KR102576635B1 - A cell line which is knock out LAMP2C gene and a method of producing target genes of interests using the same - Google Patents

Abstract

The present invention relates to a transformed human cell line in which the LAMP2C gene has been knocked out, and a method of increasing the expression level of a target gene using the cell line. The present invention improves the expression level of the target foreign gene from a plasmid introduced through intracellular transfection by knocking out the lysosomal membrane protein LAMP2C gene, which can play an important role in intracellular nucleic acid degradation, in a human cell line modeled after the HEK293 cell line. As it is introduced into the genome and stably expresses the target gene or protein, the expression efficiency of the target protein can be improved. Therefore, the present invention can be used in protein biopharmaceuticals and gene therapy products manufactured through foreign gene transfer.

Description

A transgenic human cell line in which the LAMP2C gene has been knocked out and a method for producing a target gene using the same {A cell line which is knock out LAMP2C gene and a method of producing target genes of interests using the same}

The present invention relates to a transformed human cell line in which the LAMP2C gene has been knocked out, and a method of increasing the expression level of a target gene using the cell line.

Transfection of mammalian cells using non-viral vectors is essential for transient gene expression (TGE)-based therapeutic protein production, plasmid-based site-specific gene insertion, and gene therapy. Recombinant protein production using TGE is a rapid and effective technique to screen various therapeutic protein candidates and prepare protein samples for preclinical evaluation. However, compared to using stable gene expression (SGE), there are limitations due to relatively low protein yield and production scale. Effective TGE requires that the transfected plasmid cross the plasma membrane through an entry route such as endocytosis, then escape from the endosomal/lysosomal compartment, and then efficiently overcome multiple intracellular barriers such as DNA degradation, lysosomal degradation, and the nuclear envelope. It must pass through and reach the core.

Cytoplasmic nucleases, including deoxyribonuclease (DNase) I, DNase II, and DNase I-like III, are involved in the cytoplasmic degradation of transfected or injected plasmids. Previous studies have demonstrated that transfected plasmids are located in various cellular regions such as cytoplasm, endosomes, lysosomes, and nucleus, and approximately 50% of transfected plasmids are located in the endo/lysosomal compartment and are re-exposed to the cytoplasm during mitosis. do. Extranuclear DNase is one of the causes of plasmid degradation, and its knockout (KO) can improve transfection efficiency. In addition to targeting DNase, given that a portion of intracellular plasmids migrate to lysosomes, preventing lysosomal degradation of plasmids could be another strategy to maximize plasmid availability. Recent studies have emphasized the importance of a lysosomal-mediated nucleic acid degradation system called RNautophagy/DNautophagy (RDA), in which RNA and DNA are directly transported to lysosomes and then degraded, and the lysosomal membrane proteins LAMP2C and SIDT2 are known to play a major role.

The lysosomal membrane protein LAMP2C, one of the LAMP2 isoforms, has a short C-terminal cytoplasmic tail containing basic amino acids such as arginine and lysine, and is a receptor that can bind nucleotide oligomers. On the other hand, SIDT2 is known as a lysosomal membrane transporter involved in direct uptake of DNA or RNA into lysosomes in an ATP-dependent manner. To date, the interaction between LAMP2C and SIDT2 has been shown to be involved in RDA, but nothing is known about the relationship between the two proteins and plasmid transfer.

Accordingly, in order to reduce the potential decrease in TGE efficiency due to lysosomal degradation of the plasmid and to increase the expression efficiency of foreign introduced genes, the present inventors used human embryonic kidney 293 (HEK293) and Chinese hamster ovary (CHO), representative industrial animal cell lines, in which LAMP2C and SIDT2 were removed. Cell lines were prepared, and the level of TGE-based reporter protein production and transfection efficiency of several knockout cell lines were evaluated to confirm the cell type-dependent effects of these two genes. As a result, through this invention, it was confirmed that the human cell line in which the LAMP2C gene was knocked out was superior in TGE-based recombinant protein productivity compared to the wild-type host cell line, and even when a stable cell line was manufactured, the recombinant protein protein was compared to the wild-type host cell line. The present invention was completed by confirming that productivity and mRNA expression level per unit foreign gene were excellent.

Republic of Korea Patent Publication No. 10-2022-0016957

The purpose of the present invention is to provide a transformed human cell line in which the LAMP2C gene has been knocked out.

Another object of the present invention is to provide a composition for producing a target protein comprising a transformed human cell line in which the LAMP2C gene has been knocked out.

Another object of the present invention is to knock out the LAMP2C gene; To provide an in vitro production method of a transformed human cell line in which the LAMP2C gene has been knocked out.

Another object of the present invention is (a) introducing a vector containing a base sequence encoding a target protein into a transformed human cell line in which the LAMP2C gene has been knocked out; To provide a method for producing a target protein comprising.

Another object of the present invention is to introduce a vector containing a foreign gene into a transformed human cell line in which the LAMP2C gene has been knocked out; To provide a method for increasing transfection efficiency comprising.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

The terms used herein are for descriptive purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the embodiments belong. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

Hereinafter, the present invention will be described in more detail.

The present invention provides a transformed human cell line in which the LAMP2C gene has been knocked out.

In the present invention, LAMP2C (Lysosome associated membrane protein 2) is a lysosomal membrane protein that is one of the LAMP2 isoforms, has a short C-terminal cytoplasmic tail containing basic amino acids such as arginine and lysine, and can bind to nucleotide oligomers. It is a receptor that can The LAMP2C may interact with SIDT2 in RNautophagy/DNautophagy (RDA), but is not limited thereto. The RDA refers to a lysosome-mediated nucleic acid degradation system in which RNA and DNA are directly transported to lysosomes and then degraded.

In the present invention, SIDT2 (SID1 Transmembrane Family Member 2) may be a lysosomal membrane transporter involved in directly absorbing DNA or RNA into lysosomes in an ATP-dependent manner. However, unlike LAMP2C knockout, SIDT2 knockout increases foreign gene expression. If LAMP2C and SIDT2 are knocked out simultaneously, the increase in foreign gene expression caused by LAMP2C can be reduced.

Therefore, in the present invention, the cell line may be one in which the SIDT2 gene has not been knocked out, but is not limited thereto.

In the present invention, “knockout” refers to the regulation of gene activity by deletion or substitution of all or part of a gene, or insertion of one or more nucleotides. Additionally, the knockout may use the CRISPR-Cas9 system, but is not limited thereto.

In the present invention, the "CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic, Repeat)-Cas9 (CRISPR-associated protein 9) system" is a type of immune system and gene editing tool that exists in prokaryotes, and is a type of gene editing tool that rapidly recognizes and cuts foreign invading DNA. do. CRISPR refers to a structure in which repetitive nucleotide sequences of 20-50 bp are arranged sequentially. Each pair of repetitive sequences in the CRISPR-Cas9 system is separated by an insertion sequence (spacer) with a unique sequence, and the insertion sequences are similar to parts of the bacteriophage genome, and the RNA made therefrom is a guide RNA. After binding to the invading phage DNA, the Cas9 protein (Cas nucleic acid internal hydrolase) cuts the phage DNA. Using this principle, genes can be inserted or removed from embryos, cells, etc. Since Cas9 is a component of the immune system of prokaryotes, gene knockout in eukaryotic cells involves creating a cell line expressing the Cas9 enzyme and then transfecting guide RNA or encoding Cas9 and guide RNA. This can be achieved by introducing a vector containing the base sequence into the cell line, but is not limited to this.

In the present invention, the guide RNA refers to RNA that guides the Cas protein to the targeting domain.

In the present invention, the human cell line may be the HEK293 (Human embryonic kidney 293) cell line, which is an industrial animal cell line, or the CHO (Chinese hamster ovary) cell line, but is preferably the HEK293 cell line, but is not limited thereto.

In the present invention, the cell line can increase the expression of foreign genes based on transient gene expression (TGE) or stable gene expression (SGE), but is not limited thereto. The TGE refers to the temporary expression of a gene expressed for a short period of time after the foreign gene is introduced into the cell. Since the foreign DNA is not integrated into the host genome, it is not replicated and is eventually lost through a cell division cycle that lasts several days. In addition, the SGE refers to expression in which a stable clone is created through a selection and amplification process after a foreign gene is introduced into a cell, and the foreign gene becomes part of the host genome and is replicated.

In the present invention, the cell line may have a mutation in the base sequence of the cytoplasmic tail region of the LAMP2C gene, and the mutation may be a substitution, insertion, or deletion, but is not limited thereto. Specifically, the nucleotide sequence represented by SEQ ID NO: 7 may be deleted from the LAMP2C gene, and the deleted sequence has 80%, 85%, 90%, 95%, 99%, or 100% identity with SEQ ID NO: 7. It may include, but is not limited to, a sequence.

The present invention provides a composition for producing a target protein comprising a transformed human cell line in which the LAMP2C gene has been knocked out. The target protein may be selected from the group consisting of fluorescent proteins, antibody expression proteins, antibody-derived fusion proteins, immune proteins, and protein-based hormones, but is not limited thereto.

The present invention includes the steps of knocking out the LAMP2C gene; Provided is an in vitro production method of a transformed human cell line in which the LAMP2C gene has been knocked out.

In the present invention, the knockout may be performed by introducing a vector containing a sgRNA targeting the base sequence of the cytoplasmic tail region of the LAMP2C gene and a base sequence encoding the Cas9 protein, but is not limited to this. The sgRNA may target the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

In the present invention, the production method may further include, but is not limited to, the step of selecting a cell line in which the LAMP2C gene has been knocked out from the cell line into which the vector has been introduced.

The present invention includes the steps of (a) introducing a vector containing a base sequence encoding a target protein into a transformed human cell line in which the LAMP2C gene has been knocked out; Provides a method for producing a target protein comprising.

In the present invention, the production method includes (b) culturing the cell line of step (a); and (c) separating and purifying the target protein produced in step (b), but is not limited thereto. The target protein may refer to a variety of useful proteins intended to promote production within the human cell line by transfection into the human cell line. Specifically, it may be a fluorescent protein, an antibody-expressing protein, an antibody-derived fusion protein, an immune protein, or a protein-based hormone. may, but is not limited to this. The immune protein may be, but is not limited to, a cytokine.

The step of culturing the cell line in step (b) may use an appropriate cell line culture method known in the art depending on the type of cell knocking out the LAMP2C gene, preferably when the cell line is a HEK293 (Human embryonic kidney 293) cell line. , HEK293 cells can be cultured using culture media and culture conditions known to be appropriate for culturing them.

The step (c) of isolating and purifying the target protein produced in step (b) is a step of obtaining the target protein produced in the host cell, and intracellular proteins or cell secreted proteins can be separated and purified.

In the present invention, the fluorescent protein is blue fluorescent protein (BFP), cyan fluorescent protein (CFP), green fluorescent protein (GFP), or yellow fluorescent protein (Yellow fluorescent protein). , YFP), but is not limited thereto.

The present invention includes the steps of introducing a vector containing a foreign gene into a transformed human cell line in which the LAMP2C gene has been knocked out; It provides a method for increasing transfection efficiency including.

Introduction of the vector can be performed without limitation by known methods known in the art, for example, through electroporation or lipofection methods.

According to the present invention, the transfection efficiency of the foreign gene is increased regardless of the type and type of the foreign gene, and through this, the productivity of the target protein can be increased.

The present invention improves the expression level of the target foreign gene from a plasmid introduced through intracellular transfection by knocking out the lysosomal membrane protein LAMP2C gene, which can play an important role in intracellular nucleic acid degradation, in a human cell line modeled after the HEK293 cell line. As it is introduced into the genome and stably expresses the target gene or protein, the expression efficiency of the target protein can be improved. Therefore, the present invention can be used in protein biopharmaceuticals and gene therapy products manufactured through foreign gene transfer.

Figure 1 relates to the preparation and validation of LAMP2C knockout human cell lines. Specifically, Figure 1A shows a schematic diagram of LAMP2C knockout (KO) in HEK293E wild type (WT) host cells (scissors indicate the CRISPR/Cas9 cleavage site that simultaneously cuts the genomic region of the cytoplasmic tail of LAMP2C; Red and blue arrows indicate primer sets used for RT-PCR and genotyping PCR, respectively; DNA and amino acid sequences outline the KO target region of LAMP2C in WT and KO clones). Figure 1B shows the results of qRT-PCR analysis of LAMP2A, LAMP2B, and SIDT2 in HEK293E WT cells and LAMP2C knockout clones #2, 5, 13, and 14 (mRNA levels of each gene were normalized to ACTB and HPRT1). Figure 1c shows the results of validation of the LAMP2C knockout clone through RT-PCR analysis (positive control: HEK293E WT; expected PCR product size: 110 bp).
Figure 2 relates to the preparation and validation of SIDT2 knockout human cell lines. Specifically, Figure 2a shows a schematic diagram of SIDT2 knockout (KO) in HEK293E wild type (WT) host cells (the scissors are genetic scissors (CRISPR/Cas9) that simultaneously cut the genomic region corresponding to exons 3 and 4 of SIDT2. ) indicates the cut site). Figure 2b shows four SIDT2 gene knockout clones (#3, 14, 24, 27) and five SIDT2/LAMP2C double knockout clones (#8, 11, 17, 19, 21) through RT-PCR analysis. SIDT2 knockout validation results are shown (positive control: HEK293E WT; black triangle: expected PCR product from WT and knockout clones).
Figure 3 shows the analysis results of TagRFP657 fluorescent protein TGE expression pattern in HEK293E wild type and LAMP2C knockout host cell lines through the proportion of TagRFP657+ cells, mean fluorescence intensity (MFI), and total fluorescence intensity (TFI) (proportion of TagRFP657 expression vector) : (A) 20%, (B) 50%, (C) 100%; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).
Figure 4 shows the results of analysis of TagRFP657 fluorescent protein TGE expression pattern in HEK293E wild type, SIDT2 knockout (A), LAMP2C knockout (L5), and SIDT2/LAMP2C (SL) double knockout (B) host cell lines as percentage of TagRFP657+ cells and average. It is expressed through fluorescence intensity (MFI) and total fluorescence intensity (TFI) (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001).
Figure 5 relates to the preparation and validation of the LAMP2C knockout CHO-K1 host cell line. Specifically, Figure 5a shows a schematic diagram of LAMP2C knockout (KO) in CHO-K1 wild type (WT) host cells (the scissors are the genetic scissors (CRISPR/Cas9) cutting site that simultaneously cuts the genomic region of the cytoplasmic tail of LAMP2C. indicates). Figure 5b shows the results of verifying the LAMP2C knockout clone through RT-PCR analysis (positive control: CHO-K1 WT). Figures 5c and 5d show the results of analysis of the TagRFP657 fluorescent protein TGE expression pattern in the LAMP2C knockout CHO-K1 host cell line using different transfection methods (lipofection 5c; electroporation 5d).
Figure 6 shows the results of TGE mean fluorescence intensity (MFI) analysis of EGFP fluorescent protein based on serum-free suspension culture in HEK293E wild-type and LAMP2C knockout host cell lines (relative fold change normalized to the value of each clone 1 day after transfection). ; error bars represent the mean ± standard deviation of three independent experiments; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001).
Figure 7 shows the results of serum-free suspension culture-based antibody TGE productivity (qP) analysis in HEK293E wild-type and LAMP2C knockout host cell lines (qP is based on cumulative cell concentration and measured antibody concentration based on EGFP+ cells of each clone) Calculated as; error bars represent the mean ± standard deviation of three independent experiments; *p ≤ 0.05, ***p ≤ 0.001).
Figure 8 shows the results of EGFP fluorescent protein SGE mean fluorescence intensity (MFI) analysis in EGFP cell pools based on HEK293E wild-type and LAMP2C knockout host cell lines.
Figure 9 shows the results of measuring the mRNA expression rate compared to the EGFP gene copy number in the EGFP cell pool based on the HEK293E wild-type and LAMP2C knockout host cell lines (relative fold change is normalized to the value in the EGFP cell pool based on the wild-type host cell line).

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[ Example ]

Example One. LAMP2C Knockout human cell line preparation

To prepare a human cell line in which the LAMP2C gene has been knocked out, HEK293E wild-type host cells were first attached and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% (v/v) FBS (fetal bovine serum) and L-glutamine. . In cultured HEK293E cells, a vector containing two types of genetic scissors (CRISPR/Cas9) that recognize the genomic region encoding the cytoplasmic tail region of the LAMP2C gene was used to excise 138 base pairs to delete the region. All KO clones have the same deletion in the sequence shown in red in the LAMP2C gene (SEQ ID NO: 7: GCAGTGGGTGTGGCCTTGGGCTTCCTTATAATTGTTGTCTTTATCTCTTATATGATTGGAAGAAGGAAAAGTCGTACTGGTTATCAGTCTGTGTAATCAGTTAAATCTAGTGTTTGTTTGTTTTTTTCAATTAGAAGTTACGTTTC). A schematic diagram of the process is shown in Figure 1a. Additionally, the sequence of Figure 1a is shown in Table 1 below.

base sequence/amino acid sequence sequence number WT
(base sequence) CTGAAGAATGTTCTGCTGACTCTGAACCTCAACTTTCTTATTCCTGTT GCAGTGGGTGTGGCCTTGGGCTTCCTTATAATTGTTGTCTTTATCTCTTATATGATTGGAAGAAGGAAAAGTCGTACTGGTTATCAGTCTGTGTAATCAGTTAAATCTAGTGTTTGTTTGTTTTTTTCAATTAGAAGTTACGTTTC CATTGGCTAAAAGCCAGGACATGCTGTGCAATAGATTGTTTAAGATATGCAGACTAA 8 WT
(amino acid sequence) EECSADSDLNFLIPVAVGVALGFLIIVVFISYMIG RRKSRTGYQSV 9 LAMP2C KO (nucleotide sequence) CTGAAGAATGTTCTGCTGACTCTGACCTCAACTTTCTTATTCCTGTTCATTGGCTAAAAGCCAGGACATGCTGTGCAATAGATTGTTTAAGATATGCAGACTAA 10 LAMP2C KO
(amino acid sequence) EECSADSDLNFLIPVHWLKARTCCAIDCLRYAD 11

* Bold indicates the sequence to be knocked out.

Specifically, the vector expresses sgRNA targeting the cytoplasmic tail region genomic region of the LAMP2C gene (SEQ ID NO: 1: 5'-ATTAGAAGTTACGTTTCCAT TGG ; SEQ ID NO: 2: AAGGCCACACCCACTGCAAC AGG ; PAM sequence underlined) and a vector expressing the Cas9 protein. used. The vector was introduced into HEK293E cells using Lipofectamine™ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Two days after transfection, taking advantage of the mCherry expression contained in the vector, mCherry-positive cells were sorted and recovered using a FACSAria™ III cell sorter (Becton Dickinson, NJ, USA) and then sorted by limiting dilution in a 96-well plate ( limiting dilution) was performed. Knockout of each monoclonal cell was verified through genotyping PCR using genomic DNA extracted with QuickExtract™ (Lucigen, Teddington, UK). The gene sequence of the obtained PCR product was confirmed through DNA sanger sequencing. Primer sequence information used in the experiment is shown in Table 2 below.

Primer name Purpose Sequence (5'-3') sequence number CHO_LAMP2C_val_fwd Genotyping PCR for LAMP2C KO validation of CHO-K1 GTGATCTGTGGCCGTGGACT 12 CHO_LAMP2C_val_rev AGTGTGAAGTGTAGCTTTTGTTTCA 13 HEK_LAMP2C_val_fwd Genotyping PCR for LAMP2C KO validation of HEK293E TTGGATCCAGGGGGCTTAAAATCAT 14 HEK_LAMP2C_val_rev TGCTGTCTGTTCTTTTCTAACAGC 15 HEK_SIDT2_val_fwd5 Genotyping PCR for SIDT2 KO validation of HEK293E TGGAACGAACCCTGTGTCAG 16 HEK_SIDT2_val_rev5 CACGCCTTCAGGGAACTCAT 17 cLAMP2C_qPCR_F RT-PCR for LAMP2C KO validation of CHO-K1 GTGACGAAAGGAAAGTATGC 18 cLAMP2C_qPCR_R2 CAGACTGATAGCCAGTAC 19 hLAMP2C_qPCR_F RT-PCR for LAMP2C KO validation of HEK293E CAATGTGACACAAGGAAAGT 20 hLAMP2C_qPCR_R CAATTATAAGGAAGCCCAAGG 21 hSIDT2_qPCR_F2 RT-PCR for SIDT2 KO validation of HEK293E ACCAGTCAACACCACATAC 22 hSIDT2_qPCR_R2 GCCTTCAGGGAACTCATAC 23 hLAMP2C_rev qRT-PCR for validation of LAMP2A mRNA expression from HEK293E TCAATGTGACACAAGGAAAG 24 hLAMP2A_qPCR_R GCCAGCAACACTAGAATAAG 25 hLAMP2B_qPCR_F qRT-PCR for validation of LAMP2B mRNA expression from HEK293E GGTTCAGCTTTCAATGTG 26 hLAMP2B_qPCR_R AAGACCAGCACCAACTATAA 27 hSIDT2_qPCR_F qRT-PCR for validation of SIDT2 mRNA expression from HEK293E ACCAGTCAACACCACATAC 28 hSIDT2_qPCR_R GCCTTCAGGGAACTCATAC 29

Afterwards, clones in which four LAMP2C genes were knocked out were selected. Specifically, compared to the wild type, it was confirmed that all genomic regions corresponding to the cytoplasmic tail region of the LAMP2C gene in clones 2, 5, 13, and 14 were removed, suggesting that no basic amino acids in the cytoplasmic tail region of LAMP2C remain. .

In addition, qRT-PCR analysis and reverse transcription PCR (RT-PCR) were performed on the wild type and clones 2, 5, 13, and 14. The results are shown in Figures 1b and 1c.

As shown in Figure 1b, qRT-PCR analysis confirmed that the expression of SIDT2 and other LAMP2 isoforms, LAMP2A and LAMP2B, were not affected by deletion of the genomic region of LAMP2C in the LAMP2C knockout clone.

As shown in Figure 1c, the results of reverse transcription PCR (RT-PCR) confirmed that LAMP2C mRNA expression was not detected in the LAMP2C knockout clone.

Additionally, a human cell line in which the SIDT2 gene, known to be involved in the lysosome-mediated nucleic acid degradation system, was knocked out was prepared from HEK293E wild-type host cells. In cultured HEK293E cells, vectors containing two types of genetic scissors that recognize exons 3 and 4 of the SIDT2 gene were used to excise 138 base pairs to delete the corresponding region. This is shown in Figure 2a. Specifically, the vector was used to express sgRNA targeting the SIDT2 gene (SEQ ID NO: 3: 5'-TGAGCACAAAATCGTCCATG CGG ; SEQ ID NO: 4: TACCTGGGGCTGTGCTGCTG TGG ; PAM sequence underlined) and Cas9 protein were used, and knockout clones were constructed. The process is the same as the LAMP2C knockout clone selection process. Accordingly, four SIDT2 gene knockout clones (#3, 14, 24, 27) and five SIDT2/LAMP2C double knockout clones (#8, 11, 17, 19, 21) host cell lines were selected, and RT- It was verified that it was a SIDT2 knockout clone through PCR analysis. The results are shown in Figure 2b.

As shown in Figure 2b, SIDT2 knockout was confirmed through SIDT2 mRNA expression analysis.

Example 2. LAMP2C gene knocked out human cell lines TGE Verification of gene expression level for basic purpose

To evaluate changes in the expression level of the target gene in the HEK293E host cell line in which the LAMP2C gene was knocked out, the target gene (fluorescent protein and antibody expression gene) was expressed based on TGE, and the resulting target protein production was compared with the wild-type HEK293E host cell line.

2.1. LAMP2C in knockout host cell lines. TagRFP657 fluorescent protein TGE Confirmation of expression pattern

First, HEK293E wild-type and LAMP2C knockout host cell lines were transfected with TagRFP657 expression vector via lipofection, and flow cytometry was used at 12, 24, and 48 h after transfection to determine the percentage of TagRFP657+ cells, mean fluorescence intensity ( MFI) and total fluorescence intensity (TFI) were analyzed. In this TGE experiment, the ratio of TagRFP657 expression vector to empty vector was adjusted to 20%, 50%, and 100% (w/w) so that fewer plasmids could sufficiently reach gene expression levels comparable to 100% plasmid introduction. It was analyzed whether it was present or not. The results are shown in Figure 3 (ratio of TagRFP657 expression vector: (A) 20%, (B) 50%, (C) 100%).

As shown in Figure 3, all four LAMP2C knockout clones showed a high transfection effect compared to WT, and in particular, the L5 clone showed a significant difference in MFI and transfected population ratio 24 hours after transfection regardless of the plasmid ratio. It seemed. TFI is calculated by multiplying the number of transfected cells by MFI when analyzing ³ It showed a big difference.

At 48 hours after transfection, the TFI of the L5 clone transfected with 50% TagRFP657 expression vector was higher than the TFI of WT transfected with 100% expression vector. The other three LAMP2C knockout clones also showed similar TFI when transfected with only 50% of the expression vector compared to WT transfected with 100% of the expression vector.

2.2. SIDT2 knockout and SIDT2 / LAMP2C ( SL ) TagRFP657 fluorescent protein in double knockout host cell lines. TGE Confirmation of expression pattern

The same experiment as Example 2.1 was performed on SIDT2 and SIDT2/LAMP2C knockout host cell lines. The SIDT2/LAMP2C double knockout host cell line was obtained by knocking out SIDT2 based on the L5 clone, and the L5 clone was used as a control in a TGE-based experiment to confirm the expression level of the target gene. The results are shown in Figure 4 (SIDT2 knockout (A) and SIDT2/LAMP2C (SL) double knockout (B)).

As shown in Figure 4, SIDT2 knockout clones showed inconsistent effects, with some showing lower MFI than HEK293E wild-type host cells (A). Additionally, the L5-derived SIDT2/LAMP2C double knockout clone performed worse than the L5 clone (B). These results confirmed that SIDT2 gene knockout did not contribute to increasing the TGE-based target protein expression rate regardless of the presence of LAMP2C.

2.3. LAMP2C knockout CHO -Preparation and validation of K1 host cell line

To verify whether the effect of LAMP2C knockout is independent of host cell type, a CHO host cell line in which the LAMP2C gene was knocked out was generated. To prepare a CHO cell line with the LAMP2C gene knocked out, first, CHO-K1 wild-type host cells were cultured in DMEM medium containing 10% (v/v) FBS. In cultured CHO-K1 cells, vectors containing two types of genetic scissors that recognize the genomic region encoding the cytoplasmic tail region of the LAMP2C gene were used to excise 138 base pairs to delete the region. This is shown in Figure 5a.

Specifically, the vector expresses sgRNA targeting the cytoplasmic tail region genomic region of the LAMP2C gene (SEQ ID NO: 5: 5'-CAGTGCTTCCATTGGCTAAA AGG ; SEQ ID NO: 6: AGCAATTATAAGGAAGCCCA AGG ; PAM sequence underlined) and a vector expressing the Cas9 protein. used. The vector was introduced into CHO-K1 cells using a NEPA21 electroporator (Nepagene, Chiba, Japan) or into CHO-K1 cells using Lipofectamine™ 3000 transfection reagent (Invitrogen, Carlsbad, CA, USA). Afterwards, clones in which three LAMP2C genes were knocked out (#3, 19, and 27) were selected, and the LAMP2C knockout clones were verified through RT-PCR analysis. The results are shown in Figure 5b.

As shown in Figure 5b, LAMP2C knockout was confirmed through analysis of LAMP2C mRNA expression.

Next, the expression pattern of the fluorescent protein TGE was analyzed using the obtained LAMP2C knockout CHO-K1 host cell line in the same process as the experiment in Example 2.1. The ratio of TagRFP657 expression vector to empty vector was set at 50%. At 12, 24, and 48 hours after transfection, cells were analyzed for mean fluorescence intensity (MFI), transfection efficiency (%), and total fluorescence intensity (TFI) using flow cytometry. The lipofection results are shown in Figure 5c and the electroporation results are shown in Figure 5d.

As shown in Figures 5c and 5d, unlike the HEK293E host cell line in which the LAMP2C gene was knocked out, the LAMP2C knockout CHO-K1 clone did not show a positive effect in terms of MFI and transfected population ratio. In addition, based on the results showing that the effects were similar in experiments conducted identically other than the difference in transfection method (lipofection vs. electroporation), it can be concluded that the effect of LAMP2C knockout is cell type dependent.

2.4. LAMP2C In knockout host cell lines Serum-free suspension culture based TGE Confirmation of expression pattern of protein expression gene for basic treatment

To investigate the effect of LAMP2C knockout human host cell lines on the expression patterns of TGE-based therapeutic protein expression genes, three LAMP2C knockout HEK293E host cell lines (clones #2, 5, and 14) were grown in serum-free suspension culture. First, serum-free suspension culture-adapted HEK293E wild-type and LAMP2C knockout host cell lines were washed twice with anti-aggregant-free FreeStyleTM 293 expression medium, and the washed cells were seeded in anti-aggregant-free FreeStyleTM 293 expression medium at 1.0 x 10 ⁶ cells/mL. It was inoculated at high density. Afterwards, the cells were transfected with expression vectors containing green fluorescent protein (EGFP) and antibody expression cassettes using FreeStyle™ MAX Reagent (Gibco). The EGFP expression cassette was included in the transfected population and the antibody production vector to assess the expression levels of intracellular and secreted proteins (antibodies). Batch culture was performed with shaking at 120 rpm at the same temperature and humidity in a 125 mL Erlenmeyer flask for 7 days after transfection. In addition, antibody productivity per unit cell (q _mAb ) calculated based on the antibody concentration measured through ELISA and the cumulative cell concentration based on EGFP+ cells was measured. The results are shown in Figures 6 and 7.

As shown in Figures 6 and 7, consistent with the TGE results using serum-containing adherent cultures ( Figure 3 ), the MFI of the LAMP2C clone was higher than that of the wild type 48 hours after transfection. Similar to the MFI results, antibody concentration and antibody productivity per unit cell (q _mAb ) were found to be significantly higher in the LAMP2C knockout host cell line compared to the wild-type host cell line. In particular, the q _mAb (qP) of the L2 clone was 2.4 pg/cell/day (pcd), a 2.82-fold increase compared to the wild type (0.85 pcd). These results confirmed that LAMP2C knockout contributes to increasing the productivity of the target protein regardless of the type and type of foreign gene expressed.

Example 3. LAMP2C gene knocked out human cell lines SGE Verification of gene expression level for basic purpose

To evaluate changes in the expression level of the target gene in the HEK293E host cell line in which the LAMP2C gene was knocked out, the target gene (fluorescent protein) was expressed based on SGE, and the resulting target protein production was compared with the wild-type HEK293E host cell line. HEK293E wild type and LAMP2C knockout host cell lines (clones #2, 5, and 14) used in the serum-free suspension culture in Example 2 were transfected with an expression vector containing the EGFP-2A-PuroR gene expression cassette using FreeStyle™ MAX Reagent. After infection, a selection process was performed for about 3 weeks using FreeStyleTM 293 expression medium containing puromycin at a concentration of 3μg/mL, and then a stable cell pool was created. The stable expression level of the target gene in the prepared cell pool was analyzed and compared using the EGFP fluorescent protein SGE mean fluorescence intensity (MFI). The results are shown in Figure 8.

As shown in Figure 8, the MFI of the three LAMP2C knockout clones (#2, 5, and 14) increased approximately 2-3 times that of the wild type.

In addition, the mRNA expression rate compared to the EGFP gene copy number in the corresponding cell pool was analyzed through qRT-PCR. The results are shown in Figure 9.

As shown in Figure 9, compared to the EGFP cell pool based on the HEK293E wild-type host cell line, an approximately 4-4.5-fold difference in EGFP mRNA/gDNA expression rate was measured in the EGFP cell pool based on the LAMP2C knockout host cell line.

The above results indicate that LAMP2C knockout increases not only TGE but also SGE-based foreign gene expression. This can be inferred to be due to increased stability of mRNA.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims (14)

Transformed HEK293 (Human embryonic kidney 293) cell line in which the LAMP2C gene has been knocked out.
delete According to paragraph 1,
The cell line is a cell line that increases the expression of foreign genes based on TGE (transient gene expression) or SGE (stable gene expression).
According to paragraph 1,
The cell line is a cell line in which a mutation has occurred in the base sequence of the cytoplasmic tail region of the LAMP2C gene.
According to paragraph 1,
The cell line is a cell line in which the nucleotide sequence shown in SEQ ID NO: 7 has been deleted from the LAMP2C gene.
According to paragraph 1,
A cell line in which the SIDT2 gene has not been knocked out.
A composition for producing a target protein comprising a transformed human cell line in which the LAMP2C gene has been knocked out.
Knocking out the LAMP2C gene; An in vitro production method of a transformed HEK293 (Human embryonic kidney 293) cell line in which the LAMP2C gene has been knocked out.
According to clause 8,
The knockout is performed by introducing a vector containing sgRNA targeting the base sequence of the cytoplasmic tail region of the LAMP2C gene and a base sequence encoding the Cas9 protein.
According to clause 9,
A production method wherein the sgRNA targets the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
(a) introducing a vector containing a base sequence encoding the target protein into a transformed human cell line in which the LAMP2C gene of claim 1 has been knocked out; A method for producing a target protein comprising.
According to clause 11,
Production method further comprising the following steps:
(b) culturing the cell line of step (a); and
(c) separating and purifying the target protein produced in step (b).
According to clause 11,
The production method wherein the target protein is selected from the group consisting of fluorescent proteins, antibody expression proteins, antibody-derived fusion proteins, immune proteins, and protein-based hormones.
Introducing a vector containing a foreign gene into a transformed human cell line in which the LAMP2C gene of claim 1 has been knocked out; A method for increasing transfection efficiency comprising.