Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.
CHO
CRISPR/Cas9
FluidFM nanoinjection
monoclonal cell line
Journal
Biotechnology journal
ISSN: 1860-7314
Titre abrégé: Biotechnol J
Pays: Germany
ID NLM: 101265833
Informations de publication
Date de publication:
Apr 2024
Apr 2024
Historique:
revised:
21
03
2024
received:
22
09
2023
accepted:
02
04
2024
medline:
23
4
2024
pubmed:
23
4
2024
entrez:
23
4
2024
Statut:
ppublish
Résumé
Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.
Identifiants
pubmed: 38651269
doi: 10.1002/biot.202300505
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2300505Subventions
Organisme : Fortüne Tübingen
ID : 2485-0-0
Organisme : Fortüne Tübingen
ID : 2412-0-0
Organisme : UKT-Clinician Scientist Program
ID : 440-0-0
Organisme : University Children's Hospital of Tübingen
Informations de copyright
© 2024 The Authors. Biotechnology Journal published by Wiley‐VCH GmbH.
Références
Dumont, J., Euwart, D., Mei, B., Estes, S., & Kshirsagar, R. (2016). Human cell lines for biopharmaceutical manufacturing: History, status, and future perspectives. Critical Reviews in Biotechnology, 36, 1110.
Kim, J. Y., Kim, Y.‐G., & Lee, G. M. (2012). CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Applied Microbiology and Biotechnology, 93, 917.
Yang, G., Wang, Q., Chen, L., Betenbaugh, M. J., & Zhang, H. (2021). Glycoproteomic characterization of FUT8 knock‐out CHO cells reveals roles of FUT8 in the glycosylation. Frontiers in Chemistry, 9, 7552.
Golay, J., Andrea, A. E., & Cattaneo, I. (2022). Role of Fc core fucosylation in the effector function of IgG1 antibodies. Frontiers in Immunology, 13, 929895.
Cohen Saban, N., Yalin, A., Landsberger, T., Salomon, R., Alva, A., Feferman, T., Amit, I., & Dahan, R. (2023). Fc glycoengineering of a PD‐L1 antibody harnesses Fcγ receptors for increased antitumor efficacy. Science Immunology, 8, eadd8005.
Evans, K., Albanetti, T., Venkat, R., Schoner, R., Savery, J., Miro‐Quesada, G., Rajan, B., & Groves, C. (2015). Assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging. Biotechnology Progress, 31, 1172.
Grav, L. M., Lee, J. S., Gerling, S., Kallehauge, T. B., Hansen, A. H., Kol, S., Lee, G. M., Pedersen, L. E., & Kildegaard, H. F. (2015). One‐step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnology Journal, 10, 1446.
Shen, C.‐C., Sung, L.‐Y., Lin, S.‐Y., Lin, M.‐W., & Hu, Y.‐C. (2017). Enhancing protein production yield from chinese hamster ovary cells by CRISPR interference. ACS Synthetic Biology, 6, 1509.
Antony, J. S., Latifi, N., Haque, A., Lamsfus‐Calle, A., Daniel‐Moreno, A., Graeter, S., Baskaran, P., Weinmann, P., Mezger, M., Handgretinger, R., & Kormann, M. S. D. (2018). Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors. Molecular and Cellular Pediatrics, 5, 9.
Voronina, E. V., Seregin, Y. A., Litvinova, N. A., Shvets, V. I., & Shukurov, R. R. (2016). Design of a stable cell line producing a recombinant monoclonal anti‐TNFα antibody based on a CHO cell line. Springerplus, 5, 1584.
Yang, W., Zhang, J., Xiao, Y., Li, W., & Wang, T. (2022). Screening strategies for high‐yield Chinese hamster ovary cell clones. Frontiers in Bioengineering and Biotechnology, 10, 858478.
Antony, J. S., Daniel‐Moreno, A., Lamsfus‐Calle, A., Raju, J., Kaftancioglu, M., Ureña‐Bailén, G., Rottenberger, J., Hou, Y., Santhanakumaran, V., Lee, J.‐H., Heumos, L., Böhringer, J., Krägeloh‐Mann, I., Handgretinger, R., & Mezger, M. (2022). A mutation‐agnostic hematopoietic stem cell gene therapy for metachromatic leukodystrophy. Crispr J, 5, 66.
Sun, X., Wang, J., Mou, C., Shi, K., Bao, W., & Chen, Z. (2023). Knockout of IRF3 and IRF7 genes by CRISPR/Cas9 technology enhances porcine virus replication in the swine testicular (ST) cell line. Biotechnology Journal, 19, e2300389.