Abstract
Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.
Highlights
Transient gene expression (TGE) has become an attractive tool to produce small quantities of recombinant proteins for the development of biopharmaceutical products in mammalian host cell lines at an early stage [1]
Delivery systems for artificially engineered RNAs using cationic lipids have been actively studied in both academic research and clinical trials [16], and presently, an mRNA delivery system based on cationic lipids is at the center of attention because of COVID-19 vaccines [17]
Appropriate ratios of DOPE and cationic lipids in lipoplexes were found to be a critical factor for lipoplex formation and transfection efficiency, which could be further improved when acidic liposome solution and alkaline plasmid DNA (pDNA) solution are used
Summary
Transient gene expression (TGE) has become an attractive tool to produce small quantities of recombinant proteins for the development of biopharmaceutical products in mammalian host cell lines at an early stage [1]. The complexes of cationic liposomes and nucleic acids, lipoplexes, are widely used in TGE and gene therapy technologies, along with polyplexes (polymers with nucleic acids) containing cationic polymers (e.g., polyethylenimine) or biodegradable polymers (e.g., cyclodextrin, dextran, and chitosan). Using these cationic carriers, research of the treatment of genetic disease by carrying nucleotide-based agents into the body has been actively conducted in recent years [6]. Cationic lipids and polymers have potential for future use through steady research and development to overcome their various barriers
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