Gene delivery into neuronal and glial cells by using a replication-deficient adenovirus vector: prospects for neurological gene therapy

We have used a recombinant adenovirus vector (E1−) expressing β-galactosidase to explore a novel mechanism with which to transfer genes into cells of the central nervous system (CNS). The replication-deficient adenovirus vector expressing β-galactosidase (RAd35) was propagated on a permissive helper cell line (293 cells). High level protein expression from the human cytomegalovirus immediate early promoter (hCMV IE) was obtained in a target cell population of RAd35 infected cultured neuronal and glial cell lines. Light microscopy showed that over 50% of the glial cells studied expressed β-galactosidase. Following retinoic acid treatment, RAd35 infected cell lines ND7/23, NG108 and NTera2, showed β-galactosidase expression in up to 90% of the cells. In addition, these cells showed morphological evidence of differentiation into neurons. This pattern of β-galactosidase expression was also observed in primary rat cerebella granule neuron cultures. In vivo studies were performed in Balb/c mice following direct intracranial injections of RAd35 into the brain. Cell sections showed a localised staining in the brain at the site of injection of the virus. Non-replicating adenovirus vectors are therefore highly efficient systems for delivering a transgene into brain cells. However, their broad cell tropism may limit their applications for genetic disorders in which a specific cell type is to be targeted for gene therapy. To address this problem, we have constructed adenovirus vectors which contain specific neuronal promoters and are currently assessing in vitro expression. This revised version was published online in July 2006 with corrections to the Cover Date.     

基于DNA和RNA的双功能Semliki森林病毒复制子载体的构建

以semliki森林病毒衍生的复制子载体pSFV1和辅助载体pSFV-helper2为骨架, 用CMV IE和T7启动子替换SP6启动子并在3′ UTR下游插入BGH转录终止子,构建了基于DNA和RNA的复制子表达载体pSMCTA和辅助载体pSHCTA。在DNA和RNA二种递送方式上证实该表达载体可高水平表达外源基因,与辅助载体共转染可制备具有感染能力并能表达外源基因的重组病毒颗粒。构建的基于DNA和RNA的双功能复制子载体显著地提高SFV载体应用范围,在体外可用于高水平表达外源基因及大规模制备重组病毒颗粒,在体内也可用于研制复制子疫苗和基因治疗载体。     

Modulation of T-cell activation through protein kinase C- or A-dependent signalling pathways synergistically increases human immunodeficiency virus long terminal repeat induction by cytomegalovirus immediate-early proteins.

By using human CD4+ lymphoblastoid T cells transiently cotransfected with human immunodeficiency virus (HIV) and cytomegalovirus (CMV), we tested whether modulation of T-cell activation through the protein kinase C (PKC) or the protein kinase A (PKA) pathway synergized with CMV immediate-early (IE) proteins in HIV long terminal repeat (LTR) transactivation. Stimulation with phorbol myristate acetate, tumor necrosis factor, or cross-linked antibodies to CD3 and CD28 resulted in modest enhancement (two- to fourfold) of the activity of a luciferase expression vector under control of the HIV LTR. Cotransfection of a vector expressing the CMV IE1 and IE2 proteins under the control of their own promoter enhanced HIV LTR activity 16- to 49-fold. Combination of any one of the above stimuli and CMV IE expression amplified HIV LTR activity 99- to 624-fold. Stimulation of PKA-dependent pathways with forskolin, 8-bromo cyclic AMP, or prostaglandin E2 had a minimal effect on HIV LTR activity, whereas such stimuli resulted in synergistic amplification in cells cotransfected with CMV IE (three- to fivefold increases over the effects of CMV IE alone). This synergism was independent of the NF-kappa B binding motifs within the HIV LTR. CMV IE2, but not IE1, protein induced HIV transactivation and synergized with signals modulating T-cell activation. The intense synergism observed was superior to the increase in IE protein expression following PKC activation by phorbol myristate acetate. Treatment of cells with PKC inhibitor GF109203X blocked most of the observed synergism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulated expression of the human cytomegalovirus pp65 gene: octamer sequence in the promoter is required for activation by viral gene products.

To better understand the regulation of late gene expression in human cytomegalovirus (CMV)-infected cells, we examined expression of the gene that codes for the 65-kilodalton lower-matrix phosphoprotein (pp65). Analysis of RNA isolated at 72 h from cells infected with CMV Towne or ts66, a DNA-negative temperature-sensitive mutant, supported the fact that pp65 is expressed at low levels prior to viral DNA replication but maximally expressed after the initiation of viral DNA replication. To investigate promoter activation in a transient expression assay, the pp65 promoter was cloned into the indicator plasmid containing the gene for chloramphenicol acetyltransferase (CAT). Transfection of the promoter-CAT construct and subsequent superinfection with CMV resulted in activation of the promoter at early times after infection. Cotransfection with plasmids capable of expressing immediate-early (IE) proteins demonstrated that the promoter was activated by IE proteins and that both IE regions 1 and 2 were necessary. Analysis of promoter deletion mutants indicated that the 5' minimal sequence required for activation is -61 from the CAP site (+1) and that an 8-base-pair sequence located at -51 to -58 is necessary for activation of the pp65 promoter. This sequence is repeated once at +93 and is found as an inverted repeat at +67. These studies suggest that interactions between IE proteins and this octamer sequence may be important for the regulation and expression of this CMV gene.     

转基因鸡生物反应器载体的构建及其表达特性分析

以增强型绿色荧光蛋白和萤火虫荧光素酶为报告基因,构建了鸡卵清蛋白启动子表达载体和慢病毒载体,以巨细胞病毒 (Cytomegalovirus,CMV)启动子表达载体为对照,转染或感染鸡原代输卵管上皮细胞、鸡胚成纤维细胞、鼠3T3-L1前脂肪细胞和牛乳腺上皮细胞,通过荧光和酶活性检测,旨在筛选出用于实现转基因鸡生物反应器的高效特异性表达载体。结果发现,鸡卵清蛋白启动子表达载体转染以上4种细胞后2种标记基因均有表达,没有表现出明显的细胞特异性,且荧光素酶检测结果表明其在各细胞组中表达活性都低于CMV启动子表达载体100倍以上;慢病毒载体感染以上4种细胞后2种标记基因均有表达,在鸡输卵管上皮细胞组感染单个细胞的病毒颗粒 (Multiplicity of infection,MOI) 为20时绿色荧光蛋白表达量就可以达到CMV启动子表达载体的水平。上述结果表明,基于卵清蛋白基因调控序列构建的表达载体无法实现外源基因的高效、特异性表达,而慢病毒载体在表达活性和广泛性上可以用于进行鸡输卵管生物反应器的研究。     

Ocular Localization and Transduction by Adenoviral Vectors Are Serotype-Dependent and Can Be Modified by Inclusion of RGD Fiber Modifications

Purpose

To evaluate localization and transgene expression from adenoviral vector of serotypes 5, 35, and 28, ± an RGD motif in the fiber following intravitreal or subretinal administration.

Methods

Ocular transduction by adenoviral vector serotypes ± RGD was studied in the eyes of mice receiving an intravitreous or subretinal injection. Each serotype expressed a CMV-GFP expression cassette and histological sections of eyes were examined. Transgene expression levels were examined using luciferase (Luc) regulated by the CMV promoter.

Results

GFP localization studies revealed that serotypes 5 and 28 given intravitreously transduced corneal endothelial, trabecular, and iris cells. Intravitreous delivery of the unmodified Ad35 serotype transduced only trabecular meshwork cells, but, the modification of the RGD motif into the fiber of the Ad35 viral vector base expanded transduction to corneal endothelial and iris cells. Incorporation of the RGD motif into the fiber knob with deletion of RGD from the penton base did not affect the transduction ability of the Ad5 vector base. Subretinal studies showed that RGD in the Ad5 knob shifted transduction from RPE cells to photoreceptor cells. Using a CMV-Luc expression cassette, intravitreous delivery of all the tested vectors, such as Ad5-, Ad35- and Ad28- resulted in an initial rapid induction of luciferase activity that thereafter declined. Subretinal administration of vectors showed a marked difference in transgene activity. Ad35-Luc gene expression peaked at 7 days and remained elevated for 6 months. Ad28-Luc expression was high after 1 day and remained sustained for one month.

Conclusions

Different adenoviral vector serotypes ± modifications transduce different cells within the eye. Transgene expression can be brief or extended and is serotype and delivery route dependent. Thus, adenoviral vectors provide a versatile platform for the delivery of therapeutic agents for ocular diseases.     

细菌内同源重组法制备mIL-12腺病毒载体及其体外高效表达

利用细菌内同源重组法构建含 m IL- 1 2双顺反子重组腺病毒载体 ,其中由 polio IRES连接的 m IL- 1 2 p40和 p35双亚基目的基因片段用 Not +Xho 酶自真核表达载体 pc DNA3/m IL- 1 2中切出 ,亚克隆至经同样酶切的腺病毒穿梭质粒中 ,形成转移质粒 p Adtrack- CMV/m IL- 1 2 ,对之线性化后与腺病毒基因组质粒 p Adeasy- 1共转化 BJ51 83菌 ,抽提经鉴定含目的基因的重组体质粒DNA,转染 2 93细胞 ,包装成重组体腺病毒 Ad- m IL - 1 2 . Southern杂交证实 ,p40和 p35基因已被克隆至 Ad- m IL - 1 2基因组中 ;RT- PCR结果显示 ,Ad- m IL- 1 2感染的 MM45T· Li肿瘤细胞中有p40和 p35基因的表达 ,ELISA检测感染 48h细胞的上清中 m IL- 1 2的含量达 88ng每 1 0 6细胞 ,其体外能刺激小鼠脾脏淋巴细胞的增殖 ,提高 NK细胞活性及诱导 干扰素的产生 .结果表明 ,利用细菌内同源重组法制备的 Ad- m IL- 1 2重组腺病毒载体是一种方便、高效、成功率高的方法 ,所制备的重组体腺毒在体外能有效表达具有生物活性的 m IL- 1 2 ,为今后的体内应用奠定了基础 .     

Abundant constitutive expression of the immediate-early 94K protein from cytomegalovirus (Colburn) in a DNA-transfected mouse cell line.

A 94-kilodalton phosphoprotein known as IE94 is the only viral polypeptide synthesized in abundance under immediate-early conditions after infection by cytomegalovirus (CMV) strain Colburn in either permissive primate or nonpermissive rodent cells. The IE94 gene, which maps at coordinates 0.71 to 0.73 in the viral genome, contains a large intron in the 5' leader sequence, and its promoter regulatory region contains novel, multiple-palindromic, repeated elements. Two recombinant plasmids (pTJ148 and pTJ198) containing the 10.5-kilobase-pair HindIII-H DNA fragment from CMV (Colburn) were transfected into mouse Ltk- cells, by either linked or unlinked coselection in hypoxanthine-aminopterin-thymidine medium, together with herpes simplex virus thymidine kinase genes. With both procedures, constitutive synthesis of the IE94 immediate-early protein was detected in pools of Ltk+ cells by immunoprecipitation. Subsequently, we isolated a clonal Ltk+ cell line which expressed the [35S]methionine-labeled IE94 polypeptide in sufficient abundance to be visualized directly in autoradiographs after gel electrophoresis of total-cell-culture protein extracts. The IE94 polypeptide synthesized in the transfected cells was indistinguishable in size and overall net charge from that produced in virus-infected cells. In addition, the IE94 protein expressed in LH2p198-3 cells was phosphorylated (presumably by a cellular protein kinase) and generated similar phosphopeptide patterns after partial tryptic digestion to those obtained with the CMV IE94 protein from infected cells. The cell line contained two to four stably integrated copies of the IE94 gene and synthesized a single virus-specific mRNA of 2.5 kilobases detectable on Northern blots. A new antigen, detectable by indirect anticomplement immunofluorescence with monoclonal antibody against the human CMV IE68 protein, was present in the nuclei of more than 95% of the LH2p198-3 cells. This evidence suggests that (unlike most herpesvirus genes) the CMV IE94 gene, together with its complex promoter and spliced mRNA structure, may contain all of the regulatory elements necessary for strong constitutive expression in mammalian cells in the absence of other viral factors.     

Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo.

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)     

CMV promotor activity during ES cell differentiation: potential insight into embryonic stem cell differentiation

The activity of the P(CMV IE) promoter was studied during the differentiation of ES cells into neurons. In order to do this, stable embryonic stem (ES) cell lines that express enhanced green fluorescent protein (EGFP) under the control of P(CMV IE) were created and these ES cells were differentiated by aggregation of cells in the presence of retinoic acid (RA). Based on our observations that the activity of P(CMV IE) was highest in undifferentiated cells, and that cell-cell interaction and addition of RA that lead to enhanced cell proliferation also increased expression from P(CMV IE), we hypothesized that the activity of P(CMV IE) was positively regulated in cycling cells. However, when analysis was done at the single cell level it was found that BrdU label and EGFP expression were not correlated. EGFP expression was found to be down-regulated in many cells that were BrdU positive and conversely there were significant numbers of BrdU negative cells that were EGFP positive. Further, P(CMV IE) activity was not observed in cells that were nestin positive or in differentiated neurons, but P(CMV IE) was active in cells with a fibroblast-like morphology. Finally, several proteins present in undifferentiated ES cells were found to bind to regulatory regions of P(CMV IE). These were absent when cells were aggregated in the presence of RA. The above results have implications for expression of transgenes in ES cells as well as providing new insight into the mechanism of lineage restriction.     

High-level expression of the tick-borne encephalitis virus NS1 protein by using an adenovirus-based vector: protection elicited in a murine model.

Tick-borne encephalitis virus (TBEV) encodes an abundant, highly immunogenic nonstructural glycoprotein, NS1. The function of this protein has yet to be determined. We have cloned the NS1 gene from the Neudorfl strain of TBEV under the control of the powerful constitutive cytomegalovirus major immediate-early promoter into an adenovirus E1 deletion mutant. The novel combination of the cytomegalovirus immediate-early promoter and the adenovirus vector produced extremely high levels of NS1 expression in cells which do not support replication of the adenovirus deletion mutant. The recombinant protein was shown to be indistinguishable from authentic TBEV NS1 in its (i) apparent molecular weight by polyacrylamide gel electrophoresis, (ii) glycosylation pattern, (iii) ability to form high-molecular-weight complexes, and (iv) ability to be secreted from cells. Appropriate processing of NS1 expressed by the adenovirus recombinant occurred independently of any additional TBEV-encoded gene function. When directly inoculated into mice, the recombinant adenovirus RAd51 was shown to elicit an antibody response to the TBEV NS1 protein. Immunization with RAd51 conferred protection against challenge with TBEV.     

Cardiac-specific gene expression facilitated by an enhanced myosin light chain promoter

BACKGROUND: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. METHODS: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v) gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc) reporter gene (Ad.4 x MLC250.Luc). Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 x MLC.Luc or control vectors. For in vivo testing, Ad.4 x MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. RESULTS: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells) was 20.4 versus 0.9 (Ad.4 x MLC250.Luc vs. Ad.CMV). In vivo: Ad.4 x MLC250.Luc significantly reduced luc activity in liver (38.4-fold), lung (16.1-fold), and kidney (21.8-fold) versus Ad.CMV (p =.01); whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 x MLC.Luc) versus 1:1.4 (Ad.CMV.Luc). DISCUSSION: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.     

Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber-modified adenovirus vectors enhances transgene-encoded antigen presentation to cytotoxic T cells

The efficiency of dendritic cells (DC) as immunotherapeutic vaccines critically depends on optimal delivery of target Ags. Although DC modified by subgroup C type 5 recombinant adenoviruses (rAd5) provide encouraging results, their clinical application is hampered by the need for high viral titers to achieve sufficient gene transfer, due to the lack of the Ad5 fiber receptor. We now demonstrate that rAd5 carrying subgroup B Ad fibers are up to 100-fold more potent than classical rAd5 for gene transfer and expression in human DC, rAd5 with a type 35 fiber (rAd5F35) being the most efficient vector. This improvement relates to a greater and faster virus entry and to an increased transgene expression especially following DC maturation. Furthermore, these new vectors possess enhanced synergistic effects with other activation signals to trigger DC maturation. Consequently, rAd5F35-infected DC engineered to express the gp100 melanoma-associated Ag largely exceed rAd5-infected DC in activating gp100-specific CTL. Finally, the DC infection pattern of rAd5F35 is fully conserved when DC are in the vicinity of primary skin-derived fibroblasts, suggesting this vector as a candidate for in vivo targeting of DC. Thus, subgroup B fiber-modified rAd5 constitute a major breakthrough in the exploitation of ex vivo rAd-targeted DC as clinically relevant vaccines and may also be suitable for in vivo genetic modification of DC.     

Requirements for enhanced transgene expression by untranslated sequences from the human cytomegalovirus immediate-early gene.

BACKGROUND: The cytomegalovirus immediate early (CMV IE) promoter has been widely used for heterologous expression. Further enhancements of gene expression from this potent promoter may allow for the development of improved gene transfer strategies. We aimed to determine whether inclusion of the first exon (5' untranslated) and first intron of the CMV IE gene would increase heterologous transgene expression in primary target cells and to determine the sequences required for any observed increases. MATERIALS AND METHODS: Comparisons of reporter gene expression were made following transient transfection of vascular smooth muscle cells (VSMCs) with plasmids containing the first exon and intron from the CMV IE gene or deletional mutations. Comparisons were also made using a heterologous promoter (RSV). RESULTS: Gene expression from the CMV IE promoter was increased 5.7-fold in VSMC with the inclusion of the first exon and intron. Similar increases were seen with other target cells and from the heterologous RSV promoter. This increase was associated with an increase in steady-state mRNA. Deletion analyses demonstrated that the enhancement was dependent on the presence of the 5' portion of the first exon while deletion of large segments within the intron was associated with similar levels of expression compared with the parental plasmid. CONCLUSIONS: Inclusion of the first exon and intron from the CMV IE gene increases expression from the CMV IE promoter. This enhancement is seen with the heterologous RSV promoter and is associated with an increase in steady-state mRNA. Deletion analyses suggest that this enhancement is associated with inclusion of sequences within the 5' portion of the first exon and inclusion of an intron.