Using intrinsic and extrinsic methods to engineer improved expression of recombinant proteins and retroviral vectors in mammalian cell

recombinant proteins;retroviral vectors;non-coding RNA;microRNA;Chemical & Biomolecular Engineering

Inwood, Sarah LouiseBeemon, Karen ;

Johns Hopkins University

关键词: recombinant proteins;    retroviral vectors;    non-coding RNA;    microRNA;    Chemical & Biomolecular Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/61125/INWOOD-DISSERTATION-2018.pdf?sequence=1&isAllowed=n

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Recombinant proteins, produced by introducing DNA into producer cells, are important in biotechnology, pharmaceuticals and academia. While prokaryotic cells are still most commonly used in these fields, mammalian cells are becoming more prevalent, especially for human proteins such antibodies, due to their inherent ability to correctly fold proteins, and retroviral vectors, due to their viral pseudotyping. This dissertation focuses on engineering improvement of recombinant protein expression and retroviral vector titer using both intrinsic methods such as cell engineering and extrinsic methods such as process development. To this end, multiple strategies such as non-coding RNA, stable transfections, CRISPR/Cas9 knockout, high-throughput screenings, and bioreactor perfusion processes were employed. Retroviral vectors have been of interest for some time due to their ability to modify genomes with relative ease and safety. This is increasingly so with the advancement of adoptive T-cell therapy, which is the transfer of T-Cells into a patient. These T-cells, often autologous, are typically modified, via various methods including retroviral vectors.Using mir-22-3p, which improves recombinant protein production, the first strategy was to identify gene targets of this microRNA that also improve recombinant protein expression. A microarray analysis was followed by bioinformatics; combining the results of the microarray with the predicted microRNA targets and the results of a high-throughput siRNA screen. Finally, confirmation with siRNA was performed to identify a focus gene, HIPK1. The second strategy was to create stable, high producing, recombinant protein expressing cell lines. This was achieved with a stably over-expressing microRNA, mir-22, and with a stable knockout of the gene identified earlier, HIPK1. Another strategy involved improving retroviral vector titer from PG13 cells in a bioreactor. PG13 cells are anchorage-dependent stable producing retroviral packaging cells for which scale up is difficult. It was achieved by attaching the PG13 cells to microcarriers, growing them in a suspension like environment in a continuous perfusion bioreactor. The dissertation wraps up with the method development of a high throughput RNAi screening assay to improve retroviral titer. The purpose of the screening is to identify microRNAs or siRNAs that affect vector titer. The design involves miniaturization, assay development and optimization for two transfections.