Lux Expression Research Articles

From macro to micro: a combined bioluminescence-fluorescence approach to monitor bacterial localization.

SummaryBacterial bioluminescence is widely used to study the spatiotemporal dynamics of bacterial populations and gene expression in vivo at a population level but cannot easily be used to study bacterial activity at the level of individual cells. In this study, we describe the development of a new library of mini‐Tn7‐lux and lux::eyfp reporter constructs that provide a wide range of lux expression levels, and which combine the advantages of both bacterial bioluminescence and fluorescent proteins to bridge the gap between macro‐ and micro‐scale imaging techniques. We demonstrate that a dual bioluminescence‐fluorescence approach using the lux operon and eYFP can be used to monitor bacterial movement in plants both macro‐ and microscopically and demonstrate that Pseudomonas syringae pv phaseolicola can colonize the leaf vascular system and systemically infect leaves of common bean (Phaseolus vulgaris). We also show that bacterial bioluminescence can be used to study the impact of plant immune responses on bacterial multiplication, viability and spread within plant tissues. The constructs and approach described in this study can be used to study the spatiotemporal dynamics of bacterial colonization and to link population dynamics and cellular interactions in a wide range of biological contexts.

Quorum-sensing signaling by chironomid egg masses' microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae.

Vibrio cholerae, the causative agent of cholera, is commonly isolated, along with other bacterial species, from chironomid insects (Diptera: Chironomidae). Nevertheless, its prevalence in the chironomid egg masses' microbiota is less than 0.5%. V. cholerae secretes haemagglutinin/protease (HAP) that degrades the gelatinous matrix of chironomid egg masses and prevents hatching. Quorum sensing (QS) activates HAP production in response to accumulation of bacterial autoinducers (AIs). Our aim was to define the impact of chironomid microbiota on HAP production by V.cholerae. To study QS signaling, we used V.cholerae bioluminescence reporter strains (QS-proficient O1 El-Tor wild-type and QS-deficient mutants) and different bacterial species that we isolated from chironomid egg masses. These egg mass isolates, as well as a synthetic AI-2, caused an enhancement in lux expression by a V.cholerae QS-deficient mutant. The addition of the egg mass bacterial isolate supernatant to the QS-deficient mutant also enhanced HAP production and egg mass degradation activities. Moreover, the V.cholerae wild-type strain was able to proliferate using egg masses as their sole carbon source, while the QS-deficient was not. The results demonstrate that members of the chironomid bacterial consortium produce external chemical cues that, like AI-2, induce expression of the hapA gene in V.cholerae. Understanding the interactions between V.cholerae and the insects' microbiota may help uncover the interactions between this pathogen and the human gut microbiota.

Optimizing the antimicrobial performance of metallic glass composites through surface texturing

In the present work, we analyse the influence of laser texturing on the physicochemical and bactericidal properties of Cu55Zr40Al5 Bulk Metallic Glass Composite (BMGC) to develop novel antimicrobial touch surfaces. Laser ablation was employed to increase the average roughness of BMGC samples from 0.08 ± 0.02 μm to 3.07 ± 0.96 μm using a maximum laser fluence of 2.82 J/cm2. This treatment also influenced surface chemistry causing the formation of CuO, CuO2, ZrO2, more prominent as the laser fluence was increased. Alongside chemical and topographic changes, the initial contact angle of the as-cast sample was found to increase from 85.81° to angles between 105.72° and 126.17° after texturing. The influence of these modifications on the antimicrobial performance of all rapidly solidified alloys was studied with Escherichia coli K12 modified to drive lux expression. Luminescence measurements revealed a reduction in bacterial growth as the laser fluence applied was risen. This increase in bactericidal effect as laser fluence rose was corroborated with recovery tests, which showed an increase in log reduction of E. coli K12 from 1.10 (for as-cast sample) to 2.16 (textured at 2.82 J/cm2) after 4 h of contact. Variations in bacterial morphology were observed with SEM imaging, specifically, a length increase of E. coli cells from 2 μm up to 20 μm could be observed in cells deposited on the textured surfaces. Deposited bacteria on laser treated samples revealed loss of membrane integrity, which along the aforementioned morphological changes suggest both external and DNA damage in all ablated samples. These findings reveal the possibility of tailoring the antimicrobial behaviour of BMGCs through laser texturing, which could be used as novel touch surfaces to tackle nosocomial infections along antibiotic resistance.

743. Minicircles Are Similar to Plasmids in Providing High Level, Long-Term Expression in the Lung

Background: Many gene therapy applications require persistent transgene expression to treat chronic disease. We have previously developed CpG-free plasmids capable of robust, long-term transgene expression in the mouse lung (Hyde et al; 2008 Nat. Biotech. 26:549) and confirmed their utility in clinical trials, where monthly aerosol delivery of plasmid/liposomes to patients with Cystic Fibrosis resulted in a stabilisation of their lung function over a 12-month period (Alton et al 2015 Lancet Respir. Med. 3:684). The removal of all CpGs from a plasmid, including the selection of a CpG-free promoter, can be time-consuming, therefore we evaluated which aspects of plasmid design were crucial for long-term transgene expression in the lung, including the use of minicircles instead of conventional plasmids. Results: First, we measured luciferase (Lux) expression in the lungs of BALB/c mice from a CpG-free transgene expression cassette (hCEFI-soLux) compared with a standard CpG-rich (hCEFI-Lux) cassette. Plasmid DNA was complexed with 25 kDa branched polyethylenimine (PEI) and delivered as an aerosol to the lungs of BALB/c mice (n=6 per time-point). Lux activity from the CpG-free hCEFI-soLux cassette was greater than its CpG-rich counterpart (p < 0.005) at every time-point (up to 28 days), with no loss of activity over the course of the study. Expression from the expression cassette containing CpGs, however, was not persistent, but declined to between 30% and 2% of respective day 1 levels by day 28 (p < 0.05). Next, we performed similar studies using minicircles, where the CpG-free and CpG-rich expression cassettes were manufactured as minicircles with minimal backbone sequences (209bp with 11 CpGs). When the CpG-free cassette was used there was no significant difference between Lux activity obtained from the plasmid and minicircles; in both cases robust expression persisted beyond the duration of the study (28 days). Finally, we investigated whether the detection of CpGs by the Toll-like receptor-9 (TLR9) signalling pathway played a role in the loss of expression from the CpG-rich cassette. The experiments were repeated in TLR9-deficient mice and the results showed that Lux levels were similar to those obtained in BALB/c mice. This indicated that the effect of CpGs on the in vivo expression profile (whether in the transgene cassette or backbone) is independent of the TLR9 pathway. Conclusion: These studies indicate that a CpG-free transgene cassette is crucial to achieving high levels of persistent expression in the murine lung, when delivered as a plasmid or minicircle. Minicircles have not yet been evaluated clinically, but they have several advantages over plasmids, including their reduced overall size such that a higher effective dose is delivered. These findings could also be applicable to other organs, such as muscle and liver.

A bioluminescent arsenite biosensor designed for inline water analyzer.

Whole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter PrpoD. In the arsenite biosensor, the arsenite-inducible promoter P ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4°C over a year evidenced that about 40% of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7months in storage are maintained, with a detection limit of 0.2μM arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field.

Tn5/7-lux: a versatile tool for the identification and capture of promoters in gram-negative bacteria.

BackgroundThe combination of imaging technologies and luciferase-based bioluminescent bacterial reporter strains provide a sensitive and simple non-invasive detection method (photonic bioimaging) for the study of diverse biological processes, as well as efficacy of therapeutic interventions, in live animal models of disease. The engineering of bioluminescent bacteria required for photonic bioimaging is frequently hampered by lack of promoters suitable for strong, yet stable luciferase gene expression.ResultsWe devised a novel method for identification of constitutive native promoters in Gram-negative bacteria. The method is based on a Tn5/7 transposon that exploits the unique features of Tn5 (random transposition) and Tn7 (site-specific transposition). The transposons are designed such that Tn5 transposition will allow insertion of a promoter-less bacterial luxCDABE operon downstream of a bacterial gene promoter. Cloning of DNA fragments from luminescent isolates results in a plasmid that replicates in pir+ hosts. Sequencing of the lux-chromosomal DNA junctions on the plasmid reveals transposon insertion sites within genes or operons. The plasmid is also a mini-Tn7-lux delivery vector that can be used to introduce the promoter-lux operon fusion into other derivatives of the bacterium of interest in an isogenic fashion. Alternatively, promoter-containing sequences can be PCR-amplified from plasmid or chromosomal DNA and cloned into a series of accompanying mini-Tn7-lux vectors. The mini-Tn5/7-lux and mini-Tn7-lux vectors are equipped with diverse selection markers and thus applicable in numerous Gram-negative bacteria. Various mini-Tn5/7-lux vectors were successfully tested for transposition and promoter identification by imaging in Acinetobacter baumannii, Escherichia coli, and Burkholderia pseudomallei. Strong promoters were captured for lux expression in E. coli and A. baumannii. Some mini-Tn7-lux vectors are also equipped with attB sites for swapping of the lux operon with other reporter genes using Gateway technology.ConclusionsAlthough mini-Tn5-lux and mini-Tn7-lux elements have previously been developed and used for bacterial promoter identification and chromosomal insertion of promoter-lux gene fusions, respectively, the newly developed mini-Tn5/7-lux and accompanying accessory plasmids streamline and accelerate the promoter discovery and bioluminescent strain engineering processes. Availability of vectors with diverse selection markers greatly extend the host-range of promoter probe and lux gene fusion vectors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0354-3) contains supplementary material, which is available to authorized users.

Transcriptional Regulation of LUX by CBF1 Mediates Cold Input to the Circadian Clock in Arabidopsis

Circadian clocks allow organisms to anticipate daily changes in the environment to enhance overall fitness. Transcription factors (TFs) play a prominent role in the molecular mechanism but are incompletely described possibly due to functional redundancy, gene family proliferation, and/or lack of context-specific assays. To overcome these, we performed a high-throughput yeast one-hybrid screen using the LUX ARRYHTHMO (LUX) gene promoter as bait against an Arabidopsis TF library. LUX is a unique gene because its mutation causes severe clock defects and transcript maintains high-amplitude cycling in the cold. We report the well-characterized cold-inducible C-repeat (CRT)/drought-responsive element (DRE) binding factor CBF1/DREB1b is a transcriptional regulator of LUX. We show that CBF1 binds the CRT in the LUX promoter, and both genes overlap in temporal and spatial expression. CBF1 overexpression causes upregulation of LUX and also alters other clock gene transcripts. LUX promoter regions including the CRT and Evening Element (EE) are sufficient for high-amplitude transcriptional cycling in the cold, and cold-acclimated lux seedlings are sensitive to freezing stress. Our data show cold signaling is integrated into the clock by CBF-mediated regulation of LUX expression, thereby defining a new transcriptional mechanism for temperature input to the circadian clock.

Expression of a humanized viral 2A-mediated lux operon efficiently generates autonomous bioluminescence in human cells.

BackgroundExpression of autonomous bioluminescence from human cells was previously reported to be impossible, suggesting that all bioluminescent-based mammalian reporter systems must therefore require application of a potentially influential chemical substrate. While this was disproven when the bacterial luciferase (lux) cassette was demonstrated to function in a human cell, its expression required multiple genetic constructs, was functional in only a single cell type, and generated a significantly reduced signal compared to substrate-requiring systems. Here we investigate the use of a humanized, viral 2A-linked lux genetic architecture for the efficient introduction of an autobioluminescent phenotype across a variety of human cell lines.Methodology/Principal FindingsThe lux cassette was codon optimized and assembled into a synthetic human expression operon using viral 2A elements as linker regions. Human kidney, breast cancer, and colorectal cancer cell lines were both transiently and stably transfected with the humanized operon and the resulting autobioluminescent phenotype was evaluated using common imaging instrumentation. Autobioluminescent cells were screened for cytotoxic effects resulting from lux expression and their utility as bioreporters was evaluated through the demonstration of repeated monitoring of single populations over a prolonged period using both a modified E-SCREEN assay for estrogen detection and a classical cytotoxic compound detection assay for the antibiotic Zeocin. Furthermore, the use of self-directed bioluminescent initiation in response to target detection was assessed to determine its amenability towards deployment as fully autonomous sensors. In all cases, bioluminescent measurements were supported with traditional genetic and transcriptomic evaluations.Conclusions/SignificanceOur results demonstrate that the viral 2A-linked, humanized lux genetic architecture successfully produced autobioluminescent phenotypes in all cell lines tested without the induction of cytotoxicity. This autobioluminescent phenotype allowed for repeated interrogation of populations and self-directed control of bioluminescent activation with detection limits and EC50 values similar to traditional reporter systems, making the autobioluminescent cells amenable to automated monitoring and significantly reducing the time and cost required to perform bioluminescent workflows.

Anti-Quorum Sensing Activity of the Traditional Chinese Herb, Phyllanthus amarus

The discovery of quorum sensing in Proteobacteria and its function in regulating virulence determinants makes it an attractive alternative towards attenuation of bacterial pathogens. In this study, crude extracts of Phyllanthus amarus Schumach. & Thonn, a traditional Chinese herb, were screened for their anti-quorum sensing properties through a series of bioassays. Only the methanolic extract of P. amarus exhibited anti-quorum sensing activity, whereby it interrupted the ability of Chromobacterium violaceum CVO26 to response towards exogenously supplied N-hexanoylhomoserine lactone and the extract reduced bioluminescence in E. coli [pSB401] and E. coli [pSB1075]. In addition to this, methanolic extract of P. amarus significantly inhibited selected quorum sensing-regulated virulence determinants of Pseudomonas aeruginosa PA01. Increasing concentrations of the methanolic extracts of P. amarus reduced swarming motility, pyocyanin production and P. aeruginosa PA01 lecA∷lux expression. Our data suggest that P. amarus could be useful for attenuating pathogens and hence, more local traditional herbs should be screened for its anti-quorum sensing properties as their active compounds may serve as promising anti-pathogenic drugs.

“Impossible Narrative Voices”:

“Impossible Narrative Voices”:

Autonomous Bioluminescent Expression of the Bacterial Luciferase Gene Cassette (lux) in a Mammalian Cell Line

BackgroundThe bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo.Methodology/Principal FindingsAutonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH2) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH2 supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background.Conclusions/SignificanceThe speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

Improved lux reporters for use in Staphylococcus aureus

The use of luxABCDE ( lux) offers certain advantages over other reporters, such as: lacZ and xylE. It is real time and its signal generation is produced without the requirement for any additional substrates. In some bacteria such as Staphylococcus spp, light production by luciferase is restricted because of a limited availability of endogenous substrates such as fatty acid aldehyde. We describe the construction of promoterless- lux cloning vectors, pGYlux and pAmilux. S. aureus carrying B. subtilis xyl/tetO promoter fused to the lux genes of pGYlux gave up to a 2.5-fold enhancement of luminescence over S. aureus carrying the xyl/tetO promoter fused to lux genes of the previously published parent vector pAL2. Furthermore, pAmilux showed a 6-fold enhancement of lux expression when compared to pGYlux in S. aureus. This was achieved by cloning the constitutive ami promoter upstream of the luxCDE genes to increase endogenous fatty acid aldehyde production while maintaining its reporter functionality by fusing promoters to the luxAB genes.

Effects of luxCDABEG induction in Vibrio fischeri: enhancement of symbiotic colonization and conditional attenuation of growth in culture

Production of bioluminescence theoretically represents a cost, energetic or otherwise, that could slow Vibrio fischeri growth; however, bioluminescence is also thought to enable full symbiotic colonization of the Euprymna scolopes light organ by V. fischeri. Previous tests of these models have proven inconclusive, partly because they compared nonisogenic strains, or undefined and/or pleiotropic mutants. To test the influence of the bioluminescence-producing lux operon on growth and symbiotic competence, we generated dark luxCDABEG mutants in strains MJ1 and ES114 without disrupting the luxR-luxI regulatory circuit. The MJ1 luxCDABEG mutant out-competed its visibly luminescent parent approximately 26% per generation in a carbon-limited chemostat. Similarly, induction of luminescence in the otherwise dim ES114 strain slowed growth relative to DeltaluxCDABEG mutants. Some culture conditions yielded no detectable effect of luminescence on growth, indicating that luminescence is not always growth limiting; however, luminescence was never found to confer an advantage in culture. In contrast to this conditional disadvantage of lux expression, ES114 achieved approximately fourfold higher populations than its luxCDABEG mutant in the light organ of E. scolopes. These results demonstrate that induction of luxCDABEG can slow V. fischeri growth under certain culture conditions and is a positive symbiotic colonization factor.

Bactofection of lung epithelial cells in vitro and in vivo using a genetically modified Escherichia coli.

Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.

Bioluminescence in Vibrio fischeri is controlled by the redox‐responsive regulator ArcA

Bioluminescence generated by the Vibrio fischeri Lux system consumes oxygen and reducing power, and it has been proposed that cells use this to counteract either oxidative stress or the accumulation of excess reductant. These models predict that lux expression should respond to redox conditions; yet no redox-responsive regulator of lux is known. We found that the luxICDABEG operon responsible for bioluminescence is repressed by the ArcAB system, which is activated under reducing conditions. Consistent with a role for ArcAB in connecting redox monitoring to lux regulation, adding reductant decreased luminescence in an arc-dependent manner. ArcA binds to and regulates transcription from the luxICDABEG promoter, and it represses luminescence both in the bright strain MJ1 and in ES114, an isolate from the squid Euprymna scolopes that is not visibly luminescent in culture. In ES114, deleting arcA increased luminescence in culture approximately 500-fold to visible levels comparable to that of symbiotic cells. ArcA did not repress symbiotic luminescence, but by 48 h after inoculation, ArcA did contribute to colonization competitiveness. We hypothesize that inactivation of ArcA in response to oxidative stress during initial colonization derepresses luxICDABEG, but that ArcAB actively regulates other metabolic pathways in the more reduced environment of an established infection.

475. SeV-Mediated Gene Transfer after Three Re-Administrations in Mice and Sheep

Recombinant Sendai-virus (SeV) vector transduces airway epithelium efficiently via the apical membrane and leads to high, but transient gene expression. The treatment of chronic diseases may require repeated administration of the virus. We have previously shown that three repeat administrations of recombinant Sendai virus (DF/SeV) did not lead to measurable luciferase (lux) expression after the third administration. However, as part of these experiments we noted that even after one administration, lux expression was lower than expected for a given virus dose.

842. Repeated Administration of Sendai Virus into Lung

Recombinant Sendai virus (SeV) is currently the most efficient vector for airway gene transfer. We have previously shown that after a second administration of transmission incompetent F protein-deficient DF/SeV to the lung gene expression was reduced by 60% when compared to a single administration, but was still significantly higher than in untreated mice. Here, we assessed, if this residual level is maintained after further virus administrations. Mice received three administrations one week apart of DF/SeVMtsHNtsPLmut, an F-deficient virus with mutations in the M, HN, P and L protein, that reduce cytotoxicity in vitro (week 1: SeV-GFP, week 2: SeV-GFP, week 3: SeV-lux). Because the immune response, might be depend on virus titre, individual cohorts of animals were treated with either 106, 107 or 108 pfu/mouse (n=20/dose). Luciferase (lux) expression was measured two days after SeV-lux administration. Importantly, repeat administration of DF/SeVMtsHNtsPLmut was well tolerated even at the highest titre. However, Lux expression after three administrations of SeV was significantly reduced compared to single administration, regardless of the virus titre used. A similar study in sheep has been instigated and will serve to indicate whether expression is similarly attenuated in a host in which wild type SeV infection is of no apparent significance. As a prelude to repeat administration the duration of expression after single administration was eastablished. We, therefore, aerosolised sheep with DF/SeV-lacZ (1010 pfu/sheep, n=3) using a Trudell AeroProbe catheter and compared bgal expression in bronchial biopsies (Bx, n=4/sheep from different lobes) and bronchial brushings (Br, n=4/sheep from different lobes) pre-and two days post-transfection. Despite the comparatively small amount of tissue sample bgal expression was significantly (p<0.05) increased post transfection (Bx pre: 2.1|[plusmn]|0.8, post: 253|[plusmn]|9.8, Br pre: 2.6|[plusmn]|1.2, post: 102.5|[plusmn]|72.6 LU/mg protein). We also assessed shedding of infective virus particles in oral and nasal swabs, broncho-alveolar lavage fluid (BALF) and lung tissue and importantly, did not detect any virus shedding. A second cohort of sheep was nebulised in a silimalar manner (1010 pfu/sheep, n=3) and Br, Bx, lavage fluid were collected and blood 2, 7, 14, 21 and 28 days after transfection to determine the dosing interval for repeat administration. At present only the safety data are available. DF/SeV transfection was well tolerated. There was no SeV-related increase in body temperature, or total and differential blood cell counts. However, serum haptoglobin, an inflammatory marker, increased 7 days after transfection, but had returned to baseline values by day 14 (pre: 0, day 7: 2.8|[plusmn]|0.2, day 14: 0.5|[plusmn]|0.3). Further histological and BALF analysis and bgal quantification to determine the time-course of SeV transfection in sheep is currently underway. These studies will inform repeat dosing intervals in the sheep.

356. Sonoporation Increases Non-Viral Gene Transfer to the Murine Lung

Related to our programme of cystic fibrosis gene therapy, we assessed whether ultrasound (U/S) can increase non-viral lung transfection. U/S penetration in the air-filled lung is limited due to significant energy losses at the air/tissue interfaces. Thus, using an ex vivo rat lung model, we have shown that more than 99% of the U/S energy is lost. Here, we assessed, whether the remaining energy that penetrates the tissue is sufficient to increase non-viral gene transfer. Naked plasmid DNA (pDNA) and polyethylenimine (PEI)/pDNA complexes (100 |[mu]|g pDNA and 10 |[mu]|g pDNA, respectively) were administered to Balb/c mice via intranasal instillation, in conjunction with Optison (1:1 v:v), an U/S contrast agent shown to improve sonoporation (formation of transient pores). U/S (1 MHz, 20% duty cycle, 3 W/cm2) was then applied externally onto the chest and the back. For naked pDNA, a 2-min U/S exposure, led to a significant 6-fold increase in luciferase (Lux) expression (no U/S: 1.0|[plusmn]|0.2 RLU/mg protein, 2-min U/S: 6.3|[plusmn]|0.8 RLU/mg protein; p<0.001; n=8). This was further enhanced (4-fold) after a 20-min U/S exposure (28.0|[plusmn]|7.8 RLU/mg protein; p<0.05, compared to 2-min U/S; n=8). The positive U/S effect on naked pDNA is, however, dependent on the presence of Optison. Without it even a 20-min U/S exposure has no effect (naked pDNA: 4.1|[plusmn]|0.8 RLU/mg protein, pDNA + 20-min U/S: 6.1|[plusmn]|0.8 RLU/mg protein; n=6-10). U/S exposure did not increase PEI/pDNA transfection efficiency, suggesting that U/S has a vector-specific effect (no U/S: 3.1|[plusmn]|0.9 RLU/mg protein, 20-min U/S: 3.0|[plusmn]|0.5 RLU/mg protein; n=8). While the use of Optison was not detrimental to naked pDNA gene transfer in the absence of U/S (pDNA alone: 0.9|[plusmn]|0.3 RLU/mg protein, pDNA+Optison: 1.0|[plusmn]|0.2 RLU/mg protein; n=8), when added to PEI/pDNA complexes it resulted in an 8-fold decrease in gene expression (PEI/pDNA: 24.7|[plusmn]|10.4 RLU/mg protein, PEI/pDNA+Optison: 3.1|[plusmn]|0.9 RLU/mg protein; p<0.01; n=8), again emphasising vector-specific effects. To reduce U/S energy losses and thus attempt to enhance transfection efficiency further, we are also investigating the use of perfluorocarbon (PFC) liquid. Ex vivo measurements in rat lungs showed that PFC allows 20-30% of U/S energy to penetrate the lung tissue. Magnetic resonance imaging is currently being used to determine the distribution, and the attenuation time of PFC in the lung of spontaneously breathing mice, to indicate the optimal conditions for U/S application for future gene transfer studies.

473. Magnetofection To Enhance Airway Gene Transfer

Gene delivery to the airway epithelium (AE) is very inefficient due to many extracellular barriers such as mucus, cilia, glycocalyx, and in the case of Cystic Fibrosis (CF) a thick layer of sputum. In an attempt to overcome these we assessed “magnetofection”, a method shown to increase transfection efficiency in vitro and in gut epithelium in vivo. This requires the formation of magnetic gene transfer complexes by coupling superparamagnetic iron oxide nanoparticles to vectors, which are then attracted to strong magnets. Magnetofection is thought to increase the contact time and the local concentration of the vectors on the cell surface, thus increasing gene transfer efficiency. A variety of magnetic particles have been described. Here we used TransMAGPEI (Chemicell, Germany), a polyethylenimine (PEI) – coated iron oxide particle, complexed to Genzyme Lipid 67 (GL67), which has already been used in several clinical trials. GL67 was mixed with luciferase plasmid DNA (pLux) at a previously optimised molar ratio of 1:4 (GL67:pLux). This complex was then coupled to TransMAGPEI at 1:1 up to 4:1 w/w ratios (transMAGPEI:pLux) in the presence of 0.9% NaCl. The complexes were tested in C127 cells (mouse mammary epithelial cells) in vitro (0.2 μg pDNA/well in 96-well plate). After a 15 min exposure to the magnet, lux expression was increased up to 3-fold (magnet: 45,199 ± 11,063 RLU/mg protein, no magnet: 14,102 ± 3,278 RLU/mg protein; p < 0.05; n = 6). These complexes were also tested in mouse nasal epithelium in vivo. We complexed our standard vector dose (80 μg/100 μl of GL67/pLux) to TransMAGPEI at a 4:1 w/w ratio (TransMAGPEI:pLux). The complexes were slowly perfused (6.7 μl/min) into the nasal cavity via a thin catheter placed 2.5 mm into the nostrils of anaesthetised mice. However, at this concentration the formulation precipitated resulting in a ~50-fold decrease in gene expression, when compared to the standard GL67 formulation without addition of magnetic particles (TransMAGPEI: 0.9 ± 0.7 RLU/mg protein, no TransMAGPEI: 47.7 ± 4.1; p < 0.01; n = 8). Exposure of the nostril to the magnet (NeoDelta circular disc attached to an AlNiCo rod magnet, IBS Magnet, Germany) for 15 min during perfusion did not increase gene expression. We attempted to overcome the precipitation problem by changing the ratio of TransMAGPEI to pDNA, the total volume and the mixing order. The optimised formulation (TransMAGPEI-GL67/pLux 80 μg/500 μl; 1:1 w/w TransMAGPEI:pDNA) did not precipitate and gave a 6-fold increase in gene expression in vitro in the presence of the magnet. However, Lux expression in the nasal tissue after in vivo transfection was still reduced compared to GL67/pDNA alone (TransMAGPEI: 1.3 ± 0.8 RLU/mg protein, GL67/pDNA: 26.3 ± 6.6 RLU/mg protein; p < 0.01; n = 8). Again, exposure to the magnetic field during perfusion did not make a difference. Although TransMAGPEI in combination with magnetic forces increased gene transfer in vitro, it did not improve transfection efficiency of GL67/pDNA in airway epithelial cells in vivo.

935. Magnetofection and Ultrasound To Increase “Naked” DNA Delivery to the Myocardium

We, and other have previously shown that using optimised formulations adenovirus (Ad) is several logs more efficient in myocardial gene transfer than “naked” plasmid DNA (pDNA). Here, we evaluated “magnetofection”, the use of magnetic force acting on vectors complexed to magnetic particles and ultrasound to increase non-viral gene transfer to the murine myocardium. C57BL6/J mice were intubated and a thoracotomy performed to expose the heart. The ability of the magnetic force to pass through tissue was tested ex vivo on a dissected lamb's heart. Iron filings sprinkled on the endocardial site moved and clustered together when the magnet was placed on the epicardial side of the left myocardial wall (n=3). A variety of magnetic particles have been described, here we used TransMAGPEI (Chemicell, Germany), a polyethyleneimine (PEI)–coated iron oxide particle. For the myocardial injection the total volume could not exceed 10 μl. The magnetic particle:DNA complexes were carefully optimised by altering the TransMAGPEI to pDNA w/w and v/v ratios to avoid precipitation. The complexes used for in vivo injections were made in the following way: 0.8 μl TransMAGPEI (60 μg/μl) was diluted with 41.7 μl water and mixed with 5 μl pDNA (5 μg/μl) carrying a luciferase reporter gene (pLux). 2.5 μl 0.9% NaCl (18%) was added after 15 minutes. 10 μl of this formulation containing 5 μg pLux were injected into the left ventricular wall. The hearts were exposed to the magnet (AlNiCo Rod magnet plus NeoDelta magnet, IBS) for 5 min straight after injection and for a further 10 min after re-sutering of the chest. The left ventricular wall was harvested 48 hrs after gene transfer and lux expression was measured. Although magnet exposed hearts expressed more lux than the non-magnet exposed heart, this difference was not significant (magnet: 268±124 RLU/mg protein, no magnet: 94.0±41.8 RLU/mg protein, n=8-10/group). It has previously been shown that ultrasound (US) enhances non-viral gene transfer in skeletal muscle probably through acoustic cavitation of the cell membrane. Here, US was applied for 5 minutes before and after myocardial injection of 10 μg pLux in 10 μl total volume containg 1:1 v/v Opitson microbubbles. For this purpose the chest cavity was filled with gel (Henleys Medical, Welwyn Garden City, UK) and a 0.5 cm2 probe massaged over the heart area. The Medi-Link system machine (Electro-Medical Supplies Ltd, Wantage, UK) was set to deliver 1MHz, 2 W/cm2 at 100% duty cycle continous wave. 48 hrs after gene transfer the left vetricular wall was harvested and lux expression measured. In a parallel experiment we demonstrated that ultrasound imaging (14 MHz) using a gel filled chest cavity is feasible and therefore concluded that ultrasound waves were reaching the heart. However, the US conditions described above did not increase lux expression in the mouse heart (US: 71.8±38.4 RLU/mg protein, no US:138±69.2 RLU/mg protein, n=10/group). In conclusion, although physical energy may be useful in improving non-viral gene transfer to the myocardium further optimisation of these techniques will be required.