Comparative analysis of macrophage associated vectors for use in genetic vaccine

Background

Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

Methods

Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

Results

All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

Conclusions

Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

An E8 promoter–HSP terminator cassette promotes the high-level accumulation of recombinant protein predominantly in transgenic tomato fruits: a case study of miraculin

Key message

The E8 promoter–HSP terminator expression cassette is a powerful tool for increasing the accumulation of recombinant protein in a ripening tomato fruit.

Abstract

Strong, tissue-specific transgene expression is a desirable feature in transgenic plants to allow the production of variable recombinant proteins. The expression vector is a key tool to control the expression level and site of transgene and recombinant protein expression in transgenic plants. The combination of the E8 promoter, a fruit-ripening specific promoter, and a heat shock protein (HSP) terminator, derived from heat shock protein 18.2 of Arabidopsis thaliana, produces the strong and fruit-specific accumulation of recombinant miraculin in transgenic tomato. Miraculin gene expression was driven by an E8 promoter and HSP terminator cassette (E8–MIR–HSP) in transgenic tomato plants, and the miraculin concentration was the highest in the ripening fruits, representing 30–630 μg miraculin of the gram fresh weight. The highest level of miraculin concentration among the transgenic tomato plant lines containing the E8–MIR–HSP cassette was approximately four times higher than those observed in a previous study using a constitutive 35S promoter and NOS terminator cassette (Hiwasa-Tanase et al. in Plant Cell Rep 30:113–124, 2011). These results demonstrate that the combination of the E8 promoter and HSP terminator cassette is a useful tool to increase markedly the accumulation of recombinant proteins in a ripening fruit-specific manner.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.     

Alpha-Fetoprotein Promoter-Driven Cre/LoxP-Switched RNA Interference for Hepatocellular Carcinoma Tissue-Specific Target Therapy

Backgroud

RNA interference (RNAi) has recently emerged as a potential treatment modality for hepatocellular carcinoma (HCC) therapy, but the lack of cellular targets and sustained efficacy limits its application. The purpose of this study is to develop an HCC tissue-specific RNAi system and investigate its possibility for HCC treatment.

Methods

Two different HCC-specific RNAi systems in which therapeutic miRNA or shRNA against target gene (Beclin 1) was directly or indirectly driven by alpha-fetoprotein promoter (AFP-miRNA and AFP-Cre/LoxP-shRNA) were constructed. Human HCC cell lines (HepG2, Hep3B and HCCLM3) and non-HCC cell lines (L-02, Hela and SW1116) were infected with the systems. The effectiveness and tissue-specificity of the systems were examined by Q-PCR and western blot analysis. The efficacy of the systems was further tested in mouse model of HCC by intravenous or intratumoral administration. The feasibility of the system for HCC treatment was evaluated by applying the system as adjuvant therapy to enhance sorafenib treatment. An AFP-Cre/LoxP-shRNA system targeting Atg5 gene (AFP-Cre/LoxP-shRNA-Atg5) was constructed and its efficacy in sensitizing HCC cells (MHCC97L/PLC) to sorafenib treatment was examined by apoptosis assay in vitro and tumorigenesis assay in vivo.

Results

The AFP-miRNA system could silence target gene (Beclin 1) but required a high titer which was lethal to target cells. The AFP-Cre/LoxP-shRNA system could efficiently knockdown target gene while maintain high HCC specificity. Intratumoral injection of the AFP-Cre/LoxP-shRNA system could efficiently silence target gene (Beclin 1) in vivo while intravenous administration could not. The AFP-Cre/LoxP-shRNA system target Atg5 gene could significantly sensitize MHCC97L/PLC cells to sorafenib-induced apoptosis in vitro and tumor growth suppression in vivo.

Conclusions

An efficient HCC tissue-specific RNAi system (AFP-Cre/LoxP-shRNA) was successfully established. The system provides a usable tool for HCC-specific RNAi therapy, which may serve as a new treatment modality for HCC.     

Sequence and functional analyses of the aldehyde dehydrogenase 7B4 gene promoter in Arabidopsis thaliana and selected Brassicaceae: regulation patterns in response to wounding and osmotic stress

Key message

The core promoter of the antiquitin ALDH7B4 gene was compared between selected Brassicaceae. Conserved cis elements controlling osmotic stress and wound-induced expression were identified and analysed in Arabidopsis thaliana leaves and seeds.

Abstract

Aldehyde dehydrogenases metabolise a wide range of aliphatic and aromatic aldehydes, which become cytotoxic at high levels. Family 7 aldehyde dehydrogenase genes, often described as antiquitins or turgor-responsive genes in plants, are broadly conserved across all domains. Despite the high conservation of the plant ALDH7 proteins and their importance in stress responses, their regulation has not been investigated. Here, we compared ALDH7 genes of different Brassicaceae and found that, in contrast to the gene organisation and protein coding sequences, similarities in the promoter sequences were limited to the first few hundred nucleotides upstream of the translation start codon. The function of this region was studied by isolating the core promoter of the Arabidopsis thaliana ALDH7B4 gene, taken as model. The promoter was found to be responsive to wounding in addition to salt and dehydration stress. Cis-acting elements involved in stress responsiveness were analysed and two conserved ACGT-containing motifs proximal to the translation start codon were found to be essential for the responsiveness to osmotic stress in leaves and in seeds. The integrity of an upstream ACGT motif and a dehydration-responsive element/C-repeat—low temperature-responsive element was found to be necessary for ALDH7B4 expression in seeds and induction by salt, dehydration and ABA in leaves. The comparison of the gene expression in selected Arabidopsis mutants demonstrated that osmotic stress-induced ALDH7B4 expression in leaves and seeds involves both ABA- and lipid-signalling components.     

Expression analyses of nuclear receptor genes in breast cancer cell lines exposed to soy phytoestrogens after BRCA2 knockdown by TaqMan Low-Density Array (TLDA)

Background

Most of breast cancers are considered sporadic and modulation of the two major genes BRCA1 and BRCA2 expressions caused by tissue-specific somatic mutations lead to this pathology. The nutritional intake of phytoestrogens seems to reduce the risk of breast cancer and investigation of their potential as anticancer agents has increased. However, the possible mechanisms and signalling pathways of phytoestrogen action in breast cancer prevention remains unknown.

Results

Using Taqman Low Density Array technology, we investigated the BRCA2 loss of function role in sporadic breast cancers and the links existing with soy isoflavones on a panel of nuclear receptor expression. Human breast cell lines (MCF-7, MDA-MB-231, and MCF-10a) were transfected by BRCA2-siRNA and treated with genistein (18.5 μM) or daidzein (78.5 μM) for 72 h. Generating the transitory knockdown of BRCA2 oncosuppressor, we observed different modulations in several nuclear receptor genes such as ER, RAR and RXR, as well as PPARs and VDR according to the studied breast cell line. Additional isoflavone treatments showed different nuclear receptor gene modulation profiles.

Conclusion

Our results seemed to implicate the oncosuppressor BRCA2 and the phytoestrogen pathways in different nuclear gene expressions via an ER-independent manner.