Annotation of additional evolutionary conserved microRNAs in CHO cells from updated genomic data

Diendorfer, Andreas; Hackl, Matthias; Klanert, Gerald; Jadhav, Vaibhav; Reithofer, Manuel; Stiefel, Fabian; Hesse, Friedemann; Grillari, Johannes; Borth, Nicole

doi:10.1002/bit.25539

Cited by 13 publications

(9 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous miRNA profiling studies suggested that miR‐557 is not expressed in CHO cells (Diendorfer et al, ; Hackl et al, ; Hammond et al, ; Hernandez Bort et al, ; Lin et al, ; Stiefel et al, ), which we could confirm via qRT‐PCR based quantification of mature miR‐557 in our stable negative control miRNA expressing CHO host cells. According to the latest release of the miRBase repository (Kozomara and Griffiths‐Jones, ), miR‐557 has yet only found to be expressed in four species comprising human, chimpanzee, rhesus macaque, and orangutan.…”

Section: Discussionsupporting

confidence: 78%

“…Furthermore, individual miRNAs are capable of regulating entire cellular pathways (Fischer et al, ; Hackl et al, ), and thus might have a substantial impact on CHO cell phenotypes. Currently available literature on miRNA research in CHO cells mostly focusses on the identification of miRNAs to improve recombinant protein production (Emmerling et al, ; Fischer et al, , ; Jadhav et al, ; Kelly et al, ; Loh et al, ), or cell growth (Barron et al, ; Druz et al, ; Sanchez et al, ), while other studies were dedicated to basic principles of miRNA biogenesis and function (Diendorfer et al, ; Fischer et al, ,; Hackl et al, , ; Hernandez Bort et al, ). We have previously identified miR‐557 to increase productivity of a recombinant easy‐to‐express mAb in CHO cells (Strotbek et al, ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Fischer

Marquart

Pieper

et al. 2017

Biotech & Bioengineering

View full text Add to dashboard Cite

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

show abstract

Section: Discussionsupporting

confidence: 78%

Section: Introductionmentioning

confidence: 99%

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Fischer

Marquart

Pieper

et al. 2017

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…The miR-29 family is conserved across evolution in human, mouse, rat, and hamster (Barron et al, 2011; Diendorfer et al, 2015; Hackl et al, 2011; Johnson et al, 2011; Kriegel et al, 2012). Using the miRBase (Griffiths-Jones et al, 2006) precursor sequences as a guide, in the Chinese hamster genome, miR-29a and miR-29b are separated by approximately 250 base pairs on contig KE378782 in a region with sequence homology to an intron located within the mouse angiopoietin-like 7 ( ANGPTL7 ) gene.…”

Section: Introductionmentioning

confidence: 99%

“…A third member of the miR-29 family, miR-29c, is located on contig KE382798 within an intronic region of the APH1 homolog A, gamma secretase subunit ( APH1A ) based on sequence homology to the mouse APH1A gene. Each of these precursor miRs (pre-miRs) produces a mature miR on both strands of the hairpin, resulting in the six mature miRs cgr-miR-29a-5p, cgr-miR-29a-3p, cgr-miR-29b-5p, cgr-miR-29b-3p, cgr-miR-29c-5p, and cgr-miR-29c-3p (Diendorfer et al, 2015; Hackl et al, 2011). Pre miR-29b-1 and pre-miR-29a are transcribed in the reverse direction and are 78 nt and 83 nt respectively (Hackl et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells

Muluhngwi

Richardson

Napier

et al. 2017

Molecular and Cellular Endocrinology

View full text Add to dashboard Cite

miR-29b and miR-29a transcript levels were reported to increase in exponentially growing CHO-K1 cells. Here, we examine the regulation of miR-29b-1/a in CHO-K1 cells. We observed that 4-hydroxytamoxifen (4-OHT) increased pri-miR-29b-1 and pri-miR-29a transcription in CHO-K1 cells by activating endogenous estrogen receptor α (ERα). DICER, an established, bona fide target of miR-29b-1/a, was shown to be regulated by 4-OHT in CHO-K1 cells. We showed that miR-29b-1 and miR-29a serve a repressive role in cell proliferation, migration, invasion, and colony formation in CHO-K1 cells. To identify other targets of miR-29b-1 and miR-29a, RNA sequencing was performed by transfecting cells with anti-miR-29a, which inhibits both miR-29a and miR-29b-1, pre-miR-29b-1, and/or pre-miR-29a. In silico network analysis in MetaCore™ identified common and unique putative gene targets of miR-29b-1 and miR-29a. Pathway analysis of identified putative miR-29 targets were related to cell adhesion, cytoskeletal remodeling, and development. Further inquiry revealed regulation of pathways mediating responses to growth factor stimulus and cell cycle regulation.

show abstract

“…Furthermore, the location and sequence of the miR‐2861 binding site on cgr ‐HDAC5 is strikingly similar to the mouse ortholog, which further indicates a conserved function of miR‐2861 in mammalian cells. These findings suggest that the entire CHO miRnome is still not completely uncovered and need to be investigated more thoroughly as it has recently been done by Diendorfer et al who performed in silico re‐analysis of the CHO genome in order to find novel miRNAs in CHO cells (Diendorfer et al, ).…”

Section: Discussionmentioning

confidence: 99%

miR‐2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality

Fischer

Paul

Wagner

et al. 2015

Biotech & Bioengineering

Self Cite

View full text Add to dashboard Cite

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.

show abstract

Annotation of additional evolutionary conserved microRNAs in CHO cells from updated genomic data

Cited by 13 publications

References 25 publications

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

Regulation of miR-29b-1/a transcription and identification of target mRNAs in CHO-K1 cells

miR‐2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality

Contact Info

Product

Resources

About