Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

A new inducible adenoviral expression system that responds to inflammatory stimuli in vivo

BACKGROUND: Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. METHODS: A luciferase reporter vector with a hybrid promoter containing the human IL-1beta enhancer region (-3690 to - 2720) and the human CIITA promoter IV (-399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1beta/CIITApIV promoter (Ad-IL1beta/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1beta/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1beta/CIITApIV promoter. RESULTS: The IL1beta/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1beta/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. CONCLUSIONS: Using the IL1beta/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity.     

Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

CYP7A1基因-204位点A/C变异对启动子活性的影响

CYP7A1（cholesterol 7α-hydroxylase ）在胆固醇向胆汁酸代谢途径中起着至关重要的作用.为研究该基因启动子区-204位点A/C多态性是否影响基因表达, 利用荧光素酶作为报告基因,将含有A或C等位基因的启动子区片段分别正向和反向插入不含启动子的pGL3 basic质粒载体中,再以重组体转染4种细胞株,采用双荧光素酶报告基因检测系统测定酶活性并进行比较.实验结果表明,2种基因型的正向序列启动子活性均高于相应的反向序列,含有A等位基因的启动子片段活性比含有C等位基因的片段低约1/3.TRANSFAC数据库分析显示,当-204位点等位基因为C时,可能存在1个Zic3结合位点.研究结果提示,CYP7A1基因启动子区-204位点A/C变异可减少启动子活性从而影响基因表达,其原因可能为1个潜在的Zic3结合位点的丧失.     

小鼠组蛋白H3K4甲基化酶Smyd3启动子荧光素酶报告载体的构建与活性分析

为研究小鼠(Mus musculus)组蛋白H3 K4甲基化酶基因Smyd3转录调控的分子机制,本研究首先通过PCR的方法克隆了5条不同长度的Smyd3启动子5’端缺失片段,与pMD19-T载体连接后,双酶切克隆入pGL3-Basic荧光素酶报告基因载体,构建Smyd3启动子-pGL3-Basic报告基因重组质粒,瞬时转染HEK293细胞48 h后采用双报告基因检测试剂盒检测Smyd3启动子各缺失片段的相对荧光活性.结果表明,本研究成功构建Smyd3启动子5’端缺失片段-pGL3-Basic荧光报告基因重组质粒,所构建的启动子重组子转染组与阳性对照组相比表现出荧光活性,并且pGL3-Smyd3-4的荧光活性最强,是其他的2至4倍左右,pGL3-Smyd3-5的荧光活性最弱.本研究初步确定Smyd3基因的启动子核心区域可能位于-533～-42bp之间,在-2026～-533 bp之间可能存在启动子负调控序列.     

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.     

Use of a murine secreted alkaline phosphatase as a non-immunogenic reporter gene in mice

BACKGROUND: The development of any vector system as a gene delivery system requires its optimization in vitro and in vivo. Preliminary studies frequently involve the use of a reporter gene, which allows for the rapid and simple assay of vector function through monitoring expression levels of the reporter gene. However, evaluation of vector efficacy can be compromised by immune responses directed against immunogenic reporter proteins. METHODS: We have cloned a murine secreted alkaline phosphatase (mSEAP), and explored its use as a reporter gene in the context of an early region 1 (E1)-deleted adenovirus (Ad) vector. Studies involved characterization of gene expression in vitro and in vivo, and immunological responses after gene delivery to mice. RESULTS: In tissue culture, we show that mSEAP is easily measured quantitatively using a sensitive, commercially available chemiluminescent assay, or visualized directly using histological staining. The level of transgene expression from AdmSEAP was similar to that observed for an Ad vector encoding the human placental secreted alkaline phosphatase (hSEAP). After intravenous administration in mice, AdmSEAP continued to express at high levels for the duration of the experiment (1 month), whereas expression from AdhSEAP declined to background levels over the course of the experiment. Although cytotoxic T-lymphocytes were not detected against either the murine or human SEAP proteins in mice, antibodies were readily detected against the human protein. No antibodies were detected to mSEAP. CONCLUSIONS: Taken together, these data illustrate that mSEAP is a sensitive, non-immunogenic reporter gene for preclinical mouse studies.     

一种新的重组腺病毒的构建及其对人肿瘤细胞的影响

腺病毒E1A基因诱导细胞凋亡．E1B19K基因及E1B55K基因抑制细胞凋亡,前者被克隆到腺病毒转移载体pCA13的HCMVIE启动子下游．构建成转移载体pCAE1A。采用lipofectin法将PCAE1A和含腺病毒基因组（E1、E3区缺失）的质粒pBHG11共转染293细胞,7～10d后得到重组病毒v5Ad4。用v5Ad4感染人肺腺癌细胞系A549,结果表明v5Ad4有明显杀伤和裂解肿瘤细胞功能。在人胚肺正常二倍体细胞中,v5Ad4没有表现出可见的细胞毒效应。     

Oltipraz inhibits 3-methylcholanthrene induction of CYP1A1 by CCAAT/enhancer-binding protein activation

Oltipraz, a cancer chemopreventive agent, induces CYP1A1 to a certain extent by transactivation of the gene via the Ah receptor (AhR)-xenobiotic response element (XRE) pathway. Previously, we showed that oltipraz promoted CCAAT/enhancer binding proteinbeta (C/EBPbeta) activation, which leads to the induction of glutathione S-transferase. Given that oltipraz activates C/EBPbeta for gene transactivation and that the putative C/EBP binding site is located in the CYP1A1 promoter region, this study investigated the effect of oltipraz on CYP1A1 induction by 3-methylcholanthrene (3-MC). 3-MC induced CYP1A1 in H4IIE cells in a time- and concentration-dependent manner. Gel shift analysis showed that 3-MC increased the band intensity of protein binding to the XRE. Immunocompetition analysis verified the specificity of AhR-XRE binding. Oltipraz (30 microM) induced CYP1A1 and the CYP1A1 promoter-luciferase gene and increased AhR DNA binding activity, which was 10-20% of those in 3-MC (100 nM)-treated cells. However, AhR-XRE binding was not increased after 10 microM oltipraz treatment. Oltipraz (10 microM) significantly inhibited CYP1A1 and CYP1A1-luciferase gene induction by 3-MC with no increase in AhR DNA binding. Oltipraz enhanced protein binding to the C/EBP binding site in the gene promoter and the binding complex comprised of C/EBPbeta and partly C/EBPdelta. Overexpression of dominant-negative mutant C/EBP significantly abolished the ability of oltipraz to suppress 3-MC-inducible CYP1A1 and the CYP1A1 reporter gene expression. Consistently, C/EBPbeta overexpression blocked CYP1A1 reporter gene induction by 3-MC. These results provide evidence that oltipraz suppresses 3-MC induction of CYP1A1 gene expression and that activation of C/EBPbeta by oltipraz contributes to suppression of 3-MC-inducible AhR-mediated CYP1A1 expression.     

The human CYP1A1 gene is regulated in a developmental and tissue-specific fashion in transgenic mice

Regulation and expression of human CYP1A1 is demonstrated in transgenic mice. We have developed two transgenic mouse lines. One mouse strain (CYPLucR) carries a functional human CYP1A1 promoter (-1612 to +293)-luciferase reporter gene, and the other strain (CYP1A1N) expresses CYP1A1 under control of the full-length human CYP1A1 gene and 9 kb of flanking regulatory DNA. With CYPLucR(+/-) mice, 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and several other aryl hydrocarbon receptor ligands induced hepatocyte-specific luciferase activity. When other tissues were examined, TCDD induced luciferase activity in brain with limited induction in lung and no detectable luciferase activity in kidney. Treatment of CYP1A1N(+/-) mice with TCDD resulted in induction of human CYP1A1 in liver and lung, while mouse Cyp1a1 was induced in liver, lung, and kidney. Although induced CYP1A1/Cyp1a1 could not be detected by Western blot analysis in brains from CYP1A1N(+/-) mice, induction in brain was verified by detection of CYP1A1/Cyp1a1 RNA. The administration of TCDD to nursing mothers to examine the effect of lactational exposure via milk demonstrated prominent induction of luciferase activity in livers of CYPLucR(+/-) newborn pups with limited induction in brain. However, TCDD treatment of adult CYPLucR(+/-) mice led to a 7-10-fold induction of brain luciferase activity. Combined these results indicate that tissue-specific and developmental factors are controlling aryl hydrocarbon receptor-mediated induction of human CYP1A1.