IRES-based Vectors Research Articles

Bicistronic vector-based procedure to measure correlative expression and bacteriostatic activity of recombinant neutrophil gelatinase-associated lipocalin

Neutrophil gelatinase-associated lipocalin (NGAL) is a component of the human innate immune system that exerts bacteriostatic effects. Recent research suggests that NGAL is also a promising biomarker for tubular damage and aids in the early diagnosis of acute kidney injury. Therefore, the development of a method to efficiently produce recombinant NGAL has considerable clinical value. This study aimed to analyze the correlation between fluorescence reporter (enhanced green fluorescent protein, EGFP) intensity and NGAL levels, mediated by a bicistronic expression system based on internal ribosome entry sites (IRESs). During the 14-day culture period, EGFP intensity was positively correlated with NGAL concentrations. The purified protein also exerted bacteriostatic effects, interfering with bacterial iron uptake machinery during periods of iron starvation. The purified NGAL was also applied to generate a standard curve that could be used for enzyme-linked immunosorbent assay of samples containing unknown NGAL quantities. In conclusion, we successfully developed a procedure using EGFP and IRES-based vector to predict NGAL production levels and optimize protein harvest time. Our approach is also a simple and effective method for evaluating bioactivity.

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Pushing the haystack aside for efficient gene targeting in human cells.

Gene targeting, i.e., the disruption of a specific gene via homologous recombination, is one of the key techniques to study gene function. However, researchers have faced the difficulty that, in human cells, the frequency of correct targeted integration is quite low compared to non-targeted (or random) integration. Adachi and colleagues investigated the factors that influence the ratio of targeted integration to non-targeted integration.

Construction of the IRES-based Vector for Multiple Gene Co-expression

Construction of the IRES-based Vector for Multiple Gene Co-expression

0399 : IRESs as translational enhancers of gene expression in hypoxic cardiomyocytes

Ischemic heart failure is a pathology characterized by coronary arteries occlusion resulting in lack of oxygen and energy in heart muscle. In hypoxic tissues, mRNA translation is altered: global translation is blocked whereas a small number of messengers are still translated by an alternative mechanism involving RNA structure elements called internal ribosome entry sites (IRESs). Our laboratory has identified IRESs in mRNAs of several angiogenic or lymphangiogenic growth factors. In the present study, we have addressed the regulation of IRES-dependent translation by hypoxia in vitro in mouse cardiomyocytes HL-1. Bicistronic lentivectors expressing the two renilla and firefly luciferases separated by different IRESs were constructed and produced. Cardiomyocytes were transduced and submitted to hypoxic conditions. We have identified three groups of IRESs, activated in cardiomyocytes at 4h, 8h and 24h of hypoxia. To study translational regulation of gene expression in hypoxic cardiomyocytes, we purified polysomes containing translated RNAs from hypoxic cardiomyocytes and analysed gene expression using the Fluidigm deltagenes qPCR gene expression assay. Our data show that many angiogenic growth factors, except for VEGFA, are induced at translational but not at the transcriptional level. Especially, IRES containing mRNAs are associated to the polysomes of hypoxic cells, suggesting that the IRES-dependent mechanism is a crucial mechanism of response to stress in hypoxic heart tissue. The trans-acting factors responsible for IRES activation during hypoxia are under investigation. In addition, IRESs are also promising biotechnological tools to design gene transfer vectors able to co-express several therapeutic molecules. IRESs activated in ischemic conditions will be used in gene transfer IRES-based vectors to optimize expression of therapeutic genes in the gene therapy approach of heart ischemia developed in the laboratory.

CXCL4L1–fibstatin cooperation inhibits tumor angiogenesis, lymphangiogenesis and metastasis

Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently.Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin–endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies.

Generation of stable Drosophila cell lines using multicistronic vectors

Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications.

IRES-based Vector Coexpressing FGF2 and Cyr61 Provides Synergistic and Safe Therapeutics of Lower Limb Ischemia

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia.

Development of Novel Multigene Lentiviral Vectors.

Lentiviral vectors are efficient in transducing dividing and non-dividing cells. Many gene transfer applications require vectors that express more than one protein which include but not limited to therapeutic gene plus a selectable marker gene, multiple genes encoding different subunits of a complex protein or multiple independent genes that cooperate functionally. Previous strategies in constructing multigene lentiviral vectors by other groups included splicing, multiple internal promoters, internal ribosome entry site (IRES), and fusion proteins. In this study we explored the feasibility of expressing homeobox B4 (HOXB4), O6-methylguanine-DNA-methyltransferase (MGMT), and enhanced green fluorescent protein (EGFP) under the control of a single promoter with the use of IRES sequence from encephalomyocarditis (EMCV) virus and/or foot and mouth disease virus (FMDV) 2A cleavage factor in a HIV-1 based self-inactivating lentiviral vector. In bicistronic vectors the small FMDV 2A sequence can be used efficiently as an alternative to IRES. In case of tricistronic vectors the 2A sequence can be used in combination with IRES to obtain simultaneous expression of three cDNAs under the control of a single internal promoter. Monocistronic, bicistronic and tricistronic vectors were constructed and a three-plasmid expression system was used to generate vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector particles. Viral titers were measured by quantification of HIV p24 gag by ELISA. The relative efficiency of transgene expression in transduced cells by various vectors was compared by appropriate methods including fluorescence microscopy, flow cytometry, immunocytochemistry, biochemical assay, and western blotting. All the multigene vectors produced high titer viral particles and were able to simultaneously express two or three transgenes in transduced cells. However, the expression of EGFP from monocistronic vector as measured by mean fluorescence intensity was 3–8 times and 10–20 times higher than that of bicistronic and tricistronic vectors respectively. Notably expression of second gene encoded by the bicistronic vector containing FMDV 2A was 2–3 fold higher than that of IRES-based vector. Expression of MGMT in monocistronic and bicistronic constructs was also 4 to 20 times higher than tricistronic vectors. The FMDV 2A cleavage factor efficiently mediated the generation of the expected cleavage products from the artificial fusion protein. These vectors can be used in a wide range of applications including the expression of: multiple subunits of a functional protein, tumor antigen and co-stimulatory molecule(s), multiple tumor antigens for immunotherapy applications, and co-expression of selectable marker gene/s with therapeutic gene/s.

Mammalian expression of transmembrane receptors for pharmaceutical applications.

Three mammalian expression systems suitable for expressing recombinant receptors have been described. Each is suited to a different aspect of the study of receptors and their behaviour. IRES-based vectors are ideal for creating stable mammalian cell lines suitable for screening receptors using a signalling readout. Unlike traditional vectors they result in almost 100% of cell lines generated expressing a particular receptor, thus increasing the efficiency of cell line generation and increasing the chance of higher expression-level cell lines being generated. They may also be utilized to express more than one protein of interest, for example it is possible to co-express a particular receptor with a particular signalling protein or trafficking protein from a single RNA, thus ensuring that both are expressed simultaneously in the same cell. The ecdysone-inducible expression system is ideal for studying receptor signalling and behaviour. It is possible to alter receptor expression levels in an identical cellular background thus making it possible to study phenomena such as constitutive receptor activity in the absence of agonist. The SFV expression system is ideal for expressing receptors at high levels of a mammalian cell. It is thus a good system for purifying receptors for structural analysis and for providing material for binding assays. All of the expression systems described above have been demonstrated to express seven-transmembrane receptors with the expected pharmacological and functional profile.

Efficient Gene Trap Screening for Novel Developmental Genes Using IRESβgeo Vector andin VitroPreselection

We have used different gene trap vectors andin vitropreselection of embryonic stem (ES) cells for a large scale screening of insertional mutations in developmentally regulated genes. A gene trap vector was constructed, which contains an internal ribosome entry site (IRES) upstream from a βgeo selectable-reporter fusion gene. Analysis of 801 independent integrations revealed that the IRESβgeo vector allows for a global enrichment of about 15 folds in the number of detectable gene trap events when compared with a conventional βgeo vector. Characterization ofin vitroandin vivo lacZexpression suggested that this IRES-based vector is able to capture a wide range of genes expressed in a variety of tissues and developmental stages, and it can also allow trapping of genes expressed at very low levels in ES cells. A preselection protocol was devised, where gene-trapped ES cells were grown in the presence of specific growth/differentiation factors such as follistatin, nerve growth factor, and retinoic acid. Several gene trap integrations were found to be either activated or repressed by one of these factors. Characterization oflacZexpression during embryogenesis showed a strong enrichment of restricted patternsin vivoafter ES cell preselection. These results suggest that a combination of IRESβgeo vector andin vitropreselection is more effective for the capture and mutation of a large number of developmental genes.