{"title":"Development of inducible packaging cell line for rAAV production via CRISPR-Cas9 mediated site-specific integration","authors":"","doi":"10.1016/j.bej.2024.109552","DOIUrl":null,"url":null,"abstract":"<div><div>AAV-mediated gene therapy is a quickly growing segment of the pharmaceutical market; however, the current transient transfection process to produce rAAV has several challenges. The stable cells are ideal for large-scale continuous production, overcoming the drawbacks in the current transient transfection and streamlining rAAV production. In this study, we proposed to use synthetic inducible promoters to control the viral component expression and develop the baseline of HEK293T stable cells via site-specific integration mediated with CRISPR-Cas9, targeting safe harbor sites of human genome (ROSA26, AAVS1, and CCR5 locus). With a total of three round integrations, stable cell pools were developed and evaluated at each round of integration. Single clones were further characterized for each integration round. Regarding the stable pools, the 5’ and 3’ junction PCR results confirmed the site-specific integration to each locus. The genome copy result showed that AAV components, including Rep78/68, E2A, E4orf6, Cap, and Rep52/40, were successfully integrated into the host cell genome. Genome and capsid titer after induction confirmed rAAV production for stable cell pools in each round. The packaging cell line (after 2nd round integration) was able to produce rAAV. However, it was observed that the genome titer was ten-fold lower than that of rAAV products done with triple plasmids transfection. The out-to-out PCR and qPCR assay results further confirm the site-specific integration. This research demonstrates the feasibility of developing the inducible stable cell line with the refactored viral vectors via a site-specific integration.</div></div>","PeriodicalId":8766,"journal":{"name":"Biochemical Engineering Journal","volume":null,"pages":null},"PeriodicalIF":3.7000,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemical Engineering Journal","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1369703X24003395","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
AAV-mediated gene therapy is a quickly growing segment of the pharmaceutical market; however, the current transient transfection process to produce rAAV has several challenges. The stable cells are ideal for large-scale continuous production, overcoming the drawbacks in the current transient transfection and streamlining rAAV production. In this study, we proposed to use synthetic inducible promoters to control the viral component expression and develop the baseline of HEK293T stable cells via site-specific integration mediated with CRISPR-Cas9, targeting safe harbor sites of human genome (ROSA26, AAVS1, and CCR5 locus). With a total of three round integrations, stable cell pools were developed and evaluated at each round of integration. Single clones were further characterized for each integration round. Regarding the stable pools, the 5’ and 3’ junction PCR results confirmed the site-specific integration to each locus. The genome copy result showed that AAV components, including Rep78/68, E2A, E4orf6, Cap, and Rep52/40, were successfully integrated into the host cell genome. Genome and capsid titer after induction confirmed rAAV production for stable cell pools in each round. The packaging cell line (after 2nd round integration) was able to produce rAAV. However, it was observed that the genome titer was ten-fold lower than that of rAAV products done with triple plasmids transfection. The out-to-out PCR and qPCR assay results further confirm the site-specific integration. This research demonstrates the feasibility of developing the inducible stable cell line with the refactored viral vectors via a site-specific integration.
期刊介绍:
The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery relevant to industries as diverse as medical/healthcare, industrial biotechnology, and environmental biotechnology.
The Journal welcomes full length original research papers, short communications, and review papers* in the following research fields:
Biocatalysis (enzyme or microbial) and biotransformations, including immobilized biocatalyst preparation and kinetics
Biosensors and Biodevices including biofabrication and novel fuel cell development
Bioseparations including scale-up and protein refolding/renaturation
Environmental Bioengineering including bioconversion, bioremediation, and microbial fuel cells
Bioreactor Systems including characterization, optimization and scale-up
Bioresources and Biorefinery Engineering including biomass conversion, biofuels, bioenergy, and optimization
Industrial Biotechnology including specialty chemicals, platform chemicals and neutraceuticals
Biomaterials and Tissue Engineering including bioartificial organs, cell encapsulation, and controlled release
Cell Culture Engineering (plant, animal or insect cells) including viral vectors, monoclonal antibodies, recombinant proteins, vaccines, and secondary metabolites
Cell Therapies and Stem Cells including pluripotent, mesenchymal and hematopoietic stem cells; immunotherapies; tissue-specific differentiation; and cryopreservation
Metabolic Engineering, Systems and Synthetic Biology including OMICS, bioinformatics, in silico biology, and metabolic flux analysis
Protein Engineering including enzyme engineering and directed evolution.