Short-term culture of myeloid leukemic cells allows efficient transduction by adenoviral vectors

BACKGROUND: Ex vivo gene therapy of acute myeloid leukemia (AML) requires efficient transduction of leukemic cells. Recombinant adenovirus has been reported to be a poorly efficient vector in leukemic cells. We investigated leukemic cell culture as a possible method of improving the efficacy of this vector. METHODS: Leukemic cell lines and primary cultured AML cells were incubated with adenoviral vectors carrying GFP, LacZ, or IL-12 cDNA. Transduction efficiency was evaluated by measuring adenoviral genome copy number and transgene expression in leukemic cells. The expression of the coxsackie/adenovirus receptor (CAR), CD29, CD49e, and CD51/61 was measured, as was the effect of blocking integrin on adenoviral transduction. RESULTS: Increasing the multiplicity of infection (MOI) to 300 plaque-forming units per cell enhanced transduction of leukemic cell lines and to a lesser degree of AML cells. Analysis of adenoviral genome copy per cell showed only a partial correlation between gene transfer efficiency and transgene expression. Culture of AML cells for 3 days prior to adenoviral transduction increased both adenoviral copy number per cell and the percentage of transgene-expressing cells. CD29, CD49e, and CD51/61 but not CAR expression increased in cultured AML cells between days 0 and 3 and integrin-blocking experiments showed inhibition of transduction in two of four AML samples tested. CONCLUSIONS: Efficient ex vivo gene transfer in primary cultured AML cells can be achieved by short-term culture of leukemic cells prior to gene transfer with adenoviral vectors at a high MOI. This effect appears to be at least partially mediated by enhanced integrin expression.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Replication properties of human adenovirus in vivo and in cultures of primary cells from different animal species

Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.     

Preparation of isotopically labelled recombinant β-defensin for NMR studies

Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such as recombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochondria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombinant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and on production of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental 293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP produced in vMIA-293 cell cultures was consistently higher (140%) compared to that in the parental cells. vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirus replication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parental cells.In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improve recombinant protein production in the adenovirus-293 expression system.     

Innate immune response to adenoviral vectors is mediated by both Toll-like receptor-dependent and -independent pathways

Recombinant adenoviral vectors have been widely used for gene therapy applications and as vaccine vehicles for treating infectious diseases such as human immunodeficiency virus disease. The innate immune response to adenoviruses represents the most significant hurdle in clinical application of adenoviral vectors for gene therapy, but it is an attractive feature for vaccine development. How adenovirus activates innate immunity remains largely unknown. Here we showed that adenovirus elicited innate immune response through the induction of high levels of type I interferons (IFNs) by both plasmacytoid dendritic cells (pDCs) and non-pDCs such as conventional DCs and macrophages. The innate immune recognition of adenovirus by pDCs was mediated by Toll-like receptor 9 (TLR9) and was dependent on MyD88, whereas that by non-pDCs was TLR independent through cytosolic sensing of adenoviral DNA. Furthermore, type I IFNs were pivotal in innate and adaptive immune responses to adenovirus in vivo, and type I IFN blockade diminished immune responses, resulting in more stable transgene expression and reduction of inflammation. These findings indicate that adenovirus activates innate immunity by its DNA through TLR-dependent and -independent pathways in a cell type-specific fashion, and they highlight a critical role for type I IFNs in innate and adaptive immune responses to adenoviral vectors. Our results that suggest strategies to interfere with type I IFN pathway may improve the outcome of adenovirus-mediated gene therapy, whereas approaches to activate the type I IFN pathway may enhance vaccine potency.     

SOCS3基因重组腺病毒的构建及其在猪脂肪细胞中的表达

本研究旨在构建细胞因子信号转导抑制因子3(Suppressor of cytokine signaling 3,SOCS3)的重组腺病毒表达载体,获得有感染性的病毒颗粒。以pcDNA3-SOCS3质粒为模板扩增SOCS3基因,将其亚克隆至腺病毒穿梭载体pAdTrack-CMV,经测序验证后,重组的穿梭质粒用PmeI酶切线性化后转化到BJ5183感受态细菌中与其内的骨架载体pAdEasy-1进行同源重组,获得的重组质粒pAd-SOCS3,经PacI线性化后转染至HEK293细胞中进行包装和扩增,纯化后用TCID50法测定病毒滴度。以重组的病毒感染原代培养的猪脂肪细胞后,荧光显微镜下观察报告基因GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3 mRNA和蛋白的表达。重组腺病毒载体pAd-SOCS3经酶切及PCR鉴定正确,病毒滴度为1.2×109PFU/mL;感染原代培养的猪脂肪细胞后,荧光显微镜观察可见报告基因GFP的表达;RT-PCR和Western blotting检测到细胞中SOCS3 mRNA和蛋白的表达显著提高。本研究成功构建了SOCS3基因的重组腺病毒,感染原代培养的猪脂肪细胞可稳定表达SOCS3蛋白,为深入研究SOCS3的功能奠定了基础。     

P27特异性siRNA重组腺病毒载体的构建及功能鉴定

目的构建针对P27的siRNA腺病毒载体及相应的对照病毒载体,并鉴定重组腺病毒在小鼠胰岛中对内源性皿7基因表达的影响。方法合成针对p27的siRNA的靶DNA序列及相应的阴性对照序列,连接至pShuttle-H1质粒中,然后用NotI和HindHI限制性内切酶将H1-siRNA片段从pShuttle—H1-siRNA质粒上双酶切下来,克隆至空载pAdTrack穿梭质粒上,与腺病毒骨架质粒pAdeasy-1在BJ5183细菌中进行同源重组,转染QBI-293A细胞,包装得到pAd—P27-siRNA和pAd—NC重组腺病毒。用病毒体外感染小鼠胰岛,Western印迹法检测P27蛋白表达水平。结果重组腺病毒载体经测序鉴定正确,制备的病毒感染效率高,能有效抑制小鼠胰岛中P27的表达。结论成功构建了针对P27的siRNA重组腺病毒载体,为进一步研究P27在胰岛β细胞生长中的作用提供了基础。     

Genetic incorporation of human metallothionein into the adenovirus protein IX for non-invasive SPECT imaging

As the limits of existing treatments for cancer are recognized, clearly novel therapies must be considered for successful treatment; cancer therapy using adenovirus vectors is a promising strategy. However tracking the biodistribution of adenovirus vectors in vivo is limited to invasive procedures such as biopsies, which are error prone, non-quantitative, and do not give a full representation of the pharmacokinetics involved. Current non-invasive imaging strategies using reporter gene expression have been applied to analyze adenoviral vectors. The major drawback to approaches that tag viruses with reporter genes is that these systems require initial viral infection and subsequent cellular expression of a reporter gene to allow non-invasive imaging. As an alternative to conventional vector detection techniques, we developed a specific genetic labeling system whereby an adenoviral vector incorporates a fusion between capsid protein IX and human metallothionein. Our study herein clearly demonstrates our ability to rescue viable adenoviral particles that display functional metallothionein (MT) as a component of their capsid surface. We demonstrate the feasibility of (99m)Tc binding in vitro to the pIX-MT fusion on the capsid of adenovirus virions using a simple transchelation reaction. SPECT imaging of a mouse after administration of a (99m)Tc-radiolabeled virus showed clear localization of radioactivity to the liver. This result strongly supports imaging using pIX-MT, visualizing the normal biodistribution of Ad primarily to the liver upon injection into mice. The ability we have developed to view real-time biodistribution in their physiological milieu represents a significant tool to study adenovirus biology in vivo.     

Soluble expression and purification of the functional interleukin-30 protein in Escherichia coli

Interleukin-30 (IL-30), or IL-27p28, is the α subunit of IL-27 constructed by Epstein–Barr virus-induced gene 3 (EBI3) and IL-27p28 binding via noncovalent bonds. IL-30 can be independently secreted and function independently of IL-27. Recent studies demonstrated IL-30 could concurrently antagonize T helper 1 (Th1) and Th17 responses and might have therapeutic implications for controlling autoimmune diseases. However, no reports have stated an efficient method to generate a relatively large quantity of IL-30. In this study, an Escherichia coli expression system for the rapid expression of the mouse IL-30 is developed. For the first time, IL-30 was expressed in a form of soluble fusion protein and purified using a method of simple affinity chromatography. In order to avoid the impact of minor codons on expressing eukaryotic protein in E. coli and to improve the expression quantity, the nucleotide sequence of IL-30 was optimized. The optimized gene sequence was then subcloned into the pET-44a(+) vector, which allowed expression of IL-30 with a fusion tag, NusA. The vector was transformed into E. coli and the expressed fusion protein, NusA-IL-30, was purified by Ni chromatography. Then the fusion tag was removed by cleavage with thrombin. The purity of purified IL-30 was identified using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as well as high-performance liquid chromatography (HPLC) and the purity was up to about 92%. The yield of IL-30 was 8.95 mg from 1 L of bacterial culture. Western blot confirmed the identity of the purified protein. The recombinant IL-30 showed its biological activity by inhibiting Th17 differentiating from naive CD4⁺ T cells. Therefore, this method of express and purifying IL-30 provides novel procedures to facilitate structural and functions studies of IL-30.     

Transduction with a fiber-modified adenoviral vector is superior to non-viral nucleofection for expressing tumor-associated Ag mucin-1 in human DC

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase the chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. Methodologically, several recombinant DNA delivery techniques have been used. METHODS: In this study we compared nucleofection, an optimized form of electroporation, and adenoviral transduction regarding their efficiency to transduce human monocyte-derived (Mo-) DC in vitro. Expression of the tumor-associated Ag mucin-1 (MUC1) after adenoviral transduction (rAd5Fib35-MUC1) was determined using two MAb. RESULTS: We showed that the viability of cells and percentage of green fluorescent protein (GFP)-positive cells after transduction with a fiber-modified adenoviral vector (rAd5F35-GFP) was much higher than after nucleofection. Furthermore, phenotype and function of DC were not impaired by infection with adenovirus particles. Cells matured normally; up-regulation of CD40, CD80, CD83, CD86 and HLA-DR was not affected by adenoviral transduction. The capacity to stimulate naive T-cell proliferation was preserved and no change in IL-10 production was observed. Production of IL-12 increased up to 500-fold upon adenoviral transduction, considered to contribute positively to an anti-tumor immune response. Non-transduced mature DC expressed low levels of endogenous MUC1. After transduction with the rAd5F35-MUC1 adenoviral vector, a 100-fold increase in MUC1 expression by DC was observed. DISCUSSION: The use of the fiber-modified adenoviral vector presented here may therefore be favorable compared with non-viral gene delivery systems for DC that will be used in cancer immunotherapy.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.     

共表达构建呈现人白细胞介素-13抗原肽的Qβ噬菌体病毒样颗粒

目的:构建呈现人白细胞介素-13(interleukin-13,IL-13)抗原肽的Qβ噬菌体病毒样颗粒(virus-like particles,VLPs)疫苗。方法:将人IL-13抗原肽经基因重组插入Qβ噬菌体衣壳蛋白(CP)的C端。在BL21细菌中,经IPTG诱导CP及C端接有IL-13抗原肽的CP(CP/IL-13)同时表达。以硫酸铵沉淀及蔗糖密度梯度离心进行VLPs纯化及分析嵌合VLPs的存在,以HPLC分析VLPs纯度,以电镜观察颗粒形态。小鼠经皮下免疫VLPs后采集血清,以ELISA检测人IL-13特异性Ig G抗体水平。结果:重组蛋白CP与CP/IL-13获得成功表达,两者在密度梯度离心中有一致的、与QβVLPs相同的沉降行为,而CP/IL-13单独无Qβ颗粒行为。经纯化获得了高纯度颗粒,嵌合颗粒与Qβ颗粒形态相似。此外,该VLPs疫苗诱导小鼠产生了IL-13特异的抗体应答。结论:利用共表达策略可成功构建呈现人IL-13抗原表位的嵌合VLPs,为以主动免疫方式调控IL-13在疾病中的病理作用,提供了具有临床应用潜能的疫苗形式。     

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.     

鼠重组磷脂酶D2腺病毒的构建及功能的初步研究

将磷脂酰胆碱专一性磷脂酶D2基因及其功能缺陷点突变基因 (K75 8R)从真核表达载体pCGN中克隆至带有绿色荧光标记蛋白的穿梭质粒pAdTrack CMV中 ;再与腺病毒骨架载体一起在大肠杆菌BJ5183中进行同源重组 ,成功构建磷脂酶D2重组腺病毒。该病毒颗粒感染人胚肾 2 93细胞 ,高效表达磷脂酶D2及其功能缺陷蛋白。这种表达对M3乙酰胆碱受体介导的细胞内磷脂酶D激活无影响。但磷脂酶D2功能缺陷蛋白对蛋白激酶C介导的胞内磷脂酶D激活有显著抑制作用 ;相反 ,磷脂酶D2蛋白有显著增强作用。结果表明     

TNF-alpha receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung

Recombinant adenovirus-mediated gene therapy has demonstrated great promise for the delivery of genes to the pulmonary epithelium. However, dose-dependent inflammation and local immune responses abbreviate transgene expression. The purpose of these studies was to determine the role of TNF-alpha and individual TNF receptor signaling to adenovirus clearance and immune responses, and whether coexpression of human IL-10 could reduce inflammation and extend the duration of transgene expression in the lung. beta-Galactosidase expression in mice receiving intratracheal instillation of Adv/beta-gal (adenovirus construct expressing beta-galactosidase) was transient (less than 14 days), but a significant early increase of beta-galactosidase expression was seen in mice lacking either or both TNF-alpha receptors. Absence of TNF-alpha or the p55 receptor significantly attenuated the Ab response to both adenovirus and beta-galactosidase. Human IL-10 expression in the lung suppressed local TNF-alpha production following AdV/hIL-10 (adenovirus construct expressing human IL-10) delivery, but did not lead to increased or prolonged transgene expression when coexpressed with beta-galactosidase. Expression of human IL-10 following AdV/hIL-10 instillation extended at least 14 days, was nonimmunogenic, and suppressed the development of neutralizing Abs against adenoviral proteins as well as against human IL-10. We conclude that TNF-alpha signaling through both the p55 and p75 receptor plays important roles in the clearance of adenoviral vectors and the magnitude of the humoral immune response. Additionally, although coexpression of human IL-10 with beta-galactosidase had only modest effects on transgene expression, we demonstrate that AdV/hIL-10 is well tolerated, has extended expression compared with beta-galactosidase, and is nonimmunogenic in the lung.     

Adenovirus Entry From the Apical Surface of Polarized Epithelia Is Facilitated by the Host Innate Immune Response

Prevention of viral-induced respiratory disease begins with an understanding of the factors that increase or decrease susceptibility to viral infection. The primary receptor for most adenoviruses is the coxsackievirus and adenovirus receptor (CAR), a cell-cell adhesion protein normally localized at the basolateral surface of polarized epithelia and involved in neutrophil transepithelial migration. Recently, an alternate isoform of CAR, CAR^Ex8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that the endogenous role of CAR^Ex8 may be to facilitate host innate immunity. We show that IL-8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAR^Ex8 via activation of AKT/S6K and inhibition of GSK3β. Apical CAR^Ex8 tethers infiltrating neutrophils at the apical surface of a polarized epithelium. Moreover, neutrophils present on the apical-epithelial surface enhance adenovirus entry into the epithelium. These findings suggest that adenovirus evolved to co-opt an innate immune response pathway that stimulates the expression of its primary receptor, apical CAR^Ex8, to allow the initial infection the intact epithelium. In addition, CAR^Ex8 is a new target for the development of novel therapeutics for both respiratory inflammatory disease and adenoviral infection.     

Cationic lipids and polymers are able to enhance adenoviral infection of cultured mouse myotubes

Adenovirus-mediated gene transfer has been used to promote efficient expression of various reporter and therapeutic transgenes such as minidystrophin in skeletal muscle tissue. However, down-regulation of the adenovirus internalisation receptors, alpha(v)/beta3 and alpha(v)beta5, in adult myofibres and in mature cultured myotubes makes them less susceptible to infection than neonatal muscle or cultured myoblasts. It has been reported elsewhere that adenoviral transduction of cells that are normally refractory to infection can be enhanced by complexing virus particles with cationic lipids or cationic polymers. In this study we describe increased levels of adenovirus-mediated transduction of cultured C2C12 myotubes, when the vector is complexed with either of the cationic lipids Lipofectamine or 1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide (DOSPER) or the cationic polymer polyethylenimine. The presence of polycations allowed a smaller dose of adenovirus vector to be used to attain the same level of infection seen with adenovirus alone, which has important relevance to future in vivo studies. Electron microscopic analysis of adenovirus/polycation complexes showed large aggregates as opposed to single adenovirus particles in the absence of polycations. Finally, by complexing adenovirus particles with polycations, partial protection against the neutralising effect of adenovirus antiserum was observed.     

The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors.

Systemic application of first-generation adenovirus induces pathogenic effects in the liver. To begin unraveling the mechanisms underlying early liver toxicity after adenovirus infusion, particularly the role of macrophage activation and expression of viral genes in transduced target cells, first-generation adenovirus or adenovirus vectors that lacked most early and late gene expression were administered to C3H/HeJ mice after transient depletion of Kupffer cells by gadolinium chloride treatment. Activation of NF-kappaB, and the serum levels of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-6 (IL-6) were studied in correlation with liver damage, apoptosis, and hepatocellular DNA synthesis. While Kupffer cell depletion nearly eliminated adenovirus-induced TNF release, it resulted in a more robust IL-6 release. These responses were greatly reduced in animals receiving the deleted adenovirus. Although there were quantitative differences, NF-kappaB activation was observed within minutes of first-generation or deleted adenovirus vector administration regardless of the status of the Kupffer cells, suggesting that the induction is related to a direct effect of the virus particle on the hepatocyte. Early liver toxicity as determined by serum glutamic-pyruvic transaminase elevation and inflammatory cell infiltrates appeared to be dependent on adenovirus-mediated early gene expression and intact Kupffer cell function. Kupffer cell depletion had little effect on adenovirus-mediated hepatocyte apoptosis but did increase hepatocellular DNA synthesis. Finally, Kupffer cell depletion decreased the persistence of transgene (human alpha1-antitrypsin [hAAT]) expression that was associated with a more pronounced humoral immune response against hAAT. The elucidation of these events occurring after intravenous adenovirus injection will be important in developing new vectors and transfer techniques with reduced toxicity.     

Evaluation of optimal concentration and exposure duration of valproic acid alone or in combination with ViraDuctin to augment adenovirus transduction in human adipose stem cells

Background aimsRecombinant adenoviruses have tremendous potential in both gene therapy research and therapeutic applications. Mesenchymal stromal cells have a set of several properties that make them ideally suited for both regenerative medicine and gene and drug delivery. A limitation of adenoviral-mediated gene transfer is indeed the poor transduction rate of cells with low or no levels of the specific adenoviral cell surface receptor coxsackie virus and adenovirus receptor (CAR), such as human mesenchymal stromal cells. In the present work, we tried to increase the adenovirus transduction level and mediated gene delivery of human adipose stem cells with the use of valproic acid (VPA) and determined the proper concentration and duration of treatment alone or in combination with ViraDuctin adenovirus transduction reagent.MethodsGreen fluorescent protein–expressing recombinant adenovirus was propagated. The effects of various doses and exposure periods of VPA on CAR expression in human adipose stem cells were speculated by quantitative real-time polymerase chain reaction and adenoviral transduction rate by flow cytometry in different doses and time intervals of VPA and in combination with ViraDuctin transduction reagent.ResultsCAR messenger RNA upregulation through VPA was observed in human adipose stem cells; it was a dependent factor of dose and exposure time. Consequently, adenoviral transduction level of human adipose stem cells treated with VPA was increased, and co-administration of VPA and ViraDuctin further enhanced the transduction rate.ConclusionsThese results confirm that addition of VPA to hASCs alone or in combination with ViraDuctin has enhancing effects on adenoviral transduction rate, which can be auspicious in adenoviral-mediated gene therapy.