Structural analysis of random transgene integration in CHO manufacturing cell lines by targeted sequencing

In the present work we have identified a glutathione reductase like metalloid reductase (GRLMR) responsible for mediating selenite tolerance in Pseudomonas moravenis stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (Km= 336 μM) over oxidized glutathione (Km=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC90 for selenite from 1.9 mM in cell lacking the GRLMR gene to 21.3 mM for cells containing the GRLMR gene. It was later confirmed by STEM and EDS that Se nanoparticles were absent in control cells and present in cells expressing GRLMR. Structural analysis suggests the lack of a sulfur residue in the substrate/product binding pocket may be responsible for this unique substrate specificity.

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Response to “Statistical analysis of data from limiting dilution cloning to assess monoclonality in generating manufacturing cell lines”

Biotechnology Progress ◽

10.1002/btpr.2547 ◽

2017 ◽

Vol 34 (3) ◽

pp. 558-558 ◽

Cited By ~ 2

Author(s):

James Savery ◽

Krista Evans

Keyword(s):

Statistical Analysis ◽

Cell Lines ◽

Manufacturing Cell ◽

Limiting Dilution ◽

Limiting Dilution Cloning

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Engineering Enhanced Vaccine Cell Lines To Eradicate Vaccine-Preventable Diseases: the Polio End Game

Journal of Virology ◽

10.1128/jvi.01464-15 ◽

2015 ◽

Vol 90 (4) ◽

pp. 1694-1704 ◽

Cited By ~ 20

Author(s):

Sabine M. G. van der Sanden ◽

Weilin Wu ◽

Naomi Dybdahl-Sissoko ◽

William C. Weldon ◽

Paula Brooks ◽

...

Keyword(s):

Gene Silencing ◽

Cell Line ◽

Cell Lines ◽

Enterovirus 71 ◽

Vaccine Production ◽

Wild Type ◽

Vaccine Preventable Diseases ◽

Manufacturing Cell ◽

Genome Wide ◽

A Genome

ABSTRACTVaccine manufacturing costs prevent a significant portion of the world's population from accessing protection from vaccine-preventable diseases. To enhance vaccine production at reduced costs, a genome-wide RNA interference (RNAi) screen was performed to identify gene knockdown events that enhanced poliovirus replication. Primary screen hits were validated in a Vero vaccine manufacturing cell line using attenuated and wild-type poliovirus strains. Multiple single and dual gene silencing events increased poliovirus titers >20-fold and >50-fold, respectively. Host gene knockdown events did not affect virus antigenicity, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9-mediated knockout of the top candidates dramatically improved viral vaccine strain production. Interestingly, silencing of several genes that enhanced poliovirus replication also enhanced replication of enterovirus 71, a clinically relevant virus to which vaccines are being targeted. The discovery that host gene modulation can markedly increase virus vaccine production dramatically alters mammalian cell-based vaccine manufacturing possibilities and should facilitate polio eradication using the inactivated poliovirus vaccine.IMPORTANCEUsing a genome-wide RNAi screen, a collection of host virus resistance genes was identified that, upon silencing, increased poliovirus and enterovirus 71 production by from 10-fold to >50-fold in a Vero vaccine manufacturing cell line. This report provides novel insights into enterovirus-host interactions and describes an approach to developing the next generation of vaccine manufacturing through engineered vaccine cell lines. The results show that specific gene silencing and knockout events can enhance viral titers of both attenuated (Sabin strain) and wild-type polioviruses, a finding that should greatly facilitate global implementation of inactivated polio vaccine as well as further reduce costs for live-attenuated oral polio vaccines. This work describes a platform-enabling technology applicable to most vaccine-preventable diseases.

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Development of a targeted sequencing approach to identify prognostic, predictive and diagnostic markers in paediatric solid tumours

10.1101/165746 ◽

2017 ◽

Cited By ~ 1

Author(s):

Elisa Izquierdo ◽

Lina Yuan ◽

Sally George ◽

Michael Hubank ◽

Chris Jones ◽

...

Keyword(s):

Cell Lines ◽

Personalised Medicine ◽

Unmet Need ◽

Paediatric Oncology ◽

Solid Tumours ◽

Targeted Sequencing ◽

Childhood Cancers ◽

Single Nucleotide Variants ◽

Target Sequencing ◽

Fresh Frozen

AbstractThe implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology.

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The Metalloid Reductase of Pseudomonas Moravenis Stanleyae Conveys Nanoparticle Mediated Metalloid Tolerance

10.26434/chemrxiv.6267383.v1 ◽

2018 ◽

Author(s):

Richard Nemeth ◽

Mackenzie Neubert ◽

Thomas Ni ◽

Christopher J. Ackerson

Keyword(s):

Structural Analysis ◽

Substrate Specificity ◽

Glutathione Reductase ◽

Cell Lines ◽

Bacterial Cell ◽

Binding Pocket ◽

Oxidized Glutathione ◽

Se Nanoparticles ◽

In Cells

In the present work we have identified a glutathione reductase like metalloid reductase (GRLMR) responsible for mediating selenite tolerance in Pseudomonas moravenis stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (Km= 336 μM) over oxidized glutathione (Km=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC90 for selenite from 1.9 mM in cell lacking the GRLMR gene to 21.3 mM for cells containing the GRLMR gene. It was later confirmed by STEM and EDS that Se nanoparticles were absent in control cells and present in cells expressing GRLMR. Structural analysis suggests the lack of a sulfur residue in the substrate/product binding pocket may be responsible for this unique substrate specificity.

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Faster, cheaper and under control: de-risking CMC development with transposon-derived manufacturing cell lines

10.22541/au.159609431.16553445 ◽

2020 ◽

Author(s):

Sowmya Rajendran ◽

Sowmya Balasubramanian ◽

Lynn Webster ◽

Maggie Lee ◽

Divya Vavilala ◽

...

Keyword(s):

Cell Lines ◽

Manufacturing Cell

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Development and validation of a targeted sequencing panel for application to treatment-refractory solid tumor.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.3_suppl.245 ◽

2021 ◽

Vol 39 (3_suppl) ◽

pp. 245-245

Author(s):

Woo Sun Kwon ◽

Jingmin Che ◽

Sun Young Rha ◽

Hyun Cheol Chung ◽

Hyo Jun Han ◽

...

Keyword(s):

Microsatellite Instability ◽

Cell Lines ◽

Solid Tumor ◽

Targeted Sequencing ◽

Molecular Profile ◽

Infected Cell Line ◽

Precision Oncology ◽

Rna Probes ◽

Treatment Refractory ◽

Refractory Solid Tumor

245 Background: Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted sequencing panel for application to treatment-refractory solid tumor types with particular focus on tumours of the stomach cancer, plus test for utility in FFPE, fluid sample and cancer cell lines. Methods: “CancerMaster” is custom RNA probes for target enrichment sequencing. It consists of all unions (7,811 regions, 2.5Mb) of reported exons of 524 tumor and immune related genes. The panel contains special RNA probes, which enables detection of microsatellite instability, Epstein-Barr virus and Human papillomavirus. Also, the CancerMaster panel analyzes 524 genes as well as genomic signatures including microsatellite instability (MSI), tumor mutational burden (TMB) and HLA allele. FFPE samples and 49 gastric cancer (GC) cell lines were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 117), and specificity by Sanger sequencing, pyrosequencing, FoundationOne CDx. Results: We achieved a mean coverage of 1,032x, with sensitivity and specificity of >99% and precision of >97%. In this study, we evaluated 26 Yonsei Cancer Center (YCC) GC cell lines and 23 GC cell lines from other sources (ATCC, KCLB, and JCRB). Application to 49 gastric cancers cell lines resulted in detection of copy number variant (CNV), single number variant (SNV) and small InDel aligned to whole exome sequencing data. In addition to previous novel EBV infected cell line (YCCEL1/YCC-10, J Gen Virol. 2013), we observed amplification and overexpression of receptor tyrosine kinase (RTK) including HER2 (YCC-19, 32, 33, 38), EGFR (YCC-11, 21), Met (YCC-31, 34), and FGFR2 (YCC-28, 30) in YCC GC cell lines; confirming that protein was overexpression by Western blot or Flow cytometry in 19/49 (38.8%). Interestingly, amplification of RTKs was detected mutually exclusive pattern with other RTK amplification. Conclusions: We have developed and validated an analytically-validated panel for application to cancers of treatment-refractory types.

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