Enhancing recombinant protein and viral vector production in mammalian cells by targeting the YTHDF readers of N6–methyladenosine in Mrna

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Rolling cycle translation of circularized infinite open reading frames; fooling the ribosome

Poster Number 156 ROLLING CYCLE TRANSLATION OF CIRCULARIZED INFINITE OPEN READING FRAMES; FOOLING THE RIBOSOME Alan Costello, National Institute for Cellular Biotechnology, Dublin City University [email protected] Nga Lao, National Institute for Cellular Biotechnology, Dublin City University Niall Barron, National Institute for Bioprocessing Research and Training, University College Dublin Martin Clynes, National Institute for Cellular Biotechnology, Dublin City University Key Words: Circular RNA, Translation, RNA structure, Cell engineering, Chinese Hamster Ovary (CHO) cell Recent. Please click Additional Files below to see the full abstract

Application of response surface methodology in the design of functionally graded plasma sprayed hydroxyapatite coatings

The highly complex process-property-structure relationship poses a major challenge in the optimization of plasma sprayed hydroxyapatite coatings. In addition, contradictions in relation to the ideal coating properties exist; a dense, highly crystalline coating is required for long term coating stability, whereas coatings with lower crystallinity dissolve more rapidly but have an improved osteogenic response in vivo. In this study, response surface methodology (RSM) is utilized to investigate the influences and interaction effects of current, gas flow rate, powder feed rate, spray distance and carrier gas flow rate on the roughness, crystallinity, purity, porosity and thickness of plasma sprayed HA coatings. Roughness related to the particle velocity and particle melting, and was highest at low gas flow rates and, due to the quadratic effect of current, at the central current value. High crystallinity resulted at high current and low spray distance due to the presence of bulk crystalline material and recrystallization of amorphous material. Purity was highest at low carrier gas flow rate and high gas flow rate, where particle temperature was reduced. Porosity was dependent on the degree of particle melting and was highest at low gas flow rate and powder feed rate and at high current and spray distance. Coating thickness was determined by the number of particles and the degree of flattening on impact, and was highest at high current, low gas flow rate, high powder feed rate and low spray distance. From this in-depth analysis, predictive process equations were developed and optimized to produce two distinct coatings; a stable coating and a bioactive coating, designed to form the base and surface layers of a functionally graded coating respectively, to provide enhanced osteogenesis, while maintaining long-term stability. Culture of osteoblast-like cells on the coatings demonstrated an increased osteogenic response on the bioactive coating compared to the other groups. Overall, this study identifies parameter effects and interactions leading to the development of optimized coatings with the potential to enhance the functional life of HA coated implants in vivo

miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP, in Chinese hamster ovary cells

Genetic engineering of mammalian cells, in particular Chinese hamster ovary (CHO) cells, is of critical interest to the biopharmaceutical industry as a means to further boost the yields of therapeutic proteins. Complimentary to already in place advanced bioprocesses, stable overexpression of the pro-survival X-linked inhibitor of apoptosis (XIAP) is one example of the successful manipulation of CHO cell genetics resulting in prolonged culture survival, ultimately increasing recombinant protein productivity. However, saturation or burdening of the cells translational machinery can occur in instances of forced expression of a trans-gene thereby achieving the anticipated cellular phenotype without the associated improvement in productivity. Ribosomal footprint sequencing has demonstrated that ~15% of an IgG-producing CHO cell translatome is occupied by the Neomycin selection marker. microRNAs (miRNAs) have the ability to fine tune endogenous gene expression thereby achieving elevated gene levels without the excess that could negatively impact global gene expression. Additionally, not only does a single miRNA have the capacity to regulate multiple mRNA transcripts simultaneously but individual mRNAs can be regulated by a multitude of miRNAs at the post-transcriptional level. This can facilitate the maximal translation of an endogenous gene without surpassing the superphysiological threshold associated with diminished productivity. The promiscuous nature of miRNA represented by the variety of binding patterns associated with mRNA targeting limits the predictability of high confidence miRNA regulators of attractive engineering candidates. This results in a lengthy list of falsely predicted in-silico miRNA regulators for a single gene. We explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH1 protocol. A biotin-tagged antisense DNA oligonucleotide was designed for an exposed predicted secondary structure loop of endogenous CHO XIAP. This mRNA anchor resulted in the pulldown of XIAP and all associated RNA/protein complexes thereby enriching for all bound miRNAs. Two miRNAs were chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both resulted in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increased XIAP protein levels (Fig. 1). Please click Additional Files below to see the full abstract

CHO microRNA engineering is growing up : recent successes and future challenges

microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines

Radical-induced purine lesion formation is dependent on DNA helical topology

AbstractHerein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO•) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]4 (TG4T), d[AGGG(TTAGGG)3] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)3A] (mutTel24) were exposed to HO• radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating ..

Sustained productivity in recombinant Chinese Hamster Ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype

<p>Abstract</p> <p>Background</p> <p>The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture.</p> <p>Results</p> <p>Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction.</p> <p>Conclusion</p> <p>These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.</p