Gene transfer into mouse embryonic stem cell-derived cardiac myocytes mediated by recombinant adenovirus

Summary The main purpose of this study was to examine, for the first time, the ability of recombinant adenovirus to mediate gene transfer into cardiac myocytes derived from mouse embryonic stem (ES) cells differentiating in vitro. In addition, observations were made on the effect of adenovirus infection on cardiac myocyte differentiation and contractility in this in vitro system of cardiogenesis. ES cell cultures were infected at various times of differentiation with a recombinant adenovirus vector (AdCMVlacZ) containing the bacterial lacZ gene under the control of the cytomegalovirus (CMV) promoter. Expression of the lacZ reporter gene was determined by histochemical staining for β-galactosidase activity. LacZ expression was not detected in undifferentiated ES cells infected with AdCMVlacZ. In contrast, infection of differentiating ES cell cultures showed increasing transgene expression with continued time in culture. Expression in ES-cell-derived cardiac myocytes was demonstrated by codetection of β-galactosidase activity and troponin T with indirect immunofluorescence. At 24 h postinfection, approximately 27% of the cardiac myocytes were β-galactosidase positive, and lacZ gene expression appeared to be stable for up to 21 postinfection. Adenovirus infection had no apparent effect on the onset, extent, or duration of spontaneously contracting ES-cell-derived cardiomyocytes, indicating that cardiac differentiation and contractile function were not significantly altered in the infected cultures. The demonstration of adenovirus-mediated gene transfer into ES-cell-derived cardiac myocytes will aid studies of gene expression with this in vitro model of cardiogenesis and may facilitate future studies involving the use of these myocytes for grafting experiments in vivo.     

Activity assays of nine heterogeneous promoters in neural and other cultured cells

Summary To express high levels of proteins encoded by transfected DNA constructs in a variety of cultured cells, including neuronal cells, the activities of nine different promoters were evaluated usingEscherichia coli β-galactosidase (β-gal) (LacZ) as a reporter gene. These nine promoters were categorized into three distinct groups (high, intermediate, and low expresser), in terms of the levels ofβ-gal expression. An expression vector containing the cytomegalovirus enhancer and the chickβ-actin promoter (high expresser) showed the highest levels of expression, followed by vectors containing the cytomegalovirus promoter/enhancer and the SV40 promoter/enhancer (intermediate expresser). The rest of the promoters (thymidine kinase, adenovirus, murine proliferative sarcoma virus, nerve growth factor receptor, Rous sarcoma and mouse mammary tumor virus, andβ-amyloid precursor protein) expressed low levels ofβ-gal. These results were consistent for eight different cell types. A particularly attractive model is the stem cell, P19; cultures differentiating into progeny consisting predominantly of cholinergic neurons could be readily transfected with expression vectors using liposomes and expressedβ-gal without significant morphologic changes of the differentiated neurons. The systems should be useful for the study of promoters and various expressed proteins, including those involved in axonal transport.     

Retroviral gene transfer inXenopus cell lines and embryos

Summary A new class of retroviral vector pseudotypes have an expanded host species range and can be concentrated to high titers by ultracentrifugation. These pantropic vectors contain the genome of the murine leukemia virus-based vectors and the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein. We tested (a) the ability of pseudotyped (pantropic) and unmodified (amphotropic) vectors to stably infect three diffeentXenopus laevis cell lines, including one derived from the embryonic retina; and (b) the ability of the concentrated pseudotyped virus to infect embryos and to mediate foreign gene expression in the embryonic CNS. Expression of the neomycin phosphotransferase gene and single copy integration of the provirus into the genome of the cell lines was demonstrated. Surprisingly, the amphotropic and pantropic vectors generated neomycin-resistant clones with similar efficiency. PCR amplification of genomic DNA from single stage 10, 20, and 25 embryos microinjected in the blastocoel or neural tube cavities with concentrated pantropic vector (10⁸ cfu/ml) revealed proviral DNA. Microinjection of a concentrated pantropic vector containing the coding sequence for the β-galactosidase gene into the neural tube lumen of 24-h embryos yielded β-galactosidase expressing cells in the brain. Thus, retroviral vectors provide an additional approach to existing strategies for gene transfer inXenopus embryos and cell lines.     

Attenuation of seizure in P77PMC rats with an HSV-vector expressing IL-1ra in brain

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Targeting Neurons of Rat Nucleus Tractus Solitarii with the Gene Transfer Vector Adeno-Associated Virus Type 2 to Up-Regulate Neuronal Nitric Oxide Synthase

Adeno-associated virus (AAV) has distinct advantages over other viral vectors in delivering genes of interest to the brain. AAV mainly transfects neurons, produces no toxicity or inflammatory responses, and yields long-term transgene expression. In this study, we first tested the hypothesis that AAV serotype 2 (AAV2) selectively transfects neurons but not glial cells in the nucleus tractus solitarii (NTS) by examining expression of the reporter gene, enhanced green fluorescent protein (eGFP), in the rat NTS after unilateral microinjection of AAV2eGFP into NTS. Expression of eGFP was observed in 1–2 cells in the NTS 1 day after injection. The number of transduced cells and the intensity of eGFP fluorescence increased from day 1 to day 28 and decreased on day 60. The majority (92.9 ± 7.0%) of eGFP expressing NTS cells contained immunoreactivity for the neuronal marker, protein gene product 9.5, but not that for the glial marker, glial fibrillary acidic protein. We observed eGFP expressing neurons and fibers in the nodose ganglia (NG) both ipsilateral and contralateral to the injection. In addition, eGFP expressing fibers were present in both ipsilateral and contralateral nucleus ambiguus (NA), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). Having established that AAV2 was able to transduce a gene into NTS neurons, we constructed AAV2 vectors that contained cDNA for neuronal nitric oxide synthase (nNOS) and examined nNOS expression in the rat NTS after injection of this vector into the area. Results from RT-PCR, Western analysis, and immunofluorescent histochemistry indicated that nNOS expression was elevated in rat NTS that had been injected with AAV2nNOS vectors. Therefore, we conclude that AAV2 is an effective viral vector in chronically transducing NTS neurons and that AAV2nNOS can be used as a specific gene transfer tool to study the role of nNOS in CNS neurons.     

β-Galactosidase-deficient mouse as an animal model for GM1-gangliosidosis

GM1-gangliosidosis is a progressive neurological disease in humans caused by deficiency of lysosomal acid β-galactosidase, which hydrolyses the terminal β-galactosidic residue from ganglioside GM1 and other glycoconjugates. In this study, we generated a mouse model for GM1-gangliosidosis by gene targeting in embryonic stem cells. The mouse homozygous for the disrupted β-galactosidase gene showed β-galactosidase deficiency, presented with progressive spastic diplegia, and died of emaciation at 7–10 months of age. Pathologically, PAS-positive intracytoplasmic storage was observed in neuronal cells of various areas in the brain. Biochemical analysis revealed a marked accumulation of ganglioside GM1 and asialo GM1 in brain tissue. This animal model will be useful for pathogenetic analysis and therapeutic trial of human GM1-gangliosidosis. This revised version was published online in November 2006 with corrections to the Cover Date.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Canine Adenovirus Type 2 Vector Generation via I-Sce1-Mediated Intracellular Genome Release

When canine adenovirus type 2 (CAdV-2, or also commonly referred to as CAV-2) vectors are injected into the brain parenchyma they preferentially transduce neurons, are capable of efficient axonal transport to afferent regions, and allow transgene expression for at last >1 yr. Yet, translating these data into a user-friendly vector platform has been limited because CAV-2 vector generation is challenging. Generation of E1-deleted adenovirus vectors often requires transfection of linear DNA fragments of >30 kb containing the vector genome into an E1-transcomplementing cell line. In contrast to human adenovirus type 5 vector generation, CAV-2 vector generation is less efficient due, in part, to a reduced ability to initiate replication and poor transfectibility of canine cells with large, linear DNA fragments. To improve CAV-2 vector generation, we generated an E1-transcomplementing cell line expressing the estrogen receptor (ER) fused to I-SceI, a yeast meganuclease, and plasmids containing the I-SceI recognition sites flanking the CAV-2 vector genome. Using transfection of supercoiled plasmid and intracellular genome release via 4-OH-tamoxifen-induced nuclear translocation of I-SceI, we improved CAV-2 vector titers 1,000 fold, and in turn increased the efficacy of CAV-2 vector generation.     

β-Amyloid 25-35 Peptide Reduces the Expression of Glutamine Transporter SAT1 in Cultured Cortical Neurons

β-Amyloid (Aβ) peptides may cause malfunction and death of neurons in Alzheimer’s disease. We investigated the effect of Aβ on key transporters of amino acid neurotransmission in cells cultured from rat cerebral cortex. The cultures were treated with Aβ(25-35) at 3 and 10 μM for 12 and 24 h followed by quantitative analysis of immunofluorescence intensity. In mixed neuronal–glial cell cultures (from P1 rats), Aβ reduced the concentration of system A glutamine transporter 1 (SAT1), by up to 50% expressed relative to the neuronal marker microtubule-associated protein 2 (MAP2) in the same cell. No significant effects were detected on vesicular glutamate transporters VGLUT1 or VGLUT2 in neurons, or on glial system N glutamine transporter 1 (SN1). In neuronal cell cultures (from E18 rats), Aβ(25-35) did not reduce SAT1 immunoreactivity, suggesting that the observed effect depends on the presence of astroglia. The results indicate that Aβ may impair neuronal function and transmitter synthesis, and perhaps reduce excitotoxicity, through a reduction in neuronal glutamine uptake. Special issue article in honor of Dr. Frode Fonnum.     

Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

The antisense fragments, which were available inin vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS-2-654 C→T) human β-globin gene. In these transfected cells, the level of correctly spliced β-globin mRNA in total β-globin mRNA (β/(β + β*)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β-globin gene (IVS-2-654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β-thalassaemia (IVS-2-654 C→T) by antisense RNAs. Project supported in part by the National Natural Science Foundation of China (Grant Nos. 39780019, 39392903) and the Shanghai Life Sciences Research Centre.     

Adipose differentiation related protein induces lipid accumulation and lipid droplet formation in hepatic stellate cells

Summary The function of adipose differentiation-related protein (ADRP) is known to be the uptake of long-chain fatty acids and formation of lipid droplets in lipid-accumulating cells. We hypothesized that ADRP might stimulate activated hepatic stellate cells (HSCs) to accumulate lipids, resulting in their transition to the quiescent state. In this study, cultured HSCs in fifth passages isolated from rat were infected by adenovirus vector expressing ADRP (Ad.GFP-ADRP), and morphologic and functional changes were evaluated in comparison with control HSCs infected by recombinant adenovirus-expressing β-galactosidase (Ad. LacZ). In Ad. GFP-ADRP-infected cells only, many tiny lipid droplets were apparent in the cytoplasm, while the outline of the cells was not changed. The ADRP was detected around the lipid droplets. In HSCs with intracellular actin filaments, the staining pattern of the filaments before and after infection with Ad.GFP-ADRP or Ad.LacZ did not differ. The cell proliferation rate was not influenced by infection with Ad.LacZ or Ad.GFP-ADRP. Type I collagen secretion from cells overexpressing ADRP was not significantly different from that of Ad.LacZ-infected cells. In our in vitro study, ADRP overexpression induced the formation of cytoplasmic lipid droplets in activated HSCs but could not convert other characteristics of the activated form into those of the quiescent form.     

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.     

腺病毒介导siRNA抑制全反式维甲酸诱导的骨髓间充质干细胞RARβ表达

构建携带针对大鼠维甲酸受体β(Retinoic acid receptorβ,RARβ)基因的siRNA重组腺病毒,并感染全反式维甲酸(All-trans retinoic acid,ATRA)处理的骨髓间充质干细胞(Mesenchymal stem cells,MSCs),检测其对RARβ的表达及MSCs成神经分化的影响。设计针对大鼠RARβ的4对siRNA的DNA序列,体外退火形成双链,定向克隆至含有U6/H1双启动子的腺病毒穿梭质粒pSES-HUS,随后与腺病毒骨架质粒pAd-Easy1在BJ5183细菌中同源重组,并在HEK293细胞中包装获得重组腺病毒Ad-siRARβ。腺病毒感染大鼠MSCs后经ATRA处理24 h,Real-time、Western blotting及免疫荧光检测RARβ的表达情况。改良神经诱导培养基(Modified neuronal induction medium,MNM)诱导MSCs神经分化,Real-time PCR及免疫荧光检测神经相关蛋白表达。PCR、酶切及测序鉴定均证实siRNA正确克隆至腺病毒质粒中,腺病毒感染大鼠MSCs后可观察到60%以上的细胞有红色荧光蛋白(Red fluorescent protein,RFP)表达。经ATRA处理24 h,Real-time、Westernblotting及免疫荧光检测发现RARβ表达定位于细胞核,ATRA作用后MSCs中RARβ表达增高16.5±2.34倍(P<0.05),有3组siRNA能有效抑制ATRA诱导的RARβ表达增强,抑制率分别为(66.26±9.12)%、(48.70±5.78)%、(64.09±0.53)%(P<0.05),且以pool组效果最强,抑制率为(78.09±4.24)%(P<0.01)。ATRA联合MNM诱导MSCs成神经样细胞,表达相关神经特异蛋白Nestin、NSE、MAP-2、Tau,免疫荧光结果显示神经标志蛋白Nestin、NSE、Tju1表达阳性细胞率为(50-88)%,而腺病毒介导的siRARβ能有效抑制MSCs的神经标志物表达水平及阳性细胞率(P<0.05)。成功构建了携带针对大鼠RARβ基因的siRNA重组腺病毒,能有效感染MSCs并显著抑制ATRA诱导的RARβ表达增强和MSCs的神经分化。     

GABA Synthesis in Astrocytes After Infection with Defective Herpes Simplex Virus Vectors Expressing Glutamic Acid Decarboxylase 65 or 67

Abstract: Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform-specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a β-galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein-expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector-infected cells were almost twofold higher than in GAD65 vector-infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K⁺, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.     

Galactosidase of plant fibers with gelatinous cell wall: Identification and localization

Using a comprehensive approach, we have identified a tissue-specific β-galactosidase from flax (Linum usitatissimum L.) phloem fibers forming a gelatinous cell wall. It was found that when fibers started to develop gelatinous cell wall, β-galactosidase gene expression was enhanced.. Using the antibodies against β-galactosidase, we showed that the enzyme was located in flax phloem fibers where it was detected together with tissue-specific galactan in secreted Golgi vesicles and in gelatinous secondary cell wall. Similar β-galactosidase present in gelatinous cell wall of fibers was found in plants belonging to various taxa and produced by different meristems; these data presume the identical mechanisms of gelatinous cell wall formation and an important role of β-galactosidase. The role of this enzyme in developing the supramolecular structure of gelatinous cell wall is discussed.     

New adenovirus vectors for protein production and gene transfer

Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10–15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non- complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression. This revised version was published online in August 2006 with corrections to the Cover Date.     

Adenoviral transduction of hTGF-β1 enhances the chondrogenesis of bone marrow derived stromal cells

TGF-β1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-β1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-β1 expressed and secreted more hTGF-β1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 ± 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-β1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-β1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.     

Methods for construction of adenovirus vectors

Adenoviruses are attracting increasing attention as general purpose mammalian cell expression vectors, as recombinant vaccines, and potentially as vectors for gene therapy. Not only is the adenovirus genome relatively easy to manipulate by recombinant DNA techniques, but adenovirus vectors are relatively stable, grow to high titers, and can transduce a variety of cell types in cell culture and in vivo. Vectors can be designed that are either replication competent or replication defective and, in the latter case, are highly efficient at delivering and expressing genes in mammalian cells without resulting in cell killing. Methods are described for growing, titrating, and purifying adenoviruses, for extracting viral DNA from purified virions and from infected cells, for rescuing inserts of foreign DNA into the viral genome, and for assessing expression of inserted genes in adenovirus vectors.     

Adenovirus binding to the coxsackievirus and adenovirus receptor or integrins is not required to elicit brain inflammation but is necessary to transduce specific neural cell types

Intracranial administration of adenovirus vectors elicits rapid, capsid-mediated dose-dependent brain inflammation. The mechanisms through which adenovirus capsids trigger inflammation in the brain remain unknown. We determined whether adenovirus interaction with the primary and secondary cell surface receptors for infection (CAR and alphav integrins) was necessary to trigger acute adenovirus-mediated brain inflammation, and, furthermore, whether capsid mutations that abrogated CAR and integrin binding altered vector tropism in the brain. Vectors ablated for CAR binding, but retaining integrin binding function, transduced equivalent areas of brain compared to vectors with wild-type capsids; however, vector tropsim was dramatically altered. Vectors with wild-type capsids predominantly transduced oligodendrocytes, whereas mutation of the fiber protein to ablate CAR binding resulted in a loss of oligodendrocyte transduction and a consequent redirection of transduction to neurons and other types of glial cells. Combined mutations of fiber and penton base that ablate both CAR and integrin binding almost abolished brain transduction. Thus, doubly-ablated capsids engineered to express new ligands should allow complete vector retargeting in the central nervous system. Although transduction by the doubly-ablated vectors was reduced by greater than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of adenovirus binding and infection of cells via CAR and integrin receptors.