Site-specific Integration Ushers in a New Era of Precise CHO Cell Line Engineering.
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ABSTRACT:
SUBMITTER: Hamaker NK
PROVIDER: S-EPMC6510493 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
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Site-specific Integration Ushers in a New Era of Precise CHO Cell Line Engineering.
Hamaker Nathaniel K NK Lee Kelvin H KH
Current opinion in chemical engineering 20181023
Chinese hamster ovary (CHO) cells are widely used for the production of therapeutic proteins. Customarily, CHO production cell lines are established through random integration, which requires laborious screening of many clones to isolate suitable producers. In contrast, site-specific integration (SSI) accelerates cell line development by targeting integration of transgenes to pre-validated genomic loci capable of supporting high and stable expression. To date, a relatively small number of these ...[more]
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Project description:BackgroundConventional methods applied to develop recombinant CHO (rCHO) cell line as a predominant host for mammalian protein expression are limited to random integration approaches, which can prolong the process of getting the desired clones for months. CRISPR/Cas9 could be an alternative by mediating site-specific integration into transcriptionally active hot spots, promoting homogenous clones, and shortening the clonal selection process. However, applying this approach for the rCHO cell line development depends on an acceptable integration rate and robust sites for the sustained expression.Methods and resultsIn this study, we aimed at improving the rate of GFP reporter integration to the Chromosome 3 (Chr3) pseudo-attP site of the CHO-K1 genome via two strategies; these include the PCR-based donor linearization and increasing local concentration of donor in the vicinity of DSB site by applying the monomeric streptavidin (mSA)-biotin tethering approach. According to the results, compared to the conventional CRISPR-mediated targeting, donor linearization and tethering methods exhibited 1.6- and 2.4-fold improvement in knock-in efficiency; among on-target clones, 84% and 73% were determined to be single copy by the quantitative PCR, respectively. Finally, to evaluate the expression level of the targeted integration, the expression cassette of hrsACE2 as a secretory protein was targeted to the Chr3 pseudo-attP site by applying the established tethering method. The generated cell pool reached 2-fold productivity, as compared to the random integration cell line.ConclusionOur study suggested reliable strategies for enhancing the CRISPR-mediated integration, introducing Chr3 pseudo-attP site as a potential candidate for the sustained transgene expression, which might be applied to promote the rCHO cell line development.
| S-EPMC10224643 | biostudies-literature
Project description:Background: Chinese hamster ovary (CHO) cells are the most widely used mammalian host for recombinant protein production. The primary production method is to construct stable, high-yielding cell lines to provide high-quality, low-cost products. The key to optimizing recombinant protein production is to modify engineered cell lines to obtain better growth, expression, or product quality characteristics. Site-specific integration technology can build monoclonal cell lines with ideal stability, yield, and epigenetics by integrating transgenes at stable transcriptional hotspots, becoming a potential tool for constructing genetically engineered cell lines. The previous study screened the C12orf35 site of CHO cells and built an engineered cell line expressing a mAb by CRISPR/Cas9. While the mechanism of site-specific integration of transgenes to achieve high-efficiency expression also needs to be further studied. In this study, we used the extracellular domains of CLL1 and CD33 as model proteins to construct engineered cell lines integrating at C12orf35. Then we studied the growth, expression, and passage stability of these cell lines and conducted transcriptomic studies. Method: In this study, the transgenes CLL1 and CD33 were site-specifically integrated into the C12orf35 site of the CHO-S cell by CRISPR/Cas9 technology. To obtain stable and high-yielding cell lines, we screened and identified monoclonal cell lines by 5’/3’ junction PCR, out-out PCR, and Western blot. Then we compared the growth characteristics of CLL1 and CD33 monoclonal cell lines with wild-type CHO-S cell lines to investigate the growth characteristics. We studied the protein yield and affinity of CLL1 and CD33 cell lines through protein purification, BCA quantification, and ELISA. In addition, we assessed the stability of monoclonal cell lines in long-term culture by studying the growth characteristics, protein yield, and protein quality of early (5th-10th passage) and late (about 20th-25th passage) cells. We performed transcriptomic studies on cell lines with different transgenes, including CLL1, CD33, and anti-PD-1 mAb, inserted at C12orf35. We analyzed the differential genes, GO enrichment, and KEGG enrichment of cell lines hoping to explore the reasons for the phenotypic changes of cell lines from the transcriptional level, as well as the high expression mechanism of C12orf35 site-specific integration cell lines. Results:Using the site-specific integration technology mediated by CRISPR/Cas9, we successfully built CLL1 and CD33 monoclonal cell lines integrated at the C12orf35 site. We evaluated the cell growth characteristics and protein expression properties. The results showed that the insertion of different transgenes at the C12orf35 site had no specific effect on the growth state of the cells. The proteins yields were about 120 mg/L and 160 mg/L, respectively. And the EC50 values of CLL1 and CD33 with the corresponding antibodies reached about 0.36 μg/mL and 0.1 μg/mL, respectively. In addition, the above indicators had similar properties between the 5th and 20th generations, indicating that the specific integration at C12orf35 can achieve both high expression of the target protein and production stability. Transcriptomic analysis showed that the integration of transgenes at the C12orf35 site of CHO cells mainly induced genes in the GO term of “Regulation of transcription, DNA-templated” differentially expressed. Further analysis screened that ATF4 and multiple ZFPs family members were significantly up-regulated. ATF4 and ZFPs are related to gene transcription and protein synthesis, which may be the mechanism of the stable and high expression of the cell lines. Besides, differential gene analysis showed that Car4 and CXCL12 were significantly up- and down-regulated in CD33 cells, respectively. And KEGG enrichment showed that Wnt, PI3K/AKT, and JAK/STAT pathways related to cell proliferation and apoptosis were significantly enriched in CD33 cells. These genes and pathways may be related to the growth state of CD33. In summary, this paper studied the growth characteristics, protein expression characteristics, and stability of the C12orf35 site-specific integration of recombinant CHO cell lines. Combined with transcriptomic analysis, this paper explored the stable and high expression mechanism of these cell lines, hoping to provide a theoretical reference for the subsequent construction and optimization of high-expression stable cell lines.
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Project description:We describe a simple and efficient technique that allows scarless engineering of Drosophila genomic sequences near any landing site containing an inverted attP cassette, such as a MiMIC insertion. This two-step method combines phiC31 integrase-mediated site-specific integration and homing nuclease-mediated resolution of local duplications, efficiently converting the original landing site allele to modified alleles that only have the desired change(s). Dominant markers incorporated into this method allow correct individual flies to be efficiently identified at each step. In principle, single attP sites and FRT sites are also valid landing sites. Given the large and increasing number of landing site lines available in the fly community, this method provides an easy and fast way to efficiently edit the majority of the Drosophila genome in a scarless manner. This technique should also be applicable to other species.
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Project description:Site specific integration (SSI) expression systems offer robust means of generating highly productive and stable cell lines for traditional monoclonal antibodies. As complex modalities such as antibody-like molecules comprised of greater than two peptides become more prevalent, greater emphasis needs to be placed on the ability to produce appreciable quantities of the correct product of interest (POI). The ability to screen several transcript stoichiometries could play a large role in ensuring high amounts of the correct POI. Here we illustrate implementation of an SSI expression system with a single site of integration for development and production of a multi-chain, bi-specific molecule. A SSI vector with a single copy of all of the genes of interest was initially selected for stable Chinese hamster ovary transfection. While the resulting transfection pools generated low levels of the desired heterodimer, utilizing an intensive clone screen strategy, we were able to identify clones having significantly higher levels of POI. In-depth genotypic characterization of clones having the desirable phenotype revealed that a duplication of the light chain within the landing pad was responsible for producing the intended molecule. Retrospective transfection pool analysis using a vector configuration mimicking the transgene configuration found in the clones, as well as other vector configurations, yielded more favorable results with respect to % POI. Overall, the study demonstrated that despite the theoretical static nature of the SSI expression system, enough heterogeneity existed to yield clones having significantly different transgene phenotypes/genotypes and support production of a complex multi-chain molecule.
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Project description:Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 2040 patients and caused 712 deaths since its first appearance in 2012, yet neither pathogen-specific therapeutics nor approved vaccines are available. To address this need, we are developing a subunit recombinant protein vaccine comprising residues 377-588 of the MERS-CoV spike protein receptor-binding domain (RBD), which, when formulated with the AddaVax adjuvant, it induces a significant neutralizing antibody response and protection against MERS-CoV challenge in vaccinated animals. To prepare for the manufacture and first-in-human testing of the vaccine, we have developed a process to stably produce the recombinant MERS S377-588 protein in Chinese hamster ovary (CHO) cells. To accomplish this, we transfected an adherent dihydrofolate reductase-deficient CHO cell line (adCHO) with a plasmid encoding S377-588 fused with the human IgG Fc fragment (S377-588-Fc). We then demonstrated the interleukin-2 signal peptide-directed secretion of the recombinant protein into extracellular milieu. Using a gradually increasing methotrexate (MTX) concentration to 5 μM, we increased protein yield by a factor of 40. The adCHO-expressed S377-588-Fc recombinant protein demonstrated functionality and binding specificity identical to those of the protein from transiently transfected HEK293T cells. In addition, hCD26/dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with AddaVax-adjuvanted S377-588-Fc could produce neutralizing antibodies against MERS-CoV and survived for at least 21 days after challenge with live MERS-CoV with no evidence of immunological toxicity or eosinophilic immune enhancement. To prepare for large scale-manufacture of the vaccine antigen, we have further developed a high-yield monoclonal suspension CHO cell line.
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Project description:Chinese hamster ovary (CHO) cells are the most widely used mammalian hosts for production of therapeutic proteins. However, development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. Here we demonstrate efficient targeted gene integration into site-specific loci in CHO cells using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring a gene of interest (GOI) and short homology arms. This strategy has enabled precise insertion of a 3.7-kb gene expression cassette at defined loci in CHO cells following a simple drug-selection, resulting in homogeneous transgene expression. Taken together, the results displayed here can help pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.
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Project description:Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 μM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1-11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.
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Project description:Stability and transcriptomic study of C12orf35 site-specific integrated CHO cell line
| PRJNA839086 | ENA
Project description: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 Wiley Periodicals, Inc.
| S-EPMC6084326 | biostudies-literature
Project description:BACKGROUND:CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details. METHODS:Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. RESULTS:Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. CONCLUSION:In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids.
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