕC31 integrase is a promising tool for non-viral gene therapy of various genetic disorders. We investigated the ϕC31 integrase recombination activity in T cell derived cell lines, primary T lymphocytes and CD34+ haematopoietic stem cells in comparison to mesenchymal stem cells and cell lines derived from lung-, liver- and cervix-tissue. In T cell lines, enhanced long-term expression above control was observed only with high amounts of integrase mRNA. Transfections of ϕC31 integrase plasmids were not capable of mediating enhanced long-term transgene expression in T cell lines. In contrast, moderate to high efficiency could be detected in human mesenchymal stem cells, human lung, liver and cervix carcinoma cell lines. Up to 100-fold higher levels of recombination product was found in ϕC31 integrase transfected A549 lung than Jurkat T cells. When the ϕC31 integrase activity was normalized to the intracellular integrase mRNA levels, a 16-fold difference was found. As one possible inhibitor of the ϕC31 integrase, we found 3- to 5-fold higher DAXX levels in Jurkat than in A549 cells, which could in addition to other yet unknown factors explain the observed discrepancy of ϕC31 integrase activity.

In this study lactoferrin (Lf) was investigated as a targeting ligand for receptor-mediated gene delivery to human bronchial epithelial cells. A high number of lactoferrin receptors (LfRs) were detected on bronchial epithelial (BEAS-2B), but not on alveolar epithelial (A549) cells by fluorescence microscopy and FACS measurements, suggesting potential targeting selectivity for bronchial epithelial cells. Molecular conjugates with ratios of Lf to branched polyethylenimine 25 kDa (PEI) ranging from 4:1 to 1:40 (mol/mol) were synthesized and analyzed for complexation of plasmid DNA (pDNA), transfection efficiency, and cytotoxicity. Whereas particle size increased with the degree of Lf coupling from 45 to 225 nm, surface charge was not significantly influenced. Transfection studies on BEAS-2B cells revealed that Lf-PEI 1:20 exhibited the highest luciferase gene expression which was 5-fold higher at an N/P ratio (molar ratio of PEI nitrogen to pDNA phosphate) of 4 than PEI and could be inhibited by an excess of free Lf. With A549 cells, no significant enhancement in transfection efficiency between Lf-PEI/pDNA and PEI/pDNA complexes could be observed. Increasing the degree of Lf coupling to PEI resulted in reduced transfection efficiency in both alveolar and bronchial epithelial cells. Cell viability assays resulted in significantly lower cellular toxicity of Lf-PEI/pDNA compared with PEI/pDNA complexes. We suggest that Lf represents a potent targeting ligand for receptor-mediated gene delivery to bronchial epithelial cells and might be a promising candidate for lung gene transfer in vivo.

Background It has been previously demonstrated that plasmid DNA transport into the nucleus could be increased by transcription factor binding. We chose the glucocorticoid responsive element (GRE) which binds to the glucocorticoid receptor (GR), a transcription factor which is shuttled into the nucleus upon ligand binding such as dexamethasone. Methods We cloned two, four, and eight repetitive sequences of the GRE into the reporter plasmid pEGFPLuc. Binding of the pEGFPLuc-GRE to the GR was examined by electrophoretic mobility shift assay (EMSA) experiments. GR expression in bronchiolar and alveolar epithelial cells was confirmed by Western blotting. Intracellular trafficking of GR was examined using a fusion protein of cyano-fluorescent protein (CFP) and GR. Transfection efficiencies of pEGFPLuc compared to pEGFPLucGRE2–8 were examined in vitro and in vivo upon tail vein injection of cationic liposome gene vectors containing dexamethasone (safeplexes) and aerosol application of polyethylenimine (PEI)-pDNA particles. Results Binding of GRE containing plasmids to the GR was shown in EMSA experiments and intranuclear shuttling of CFP-GR after ligand stimulation was confirmed. Enhanced gene transfer efficiency of pEGFPLucGRE2in vitro was only observed on confluent cells. A 2.5-fold increase in gene expression in the lungs of mice after tail vein injection of pEGFPLucGRE2 complexed with safeplexes compared with pEGFPLuc was observed. PEI-mediated aerosol gene delivery of pEGFPLucGRE2 was 4.7-fold higher than pEGFPLuc only after intraperitoneal dexamethasone. Conclusion The results suggest that inclusion of GRE sequences into plasmid DNA vectors combined with dexamethasone application could improve transgene expression in the lungs in vivo. Copyright © 2007 John Wiley & Sons, Ltd.

In this study, we investigated galacturonic (GalAc)- and mannuronic (ManAc) acids as novel targeting ligands for receptor-mediated gene delivery. GalAc and ManAc were coupled to either polyethyleneimine (PEI) or PEI-polyethyleneglycol (PEG). Furthermore, lactobionic acid (LacAc), which comprises a GalAc-related carbohydrate ring, was coupled to each of the polymers through its open-chain gluconic acid moiety. The molar mass distributions of the polymers were characterized by analytical ultracentrifugation and size exclusion chromatography. PEI-conjugate-pDNA complexes were transfected into HepG2-, HeLa-, and 16HBE14o(-)-cells. Gene expression mediated by GalAc- and LacAc-functionalized PEI-conjugates was lower than for PEI. In contrast, gene expression mediated by ManAc-functionalized PEI-conjugates was up to three orders of magnitude higher than for the other tested PEI-conjugates, in particular for negatively charged gene vectors at low N/P ratios, independent of the cell line. Pre-incubation of cells with an excess of ManAc before transfection significantly inhibited transfection rates only for ManAc-functionalized PEI-conjugates. Coupling of methyl-alpha-d-mannuronic acid to PEI resulted in significantly lower transfection rates than for ManAc-PEI based complexes. Together with fluorescence microscopy images of fluorescein-labelled ManAc-functionalized dextrans and FACS analyses of cells, these results demonstrate that receptor-mediated endocytosis of ManAc-PEI-conjugate-pDNA complexes via ManAc-specific receptors was involved in gene transfer. In conclusion, ManAc-modification of PEI-polymers represents a novel strategy for receptor-mediated gene delivery which could be promising for in vivo application.

Since induced pluripotent stem cells were first generated from mouse embryonic fibroblasts in 2006, somatic cell reprogramming has become a powerful and valuable tool in many fields of biomedical research, with the potential to lead to the development of in vitro disease models, cell-based drug screening platforms, and ultimately novel cell therapies. Recent research has now demonstrated the direct conversion of fibroblasts into stem, precursor, or mature cell types that are committed in their fate within a specific lineage, such as hematopoietic precursors or mature neurons. This has been achieved by ectopic expression of defined, tissue-specific transcription factors. Several studies have demonstrated direct reprogramming of mouse and human fibroblasts into immature neural stem or precursor cells, either by transient expression of the four pluripotency genes OCT3/4, KLF4, SOX2, and C-MYC or by application of different combinations of up to 11 neural transcription factors. Interestingly, in all of these studies SOX2 was introduced alone or in combination with other transcription factors. In this review we discuss the different combinations of ectopic transcription factors used to generate neural stem/precursor cells from somatic cells, with particular emphasis on SOX2 and its potential to act as a master regulator for reprogramming to a neural precursor state.

Huntington's disease (HD) is a late-onset neurodegenerative disease characterized by a progressive loss of medium spiny neurons in the basal ganglia. The development of stem cell-based therapies for HD aims to replace lost neurons and/or to prevent cell death. This review will discuss pre-clinical studies which have utilized stem or progenitor cells for transplantation therapy using HD animal models. In several studies, neural stem and progenitor cells used as allotransplants and xenografts have been shown to be capable of surviving transplantation and differentiating into mature GABAergic neurons, resulting in behavioral improvements. Beneficial effects have also been reported for transplantation of stem cells derived from non-neural tissue, for example, mesenchymal- and adipose-derived stem cells, which have mainly been attributed to their secretion of growth and neurotrophic factors. Finally, we review studies using stem cells genetically engineered to over-express defined neurotrophic factors. While these studies prove the potential of stem cells for transplantation therapy in HD, it also becomes clear that technical and ethical issues regarding the availability of stem cells must be solved before human trials can be conducted.

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.

The presence of both neural and glial precursor cells in the adult central nervous system (CNS) and the capacity of these cells to migrate through this mature structure to areas of pathological damage and injury raises hope for the development of new therapeutic strategies to treat brain injury and disease. Although at present time, the compensatory neurogenesis described after various types of brain pathologies appears to be modest, the development of a strategy promoting the directed mobilization and phenotypic induction of endogenous precursor cells to areas of neural cell loss remains of high interest. The development of such a strategy however is currently thwarted by a limited understanding of the process and factors influencing precursor cell migration. In this review, we will discuss the current knowledge around precursor cell migration in the pathological adult brain with particular focus on the response and fate of precursor sub-populations to neural cell loss and the role of the inflammatory system in mediating precursor cell migration. Through this discussion we will identify particular areas in which further detailed research is required in order to expand our current understanding and aid in the eventual development of a novel therapeutic application.

Background Supercoiled topology of transfected plasmid DNA (pDNA) is critical for transgene expression in mammalian cells. In the present study, we analysed transgene expression of transfected supercoiled pDNA concatemers. Methods Jurkat T cells were transfected with a supercoiled 4.7-kb monomeric and, in parallel, a 9.4-kb dimeric pEGFP plasmid concatemer using electroporation. The absolute amounts of pDNA delivered into the cytoplasm and the nucleus were quantified by quantitative real-time polymerase chain reaction. Further, the number and mean fluorescent intensity (MFI) of enhanced green fluorescent protein (EGFP) expressing cells and the relative amounts of TOTO-1 fluorescently-labeled pDNA associated with the cell, located in the cytoplasm, and in the nucleus, were analysed by flow cytometry. Results For both constructs, significantly higher amounts of pDNA were detected in the cytoplasm compared to the nucleus. Furthermore, from FACS analysis, we could infer the relative gene copy (Egene) and plasmid expression efficiency (Eplasmid) by determining the ratio of the EGFP MFI of the transfected cells to TOTO-1 MFI per nucleus on the single cell level. Egene and Eplasmid were significantly 1.6-and 3.5-fold higher for EGFP-dimer than for EGFP-monomer, although the transfection rates considering the number of transfected cells were significantly lower for EGFP-dimer than for EGFP-monomer. Together with hydrodynamic plasmid diameter measurements, these observations suggest that concatemer arrangement increases relative gene expression efficiency, whereas plasmid size is important for cell and nucleus entry after electroporation. Conclusions We propose using preferably small supercoiled plasmid concatemers as the ideal plasmid vectors to maximize both transgene expression and the number of transfected target cells. Copyright © 2009 John Wiley & Sons, Ltd.

Enhancing gene delivery and expression in alveolar epithelial cells could offer the opportunity for the treatment of acquired and inherited lung diseases. Here, we show that particle adsorption of human insulin (INS) is capable of increasing plasmid DNA (pDNA) delivery from polyethylenimine (PEI) nanoparticles specifically in alveolar epithelial cells. INS receptors were predominantly detected on alveolar but not on bronchial epithelial cells. INS was adsorbed on the surface of PEI gene vectors by spontaneous self-assembly resulting in ternary PEI-pDNA-INS nanoparticles. Surface adsorption was confirmed by particle size, surface charge, and fluorescence resonance energy transfer (FRET) measurements. INS adsorption significantly increased gene expression of PEI-pDNA nanoparticles up to 16-fold on alveolar epithelial cells but not on bronchial epithelial cells. This increased gene expression was INS receptor specific. Our results demonstrate that targeting INS receptor for gene delivery in alveolar epithelial cells represents a promising approach for enhanced gene delivery and expression.

phi C31 integrase has emerged as a potent tool for achieving long-term gene expression in different tissues. The present study aimed at optimizing elements of phi C31 integrase system for alveolar type II cells. Luciferase and beta-galactosidase activities were measured at different time points post transfection. 5-Aza-2'deoxycytidine (AZA) and trichostatin A (TSA) were used to inhibit DNA methyltransferase and histone deacetylase complex (HDAC) respectively. In A549 cells, expression of the integrase using a CMV promoter resulted in highest integrase activity, whereas in MLE12 cells, both CAG and CMV promoter were equally effective. Effect of polyA site was observed only in A549 cells, where replacement of SV40 polyA by bovine growth hormone (BGH) polyA site resulted in an enhancement of integrase activity. Addition of a C-terminal SV40 nuclear localization signal (NLS) did not result in any significant increase in integrase activity. Long-term expression studies with AZA and TSA, provided evidence for post-integrative gene silencing. In MLE12 cells, both DNA methylases and HDACs played a significant role in silencing, whereas in A549 cells, it could be attributed majorly to HDAC activity. Donor plasmids comprising cellular promoters ubiquitin B (UBB), ubiquitin C (UCC) and elongation factor 1 alpha (EF1 alpha) in an improved backbone prevented post-integrative gene silencing. In contrast to A549 and MLE12 cells, no silencing could be observed in human bronchial epithelial cells, BEAS-2B. Donor plasmid coding for murine erythropoietin under the EF1 alpha promoter when combined with phi C31 integrase resulted in higher long-term erythropoietin expression and subsequently higher hematocrit levels in mice after intravenous delivery to the lungs. These results provide evidence for cell specific post integrative gene silencing with C31 integrase and demonstrate the pivotal role of donor plasmid in long-term expression attained with this system.