Download PDF (all abstracts) - BioMed Central

More documents

Recommendations

Info

BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 14 of 151 expression of a challenging to express “novel therapeutic protein”. The expression of the “novel therapeutic protein” in CHO cells resulted in significant reduced cell growth as well as low productivity. Results: Transcriptomics analysis: Using customised CHO specific microarrays the gene expression profile of CHO cells expressing the “novel therapeutic protein” was analysed. The expression of the “novel therapeutic protein” resulted in a significant downregulation of all mitochondria encoded genes. The downregulation was more than 40 fold for some of these genes (Figure 1A). This massive reduced transcription of mitochondrial encoded genes was very likely causing the reduced cell growth and reduced expression of the “novel therapeutic protein”. A decrease in mitochondrial function reduces overall metabolic efficiency and a change of metabolic pathways could also be detected on gene expression level. Additionally the expression of “gene A” was detected in the applied CHO cell line, which might have the potential to trigger the down regulation of the mitochondrial encoded genes in the presence of the “novel therapeutic protein”. Gene knockdown using shRNA (short hairpin RNA) technique: A variety of cell line engineering approaches were performed to circumvent cell growth inhibition caused by down regulation of mitochondrial encoded genes with the aim to improve expression of the “novel therapeutic protein”. In the first approach the expression of “gene A”, whichwas assumed to trigger the down regulation of the mitochondrial encoded genes, was repressed more than 10 fold using shRNA technique. shRNA is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference. Expression of shRNA was accomplished by delivery of stable integrated plasmids. Cells with reduced expression of “gene A” showed an improved cell growth and higher expression of the “novel therapeutic protein” (Figure 1 B). However cell growth was still repressed, although to a lower extent, and titers were still lower in comparison to other therapeutic protein formats. Despite the significant decrease in the expression of “gene A”, the remaining “protein A” seemed to be sufficient to trigger these effects although to a lower magnitude. Gene knockout using zinc finger nucleases (ZFN): To completely eliminate the cell growth inhibition a knockout of “gene A” was performed using ZFN technique. ZFNs are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNAcleavage domain. Plasmids encoding ZFNs (specifically designed to detect and cleave “gene A”) were transiently transfected in the parental CHO cell line. ZFN cleaves “gene A” which is then repaired by non-homologous end joining. This is often error prone and resulted in the generation of mutant alleles. Three clones were identified with mutation in both alleles of “gene A” resulting in shifts of the reading frame and therefore only nonfunctional premature termination products are encoded. Knockout of “gene A” resulted in complete elimination of cell growth inhibition and the expression of mitochondria encoded genes (Figure 1D) was restored to levels comparable to parental CHO cells. In addition there was no change in the expression of genes that are involved in metabolic pathways. Most striking is the significant improved cell growth and productivity resulting in a 6-7 fold titer increase using this genetically engineered knockout cell line (Figure 1B and 1C). Conclusion: This example illustrates that transcriptomic analysis can support and facilitate the understanding and solving of specific issues during the expression of therapeutic proteins. Novel cell line engineering methods as ZFN technique are powerful tools to solve definite issues in production of therapeutic proteins in biopharmaceutical industry. P2 Expansion of mesenchymal adipose-tissue derived stem cells in a stirred single-use bioreactor under low-serum conditions Carmen Schirmaier 1* , Stephan C Kaiser 1 , Valentin Jossen 1 , Silke Brill 2 , Frank Jüngerkes 2 , Christian van den Bos 2 , Dieter Eibl 1 , Regine Eibl 1 1 Zurich University of Applied Sciences, Institute of Biotechnology, Biochemical Engineering and Cell Cultivation Technique, 8820 Wädenswil, Switzerland; 2 Lonza Cologne GmbH, 50829 Cologne, Germany E-mail: *carmen.schirmaier@zhaw.ch BMC Proceedings 2013, 7(Suppl 6):P2 Background: The need for human mesenchymal stem cells (hMSCs) has increased enormously in recent years due to their important therapeutic potential. Efficient cell expansion is essential to providing clinically relevant cell numbers. Such cell quantities can be manufactured by means of scalable microcarrier (MC)-supported cultivations in stirred single-use bioreactors. Materials and methods: Preliminary tests in disposable-spinners (100 mL culture volume, Corning) were used to determine two suitable media and MC-types for serum reduced expansions (< 10%) of human adipose tissuederived stem cells (hADSCs; passage 2, Lonza). Using such optimized media-MC-combinations, hADSCs expanded 30 to 40-fold, which compares well with expansion rates in planar culture. Based on computational fluid dynamics simulations and suspension analyses in spinners [1], optimal operating parameters were determined in a BIOSTAT® UniVessel® SU 2 L (2 L culture volume, Sartorius Stedim Biotech). Results: In subsequent batch tests with the BIOSTAT UniVessel® SU 2 L, expansion rates of between 30 and 40-fold were reached and up to 4.4·10 8 cells with a cell viability exceeding 98% were harvested. Flow cytometry tests demonstrated typical marker profiles following cell expansion and harvest. A 40-fold expansion rate delivered a total of 1·10 10 cells in a first cultivation with the BIOSTAT® CultiBag STR 50 L (35 L culture volume, Sartorius Stedim Biotech). Conclusions: In summary, the foundations for successfully expanding therapeutic stem cells in truly scalable systems have been laid. Strategies ensuring expansion rates between 60 and 70-fold and, thus, generating cell amounts over 10 10 are now in preparation. Acknowledgements: This work is part of the project “Development of a technology platform for a scalable production of therapeutically relevant stem cells” (No. 12893.1 VOUCH-LS). It is supported by the Commission for Technology and Innovation (CTI, Switzerland). The authors would like to thank the CTI for partially financing the investigations presented. Reference 1. Kaiser S C, Jossen V, Schirmaier C, Eibl D, Brill S, van den Bos C, Eibl R: Investigations of fluid flow and cell proliferation of mesenchymal adipose-derived stem cells in small-scale, stirred, single-use bioreactors. Chem Ing Tech 2013, 85:95-102. P3 Evaluating the effect of chromosomal context on zinc finger nuclease efficiency Scott Bahr * , Laura Cortner, Sara Ladley, Trissa Borgschulte CHOZN® Platform Development Team, SAFC/Sigma-Aldrich, St Louis, MO 63103, USA E-mail: scott.bahr@sial.com BMC Proceedings 2013, 7(Suppl 6):P3 Introduction: Zinc Finger Nuclease (ZFN) technology has provided researchers with a tool for integrating exogenous sequences into most cell lines or genomes in a precise manner. Using current methods, the efficiency of targeted integration (TI) into the host genome is generally low and is highly dependent on the ZFN activity at the genomic locus of interest. It is unknown if the ZFN binding and cutting efficiency is more dependent on the nucleotide recognition sequence or the chromosomal context in which the sequence is located. We have taken a highly efficient ZFN pair (hAAVS1) from human studies and introduced the exogenous DNA sequence into the Chinese Hamster Ovary (CHO) genome in an attempt to improve the efficiency of targeted integration. A “Landing Pad” comprised of human AAVS1 sequence has been integrated into the CHO genome at 3 separate loci to determine if the ZFN’s will work across species and if the cutting efficiency is affected by chromosomal context. The results of this study will help us to improve the overall efficiency of TI by using Landing Pads, particularly for genomic targets in which suitable ZFN’s may not be available. Methods: 3 CHO Loci were chosen for this study based on previous gene expression studies. Rosa26 and Neu3 show consistent but low levels of expression while Site #1 appears to have no known coding sequence. Additionally, Rosa26 and Site#1 were chosen as potential safe harbor sites in CHO. The ZFN cutting efficiency at the endogenous CHO loci Rosa26, Site #1 and Neu3 are approximately 15%, 30% and 40% respectively. Based on other studies the cutting efficiency of human AAVS1 ZFN’s was as high as 50% depending on the human cell line used. A plasmid donor carrying the hAAVS1 ZFN recognition sequence Landing Pad was introduced into CHO Rosa26, Site #1, and Neu3 via targeted integration (Figure 1).
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 15 of 151 Figure 1(abstract P1) A highlights the reduced expression of mitochondria encoded genes in CHO cells expressing the “novel therapeutic protein” in comparison to parental CHO cells. The y-axis shows the gene expression values in signal intensities. B: Batch culture titers of the “novel therapeutic protein” in shake flask are shown. Titer for CHO WT cells are labeled in red, titer for CHO cells with reduced expression of gene A (shRNA approach) are labeled in yellow and titer for CHO cells with non-functional gene A (knockout) are labeled in green. C: Cell growth of CHO WT cells and CHO knockout (KO) cells with and without “novel therapeutic protein” in shake flasks. Y-axis shows viable cell density and x-axis cultivation time. D: Gene expression of mitochondrial encoded genes is not reduced in all three generated CHO KO cell lines with the “novel therapeutic protein”. Hierarchical clustering reveals that the “novel therapeutic protein” does not affect the gene expression profile of the KO cell lines. In contrast the “novel therapeutic protein” has a clear effect on the WT cell line.
Page 1 and 2: BMC Proceedings 2013, Volume 7 Supp
Page 13: BMC Proceedings 2013, Volume 7 Supp
Page 65 and 66:
BMC Proceedings 2013, Volume 7 Supp
Page 67 and 68:
Page 69 and 70:
Page 71 and 72:
Page 73 and 74:
Page 75 and 76:
Page 77 and 78:
Page 79 and 80:
Page 81 and 82:
Page 83 and 84:
Page 85 and 86:
Page 87 and 88:
Page 89 and 90:
Page 91 and 92:
Page 93 and 94:
Page 95 and 96:
Page 97 and 98:
Page 99 and 100:
Page 101 and 102:
Page 103 and 104:
Page 105 and 106:
Page 107 and 108:
Page 109 and 110:
Page 111 and 112:
Page 113 and 114:
Page 115 and 116:
Page 117 and 118:
Page 119 and 120:
Page 121 and 122:
Page 123 and 124:
Page 125 and 126:
Page 127 and 128:
Page 129 and 130:
Page 131 and 132:
Page 133 and 134:
Page 135 and 136:
Page 137 and 138:
Page 139 and 140:
Page 141 and 142:
Page 143 and 144:
Page 145 and 146:
Page 147 and 148:
Page 149 and 150:
Page 151:
show all

Download PDF (all abstracts) - BioMed Central

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?