Luc Expression Research Articles

Expression of Long Anti-HIV-1 Hairpin RNAs for the Generation of Multiple siRNAs: Advantages and Limitations

Promoter expressed long-hairpin RNAs (lhRNAs) that can be processed into multiple small interfering RNA (siRNAs) are being considered as effective agents for treating rapidly mutating viruses such as human immunodeficiency virus (HIV). In the present study, we have generated human U6 promoter-driven lhRNAs of 50, 53, and 80 base pairs (bp) targeting contiguous sequences within the tat and rev genes of HIV-1 and evaluated the efficacy of these lhRNAs as well as their processing in cells. By using multiple G:U mismatches in the stems, we have been able to readily incorporate the long-hairpin structures into a lentiviral vector transduction system. Here we show that such long hairpins can be stably and functionally expressed for a long term in HIV-1 susceptible T cells, where they provide potent inhibition of HIV replication against both non-mutant and mutant variants of HIV-1. Our studies provide strong support for the use of the G:U wobble pair containing lhRNAs to generate multiple siRNAs from a single transcript, but we also show that lhRNAs of 80 bp may be the upper size limit for effectively producing multiple, functional siRNAs.

Cre‐loxP system as a versatile tool for conferring increased levels of tissue‐specific gene expression from a weak promoter

Attempts to image reporter gene expression driven by weak promoters are often hampered by the poor transcriptional activity of such promoters. Most tissue-specific promoters are weak compared with stronger but constitutively expressing viral promoters. In this study, we validated methods of enhancing the transcriptional activity of weak promoters using a Cre-loxP system in vitro and in vivo. We constructed a tester vector, pCTL, which carries a strong systemic cytomegalovirus enhancer/chicken beta-actin promoter (CAG), loxP-flanked CAT, and firefly luciferase (luc) cDNAs. Herpes simplex virus-thymidine kinase (HSV-tk) promoter was used as a weak and systemic promoter and ligated to Cre for construction of pTC. Luc activity was higher (about 10-fold enhancement) in co-transfected (with pCTL and pTC) than in singly (with HSV-tk promoter-driven luc expression vector pTL) transfected NIH3T3 cells. In vivo electroporation-mediated gene delivery of both pCTL and pTC into murine oviductal epithelium yielded results (about 16-fold enhancement) similar to those obtained with in vitro-transfected NIH3T3 cells. To evaluate tissue-specific enhancement of gene expression, podocyte (glomerular visceral epithelial cell)-specific nephrin promoter was ligated to the Cre gene or luc cDNA to create pNC and pNL, respectively. We achieved 2.4-fold improvement of luc gene expression in the mouse kidney in vivo when pCTL and pNC were co-transfected via the tail vein via the lipoplex method. The combination of a weak tissue-specific promoter with the Cre-loxP system could thus be used to enhance the strength of tissue-specific promoters in vitro and in vivo.

Ultrasound-based nonviral gene delivery induces bone formation in vivo

Nonviral gene delivery is a promising, safe, therapeutic tool in regenerative medicine. This study is the first to achieve nonviral, ultrasound-based, osteogenic gene delivery that leads to bone tissue formation, in vivo. We hypothesized that direct in vivo sonoporation of naked DNA encoding for the osteogenic gene, recombinant human bone morphogenetic protein-9 (rhBMP-9) would induce bone formation. A luciferase plasmid (Luc), encoding rhBMP-9 or empty pcDNA3 vector mixed with microbubbles, was injected into the thigh muscles of mice. After injection, noninvasive sonoporation was applied. Luc activity was monitored noninvasively, and quantitatively using bioluminescence imaging in vivo, and found for 14 days with a peak expression on day 7. To examine osteogenesis in vivo, rhBMP-9 plasmid was sonoporated into the thigh muscles of transgenic mice that express the Luc gene under the control of a human osteocalcin promoter. Following rhBMP-9 sonoporation, osteocalcin-dependent Luc expression lasted for 24 days and peaked on day 10. Bone tissue was formed in the site of rhBMP-9 delivery, as was shown by micro-computerized tomography and histology. The sonoporation method was also compared with previously developed electrotransfer-based gene delivery and was found significantly inferior in its efficiency of gene delivery. We conclude that ultrasound-mediated osteogenic gene delivery could serve as a therapeutic solution in conditions requiring bone tissue regeneration after further development that will increase the transfection efficiency.

In Situ Pulmonary Localization of Air Pollution Particle-induced Oxidative Stress

Exposure to air particulate matter (PM) may be associated with increased morbidity and mortality. An improved understanding of the mechanism(s) by which PM induces adverse effects is needed. This preliminary study examined the ability to use unique bioluminescent technologies to identify acute localized areas of residual oil fly ash (ROFA)-induced, oxidative lung injury. Transgenic mice, in which luciferase (luc) expression was regulated by the heme oxygenase (HO)-1 promoter, were exposed by pharyngeal aspiration to either saline or 50 μg ROFA/mouse. HO-1-luc expression was determined at 2, 6, 12, and 24 h postexposure using luminescent quantification and Western blot analysis of lung protein extracts, as well as with a novel in situ pulmonary bioluminescence imaging approach. The different approaches for the detection of luciferase in lung protein extracts recovered from ROFA exposed HO-1-luc transgenic mice gave variable results. Pulmonary homogenate HO-1-luc levels were increased at 2 h and 24 h postexposure to ROFA when examined by chemilumescent and Western blot analyses, respectively. In situ bioluminescent imaging of pulmonary tissue sections detected ROFA-induced pulmonary luciferase expression by identifying highly localized increases in HO-1-luc expression at 12 h and 24 h postexposure. These results suggest that the variability observed in the methods of detection for luciferase may be due to a localization of cells expressing luciferase within tissue samples, demonstrating that the HO-1-luc transgenic mouse model is the preferred method to detect and pinpoint in vivo particle-induced, oxidative lung injury. The feasibility of using this in situ approach is a unique proof-of-concept application for the identification of localized sites of oxidative injury induced by environmental pollutants.

Transcriptional Specificity of Drosophila Dysfusion and the Control of Tracheal Fusion Cell Gene Expression

The Drosophila Dysfusion basic-helix-loop-helix-PAS (bHLH-PAS) protein controls the transcription of genes that mediate tracheal fusion. Dysfusion is highly related to the mammalian Nxf protein that has been implicated in nervous system gene regulation. Toward the goal of understanding how Dysfusion controls fusion cell gene expression, the biochemical properties of Dysfusion were investigated using protein interaction experiments, cell culture-based transcription assays, and in vivo transgenic analyses. Dysfusion dimerizes with the Tango bHLH-PAS protein, and together they act as a DNA binding transcriptional activator. Dysfusion/Tango binds multiple NCGTG binding sites, with the following preference: TCGTG > GCGTG > ACGTG > CCGTG. This binding site promiscuity differs from the restricted binding site preferences of other bHLH-PAS/Tango heterodimers. However, it is identical to the binding site preferences of mammalian Nxf/Arnt, indicating that the specificity is evolutionarily conserved. Germ line transformation experiments using a fragment of the CG13196 Dysfusion target gene allowed identification of a fusion cell enhancer. Experiments in which NCGTG sites were mutated individually and in combination revealed that TCGTG sites were required for fusion cell expression but that the single ACGTG and GCGTG sites present were not. Finally, a reporter transgene containing four tandemly arranged TCGTG elements has strong expression in tracheal fusion cells. Transgenic misexpression of dysfusion further revealed that Dysfusion has the ability to activate transcription in multiple cell types, although it does this most effectively in tracheal cells and can only function at mid-embryogenesis and later.

E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells

The pituitary gland's ability to respond to the hypothalamic hormone GnRH (gonadotropin-releasing hormone) depends directly on the gonadotrope-specific expression of the GnRH receptor (GnRHR), a G-protein coupled transmembrane protein coded by the GnRHR gene. In the present study, we have investigated the potential regulatory role of seven noncanonical E-box enhancer sequences within the 856 bp proximal 5′-flanking region of the mGnRHR gene in regulating transcription. These sequences are known to mediate the action of clock gene proteins on the expression of a diverse array of genes both central and peripheral. In the present studies the expression of all of the cognate clock genes was identified in the αT3-1 gonadotrope cell line. Additionally, luteinizing hormone-immunoreactive cells in the adult rodent pituitary gland were also shown to co-express the PERIOD-1 protein. By means of chromatin immunoprecipitation of αT3-1 nuclear extracts we were able to capture promoter fragments of the GnRHR and Period-1 genes, indicating that E-boxes in these promoters bind the CLOCK protein. RNA interference experiments with αT3-1 cells in which Bmal1 expression was attenuated also confirmed the involvement of E-boxes in transcriptional regulation of the mGnRHR gene. Subsequent luciferase reporter assay experiments with GnRHR constructs possessing intact or mutated E-boxes confirmed the use of these sequences for the regulation of mGnRH-R/ luc expression. Transient overexpression of the dominant negative E-box-binding factor CLOCK-Δ19, or the inhibitory clock protein mPER1, markedly reduced CLOCK/BMAL1-driven mGnRH-R/ luc expression in a dose-dependent fashion. Our data implicate the clock genes as important factors controlling GnRHR expression in murine gonadotrope cells.

Efficient and Stable Transgene Expression in Human Embryonic Stem Cells Using Transposon-Mediated Gene Transfer

Efficient and stable genetic modification of human embryonic stem (ES) cells is required to realize the full scientific and potential therapeutic use of these cells. Currently, only limited success toward this goal has been achieved without using a viral vector. The Sleeping Beauty (SB) transposon system mediates nonviral gene insertion and stable expression in target cells and tissues. Here, we demonstrate use of the nonviral SB transposon system to effectively mediate stable gene transfer in human ES cells. Transposons encoding (a) green fluorescent protein coupled to the zeocin gene or (b) the firefly luciferase (luc) gene were effectively delivered to undifferentiated human ES cells with either a DNA or RNA source of transposase. Only human ES cells cotransfected with transposon- and transposase-encoding sequences exhibited transgene expression after 1 week in culture. Molecular analysis of transposon integrants indicated that 98% of stable gene transfer resulted from transposition. Stable luc expression was observed up to 5 months in human ES cells cotransfected with a transposon along with either DNA or RNA encoding SB transposase. Genetically engineered human ES cells demonstrated the ability to differentiate into teratomas in vivo and mature hematopoietic cells in vitro while maintaining stable transgene expression. We conclude that the SB transposon system provides an effective approach with several advantages for genetic manipulation and durable gene expression in human ES cells.

Altered ABA, proline and hydrogen peroxide in an Arabidopsis glutamate:glyoxylate aminotransferase mutant

Plant responses to abiotic stress are determined both by the severity of the stress as well as the metabolic status of the plant. Abscisic acid (ABA) is a key component in integrating these various signals and controlling downstream stress responses. By screening for plants with decreased RD29A:LUC expression, we isolated two alleles, glutamate:glyoxylate transferase1-1 (ggt1-1) and ggt1-2, of a mutant with altered ABA sensitivity. In addition to reduced ABA induction of RD29A, ggt1-1 was altered in ABA and stress regulation of Delta1-pyrroline-5-carboxylate synthase, proline dehydrogenase and 9-cis-epoxycarotenoid dioxygenase 3, which encode enzymes involved in Pro and ABA metabolism, respectively. ggt1-1 also had altered ABA and Pro contents after stress or ABA treatments while root growth and leaf water loss were relatively unaffected. A light-dependent increase in H2O2 accumulation was observed in ggt1-1 consistent with the previously characterized role of GGT1 in photorespiration. Treatment with exogenous H2O2, as well as analysis of a mutant in nucleoside diphosphate kinase 2 which also had increased H2O2 content but is not involved in photorespiration or amino acid metabolism, demonstrated that the greater ABA stimulation of Pro accumulation in these mutants was caused by altered H2O2 content as opposed to other metabolic changes. The results suggest that metabolic changes that alter H2O2 levels can affect both ABA accumulation and ABA sensitivity.

Testing of internal translation initiation via dicistronic constructs in yeast is complicated by production of extraneous transcripts

Introduction of sequences of interest into an intercistronic spacer (ICS) of dual reporter plasmids is the main experimental set-up used to identify and study internal ribosome entry sites (IRESs). We studied internal initiation of translation in yeast using the dicistronic approach. Three viral sequences and a polylinker-derived reference sequence were inserted into the ICS of a dual reporter plasmid upstream of the firefly luciferase gene, luc. LUC expression was taken as a putative indication of internal translation initiation from the studied sequences. Interestingly, all sequences mediated 3′ LUC expression. However, northern blot analysis revealed that in addition to the dicistronic mRNAs, transcripts containing only the LUC-encoding sequences were produced from the plasmids. Electroporation studies with in vitro synthesized mRNAs showed that expression from the 3′ cistron of the dicistronic mRNAs was below the level of detection. This suggested that the observed LUC expression from yeast transformed with the dicistronic expression plasmids did not originate from dicistronic messages. Deletion of the promoter increased 3′ LUC expression. Similarly, repression of transcription prevented 5′ cistron expression whereas 3′ LUC expression was stimulated. These results suggested that the observed LUC expression did not result from partially degraded or spliced dicistronic RNAs but rather from transcripts synthesized from cryptic promoters. Despite high LUC activity, northern blot analysis detected few transcripts from yeast transformed with the promoterless constructs. Therefore, our data indicate that the functional assay is more revealing than RNA analysis in the case of very sensitive reporter genes generally used in IRES studies. Furthermore, our studies show that there is a clear need for detailed analysis prior to concluding the mechanism of 3′ cistron expression.

Control of gag-pol gene expression in the Candida albicans retrotransposon Tca2

BackgroundIn the C. albicans retrotransposon Tca2, the gag and pol ORFs are separated by a UGA stop codon, 3' of which is a potential RNA pseudoknot. It is unclear how the Tca2 gag UGA codon is bypassed to allow pol expression. However, in other retroelements, translational readthrough of the gag stop codon can be directed by its flanking sequence, including a 3' pseudoknot.ResultsThe hypothesis was tested that in Tca2, gag stop codon flanking sequences direct translational readthrough and synthesis of a gag-pol fusion protein. Sequence from the Tca2 gag-UGA-pol junction (300 nt) was inserted between fused lacZ and luciferase (luc) genes in a Saccharomyces cerevisiae dual reporter construct. Although downstream of UGA, luc was expressed, but its expression was unaffected by inserting additional stop codons at the 3' end of lacZ. Luc expression was instead being driven by a previously unknown minor promoter activity within the gag-pol junction region. Evidence together indicated that junction sequence alone cannot direct UGA readthrough. Using reporter genes in C. albicans, the activities of this gag-pol junction promoter and the Tca2 long terminal repeat (LTR) promoter were compared. Of the two promoters, only the LTR promoter was induced by heat-shock, which also triggers retrotransposition. Tca2 pol protein, epitope-tagged in C. albicans to allow detection, was also heat-shock induced, indicating that pol proteins were expressed from a gag-UGA-pol RNA.ConclusionThis is the first demonstration that the LTR promoter directs Tca2 pol protein expression, and that pol proteins are translated from a gag-pol RNA, which thus requires a mechanism for stop codon bypass. However, in contrast to most other retroelement and viral readthrough signals, immediate gag UGA-flanking sequences were insufficient to direct stop readthrough in S. cerevisiae, indicating non-canonical mechanisms direct gag UGA bypass in Tca2.

Lack of Noggin Expression by Cancer Cells Is a Determinant of the Osteoblast Response in Bone Metastases

Prostate and mammary cancer bone metastases can be osteoblastic or osteolytic, but the mechanisms determining these features are unclear. Bone morphogenetic and Wnt proteins are osteoinductive molecules. Their activity is modulated by antagonists such as noggin and dickkopf-1. Differential expression analysis of bone morphogenetic and Wnt protein antagonists in human prostate and mammary cancer cell lines showed that osteolytic cell lines constitutively express in vitro noggin and dickkopf-1 and at least one of the osteolytic cytokines parathyroid hormone-related protein, colony-stimulating factor-1, and interleukin-8. In contrast, osteoinductive cell lines express neither noggin nor dickkopf-1 nor osteolytic cytokines in vitro. The noggin differential expression profile observed in vitro was confirmed in vivo in prostate cancer cell lines xenografted into bone and in clinical samples of bone metastasis. Forced noggin expression in an osteoinductive prostate cancer cell line abolished the osteoblast response induced in vivo by its intraosseous xenografts. Basal bone resorption and tumor growth kinetics were marginally affected. Lack of noggin and possibly dickkopf-1 expression by cancer cells may be a relevant mechanism contributing to the osteoblast response in bone metastases. Concomitant lack of osteolytic cytokines may be permissive of this effect. Noggin is a candidate drug for the adjuvant therapy of bone metastasis.

Analysis of genetic elements regulating the methionine adenosyltransferase gene in Leishmania infantum

Methionine adenosyltransferase (MAT) is an important enzyme for metabolic processes, inasmuch as its product, S-adenosylmethionine (AdoMet), plays a key role in trans-methylation, trans-sulphuration and polyamine synthesis. Our prior studies have shown that the Leishmania infantum genome contains two identical copies of the gene encoding MAT ( MAT2 gene), arranged in head-to-tail configuration and alternating with another gene, called LORIEN that contains a zinc-finger motif. Both genes are constitutively expressed throughout the promastigote stage of the parasite cell cycle, and their flanking regions were detected by RT-PCR. Luciferase (luc) reporter assays indicated the presence of regulatory elements within the MAT2 3′UTR and intergenic region, and fragments responsible for such regulation were identified by deletional analysis. By site-directed mutagenesis of the wild-type − 42 AG recognized in the trans-splicing of the MAT2 gene, the AG slightly downstream (position − 36) was observed to be able to generate the same levels of luc expression, thus suggesting that potentially this gene has alternative spliced leader acceptor sites. The stability of MAT2 and LORIEN transcripts was very similar in both logarithmic and stationary phases. However, cycloheximide (CHX) inhibition of protein synthesis increased MAT2 and LORIEN mRNA levels in the logarithmic phase only, an indication that these genes are regulated in promastigotes at the post-transcriptional level by protein factors that targets both transcripts for degradation. However, during the stationary phase, another CHX-independent factor also led to MAT2 and LORIEN mRNAs degradation, indicating the existence of different mechanisms operating on the post-transcriptional regulation of these two genes.

In vivo gene delivery and expression by bacteriophage lambda vectors

AimsBacteriophage vectors have potential as gene transfer and vaccine delivery vectors because of their low cost, safety and physical stability. However, little is known concerning phage-mediated gene transfer in mammalian hosts. We therefore performed experiments to examine phage-mediated gene transfer in vivo.Methods and ResultsMice were inoculated with recombinant lambda phage containing a mammalian expression cassette encoding firefly luciferase (luc). Efficient, dose-dependent in vivo luc expression was detected, which peaked within 24 h of delivery and declined to undetectable levels within a week. Display of an integrin-binding peptide increased cellular internalization of phage in vitro and enhanced phage-mediated gene transfer in vivo. Finally, in vivo depletion of phagocytic cells using clodronate liposomes had only a minor effect on the efficiency of phage-mediated gene transfer.ConclusionsUnmodified lambda phage particles are capable of transducing mammalian cells in vivo, and may be taken up – at least in part – by nonphagocytic mechanisms. Surface modifications that enhance phage uptake result in more efficient in vivo gene transfer.Significance and Impact of the StudyThese experiments shed light on the mechanisms involved in phage-mediated gene transfer in vivo, and suggest new approaches that may enhance the efficiency of this process.

Differential Stability of Thymidylate Synthase 3′-Untranslated Region Polymorphic Variants Regulated by AUF1

A 6-nucleotide insertion (I)/deletion (D) polymorphism in the 3'-untranslated region of the thymidylate synthase gene was shown to influence mRNA stability, but the molecular basis of this effect has not been elucidated. Here, studies of both endogenous and ectopically expressed thymidylate synthase alleles revealed that the mRNA-binding, decay-promoting protein AUF1 has higher affinity for allele D mRNA. AUF1 overexpression preferentially suppressed D allele mRNA levels, whereas AUF1 silencing selectively elevated D allele mRNA levels. Our results illustrate the functional consequences of ribonucleoprotein associations involving a polymorphic RNA sequence and uncover a novel mechanism of action for non-coding RNA polymorphisms.

Functional analysis of the sea urchin‐derived arylsulfatase (Ars)‐element in mammalian cells

An insulator is a DNA sequence that has both enhancer-blocking activity, through its ability to modify the influence of neighboring cis-acting elements, and a barrier function that protects a transgene from being silenced by surrounding chromatin. Previously, we isolated and characterized a 582-bp-long element from the sea urchin arylsulfatase gene (Ars). This Ars-element was effective in sea urchin and Drosophila embryos and in plant cells. To investigate Ars-element activity in mammalian cells, we placed the element between the cytomegalovirus enhancer and a luciferase (luc) expression cassette. In contrast to controls lacking the Ars-element, NIH3T3 and 293T cells transfected with the element-containing construct displayed reduced luciferase activities. The Ars-element therefore acts as an enhancer-blocking element in mammalian cells. We assessed the barrier activity of the Ars-element using vectors in which a luc expression cassette was placed between two elements. Transfection experiments demonstrated that luc activity in these vectors was approximately ten-fold higher than in vectors lacking elements. Luc activities were well maintained even after 12 weeks in culture. Our observations demonstrate that the Ars-element has also a barrier activity. These results indicated that the Ars-element act as an insulator in mammalian cells.

Dexamethasone inhibits the action of TNF on ENaC expression and activity

We have reported that TNF, a proinflammatory cytokine present in several lung pathologies, decreases the expression and activity of the epithelial Na(+) channel (ENaC) by approximately 70% in alveolar epithelial cells. Because dexamethasone has been shown to upregulate ENaC mRNA expression and is well known to downregulate proinflammatory genes, we tested if it could alleviate the effect of TNF on ENaC expression and activity. In cotreatment with TNF, we found that dexamethasone reversed the inhibitory effect of TNF and upregulated alpha, beta, and gammaENaC mRNA expression. When the cells were pretreated for 24 h with TNF before cotreatment, dexamethasone was still able to increase alphaENaC mRNA expression to 1.8-fold above control values. However, in these conditions, beta and gammaENaC mRNA expression was reduced to 47% and 14%, respectively. The potential role of TNF and dexamethasone on alphaENaC promoter activity was tested in A549 alveolar epithelial cells. TNF decreased luciferase (Luc) expression by approximately 25% in these cells, indicating that the strong diminution of alphaENaC mRNA must be related to posttranscriptional events. Dexamethasone raised Luc expression by fivefold in the cells and augmented promoter activity by 2.77-fold in cotreatment with TNF. In addition to its effect on alphaENaC gene expression, dexamethasone was able to maintain amiloride-sensitive current as well as the liquid clearance abilities of TNF-treated cells within the normal range. All these results suggest that dexamethasone alleviates the downregulation of ENaC expression and activity in TNF-treated alveolar epithelial cells.

Heterogeneous Nuclear Ribonucleoprotein-A2/B1 Modulate Collagen Prolyl 4-Hydroxylase, α (I) mRNA Stability

Collagen prolyl 4-hydroxylase (C-P4H) alpha-subunit is of regulatory importance in the assembling of C-P4H tetramers, which are necessary for the hydroxylation of procollagen chains. Change in collagen expression by hypoxia or iron diminishment is a significant issue in extracellular matrix remodeling. It was proposed that C-P4H-alpha (I) is regulated at the posttrancriptional level under these conditions. Here we report that the induction of C-P4H-alpha (I) in human fibrosarcoma cells HT1080 by the iron chelator 2,2-dipyridyl is predominantly caused by an enhancement of mRNA stability. This effect is mediated by an increased synthesis and binding of heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1, which interacts with a (U)(16) element located in the 3'-untranslated region of C-P4H-alpha (I) mRNA. Luciferase reporter gene assays depending on C-P4H-alpha (I) 3'-untranslated region and co-transfection with hnRNP-A2/B1 provide evidence that the (U)(16) element is necessary and sufficient for posttranscriptional control of C-P4H-alpha (I) synthesis under the analyzed conditions. Further indication for the significance of hnRNP-A2/B1 in C-P4H-alpha (I) induction was obtained by micro array experiments. In a data set representing 686 independent physiological conditions, we found a significant positive correlation between hnRNP-A2/B1 and C-P4H-alpha (I) mRNAs.

Hypersensitivity and Loss of Disease Site Targeting Caused by Antibody Responses to PEGylated Liposomes

The systemic application of nucleic acid drugs requires delivery systems that overcome the poor pharmacokinetics, limited biodistribution, and inefficient uptake of nucleic acids. PEGylated liposomes show considerable promise because of their intrinsic ability to accumulate at disease sites and facilitate transfection of target cells. Unlike many viral vectors, PEGylated liposomes are generally considered to be nonimmunogenic. We have developed a PEGylated liposome for the systemic administration of plasmid DNA that achieves high levels of selective gene expression at distal tumor sites. Here we report that the in vivo efficacy and safety of these systems can be severely compromised following repeat administration. This phenomenon is characterized by a loss of disease site targeting, accelerated clearance from the blood, and acute hypersensitivity. These effects are fully attributable to a surprisingly robust, long-lived antibody response generated against polyethylene glycol (PEG) that results from the strong adjuvant effect of the plasmid payload. Importantly, immunogenicity may be substantially reduced by modifying the alkyl chain of the PEG-lipid conjugate, thereby allowing successful repeat dosing of the modified plasmid formulations without adverse side effects. Immunogenicity is a relevant concern for a number of nonviral delivery systems given the potent immunostimulatory properties of many nucleic acid drugs.

95. Effective Genetic Engineering of Human Embryonic Stem Cells Using the Sleeping Beauty Transposon System

Human embryonic stem (ES) cells hold great promise for the study of human development and the generation of new therapeutic approaches to tissue regeneration and their genetic modification will play a key role in this development. While viral vectors have been readily used for gene transfer into human ES cells, only limited success has been achieved using non-viral vectors for this purpose. The Sleeping Beauty (SB) transposon system mediates gene insertion into chromosomes, providing a means of durable gene expression in target cells and tissues. To determine whether the SB transposon system can mediate stable gene expression in human ES cells, undifferentiated H9 human ES cells were nucleofected with an SB transposon encoding the firefly luciferase (luc) gene. The vector was designed to be transcriptionally regulated by promoters containing or depleted of CpG dinucleotides, and was delivered with or without a DNA or RNA source of transposase. Transfected human ES cells were maintained in culture, and luc expression was monitored by bioluminescence imaging. Only human ES cells cotransfected with transposon and transposase encoding sequences exhibited luc expression after 2 weeks. Importantly, we found that after 6 weeks, incorporation of a CpG-free promoter increased the average luc activity by 6- to 10-fold compared to a promoter containing CpG sequences. After 5 months, stable expression of luc was observed in human ES cells co-transfected with a transposon containing the CpG-free promoter along with transposase-encoding DNA or RNA. Human ES cells engineered with the CpG-free promoter demonstrated the ability to differentiate into hematopoietic cells in vitro and into teratomas in vivo and maintained luc expression with minimal incidence of epigenetic silencing. We conclude that the SB transposon system provides an effective approach for genetic manipulation of human ES cells and for durable expression using appropriately engineered transposon cargo. Proof-of-principal studies are currently in progress to test gain-of-function and loss-of-function outcomes using the SB system in human ES cells whereby any candidate gene can be evaluated for its role in the differentiation of human ES cells into committed lineages.

405. Correction of the Murine Model of Hereditary Tyrosinemia Type I Using Messenger RNA as a Source of Transposase for Sleeping Beauty Mediated Integration of the FAH Gene

Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the transposase component has been provided as a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to remobilization and reintegration of transposons. Such continued expression of transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of transposase, we have previously shown that in vitro transcribed transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of transposase- encoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time that transposase-encoding mRNA can be used to mediate non-viral gene therapy resulting in complete phenotypic correction that is stable and not associated with any incidents of cellular transformation.