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Engineering Strategies for Intracellular Plasmid Delivery in Mammalian Cells

  • 김동우 (Dongwoo Kim, 아주대학교 일반대학원)

초록/요약

Plasmid-based transfection can be utilized in many applications such as plasmid-based gene integration and transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require the translocation of the transfected plasmids into the nucleus. However, the transfected plasmids should overcome intracellular barriers such as DNA degradation and the nuclear envelope. Here, I applied a modified plasmid system and a cell engineering approach to enhance targeted integration and TGE in two mammalian cell lines, Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells. First, a multi-component (MC) system consisting of a single-guide RNA (sgRNA)/double-cut donor (DCD) vector and a Cas9 expression vector was developed to control the ratios of the sgRNA expression cassette and DCD. The MC system increased the knock-in (KI) efficiency of CHO cell lines by approximately 1.5-fold compared to the DCD system by concurrently increasing sgRNA and DCD components relative to Cas9. Second, the Cas9-ZF system was applied to co-localize the CRISPR/Cas9-mediated DNA double-strand breaks and the donor template to increase KI efficiency in CHO cell lines. However, the Cas9-ZF system showed no significant increase in KI efficiency compared to the wild-type Cas9. Third, lysosome engineering was performed in CHO and HEK cell lines to alleviate lysosome-mediated nucleic acid degradation by targeting the RNautophagy/DNautophagy (RDA) pathway. By knocking out LAMP2C and SIDT2, the main players of RDA, we showed that LAMP2C KO effectively improved TGE-based mAb production by up to 2.82-fold increase in specific mAb productivity. This study demonstrated the importance of translocating transfected plasmids into the nucleus and provided a novel plasmid system and cell lines for the effective delivery of plasmids and relevant components for gene integration and expression.

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목차

Chapter I. Plasmid Engineering for Improving HDR-mediated Knock-In 1
I-1. Introduction 2
I-2. Materials and Methods 5
I-2.1. Plasmid Design and Construction 5
I-2.2. Cell Lines and Cell Culture 5
I-2.3. KI Efficiency Measurement Using Flow Cytometry 6
I-2.4. TIDE assay 7
I-2.5. Western Blotting Analysis 7
I-2.6. Plasmid Copy Number Calculation 8
I-2.7. Statistical Analysis 8
I-3. Results and Discussion 19
I-3.1. Effect of Different CRISPR-HDR Vector Configuration on KI Efficiency in CHO Cells 19
I-3.2. CRISPR-HDR Component Titration and Ratio Optimization in the DCD and MC Systems 22
I-3.3. Generation of the Cas9-ZF system 28
I-3.4. Estimation of the Cas9-ZF system 32
I-4. Reference 41
Chapter II. Improved Transient Gene Expression via Efficient Intracellular Plasmid Delivery in LAMP2C Knockout HEK293 Cell Line 47
II-1. Introduction 48
II-2. Materials and Methods 50
II-2.1. Plasmid construction 50
II-2.2. Cell line development and suspension adaptation 50
II-2.3. Cell culture maintenance 51
II-2.4. Quantitative real-time PCR (qRT-PCR) 51
II-2.5. Transient gene expression of TagRFP657 fluorescent protein in adherent WT and KO variants of HEK293E and CHO-K1 cells 52
II-2.6. Transient gene expression of mAb in suspension-adapted WT and KO cells 52
II-2.7. Measurement of mAb concentration by ELISA 53
II-2.8. Measurement of KI efficiency using flow cytometry 53
II-2.9. Measurement of mitochondrial, lysosomal, and ER content using flow cytometry 53
II-2.10. Statistical analysis 53
II-3. Results 61
II-3.1. Generation of LAMP2C- cell lines and evaluation of the gene KO effect using TGE of TagRFP657 fluorescent protein 61
II-3.2. Assessment of TGE of mAb in suspension-adapted LAMP2C- HEK293E cells 71
II-3.3. CRISPR/Cas9-mediated targeted integration in suspension-adapted LAMP2C- HEK293E cells 76
II-3.4. Estimation of the mitochondrial, lysosomal, and ER content in LAMP2C KO cells 79
II-4. Discussion 82
II-5. Reference 86

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