Adenovirus Purification Research Articles

Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy.

Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography

Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.

Impact of grafting on the design of new membrane adsorbers for adenovirus purification

The impacts of quaternary amine ligand density and matrix structure, namely hydrogel grafted and directly grafted, on state-of-the-art chromatographic membranes operated in bind-and-elute mode were evaluated for the purification of adenovirus serotype 5. The experiments were performed on a 96-well plate membrane holder, which is a convenient high-throughput screening tool for obtaining the best operating conditions for a process yield optimization. The results show that the hydrogel-grafted membranes are more suitable for virus purification than the directly grafted ones. By reducing the number of grafted ligands to low (1.7μmol/cm2) or medium (2.4μmol/cm2) density, it is possible to increase the recovery of purified virus by 60% compared to a highly charged membrane (3.3μmol/cm2) that yielded a recovery rate lower than 30%. In the reported experiments, Sartobind® Q, chosen as benchmark comparison, provides a better compromise between high recovery and large dynamic binding capacity. Overall, this work contributes to the understanding and development of new membrane adsorbers specifically designed for virus purification.

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

Development of a platform process for adenovirus purification that removes human set and nucleolin and provides high purity vector for gene delivery

The manufacturing of virus occurs at a modest scale in comparison to many therapeutic proteins mainly because a gene therapy dose is typically only µg of vector. Although modest in scale the generation of high purity virus is challenging due to low viral expression levels and the difficulties in adequately characterizing such a large and complex molecule. A 100 L bioreactor might produce only 100 mg of virus that must be separated from host and process impurities that are typically greater by several orders of magnitude. Furthermore, in the later purification stages the main milieu component is often virus at low concentration (µg/mL) which may non-specifically adsorb to purification surfaces resulting in a lowered virus recovery. This study describes our approach to develop a scalable, manufacturable robust process for an Adenovirus (Ad) gene therapy vector. A number of analytical tools were developed to guide the purification design. During process development, two human proteins, SET and nucleolin, were identified in viral preparations. To our knowledge, this is the first time that SET and nucleolin have been described in Ad. In this report we detail a process for their removal and the robust removal of all process, product and host cell impurities.

Purification of Adenovirus by Cesium Chloride Density Gradients

Adenovirus are efficient gene delivery systems. The standard method for purification of adenoviral vectors is based on using a cesium chloride (CsCl) density gradient combined with ultracentrifugation. This method is suitable for small-scale purification and is less expensive than column chromatography or commercial purification kits., 腺病毒是有效的基因递送系统。 用于纯化腺病毒载体的标准方法基于使用氯化铯（CsCl）密度梯度与超速离心的组合。 该方法适合于小规模纯化，并且比柱层析或商业纯化试剂盒更便宜。

Insulin-like Growth Factor-binding Protein-3 (IGFBP-3) Blocks the Effects of Asthma by Negatively Regulating NF-κB Signaling through IGFBP-3R-mediated Activation of Caspases

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein known for modulating mitogenic and metabolic actions of IGFs as well as exerting a variety of biological actions not involving IGFs. Here, we show that IGFBP-3 blocks specific physiological consequences of asthma in an IGF-independent manner in vitro and in vivo. IGFBP-3 treatment effectively reduced all physiological manifestations of asthma examined in vivo (airway hyper-responsiveness, cellular and pathological changes in bronchoalveolar lavage fluid and lung tissue, and expression of numerous proinflammatory molecules). These unique IGFBP-3 effects were further confirmed in IGFBP-3-transgenic mice, thus strengthening the notion of IGFBP-3 actions within the respiratory system. Using human epithelial cells, we demonstrated the following: 1) IGFBP-3 blocks TNF-α-induced expression of proinflammatory molecules; 2) IGFBP-3 attenuates the TNF-α-induced migratory response of eosinophils; and 3) IGFBP-3 negatively regulates TNF-α-induced expression of the key NF-κB regulatory molecules IκBα and p65-NF-κB at the post-translational level. We identified that IGFBP-3 degrades IκBα and p65-NF-κB proteins through IGFBP-3 receptor (IGFBP-3R)-mediated activation of caspases thereby inhibiting TNF-α-induced activation of NF-κB signaling cascades. This unique IGFBP-3/IGFBP-3R action was further confirmed by demonstrating complete inhibition of IGFBP-3 action in the presence of caspase inhibitors as well as IGFBP-3R siRNAs. Non-IGF-binding IGFBP-3 mutants further proved the IGF-independent action of IGFBP-3. Our findings indicate that IGFBP-3 inhibits airway inflammation and hyper-responsiveness via an IGF-independent mechanism that involves activation of IGFBP-3R signaling and cross-talk with NF-κB signaling. The IGFBP-3/IGFBP-3R system therefore plays a pivotal role in the pathogenesis of asthma and can serve as a newly identified potential therapeutic target for this debilitating disease.

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery

In vitro and in vivo evaluation of adenovirus combined silk fibroin scaffolds for BMP-7 gene delivery

Enzymatic activity of the human 1-acylglycerol-3-phosphate-O-acyltransferase isoform 11: upregulated in breast and cervical cancers

The conversion of lysophosphatidic acid (LPA) to phosphatidic acid is carried out by the microsomal enzymes 1-acylglycerol-3-phosphate-O-acyltransferases (AGPATs). These enzymes are specific for acylating LPA at the sn-2 (carbon 2) position on the glycerol backbone and are important, because they provide substrates for the synthesis of phospholipids and triglycerides. At least, mutations in one isoform, AGPAT2, cause near complete loss of adipose tissue in humans. We cloned a cDNA predicted to be an AGPAT isoform, AGPAT11. This cDNA has been recently identified also as lysophosphatidylcholine acyltransferase 2 (LPCAT2) and lyso platelet-activating factor acetyltransferase. When AGPAT11/LPCAT2/lyso platelet-activating factor acetyltransferase cDNA was expressed in CHO and HeLa cells, the protein product localized to the endoplasmic reticulum. In vitro enzymatic activity using lysates of Human Embryonic Kidney-293 cells infected with recombinant AGPAT11/LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA adenovirus show that the protein has an AGPAT activity but lacks glycerol-3-phosphate acyltransferase enzymatic activity. The AGPAT11 efficiently uses C18:1 LPA as acyl acceptor and C18:1 fatty acid as an acyl donor. Thus, it has similar substrate specificities for LPA and acyl-CoA as shown for AGPAT9 and 10. Expression of AGPAT11 mRNA was significantly upregulated in human breast, cervical, and colorectal cancer tissues, indicating its adjuvant role in the progression of these cancers. Our enzymatic assays strongly suggest that the cDNA previously identified as LPCAT2/lyso platelet-activating factor-acetyltransferase cDNA has AGPAT activity and thus we prefer to identify this clone as AGPAT11 as well.

Role of Calcium-independent Phospholipase A2β in High Glucose-induced Activation of RhoA, Rho Kinase, and CPI-17 in Cultured Vascular Smooth Muscle Cells and Vascular Smooth Muscle Hypercontractility in Diabetic Animals

Previous studies suggest that high glucose-induced RhoA/Rho kinase/CPI-17 activation is involved in diabetes-associated vascular smooth muscle hypercontractility. However, the upstream signaling that links high glucose and RhoA/Rho kinase/CPI-17 activation is unknown. Here we report that calcium-independent phospholipase A(2)beta (iPLA(2)beta) is required for high glucose-induced RhoA/Rho kinase/CPI-17 activation and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility. We demonstrate that high glucose increases iPLA(2)beta mRNA, protein, and iPLA(2) activity in a time-dependent manner. Protein kinase C is involved in high glucose-induced iPLA(2)beta protein up-regulation. Inhibiting iPLA(2)beta activity with bromoenol lactone or preventing its expression by genetic deletion abolishes high glucose-induced RhoA/Rho kinase/CPI-17 activation, and restoring expression of iPLA(2)beta in iPLA(2)beta-deficient cells also restores high glucose-induced CPI-17 phosphorylation. Pharmacological and genetic inhibition of 12/15-lipoxygenases has effects on high glucose-induced CPI-17 phosphorylation similar to iPLA(2)beta inhibition. Moreover, increases in iPLA(2) activity and iPLA(2)beta protein expression are also observed in both type 1 and type 2 diabetic vasculature. Pharmacological and genetic inhibition of iPLA(2)beta, but not iPLA(2)gamma, diminishes diabetes-associated vascular smooth muscle hypercontractility. In summary, our results reveal a novel mechanism by which high glucose-induced, protein kinase C-mediated iPLA(2)beta up-regulation activates the RhoA/Rho kinase/CPI-17 via 12/15-lipoxygenases and thereby contributes to diabetes-associated vascular smooth muscle hypercontractility.

Suppression of 2,3-Oxidosqualene Cyclase by High Fat Diet Contributes to Liver X Receptor-α-mediated Improvement of Hepatic Lipid Profile

The liver X receptors (LXRs) sense oxysterols and regulate genes involved in cholesterol metabolism. Synthetic agonists of LXRs are potent stimulators of fatty acid synthesis, which is mediated largely by sterol regulatory element-binding protein-1c (SREBP-1c). Paradoxically, an improved hepatic lipid profile by LXR was observed in mice fed a Western high fat (HF) diet. To explore the underlying mechanism, we administered mice normal chow or an HF diet and overexpressed LXRalpha in the liver. The HF diet with tail-vein injection of adenovirus of LXRalpha increased the expression of LXR-targeted genes involved in cholesterol reverse transport but not those involved in fatty acid synthesis. A similar effect was also observed with the use of 22R-hydroxycholesterol, an LXR ligand, in cultured hepatocytes. Consequently, SREBP-1c maturation was inhibited by the HF diet, which resulted from the induction of Insig-2a. Importantly, increased cholesterol level suppressed the expression of 2,3-oxidosqualene cyclase (OSC), which led to an increase in endogenous LXR ligand(s). Furthermore, siRNA-mediated knockdown of OSC expression enhanced LXR activity and selectively up-regulated LXR-targeted genes involved in cholesterol reverse transport. Thus, down-regulation of OSC may account for a novel mechanism underlying the LXR-mediated lipid metabolism in the liver of mice fed an HF diet.

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 Zn 2+ 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.

Towards purification of adenoviral vectors based on membrane technology

Given increasing applications of recombinant adenoviruses for gene therapy and vaccination, there is a need for highly robust and fast purification platforms for their large scale manufacture. Traditional chromatographic methods using resins as matrices have several limitations such as high-pressure drops and slow processing rates due to pore diffusion and channelling of the feed through the bed. In contrast, membrane adsorbers offer the advantage of fast, gentle, and effective isolation. Furthermore, membranes are easy to use, no column packing is needed and, when used as disposables, no cleaning validation is necessary, representing a substantial advantage to meet cGMP requirements. In this work, a strategy for purification of adenovirus vectors from cell-culture bulks fully based on membrane devices is presented. Ultrafiltration membranes with molecular weight cutoffs of 300, 500, and 750 kDa were tested for the concentration of cell-culture supernatant after an initial clarification step. The results show that the use of ultrafiltration/diafiltration membranes not only concentrates the virus but also leads to the removal of 90% of host cell DNA and proteins in the retentate. Two membrane adsorbers (Sartobind Q and Sartobind anion direct) were evaluated for adenovirus vectors capture and purification. To define the best operating conditions, the effect of pH, conductivity, and recirculation of load bulk on the recovery yield of infectious adenoviruses were evaluated. Sartobind anion direct allows for higher recovery yields (up to 62%) of infectious adenoviruses than Sartobind Q; identical ratios between total and infectious adenoviruses (TP/IP) were achieved for both membrane adsorbers. The overall recovery yield of the process is approximately 52%; this work credits membrane technology as an alternative for the concentration and purification of adenoviruses and as a promising solution for downstream processing of other viral vectors.

291. A New Vacuum-Based Method for Adenovirus Purification and Concentration

291. A New Vacuum-Based Method for Adenovirus Purification and Concentration

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Corrigendum

CORRIGENDACorrigendumPublished Online:01 Jan 2007https://doi.org/10.1152/ajpendo.zh1-4813-corr.2007Original articleMoreSectionsPDF (26 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Volume 291, September 2006 Volume 54, September 2006 Pages E536–E543: Chaodong Wu, Salmaan A. Khan, Li-Jen Peng, Honggui Li, Steven G. Carmella, and Alex J. Lange. Perturbation of glucose flux in the liver by decreasing fructose-2,6-bisphosphate levels causes hepatic insulin resistance and hyperglycemia.The authors thank an alert reader for pointing out the missing information on the generation of a kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PFK2/FBP2 or Bif-KD). In the text of the paper, the authors cited their paper in Endocrinology (5), mainly for adenovirus preparation, i.e., amplification and purification. Strictly speaking, what was not provided was the information on the generation of the mutated cDNA coding for the kinase-deficient 6PFK2/FBP2. This information was presented at the 2002 American Diabetes Association Scientific Meeting, and the abstract was published in the supplemental issue of Diabetes (4). This abstract was cited in their Endocrinology paper, as well as in a paper from their collaborator (1). It appears that this abstract was not accessible.Herein is provided the information for generation of the mutated cDNA coding for the kinase-deficient 6PFK2/FBP2. One mutation was made that changed Ser32 to asparatate; another mutation was made that changed Thr55 to valine. These manipulations were chosen on the basis of papers published in J Biol Chem (2, 3). The mutated cDNA was then packaged into adenovirus using a commercial kit from Qbiogene (Carlsbad, CA). This mutated cDNA was also overexpressed in heart of transgenic mice (1).The authors want to point out that they did not publish any paper in Molecular Cell.REFERENCES1 Donthi RV, Ye G, Wu C, McClain DA, Lange AJ, Epstein PN. Cardiac expression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase inhibits glycolysis, promotes hypertrophy, impairs myocyte function, and reduces insulin sensitivity. J Biol Chem 279: 48085–48090, 2004.Crossref | PubMed | ISI | Google Scholar2 Kurland IJ, El-Maghrabi MR, Correia JJ, Pilkis SJ. Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho-forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis. J Biol Chem 267: 4416–4423, 1992.Crossref | PubMed | ISI | Google Scholar3 Vertommen D, Bertrand L, Sontag B, Di Pietro A, Louckx MP, Vidal H, Hue L, Rider MH. The ATP-binding site in the 2-kinase domain of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Study of the role of Lys-54 and Thr-55 by site-directed mutagenesis. J Biol Chem 271: 17875–17880, 1996.Crossref | PubMed | ISI | Google Scholar4 Wu C, Okar DA, Peng L, Lange AJ. Decreasing fructose-2,6-bisphosphate leads to diabetic phenotype in normal mice (Abstract). Diabetes 51, Suppl 2: 1300, 2002.Google Scholar5 Wu C, Okar DA, Stoeckman AK, Peng L, Herrera AH, Herrera JE, Towle HC, Lange AJ. A potential role for fructose-2,6-bisphosphate in the stimulation of hepatic glucokinase gene expression. Endocrinology 145: 650–658, 2004.Crossref | PubMed | ISI | Google Scholar Download PDF Previous Back to Top FiguresReferencesRelatedInformationRelated ArticlesPerturbation of glucose flux in the liver by decreasing F26P2 levels causes hepatic insulin resistance and hyperglycemia 01 Sep 2006American Journal of Physiology-Endocrinology and Metabolism More from this issue > Volume 292Issue 1January 2007Pages E370-E370 Copyright & PermissionsCopyright © 2007 by American Physiological Societyhttps://doi.org/10.1152/ajpendo.zh1-4813-corr.2007History Published online 1 January 2007 Published in print 1 January 2007 Metrics

Calcium-mediated Transient Phosphorylation of Tau and Amyloid Precursor Protein Followed by Intraneuronal Amyloid-β Accumulation

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Fast, effective and safe adenovirus purification with Vivapure AdenoPACK kits

The use of Sartorius membrane adsorbers incorporated in AdenoPACK units facilitates and speeds up the purification step for adenovirus preparation, which can take days using traditional methods. Ready-to-use kits are available for processing cell culture volumes from 20 ml to 500 ml, permitting preparation of up to 3 × 1013 purified viral particles in only 2 hours. Plus, the method can be scaled up for adenovirus purification in process scale.

A rapid and efficient method for purification of recombinant adenovirus with arginine–glycine–aspartic acid-modified fibers

Recombinant adenoviral vectors (adenovectors) have been subject to various genetic modifications to improve their transduction efficiency and targeting capacity. Production and purification of adenovectors with modified capsid proteins can be problematic using conventional two-cycle CsCl gradient ultracentrifugation. We have developed a new method for purifying recombinant adenovectors in two steps: iodixanol discontinuous density gradient ultracentrifugation and size exclusion column chromatography. The purity and infectious activity of adenovectors isolated by the two methods were comparable. The new method yielded three to four times more adenovectors with arginine–glycine–aspartic acid (RGD)-modified fiber proteins than did the conventional CsCl method. For other fiber-modified and wild-type adenovectors, the yields of the two methods were comparable. Thus, the iodixanol-based method can be used not only to improve the production of RGD-modified adenovectors but also to purify adenovectors with or without fiber modifications. Moreover, the whole procedure can be completed in 3 h. Therefore, this method is rapid and efficient for production of recombination adenovectors, especially those with RGD-modified fibers.

Serotype Specificity of Adenovirus Purification Using Anion- Exchange Chromatography

Serotype Specificity of Adenovirus Purification Using Anion- Exchange Chromatography