Production of adenoviral vectors and its recovery

The current demands for adenoviral vectors are increasing to satisfy pre-clinical and clinical gene therapy protocols. Consequently, there is a necessity of methodologies to improve production and recovery of intact particles with the minimum effect upon bioactivity. The production of adenoviral vectors in HEK 293 cells and the potential of an alternative aqueous two-phase system (ATPS) composed of PEG 300-phosphate in recovery of adenoviral vectors were investigated. The production of adenoviral vectors was carried out using a 2 L bioreactor equipped with two Rushton impellers. Different parameters including initial cell density, harvesting time and the addition of a buffer (HEPES) were studied in order to improve the production of adenoviral vectors in HEK 293 cells. A yield of 8 × 10¹¹ infective particles was achieved under the conditions characterized by the addition of Pluronic F-68, inoculation at an initial cell density of 3.5 × 10⁵ cells/mL and harvest of infected cells at 48 h post infection (hpi). This material was used for the evaluation of the ATPS recovery processes. It was demonstrated that the chemical components of the ATPS did not have a significant effect upon the infectivity of the adenoviral vectors and a total recovery of approximately 90% was obtained. These findings contribute to the process development for the manufacture of adenoviral vectors and other nanoparticulate bioproducts.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor.

Tagged G-protein-coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N-terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C-terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N-terminus, or EGFP located at the C-terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells.     

Analysis of the energetic state of heart cells after adenovirus-mediated expression of hALC-1

Expression of the human atrial myosin light chain 1 (hALC-1) in the cardiac ventricle in vivo as well as in primary cultivated adult cardiomyocytes caused a pronounced positive inotropic effect. Therefore, it is one of the most promising candidate gene to treat congestive heart failure (CHF). In this work, we investigated, whether hALC-1 expression also modifies the energetic state of cardiomyocytes. Primary cultivated neonatal rat hearts cells (NRHC) were infected with adenoviral vectors (Ad vectors) containing a hALC-1 cDNA (AdCMV.hALC-1) or a control Ad vector. Infection efficiency of NRHC reached 100% at 50 multiplicity of infection (MOI). Interestingly and in contrast to primary cultures of liver cells, there were no cytotoxic side effects or induction of apoptosis up to MOI 50 in Ad vector infected NRHC. NRHC expressed large amounts of hALC-1 upon infection with AdCMV.hALC-1 which could easily been detected by protein staining and Western blot analysis. Analysis of intracellular hALC-1 localization by double-labeling immunofluorescence of AdCMV.hALC-1 infected cardiomyocytes revealed the typical myofibrillar striation pattern, as well as co-localization of hALC-1 with myosin heavy chains. There was no difference in the oxygen consumption between controls and AdCMV.hALC-1 infected NRHC. These data suggest that first: adenoviral vectors could be used as a safe and effective tool for gene transfer to cardiomyocytes, and second: that a positive inotropic effect of hALC-1 is not associated with enhanced oxygen consumption.     

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.     

Intracellular trafficking of a tagged and functional mammalian olfactory receptor

Tagged G‐protein‐coupled receptors (GPCRs) have been used to facilitate intracellular visualization of these receptors. We have used a combination of adenoviral vector gene transfer and tagged olfactory receptors to help visualize mammalian olfactory receptor proteins in the normal olfactory epithelium of rats, and in cell culture. Three recombinant adenoviral vectors were generated carrying variously tagged versions of rat olfactory receptor I7. The constructs include an N‐terminal Flag epitope tag (Flag:I7), enhanced green fluorescent protein (EGFP) fusion protein (EGFP:I7), and a C‐terminal EGFP fusion (I7:EGFP). These receptor constructs were assayed in rat olfactory sensory neurons (OSNs) and in a heterologous system (HEK 293 cell line) for protein localization and functional expression. Functional expression of the tagged receptor proteins was tested by electroolfactogram (EOG) recordings in the infected rat olfactory epithelium, and by calcium imaging in single cells. Our results demonstrate that the I7:EGFP fusion protein and Flag:I7 are functionally expressed in OSNs while the EGFP:I7 fusion is not, probably due to inappropriate processing of the protein in the cells. These data suggest that a small epitope tag (Flag) at the N‐terminus, or EGFP located at the C‐terminus of the receptor, does not affect ligand binding or downstream signaling. In addition, both functional fusion proteins (Flag:I7 and I7:EGFP) are properly targeted to the plasma membrane of HEK 293 cells. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 56–68, 2002     

一种高效感染血液细胞的新型靶向性腺病毒载体的构建

人C组5型腺病毒(Ad5)载体能够有效感染上皮来源的细胞,但对造血细胞的感染效率很低,限制了其在造血调控基础研究以及血液病基因治疗中的应用。为了建立高效感染血液细胞的新型靶向性腺病毒载体系统,对5型腺病毒载体的纤维顶球进行了改造,以AdEasy系统为基础,应用递归PCR的方法人工合成人B组11p型腺病毒的部分纤维(fiber)基因,采用一系列分子生物学方法将其替换AdEasy骨架质粒中的人5型腺病毒的fiber基因,得到新的腺病毒骨架质粒命名为pAdEasy-1/F11p,应用带有GFP报告基因的穿梭质粒pShuttle-GFP与AdEasy-1/F11p腺病毒DNA在BJ5183细菌内重组得到重组腺病毒质粒,将其转染293细胞获得重组腺病毒,命名为Ad5F11p-GFP。以Ad5-GFP作对照,同时感染K562、U937等白血病细胞系,流式细胞仪检测GFP的表达。初步检测结果显示:在10MOI时,Ad5F11p-GFP能够有效感染K562、U937等白血病细胞系,感染细胞效率>90%,对照Ad5-GFP感染细胞效率<30%,这表明改建后的腺病毒AdEasy-1/F11p可以高效介导基因转移到血液细胞,是一种很好的血液细胞靶向性腺病毒载体。     

Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells

This article describes the step-wise approach undertaken to select a serum-free medium (SFM) for the efficient production of a recombinant adenoviral vectors expressing beta-galactosidase (Ad5 CMV-LacZ), in the complementing human embryonic kidney 293S cells. In the first step, a 293S-derived transfectoma, secreting a soluble epidermal growth factor receptor sEGFr (D2-22), was used to estimate the potential of selected serum-free formulations to support the production of a recombinant protein as compared to serum-containing medium. Assays showed that only one among six commercial serum-free formulations could support both sEGFr production and cell growth in static or suspension culture. In commercially available calcium-containing serum-free formulations, the cell aggregates reached up to 3 mm in diameter. In the second step, 293S cells were gradually adapted to a low-calcium version of the selected medium (LC-SFM). Cells were cloned, then screened according to their ability to grow at a rate and an extent comparable to parental cells in serum-containing medium (standard) as single cells or small aggregates. The 293SF-3F6 clone, first adapted to and then cloned in the selected serum-free medium, was selected for further experiments. Bioreactor run performed with the 293SF-3F6 clone showed similar growth curve as in the shake-flask controls. In the final step, the recombinant viral vector productivity of the 293S cells and the 293SF-3F6 clone was tested. The 293SF-3F6 cells infected by Ad5 CMV-LacZ in 3 L-scale bioreactor maintained the specific productivities of both beta-galactosidase and adenoviral vector equivalent to the shake-flask controls in suspension culture. Results from this study clearly demonstrate that the 293SF-3F6 cell line thus selected may be used either for establishing stable transfected cell line or for the production of adenoviral vectors required for gene therapy studies.     

慢病毒介导的大鼠肝BRL-3A 细胞Tmub1 基因沉默及稳定感染细胞 系的建立

目的:构建Tmub1基因慢病毒干扰载体,建立稳定转染细胞系,检测大鼠肝BRL-3A细胞中Tmub1基因表达的干扰效果。方法:设计并构建4对针对大鼠Tmub1基因的特异性shRNA干扰质粒,酶切鉴定、DNA测序所得质粒。将由pRSV-Rev、pMDLg-pRRE、pMD2G和pll3.7干扰质粒组成的包装系统共转染293T细胞,产生慢病毒。所得慢病毒感染大鼠正常肝细胞BRL-3A,Western Blot检测不同靶点RNAi后Tmub1蛋白表达情况,确定有效靶点。针对有效靶点大量包装慢病毒。测定病毒滴度并以最适感染复数(multiplicity of infection,MOI)感染BRL-3A细胞后,G418抗生素筛选稳定感染细胞系BRL-3A/256。RT-PCR和Western Blot检测各组细胞Tmub1 mRNA和蛋白质的表达差异。结果:结果显示Tmub1 RNAi慢病毒载体构建成功,C0020Sh2-Hops-256干扰靶点RNAi效果最强。成功包装Tmub1基因RNAi慢病毒,测定病毒滴度为2.3×108TU/ml,对293T细胞的最适感染复数为60。成功建立Tmub1 RNAi慢病毒载体稳定感染细胞系BRL-3A/256,且在该细胞系中Tmub1 mRNA和蛋白质表达明显降低。结论:成功构建Tmub1 RNAi慢病毒载体,有效干扰BRL-3A细胞中Tmub1 mRNA和蛋白表达;成功筛选出Tmub1RNAi慢病毒稳定感染细胞系BRL-3A/256。     

Integrin alpha(v)beta1 is an adenovirus coreceptor

The human embryonic kidney (HEK293) cell line, commonly used for recombinant adenovirus (Ad) propagation, does not express the Ad coreceptor alpha(v)beta3 or alpha(v)beta5 integrins, yet these cells are efficiently infected by Ad vectors. Here we demonstrate that Ad binds to HEK293 cells via the fiber receptor CAR and is subsequently internalized via interaction with integrin alpha(v)beta1. Function-blocking antibodies directed against alpha(v) or beta1, but not beta3, beta5, or alpha5, integrin subunits block Ad infection and viral endocytosis. Therefore, alpha(v)beta1 serves as a coreceptor for Ad infection, and the lack of beta3 and/or beta5 but the relatively high expression of alpha(v)beta1 integrins on certain tumor cell types may explain why these cells are readily transduced by Ad vectors.     

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.     

人SPRED2 基因重组腺病毒载体的制备及其对K562 细胞

目的:构建携带SPRED2的质粒载体与重组腺病毒载体,并观察其在K562细胞的表达及对ERK信号通路的作用,为Spred2在造血细胞中的作用的研究奠定基础。方法:以HepG2细胞cDNA为模板,RT-PCR克隆SPRED2全长CDS序列,并亚克隆到pCDNA3.0和pshuttle-CMV质粒载体,构建携带SPRED2的真核表达载体pCDNA3.0-Spred2与穿梭载体pshuttle-CMV-Spred2;将线性化pshuttle-CMV-Spred2与腺病毒骨架质粒Adf11p在感受态细胞BJ5183中进行同源重组,产生重组质粒Adf11p-Spred2;后者经线性化后转染至HEK293细胞进行病毒包装;在HEK293细胞扩增病毒颗粒,以CsCl密度梯度离心法进行纯化,TCID50法测定病毒滴度;将病毒颗粒以100MOI感染K562细胞,Western blot检测Spred2过表达情况及Spred2对细胞ERK的影响。结果:经酶切、DNA测序、Western blot检测等方法鉴定,证明pCDNA3.0-Spred2与Adf11p-Spred2携带Spred2序列正确,能够在HEK293细胞、K562细胞正确表达,Spred2过表达能够显著抑制K562细胞ERK活性。结论:成功构建对K562细胞有高感染效率的SPRED2重组腺病毒载体,且Spred2对K562细胞ERK信号通路有显著抑制作用。     

Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system

A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductions were carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.     

Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vectors

First-generation, E1/E3-deleted adenoviral vectors with diverse transgenes are produced routinely in laboratories worldwide for development of novel prophylactics and therapies for a variety of applications, including candidate vaccines against important infectious diseases, such as HIV/AIDS, tuberculosis, and malaria. Here, we show, for two different transgenes (both encoding malarial antigens) inserted at the E1 locus, that rare viruses containing a transgene-inactivating mutation exhibit a selective growth advantage during propagation in E1-complementing HEK293 cells, such that they rapidly become the major or sole species in the viral population. For one of these transgenes, we demonstrate that viral yield and cytopathic effect are enhanced by repression of transgene expression in the producer cell line, using the tetracycline repressor system. In addition to these transgene-inactivating mutations, one of which occurred during propagation of the pre-viral genomic clone in bacteria, and the other after viral reconstitution in HEK293 cells, we describe two other types of mutation, a small deletion and a gross rearranging duplication, in one of the transgenes studied. These were of uncertain origin, and the effects on transgene expression and viral growth were not fully characterized. We demonstrate that, together with minor protocol modifications, repression of transgene expression in HEK293 cells during viral propagation enables production of a genetically stable chimpanzee adenovirus vector expressing a malarial antigen which had previously been impossible to derive. These results have important implications for basic and pre-clinical studies using adenoviral vectors and for derivation of adenoviral vector products destined for large-scale amplification during biomanufacture.     

Development and improvement of a serum-free suspension process for the production of recombinant adenoviral vectors using HEK293 cells

Human Embryonic Kidney 293 (HEK293) cells were adapted into a serum-free suspension medium through steps of gradual serum weaning for the production of adenoviral (AdV) gene therapy vectors. The presence of sodium heparin in the medium formulation reduced cell clumping dramatically in suspension culture. The adapted cells were ready to grow either in serum-containing medium as an attached culture or in serum-free medium in suspension culture. A scalable production process was developed in shake flasks and was then evaluated in stirred tank bioreactors. This process includes a growth phase in batch-mode followed by a production phase involving medium perfusion and supplementation. Fortification with calcium chloride post viral inoculation resulted in an increase in virus production by at least one fold. Addition of stimulating agents such as sodium butyrate, N-acetyl-L-cysteine (NAC), dimethyl sulfoxide(DMSO), or ethyl alcohol post infection was shown to further improve virus production in a dose-dependent manner. The serum-free suspension process described here should be suitable for the manufacturing of other E1-deleted AdV vectors and could potentially be used for the production of recombinant proteins by HEK293 cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

应用Surrogate 报告载体提高RISPR/Cas9 对TMEM215基因的打靶效率*

目的:构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因打靶效率。方法:构建针对人TMEM215的CRISPR/Cas9表达载体及相应Surrogate报告载体,两者共转HEK293T细胞,通过流式分析、T7EI检测、TA克隆测序等明确Surrogate报告载体对不同sgRNA打靶效率的检测及对基因修饰细胞的筛选富集作用。结果:流式分析结果表明,Surrogate报告载体成功检测出不同sgRNA的打靶效率,并筛选出高效率sgRNA;T7EI检测及TA克隆测序显示,外加嘌呤霉素抗性筛选时,Surrogate报告载体可有效富集基因修饰细胞。结论:成功构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因的打靶效率。     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

Development of an adenoviral vector system with adenovirus serotype 35 tropism; efficient transient gene transfer into primary malignant hematopoietic cells

BACKGROUND: A paucity of coxsackie adenovirus receptor (CAR) hampers the adenovirus serotype 5 (Ad5)-based vector-mediated gene transfer into malignant hematopoietic cells. Fiber-retargeted adenoviral vectors with species B tropism can potentially bypass the CAR requirement and facilitate efficient gene transfer into malignant hematopoietic cells. METHODS: For feasible generation of fiber-retargeted adenoviral vectors, we have modified the versatile AdEasy system with a chimeric fiber gene encoding the Ad5 fiber tail domain and Ad35 fiber shaft and knob domains. An Ad5-based vector encoding the green fluorescent protein (GFP) gene under the control of the PGK promoter with Ad35 fiber receptor specificity was generated (Ad5F35-GFP). The Ad5F35-GFP vector-mediated gene transfer efficiency was compared with a fiber non-modified Ad5-GFP vector, which also encodes the GFP gene under the control of the PGK promoter. RESULTS: We demonstrated that a variety of Ad5-refractory malignant myeloid and B lymphoid cell lines were highly permissive to the Ad5F35-GFP vector infection. Importantly, primary chronic myeloid leukemic (CML) cells and chronic lymphocytic leukemia (CLL) B cells were superiorly transduced by the Ad5F35-GFP vector at a multiplicity of infection (MOI) of 100 compared with the Ad5-GFP vector. CONCLUSIONS: Our study will facilitate the generation of fiber-retargeted adenoviral vectors and enable transient genetic manipulation of primary malignant hematopoietic cells.