Optimization of HEK293T suspension cultivation with a DoE-approach in ambr®15 microbioreactor

Cell and gene therapies present a new treatment paradigm that have the potential to address clinical needs that are unmet by current small molecule and biotherapeutic approaches. Viral vectors such as adenoviruses, adeno-associated viruses and retroviruses are effective delivery systems for genetic material used in cell and gene therapies. Especially lentiviruses are used for example for the transfer of genetic information for novel cellular immunotherapy like CAR-T cell therapy. These novel approaches promise to be a substantial part of next-generation therapies with the potential to cure devastating diseases. HEK293T cells are a workhorse cell line for lentiviral vector production for both cell and gene therapy applications. A significant challenge for the cell and gene therapy industry is to develop a HEK293T suspension cell culture processes that is well characterized and can be scaled-up for production whilst ensuring clinical and commercial success. Ambr® 15 is an automated micro-scale bioreactor system that mimics the features and process control (pH, DO, temperature, stirring rate) provided by much larger scale bioreactors, in a volume of 10 - 15 ml. Parallel processing capability and excellent reproducibility enable rapid, high throughput process improvement and optimization, including DoE studies. High-throughput tools with parallel processing, such as ambr® 15, help to address a major manufacturing bottleneck. They can be used as a scale-down model for process screening, clone selection and effective media optimization in less time with reduced reagents use and labour savings. In the study presented we used ambr® 15 for the optimization of the HEK293T culture in suspension. We identified optimal stirring speed, DO and pH value by performing a DoE approach with the use of MODDE® software for experiment planning. Viable Cell Concentration (VCC), viability and generation time have been monitored and compared to standard shake flask culture. We observed that cultivation of HEK293T cells in the ambr® 15 microbioreactor yields improved cell growth and viability as compared to standard shake flask culture. We identified that pH was the most significant factor - besides stirring speed - which has a lesser significant impact on cell health and growth. By using the MODDE® software we were able to determine an optimal set-point for improved cell growth that can be used for scaling-up studies in stirred tank reactors. Please click Additional Files below to see the full abstract

A new simplified clarification approach for lentiviral vectors using diatomaceous earth improves throughput and safe handling

Lentiviral vectors have proven their great potential to serve as a DNA delivery tool for gene modified cell therapy and gene therapy applications. The downstream processing of these vectors is however still a great challenge, particularly because of the low stability of the virus. Harvesting and clarification are critical and until now insufficiently characterized steps for lentivirus processing. To address this bottleneck, we analyzed whether lentiviral vectors produced by transient transfection of HEK293 T/17 SF suspension cells can be efficiently clarified with a lab-scale method with the filter aid diatomaceous earth (DE) and bioburden reducing membrane filters achieving high lentivirus recoveries. Using a design of experiment approach we found that higher DE concentrations are advantageous for a higher turbidity reduction and shorter filtration times, but at the same time LV titer decreases with increasing DE concentration. A DE concentration of 9 g/L was identified with a DoE as a robust set-point. Clarification with DE was compared with for lab-scale traditionally employed centrifugation and subsequent bioburden reduction filtration of viral vectors. The use of DE allows to perform a harvest and clarification process, which does not only facilitate faster and safer virus handling, but enables a lower material consumption due to the extremely increased filter capacity, thus representing an efficient and robust lab-scale clarification process

Heme Oxygenase-1 Induction and Organic Nitrate Therapy: Beneficial Effects on Endothelial Dysfunction, Nitrate Tolerance, and Vascular Oxidative Stress

Organic nitrates are a group of very effective anti-ischemic drugs. They are used for the treatment of patients with stable angina, acute myocardial infarction, and chronic congestive heart failure. A major therapeutic limitation inherent to organic nitrates is the development of tolerance, which occurs during chronic treatment with these agents, and this phenomenon is largely based on induction of oxidative stress with subsequent endothelial dysfunction. We therefore speculated that induction of heme oxygenase-1 (HO-1) could be an efficient strategy to overcome nitrate tolerance and the associated side effects. Indeed, we found that hemin cotreatment prevented the development of nitrate tolerance and vascular oxidative stress in response to chronic nitroglycerin therapy. Vice versa, pentaerithrityl tetranitrate (PETN), a nitrate that was previously reported to be devoid of adverse side effects, displayed tolerance and oxidative stress when the HO-1 pathway was blocked pharmacologically or genetically by using HO-1+/– mice. Recently, we identified activation of Nrf2 and HuR as a principle mechanism of HO-1 induction by PETN. With the present paper, we present and discuss our recent and previous findings on the role of HO-1 for the prevention of nitroglycerin-induced nitrate tolerance and for the beneficial effects of PETN therapy

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity

Steric exclusion chromatography of lentiviral vectors using hydrophilic cellulose membranes

Enveloped viral vectors like lentiviral vectors pose purification challenges due to their low stability. A gentle purification method is considered one of the major bottlenecks for lentiviral vector bioprocessing. To overcome these challenges, a promising method is steric exclusion chromatography which has been used to purify a variety of target molecules. In this study, we successfully identified optimal process parameters for steric exclusion chromatography to purify lentiviral vectors. Lentiviral vector particle recoveries and infectious recoveries of 86% and 88%, respectively, were achieved. The process parameters optimal for steric exclusion chromatography were determined as follows: polyethylene glycol with a molecular weight of 4000 Da, a polyethylene glycol concentration of 12.5%, and a flow rate of 7 mL⋅min$^{−1}$ using 5 layers of stabilized cellulose membranes as a stationary phase. High protein and dsDNA removal of approximately 80% were obtained. The remaining polyethylene glycol concentration in the eluate was determined. We defined the maximum loading capacity as 7.5 × 10$^{12}$ lentiviral particles for the lab device used and provide deeper insights into loading strategies. Furthermore, we determined critical process parameters like pressure. We demonstrated in our experiments that steric exclusion chromatography is a gentle purification method with high potential for fragile enveloped viral vectors as it yields high recoveries while efficiently removing impurities

Differentially Tolerized Mouse Antigen Presenting Cells Share a Common miRNA Signature Including Enhanced mmu-miR-223-3p Expression Which Is Sufficient to Imprint a Protolerogenic State

Dendritic cells (DCs) are pivotal for the induction and maintenance of antigen-specific tolerance and immunity. miRNAs mediate post-transcriptional gene regulation and control in part the differentiation and stimulation-induced immunogenic function of DCs. However, the relevance of miRNAs for the induction and maintenance of a tolerogenic state of DCs has scarcely been highlighted yet. We differentiated mouse bone marrow cells to conventional/myeloid DCs or to tolerogenic antigen presenting cells (APCs) by using a glucocorticoid (dexamethasone) or interleukin-10, and assessed the miRNA expression patterns of unstimulated and LPS-stimulated cell populations by array analysis and QPCR. Differentially tolerized mouse APCs convergingly down-regulated a set of miRNA species at either state of activation as compared with the corresponding control DC population (mmu-miR-9-5p, mmu-miR-9-3p, mmu-miR-155-5p). These miRNAs were also upregulated in control DCs in response to stimulation. In contrast, miRNAs that were convergingly upregulated in both tolerized APC groups at stimulated state (mmu-miR-223-3p, mmu-miR-1224-5p) were downregulated in control DCs in response to stimulation. Overexpression of mmu-miR-223-3p in DCs was sufficient to prevent stimulation-associated acquisition of potent T cell stimulatory capacity. Overexpression of mmu-miR-223-3p in a DC line resulted in attenuated expression of known (Cflar, Rasa1, Ras) mRNA targets of this miRNA species shown to affect pathways that control DC activation. Taken together, we identified sets of miRNAs convergingly regulated in differentially tolerized APCs, which may contribute to imprint stimulation-resistant tolerogenic function as demonstrated for mmu-miR-223-3p. Knowledge of miRNAs with protolerogenic function enables immunotherapeutic approaches aimed to modulate immune responses by regulating miRNA expression

Rheumatoide Arthritis und Arteriosklerose : Bedeutung der zu Grunde liegenden chronisch entzündlichen Prozesse für die endotheliale Dysfunktion ; Wirkung der antiinflammatorischen Substanz Galiellalacton in murinen Arteriosklerosemodellen

Erhöhte arteriosklerotische und thrombotische Vorfälle sind ein Hauptgrund für die gesteigerten Zahlen kardiovaskulärer Todesfälle von Patienten mit chronisch entzündlichen Erkrankungen wie der rheumatoiden Arthritis (RA). Diese erhöhte Mortalität ist nicht auf die traditionellen Risikofaktoren, wie Alter, Geschlecht, Bluthochdruck oder Diabetes zurückzuführen. Man nimmt an, dass die systemische Entzündung einen nicht-traditionellen Risikofaktor für die erhöhten kardiovaskulären Todesfälle von RA-Patienten darstellt. Da die derzeitige Behandlung der RA zum Teil schwere Nebenwirkungen zur Folge haben kann, war es das Ziel dieser Doktorarbeit, die Zusammenhänge zwischen RA und Arteriosklerose (AS) näher zu untersuchen, sowie die neue antiinflammatorische Substanz Galiellalacton (Gal) für die Behandlung der AS zu charakterisieren.rnIn dem chronisch inflammatorischen Tiermodell der TTP-defizienten Mäuse, dessenrnPhänotyp dem einer humanen RA-Erkrankung ähnelt, konnte eine verschlechterternEndothelfunktion, die als ein erstes Symptom einer erworbenen AS gilt, nachgewiesen werden. Dies konnte auf eine erhöhte Stabilität der Nox2-mRNA zurückgeführt werden, die unabhängig von der erhöhten Expression des Entzündungsmarkers TNFα war. Diese gesteigerte Nox2-Menge führte wiederum zu einer erhöhten Bildung von reaktiven Sauerstoff- und Stickstoffspezies und somit zu einer verringerten Menge an bioaktivem Stickstoffmonoxid, welches die endotheliale Dysfunktion (eDF) bedingte.rnAls ein traditioneller Risikofaktor für das Auftreten von kardiovaskulären Ereignissen gilt unter anderem eine Diabeteserkrankung. Durch die Ausbildung einer Nitrattoleranz bei der Therapie mit organischen Nitraten wie NTG, ISMN oder ISDN kommt es zu der Entwicklung einer eDF. PETN, ein weiteres organisches Nitrat zeigt diese Nebenwirkung nicht. PETN, vermittelt seinen antioxidativen Effekt über die Nrf2-abhängige Induktion der HO-1-Promotoraktivität.rnDie Behandlung von arteriosklerotischen Mäusen (ApoE-/-- und ApoE-/-TFPI+/--Mäuse) mit dem antiinflammatorischen Pilzsekundärmetaboliten Gal zeigte eine verringerte mRNA-Expression von arteriosklerotischen und inflammatorischen Mediatoren, sowie eine reduzierte Thrombenbildung durch eine verringerte Plättchenadhäsion.rnZusammenfassend konnte gezeigt werden, dass inflammationsabhängiger oxidativerrnStress ein Hauptgrund für die entzündungsgetriebene Artheriogenese ist und Galrneine neue Leitsubstanz für die Behandlung dieser Erkrankung ist.Atherosclerotic events are one of the major risk factors for cardiovascular death in patients suffering from chronic inflammatory diseases as rheumatoid arthritis (RA). This elevated mortality is independent of the traditional risk factors, as age, gender, hypertension, or diabetes. So far, it is believed that the systemic inflammation confers a non-traditional risk factor for the cardiovascular morbidity of RA-patients. Since the current treatment can have severe side effects, the aim of this study was to analyse the correlation between RA and atherosclerosis (AS), as well as to characterisernthe new anti-inflammatory fungal substance Galiellalactone (Gal) for the treatment of AS.rnIn the chronic inflammatory TTP deficient mouse model that resembles the humanrnRA, the endothelial function is impaired, which is known to be a first symptom of a beginning AS. This correlates with an increased Nox2 mRNA stability that is independent of the augmented TNFα expression. The amplified Nox2 amount leads to increased levels of reactive oxygen and nitrogen species, in turn indicating decreased amounts of bioactive NO that attributes to the endothelial dysfunction (eDF).rnOne traditional risk factor for cardiovascular death is diabetes. The therapy with organic nitrates results in the development of a nitrate tolerance-induced eDF. PETN, another organic nitrate, does not have this adverse effect. PETN mediates its antioxidative effects via an Nrf2-dependent induction of the HO-1 promoter activity.rnThe treatment of atherosclerotic-prone mice (ApoE-/- and ApoE-/-TFPI+/- mice) with the anti-inflammatory fungal metabolite Gal decreases the mRNA expression of atherosclerotic and inflammatory marker genes and reduces the thrombus formation due to an impaired platelet adhesion.rnIn summary, the inflammation-related oxidative stress is a principal reason for inflammation-driven atherogenesis. Furthermore Gal is a promising new leading substance for the treatment of AS

Tristetraprolin regulation of interleukin-22 production

Interleukin (IL)-22 is a STAT3-activating cytokine displaying characteristic AU-rich elements (ARE) in the 3'-untranslated region (3'-UTR) of its mRNA. This architecture suggests gene regulation by modulation of mRNA stability. Since related cytokines undergo post-transcriptional regulation by ARE-binding tristetraprolin (TTP), the role of this destabilizing protein in IL-22 production was investigated. Herein, we demonstrate that TTP-deficient mice display augmented serum IL-22. Likewise, IL-22 mRNA was enhanced in TTP-deficient splenocytes and isolated primary T cells. A pivotal role for TTP is underscored by an extended IL-22 mRNA half-life detectable in TTP-deficient T cells. Luciferase-reporter assays performed in human Jurkat T cells proved the destabilizing potential of the human IL-22-3'-UTR. Furthermore, overexpression of TTP in HEK293 cells substantially decreased luciferase activity directed by the IL-22-3'-UTR. Transcript destabilization by TTP was nullified upon cellular activation by TPA/A23187, an effect dependent on MEK1/2 activity. Accordingly, IL-22 mRNA half-life as determined in TPA/A23187-stimulated Jurkat T cells decreased under the influence of the MEK1/2 inhibitor U0126. Altogether, data indicate that TTP directly controls IL-22 production, a process counteracted by MEK1/2. The TTP-dependent regulatory pathway described herein likely contributes to the role of IL-22 in inflammation and cancer and may evolve as novel target for pharmacological IL-22 modulation

T cell-specific overexpression of TGFß1 fails to influence atherosclerosis in ApoE-deficient mice.

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE(-/-) females and isogenic ApoE(-/-) controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells