RESUMEN
MicroRNAs (miRNAs) regulate approximately one-third of all human genes. The dysregulation of miRNAs has been implicated in the development of numerous human diseases, including cancers. In our investigation focusing on altering specific miRNA expression in human pancreatic cancer cells, we encountered an interesting finding. While two expression vector designs effectively enhanced miR-708 levels, they were unable to elevate mature forms of miR-29b, -1290, -2467, and -6831 in pancreatic cancer cell lines. This finding was also observed in a panel of other non-pancreatic cancer cell lines, suggesting that miRNA processing efficiency was cell line specific. Using a step-by-step approach in each step of miRNA processing, we ruled out alternative strand selection by the RISC complex and transcriptional interference at the primary miRNA (pri-miRNA) level. DROSHA processing and pri-miRNA export from the nucleus also appeared to be occurring normally. We observed precursor (pre-miRNA) accumulation only in cell lines where mature miRNA expression was not achieved, suggesting that the block was occurring at the pre-miRNA stage. To further confirm this, synthetic pre-miRNA mimics that bypass DICER processing were processed into mature miRNAs in all cases. This study has demonstrated the distinct behaviours of different miRNAs with the same vector in the same cell line, the same miRNA between the two vector designs, and with the same miRNA across different cell lines. We identified a stable vector pre-miRNA processing block. Our findings on the structural and sequence differences between successful and non-successful vector designs could help to inform future chimeric miRNA design strategies and act as a guide to other researchers on the intricate processing dynamics that can impact vector efficiency. Our research confirms the potential of miRNA mimics to surmount some of these complexities.
Asunto(s)
MicroARNs , Neoplasias Pancreáticas , Procesamiento Postranscripcional del ARN , MicroARNs/genética , MicroARNs/metabolismo , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Procesamiento Postranscripcional del ARN/genética , Línea Celular Tumoral , Ribonucleasa III/metabolismo , Ribonucleasa III/genética , Regulación Neoplásica de la Expresión Génica , Transfección , Precursores del ARN/genética , Precursores del ARN/metabolismo , AnimalesRESUMEN
Gene therapy holds great promise for the treatment of severe diseases, and adeno-associated virus (AAV) vectors have emerged as valuable tools in this field. However, challenges such as immunogenicity and high production costs complicate the commercial viability of AAV-based therapies. To overcome these barriers, improvements in production yield, driven through the availability of robust and sensitive characterization techniques that allow for the monitoring of critical quality attributes to deepen product and process understanding are crucial. Among the main attributes affecting viral production and performance, the ratio between empty and full capsids along with capsid protein stoichiometry are emerging as potential parameters affecting product quality and safety. This study focused on the production of AAV vectors using the baculovirus expression vector system (BEVS) in Sf9 cells and the complete characterization of AAV5 variants using novel liquid chromatography and mass spectrometry techniques (LC-MS) that, up to this point, had only been applied to reference commercially produced virions. When comparing virions produced using ATG, CTG or ACG start codons of the cap gene, we determined that although ACG was the most productive in terms of virus yield, it was also the least effective in transducing mammalian cells. This correlated with a low VP1/VP2 ratio and a higher percentage of empty capsids. Overall, this study provides insights into the impact of translational start codon modifications during rAAV5 production using the BEVS, the associated relationship with capsid packaging, capsid protein stoichiometry and potency. The developed characterization workflow using LC-MS offers a comprehensive and transferable analysis of AAV-based gene therapies, with the potential to aid in process optimization and facilitate the large-scale commercial manufacturing of these promising treatments.
Asunto(s)
Proteínas de la Cápside , Dependovirus , Animales , Proteínas de la Cápside/genética , Dependovirus/genética , Cromatografía Liquida , Cromatografía Líquida con Espectrometría de Masas , Flujo de Trabajo , Vectores Genéticos , Espectrometría de Masas en Tándem , Baculoviridae/genética , Mamíferos/metabolismoRESUMEN
A variety of mechanisms including transcriptional silencing, gene copy loss, and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilized single-cell RNA-seq (scRNA-seq) to study a clonally derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA-seq data, we identified subclusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Animales , Anticuerpos Monoclonales/análisis , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células CHO , Cricetinae , Cricetulus , Secuenciación de Nucleótidos de Alto Rendimiento , Transgenes/genéticaRESUMEN
OBJECTIVES: We used miRNA and proteomic profiling to understand intracellular pathways that contribute to high and low specific productivity (Qp) phenotypes in CHO clonally derived cell lines (CDCLs) from the same cell line generation project. RESULTS: Differentially expressed (DE) miRNAs were identified which are predicted to target several proteins associated with protein folding. MiR-200a was found to have a number of predicted targets associated with the unfolded protein response (UPR) which were shown to have decreased expression in high Qp CDCLs and have no detected change at the mRNA level. MiR-200a overexpression in a CHO CDCL was found to increase recombinant protein titer by 1.2 fold and Qp by 1.8 fold. CONCLUSION: These results may suggest a role for miR-200a in post-transcriptional regulation of the UPR, presenting miR-200a as a potential target for engineering industrially attractive CHO cell phenotypes.
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Fragmentos Fc de Inmunoglobulinas , MicroARNs , Proteínas Recombinantes de Fusión , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , MicroARNs/química , MicroARNs/genética , MicroARNs/metabolismo , Pliegue de Proteína , Proteómica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.
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Dependovirus/genética , Regulación de la Expresión Génica/genética , Proteoma/genética , Proteínas Recombinantes/biosíntesis , Línea Celular , Cromatografía de Fase Inversa , Endocitosis/genética , Endocitosis/fisiología , Expresión Génica/genética , Ontología de Genes , Terapia Genética/métodos , Vectores Genéticos/genética , Células HEK293 , Humanos , Lisosomas/metabolismo , Proteolisis , Proteínas Recombinantes/genética , Transducción de Señal/genética , Espectrometría de Masas en Tándem , Transfección/métodosRESUMEN
Polyamines have essential roles in cell proliferation, DNA replication, transcription, and translation processes, with intracellular depletion of putrescine, spermidine, and spermine resulting in cellular growth arrest and eventual death. Serum-free media for CHO-K1 cells require putrescine supplementation, because these cells lack the first enzyme of the polyamine production pathway, arginase. On the basis of this phenotype, we developed an arginase-based selection system. We transfected CHO-K1 cells with a bicistronic vector co-expressing GFP and arginase and selected cells in media devoid of l-ornithine and putrescine, resulting in mixed populations stably expressing GFP. Moreover, single clones in these selective media stably expressed GFP for a total of 42 generations. Using this polyamine starvation method, we next generated recombinant CHO-K1 cells co-expressing arginase and human erythropoietin (hEPO), which also displayed stable expression and healthy growth. The hEPO-expressing clones grew in commercial media, such as BalanCD and CHO-S serum-free media (SFM)-II, as well as in a defined serum-free, putrescine-containing medium for at least 9 passages (27 generations), with a minimal decrease in hEPO titer by the end of the culture. We observed a lack of arginase activity also in several CHO cell strains (CHO-DP12, CHO-S, and DUXB11) and other mammalian cell lines, including BHK21, suggesting broader utility of this selection system. In conclusion, we have established an easy-to-apply alternative selection system that effectively generates mammalian cell clones expressing biopharmaceutically relevant or other recombinant proteins without the need for any toxic selective agents. We propose that this system is applicable to mammalian cell lines that lack arginase activity.
Asunto(s)
Arginasa/metabolismo , Animales , Células CHO , Línea Celular , Cricetulus , Eritropoyetina/metabolismo , Poliaminas/metabolismo , Putrescina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade following the development of next generation sequencing technology and publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small noncoding RNAs. The annotation of long noncoding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high-resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHO K1 genome. In addition, the utilisation of strand-specific sequencing enabled the identification of more than 1,000 new antisense and divergent lncRNAs. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.
Asunto(s)
Biología Computacional/métodos , ARN Largo no Codificante/genética , Análisis de Secuencia de ARN/métodos , Animales , Células CHO , Cricetinae , Cricetulus , Genoma , TranscriptomaRESUMEN
RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb-producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.
Asunto(s)
Empalme Alternativo , ARN Mensajero , Transcriptoma , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células CHO , Frío , Cricetinae , Cricetulus , Perfilación de la Expresión Génica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ARN , Transcriptoma/genética , Transcriptoma/fisiologíaRESUMEN
OBJECTIVES: This study aims to provide insights into the molecular mechanisms underlying adaptation of CHO-K1 cells to growth in glutamine-free media and potentially identifying critical signalling proteins and pathways involved in this phenotype. RESULTS: A CHO-K1 cell line adapted to growth in glutamine-free media was established using a straightforward one-step glutamine reduction strategy. The adapted cell line had a comparable phenotype to the parental cells in terms of cell growth and viability. Global quantitative proteomic and phosphoproteomic analysis was carried out to compare the cells adapted to growth in glutamine-free media to parental cells grown in media containing 8 mM L-glutamine. The adaptation process was accompanied by changes in proteins associated with cytoskeleton rearrangement and mRNA splicing as evidenced via functional analysis of 194 differentially expressed proteins between the two cell lines. 434 phosphoproteins with altered abundance were also identified as a result of adaptation to L-glutamine-free conditions with an associated enrichment of pathways associated with MAPK and calcium signalling. CONCLUSIONS: This work provides a comprehensive proteomic and phosphoproteomic analysis of protein expression changes after adaptation to glutamine-free growth conditions highlighting critical pathways to consider in the rational design of improved feeding strategies or in cell line engineering to improve bioprocess phenotypes.
Asunto(s)
Procesamiento Proteico-Postraduccional/genética , Proteoma/genética , Proteómica , Espectrometría de Masas en Tándem/métodos , Animales , Células CHO , Ciclo Celular/genética , Proliferación Celular/genética , Cromatografía Liquida/métodos , Cricetinae , Cricetulus , Medios de Cultivo/farmacología , Glutamina/genética , Fosfoproteínas/genéticaRESUMEN
AIM: To investigate the impact of polyamine deprivation on the transcriptome of CHO cells RESULTS: Polyamines play a central but poorly-understood role in cell proliferation. Most studies to date have utilised chemical inhibitors to probe polyamine function. Here we exploit the fact that CHO cells grown in serum-free medium have an absolute requirement for putrescine supplementation due to their deficiency in activity of the enzyme arginase. A gene expression microarray (Affymetrix) analysis of CHO-K1 cells starved of polyamines for 3 days showed that cessation of growth, associated with increased G1/S transition and inhibition of M/G1 transition was accompanied by increased mRNA levels of mitotic complex checkpoint genes (Mad2l1, Tkk, Bub1b) and in the transition of G1- to S-phase (such as Skp2 and Tfdp1). mRNAs associated with DNA homologous recombination and repair (including Fanconi's anaemia-related genes) and with RNA splicing were consistently increased. Alterations in mRNA levels for genes related to protein processing in the ER, to ER stress, and to p53-related and apoptosis pathways were also observed. mRNAs showing highest levels of fold-change included several which code for membrane-localised proteins and receptors (Thbs1, Tfrc1, Ackr3, Extl1). CONCLUSIONS: Growth-arrest induced by polyamine deprivation was associated with significant alterations in levels of mRNAs associated with cell cycle progression, DNA repair, RNA splicing, ER trafficking and membrane signalling as well as p53 and apoptosis-related pathways.
Asunto(s)
Medios de Cultivo/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Putrescina/farmacología , Transcriptoma/efectos de los fármacos , Animales , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Medios de Cultivo/químicaRESUMEN
Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2â¯A "self-cleavage" peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.
Asunto(s)
ARN/genética , Proteínas Recombinantes/biosíntesis , Animales , Células CHO , Sistema Libre de Células , Codón de Terminación/genética , Cricetinae , Cricetulus , Eritropoyetina/biosíntesis , Eritropoyetina/genética , Glicosilación , Humanos , Ingeniería Metabólica , Sistemas de Lectura Abierta/genética , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Péptidos/química , Procesamiento Proteico-Postraduccional , ARN Circular , Proteínas Recombinantes/análisisRESUMEN
In this study, we report an investigation of a panel of clonally-derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full-length IgG4 Fc-fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a "high" and "low" clipping phenotype. From this analysis, six protease-encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full-length IgG4 Fc-fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell-free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33-39% increase in complete IgG4 Fc-fusion protein when compared with untreated samples.
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Inmunoglobulina G/genética , Péptido Hidrolasas/genética , Animales , Células CHO , Cricetulus , Furina/genética , Furina/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoglobulina G/metabolismo , Péptido Hidrolasas/metabolismo , Proteolisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TranscriptomaRESUMEN
The impact of local structure on mRNA translation is not well-defined pertaining to the 5' UTR. Reports suggest structural remodelling of the 5' UTR can significantly influence mRNA translation both in cis and trans however a new layer of complexity has been applied to this model with the now known reversible post-transcriptional chemical modification of RNA. N6-methyladenosine (m6A) is the most abundant internal base modification in mammalian mRNA. It has been reported that mRNAs harbouring m6A motifs in their 5' UTR have improved translation efficiency. The present study evaluated the addition of putative m6A motifs to the 5' UTR of a model recombinant human therapeutic glycoprotein, Erythropoietin (EPO), in a direct comparison with an A to T mutant and a no adenosine control. The m6A construct yielded significantly improved EPO titer in transient batch culture over no adenosine and m6T controls by 2.84 and 2.61-fold respectively. This study highlights that refinement of transgene RNA elements can yield significant improvements to protein titer.
Asunto(s)
Regiones no Traducidas 5' , Eritropoyetina/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animales , Técnicas de Cultivo Celular por Lotes , Células CHO , Cricetulus , Eritropoyetina/metabolismo , Mutación , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas Recombinantes/genética , Biología Sintética/métodosRESUMEN
Recent sequencing of the Chinese hamster ovary (CHO) cell and Chinese hamster genomes has dramatically advanced our ability to understand the biology of these mammalian cell factories. In this study, we focus on the powerhouse of the CHO cell, the mitochondrion. Utilizing a high-resolution next generation sequencing approach we sequenced the Chinese hamster mitochondrial genome for the first time and surveyed the mutational landscape of CHO cell mitochondrial DNA (mtDNA). Depths of coverage ranging from ~3,319X to 8,056X enabled accurate identification of low frequency mutations (>1%), revealing that mtDNA heteroplasmy is widespread in CHO cells. A total of 197 variants at 130 individual nucleotide positions were identified across a panel of 22 cell lines with 81% of variants occurring at an allele frequency of between 1% and 99%. 89% of the heteroplasmic mutations identified were cell line specific with the majority of shared heteroplasmic SNPs and INDELs detected in clones from 2 cell line development projects originating from the same host cell line. The frequency of common predicted loss of function mutations varied significantly amongst the clones indicating that heteroplasmic mtDNA variation could lead to a continuous range of phenotypes and play a role in cell to cell, production run to production run and indeed clone to clone variation in CHO cell metabolism. Experiments that integrate mtDNA sequencing with metabolic flux analysis and metabolomics have the potential to improve cell line selection and enhance CHO cell metabolic phenotypes for biopharmaceutical manufacturing through rational mitochondrial genome engineering.
Asunto(s)
Alelos , Frecuencia de los Genes , Genoma Mitocondrial , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación INDEL , Polimorfismo de Nucleótido Simple , Animales , Células CHO , Cricetinae , CricetulusRESUMEN
The use of microRNAs (miRNAs) for improving the efficiency of recombinant protein production by CHO cells is gaining considerable interest for their ability to regulate entire molecular networks. Differential miRNA expression profiling and large-scale transient screening have been the prerequisite for the selection of miRNA candidates for stable manipulation, reported in CHO cells expressing a range of recombinant products. We selected a potent and well characterised tumour suppressor miRNA, miR-34a, as a high priority candidate for CHO cell engineering based on the conservation of both its sequence and function across species and cell type. Ectopic expression of miR-34a retained its functional conservation in CHO-SEAP cells by inhibiting growth by 90% in addition to decreasing the viable cell population by 30% when compared to controls. When the miR-34 family was stably depleted using a miRNA sponge decoy vector, the overall product yield was enhanced by ~2-fold in both fed-batch and small scale clonal batch cultures, despite having a negative impact on cell growth. These findings further strengthen the utility of miRNAs as engineering tools to modify and improve CHO cell performance.
Asunto(s)
Ingeniería Celular/métodos , Expresión Génica , MicroARNs/biosíntesis , MicroARNs/genética , Animales , Células CHO , Proliferación Celular , Supervivencia Celular , Cricetulus , FemeninoRESUMEN
Here we report the synthesis and isolation of a series of bis-chelate Cu(2+) phenanthroline-phenazine cationic complexes of [Cu(DPQ)(Phen)](2+), [Cu(DPPZ)(Phen)](2+), and [Cu(DPPN)(Phen)](2+) (where Phen = 1,10-phenanthroline, DPQ = dipyridoquinoxaline, DPPZ = dipyridophenazine, and DPPN = benzo[i]dipyridophenazine). These compounds have enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2](2+) (bis-Phen), with calf thymus DNA binding constants of DPQ and DPPZ agents (â¼10(7) M(bp)(-1)) being the highest currently known for Cu(2+) phenanthrene compounds. Complex DNA binding follows DPQ ≈ DPPZ > DPPN > bis-Phen, with fluorescence quenching and thermal melting experiments on poly[d(A-T)2] and poly[d(G-C)2] supporting intercalation at both the minor and major groove. Phenazine complexes, however, show enhanced targeting and oxidative cleavage on cytosine-phosphate-guanine-rich DNA and have comparable in vitro cytotoxicity toward the cisplatin-resistant ovarian cancer line, SKOV3, as the clinical oxidative DNA-damaging drug doxorubicin (Adriamycin). In this study we also describe how a novel "on-chip" method devised for the Bioanalyser 2100 was employed to quantify double-stranded DNA damage, with high precision, by the complex series on pUC19 DNA (49% A-T, 51% G-C). Both DPQ and bis-Phen complexes are highly efficient oxidizers of pUC19, with DPQ being the most active of the overall series. It is apparent, therefore, that oxidative chemical nuclease activity on homogeneous canonical DNA is not entirely dependent on dynamic nucleotide binding affinity or intercalation, and this observation is corroborated through catalytic interactions with the superoxide anion radical and Fenton breakdown of hydrogen peroxide.
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Cobre/química , ADN/química , Compuestos Organometálicos/síntesis química , Fenantrenos/química , Animales , Bovinos , Fluorescencia , Humanos , Conformación Molecular , Compuestos Organometálicos/química , Compuestos Organometálicos/aislamiento & purificación , Oxidación-Reducción , Células Tumorales CultivadasRESUMEN
MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.
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Sistemas CRISPR-Cas , Cricetulus , MicroARNs , Proteínas Recombinantes , Animales , Células CHO , MicroARNs/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Edición Génica/métodos , Eliminación de GenRESUMEN
Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells.
RESUMEN
N6 -methyladenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study, the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2 and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation were evaluated. Using siRNA-mediated gene depletion, cDNA over-expression, and methylation-specific RNA immunoprecipitation, it is demonstrated that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated; and (iii) YTHDF2 depletion, or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral (LV) particles (~2-3-fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epitranscriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.
Asunto(s)
Proteínas de Unión al ARN , Animales , Cricetinae , Humanos , ARN Mensajero/genética , Cricetulus , Células HEK293 , Proteínas de Unión al ARN/genética , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: To study the role of microRNA (miRNA) in the regulation of Chinese hamster ovary (CHO) cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates. RESULTS: 51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated). Gene ontology (GO) analysis of genes (n=432) and proteins (n=285) found to be differentially expressed (DE) identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158). The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515). The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs. CONCLUSIONS: The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of mRNA/proteins involved in CHO cell growth rate. These data have allowed us to prioritise candidates for cell engineering and/or biomarkers relevant to industrial cell culture. We also expect the knowledge gained from this study to be applicable to other fields investigating the role of miRNAs in mammalian cell growth.