Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

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Quality Verification with a Cluster−Controlled Manufacturing System to Generate Monocyte−Derived Dendritic Cells

Vaccines ◽

10.3390/vaccines9050533 ◽

2021 ◽

Vol 9 (5) ◽

pp. 533

Author(s):

Haruhiko Kawaguchi ◽

Takuya Sakamoto ◽

Terutsugu Koya ◽

Misa Togi ◽

Ippei Date ◽

...

Keyword(s):

Cell Culture ◽

Dna Microarrays ◽

Cluster Formation ◽

Human Leukocyte ◽

Leukocyte Antigen ◽

Dc Vaccines ◽

Survival Gene ◽

Adherent Culture ◽

Ctl Induction

Dendritic cell (DC) vaccines for cancer immunotherapy have been actively developed to improve clinical efficacy. In our previous report, monocyte−derived DCs induced by interleukin (IL)−4 with a low−adherence dish (low−adherent IL-4−DCs: la−IL-4−DCs) improved the yield and viability, as well as relatively prolonged survival in vitro, compared to IL-4−DCs developed using an adherent culture protocol. However, la−IL-4−DCs exhibit remarkable cluster formation and display heterogeneous immature phenotypes. Therefore, cluster formation in la−IL-4−DCs needs to be optimized for the clinical development of DC vaccines. In this study, we examined the effects of cluster control in the generation of mature IL-4−DCs, using cell culture vessels and measuring spheroid formation, survival, cytokine secretion, and gene expression of IL-4−DCs. Mature IL-4−DCs in cell culture vessels (cluster−controlled IL-4−DCs: cc−IL-4−DCs) displayed increased levels of CD80, CD86, and CD40 compared with that of la−IL-4−DCs. cc−IL-4−DCs induced antigen−specific cytotoxic T lymphocytes (CTLs) with a human leukocyte antigen (HLA)−restricted melanoma antigen recognized by T cells 1 (MART−1) peptide. Additionally, cc−IL-4−DCs produced higher levels of IFN−γ, possessing the CTL induction. Furthermore, DNA microarrays revealed the upregulation of BCL2A1, a pro−survival gene. According to these findings, the cc−IL-4−DCs are useful for generating homogeneous and functional IL-4−DCs that would be expected to promote long−lasting effects in DC vaccines.

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Efficient and sustained gene expression in primary T lymphocytes and primary and cultured tumor cells mediated by adeno-associated virus plasmid DNA complexed to cationic liposomes

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2411-2418.1994 ◽

1994 ◽

Vol 14 (4) ◽

pp. 2411-2418

Author(s):

R Philip ◽

E Brunette ◽

L Kilinski ◽

D Murugesh ◽

M A McNally ◽

...

Keyword(s):

Gene Expression ◽

Tumor Cells ◽

Plasmid Dna ◽

Interleukin 2 ◽

Cationic Liposomes ◽

Lymphoid Cells ◽

Chromosomal Dna ◽

Adeno Associated Virus ◽

Cultured Tumor Cells

We have used cationic liposomes to facilitate adeno-associated virus (AAV) plasmid transfections of primary and cultured cell types. AAV plasmid DNA complexed with liposomes showed levels of expression several fold higher than those of complexes with standard plasmids. In addition, long-term expression (> 30 days) of the gene, unlike the transient expression demonstrated by typical liposome-mediated transfection with standard plasmids, was observed. Southern analysis of chromosomal DNA further substantiated the hypothesis that the long-term expression was due to the presence of the transgene in the AAV plasmid-transfected group and not in the standard plasmid-transfected group. AAV plasmid-liposome complexes induced levels of transgene expression comparable to those obtained by recombinant AAV transduction. Primary breast, ovarian, and lung tumor cells were transfectable with the AAV plasmid DNA-liposome complexes. Transfected primary and cultured tumor cells were able to express transgene product even after lethal irradiation. High-level gene expression was also observed in freshly isolated CD3+, CD4+, and CD8+ T cells from normal human peripheral blood. Transfection efficiency ranged from 10 to 50% as assessed by intracellular interleukin-2 levels in interleukin-2-transfected cells. The ability to express transgenes in primary tumor and lymphoid cells may be applied toward tumor vaccine studies and protocols which may eventually permit highly specific modulation of the cellular immune response in cancer and AIDS.

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An Eco-friendly Fabrication of Silver Chloride Nanoparticles (AgCLNPs) using Onopordum acanthium L extract Induces Apoptosis in Breast Cancer MDA-MB-232 Cells

10.21203/rs.3.rs-303501/v1 ◽

2021 ◽

Author(s):

Seyedeh Negin Shahcheraghi ◽

Seyed Ataollah Sadat Shandiz ◽

Bahareh Pakpour

Keyword(s):

Breast Cancer ◽

Real Time ◽

Cell Lines ◽

Reverse Transcription ◽

Hek293 Cell ◽

Silver Chloride ◽

Hek293 Cells ◽

Qrt Pcr ◽

Reverse Transcription Pcr ◽

Onopordum Acanthium

Abstract In the current experimental work, silver chloride nanoparticles (AgClNPs) were fabricated using Onopordum acanthium L extract and their apoptotic and cytotoxicity properties on breast cancer MDA_MB232 and normal HEK293 cell lines were also evaluated. AgClNPs formation was determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) profile. Effect of fabricated AgClNPs on MDA_MB232 and HEK293 cells viability was performed using colorimetric MTT assay. Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using quantitative real-time reverse transcription-PCR (qRT-PCR) method. Subsequently, apoptotic properties were determined using flow cytometry and fluorescence microscopy studies. MTT results investigated that AgCLNPs have a significant dose-dependent lethal activity on MDA_MB232 compared to HEK293 cell lines. Quantitative real-time reverse transcription-PCR (qRT-PCR) results have also shown that AgCLNPs could up-regulate the apoptotic Bax and CAD gene expressions in the MDA_MB232 cells. Additionally, apoptotic assessment was performed by cell cycle analysis, annexin V/PI test, Hoescht 33258 dye, acridine orange and ethidium bromide (AO/EB) staining along with the detection of the reactive oxygen species (ROS) generation. Our results suggest that novel silver chloride nanoparticles fabricated by Onopordum acanthium L extract can display some promising cytotoxic properties through inducing apoptosis pathway.

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Clonal selection of high producing, stably transfected HEK293 cell lines utilizing modified, high-throughput FACS screening

Journal of Chemical Technology & Biotechnology ◽

10.1002/jctb.2618 ◽

2011 ◽

Vol 86 (7) ◽

pp. 935-941 ◽

Cited By ~ 12

Author(s):

Michael Song ◽

Kristin Raphaelli ◽

Martina L. Jones ◽

Khosrow Aliabadi-Zadeh ◽

Kar Man Leung ◽

...

Keyword(s):

Cell Lines ◽

High Throughput ◽

Hek293 Cell ◽

Clonal Selection ◽

Transfected Hek293 Cell ◽

Selection Of

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Purification of a Chimeric Simian – Human Immunodeficiency Virus-Like Nanoparticle from HEK293 Cell Culture

Cells and Culture ◽

10.1007/978-90-481-3419-9_92 ◽

2010 ◽

pp. 521-527

Author(s):

Luísa Pedro ◽

Guilherme N. M. Ferreira

Keyword(s):

Cell Culture ◽

Human Immunodeficiency Virus ◽

Hek293 Cell ◽

Immunodeficiency Virus

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Comparative Genomics, Infectivity and Cytopathogenicity of American Isolates of Zika Virus that Developed Persistent Infections in Human Embryonic Kidney (HEK293) Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20123035 ◽

2019 ◽

Vol 20 (12) ◽

pp. 3035 ◽

Cited By ~ 4

Author(s):

Hebing Liu ◽

Hsiao-Mei Liao ◽

Bingjie Li ◽

Shien Tsai ◽

Guo-Chiuan Hung ◽

...

Keyword(s):

Cell Line ◽

Zika Virus ◽

Hek293 Cell ◽

Clonal Selection ◽

Gene Mutations ◽

Host Cells ◽

Hek293 Cells ◽

Specific Gene ◽

Human Embryonic Kidney ◽

Hek293 Cell Line

Zika virus (ZIKV) transmission can cause serious fetal neurological abnormalities. ZIKV persistence in various human cells and tissues can serve as infectious reservoirs and post serious threats to public health. The human embryonic kidney (HEK293) cell line with known neuronal developmental properties was readily infected by ZIKV in a strain-dependent fashion. Significant cytopathic effect in HEK293 cells infected by the prototype MR 766 strain of ZIKV resulted in complete loss of cells, while small numbers of HEK293 cells infected by contemporary ZIKV isolates (PRV or FLR strain) continued to survive and regrow to confluency in the culture around two months after initial infection. Most, if not all, of the cells in the two resulting persistently ZIKV-infected HEK293 cell lines tested positive for ZIKV antigen. Compared to HEK293 control cells, the persistently ZIKV-infected HEK293 cells had slower growth rates with some cells undergoing apoptosis in culture. The “persistent ZIKVs” produced constitutively by both PRV and FLR strains ZIKV-infected HEK293 cells had significantly attenuated cell infectivity and/or cytopathogenicity. Comparative genome sequence analyses between the persistent ZIKVs and the original inoculum ZIKVs showed no clonal selection with specific gene mutations in the prolonged process of establishing persistently PRV strain ZIKV-infected HEK293 cells; while selection of ZIKV subclones with mutations in the envelope, protein pr and multiple NS genes was evident in developing persistently FLR strain ZIKV-infected HEK293 cell line. Our study provides molecular insights into the complex interplays of ZIKV and human host cells in establishing ZIKV persistence.

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