KR102560588B1 - HLA class I and MICA/MICB null cell line constructed from multiplex CRISPR-Cas9 system and use thereof - Google Patents

Abstract

The present invention relates to an HLA class I and MICA/MICB gene deficient cell line using a multiplex CRISPR-Cas9 system and a use thereof, and more specifically, the present invention relates to HLA class I and MICA/MICB completely removed H1ME- 5 cell line is provided, and the H1ME-5 cell line does not induce an immune response and thus can be used to prepare an immunomodulatory formulation with lower immunogenicity.

Description

Multiplex CRISPR-Cas9 system using multiplex CRISPR-Cas9 system and ＨＬＡ class I and ＭＩＣＡ／ＭＩＣＢ gene deficient cell line and use thereof

The present invention relates to HLA class I and MICA/MICB gene deficient cell lines using a multiplex CRISPR-Cas9 system and uses thereof.

Human leukocyte antigen (HLA) is the major histocompatibility complex in humans and is the most important immunological molecule. The two main sets of these are HLA class I (HLA-A, -B, -C), which present antigen to CD8+ T cells, and HLA class II (HLA-DR, -DP, which present antigen to CD4+ T cells). ,-DQ). A high level of polymorphism has been revealed in HLA molecules, which can present unique peptides or induce alloreactive immune responses. In addition, various subtypes exist due to the polymorphism described above, and at least 3 to 6 types of HLA class I molecules may exist in one cell. In addition, mismatch of HLA alleles can cause transplant rejection during organ transplantation or graft-versus-host disease during hematopoietic stem cell transplantation.

In order to overcome many limitations of transplantation and cell therapy, many studies have been conducted to modulate HLA expression. Modulation of HLA has been studied using a leukemia cell line and a lymphoblastoid cell line (LCL) mutated with γ-rays and hybridized with somatic cells. These HLA-deficient cell lines have been used as base cells for artificial antigen-presenting cells for reliable research on the structure and function of specific HLA molecules and efficient adaptive cell therapy. In particular, artificial antigen presenting cells are found in Drosophila, It has been developed by delivering genes for HLA and co-stimulatory molecules to mouse and human leukemia cell lines.

As genetic control techniques have developed, many studies on HLA regulation have been reported. Short hairpin RNA (shRNA) was used to lower HLA expression in various cells such as Jurkat, T cells, HeLa, B-LCL, 293T cells, hematopoietic stem cells, human embryonic stem cell lines, and human cord blood-derived endothelial cells. These reports demonstrated HLA silencing by targeting specific HLA alleles or by targeting beta-2-microglobulin, a heterodimer of the HLA class I molecule. And they each suggested the use of allograft rejection bypass, cell therapy, and platelet transfusion management. The loxP/Cre system was also used for B2M removal for HLA-negative embryonic stem cell lines.

Recently, HLA manipulation using gene editing tools such as zinc finger nucleases (ZFNs) and the CRISPR/Cas9 system has been reported. In particular, the CRISPR/Cas9 system is a gene editing technology suitable for multiple gene editing. Moreover, the CRISPR/Cas9 system can induce large deletions using multiple guide RNAs (gRNAs). ZFN was used to delete the HLA-A gene. And the CRISPR/Cas9 system was used to delete the B2M gene and the class II transactivator (CllTA) to regulate HLA class II expression.

The present invention induces a large deletion between exon 2 and exon 3 of HLA class I genes using a multiplex CRISPR/Cas9 system in HEK 293T cells to prepare an HLA class I deficient-293T cell line in which the HLA class I gene is completely removed. and completed the present invention by using them for preparing formulations for immunosuppression or immunotherapy.

1. Cong, L., et al., Science, 2013. 541 339(6121): p. 819-23. 2. Ran, F.A., et al., Nature Protocols, 2013. 8(11): p. 2281-2308. 3. Hong, C.H., et al., J Immunother, 2017. 40(6): p. 201-210.

An object of the present invention is to provide an HLA class I and MICA/MICB deficient 293T cell line in which HLA class I and MICA/MICB genes are completely removed.

Another object of the present invention is to provide a pharmaceutical use of the HLA class I and MICA/MICB deficient 293T cell line.

In order to achieve the above object, the present invention provides an HLA class I and MICA/MICB gene deficient Null-293T (H1ME-5) cell line (Accession Number: KCTC 13602BP).

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); And a composition for detecting a MICA/MICB-specific antibody in serum comprising an expression vector into which the nucleic acid sequence of the MICA/MICB gene has been inserted.

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); and an expression vector into which a nucleic acid sequence of an immunosuppression-related protein is inserted.

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); an expression vector into which a nucleic acid sequence of a co-stimulatory molecule is inserted; and an expression vector into which a nucleic acid sequence of an HLA class I gene is inserted.

The present invention provides an H1ME-5 cell line in which both HLA class I and MICA/MICB are completely removed, and since the H1ME-5 cell line does not induce an immune response, it can be used to prepare an immunomodulatory formulation with lower immunogenicity.

Figure 1 depicts a process for producing and selecting distinct MIC-deficient cells using multiplex removal of individual MIC types using a multiplex CRISPR/Cas9 system.
Figure 2 shows the expression and negative cell distribution of MIC molecules after multiplex CRISPR-Cas9 system transfection.
Figure 3 shows the results of analysis of genotype change patterns in HLA-negative single cell clones.
Figure 4 shows the sequencing results (upper figure) and expression level measurement results (lower figure) of a monoclonal cell line in which exons 2 to 3 of the MICA/MICB gene in the genome were completely removed.
Figure 5 shows the results of measuring expression levels in HLA_MIC deficient single cells and single MICA expressing cells.
6 shows the results of MICA-specific antibody detection in serum.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to an HLA class I and MICA/MICB gene deficient Null-293T (H1ME-5) cell line (Accession Number: KCTC 13602BP).

The H1ME-5 cell line of the present invention was developed using the multiplex CRISPR-Cas9 system to detect exons 2 and 3 of HLA-A*02:01, exons 2 and 3 of HLA-B*07:02, and HLA-C*07:02 It is characterized in that it is a HEK 293T cell line in which these genes are knocked out by targeting exons 2 and 3 of MICA, exons 2 and 3 of MICA, and exons 2 and 3 of MICB. Conventional HEK 293T cells express HLA class I, so it is difficult to determine which HLA-specific response is present even when there is a response to an antigen. However, the H1ME-5 cell line of the present invention is deficient in HLA class I and MICA/MICB genes, and thus has no response to antigen. In addition, the H1ME-5 cell line of the present invention can be established as a cell line expressing a single MICA/MICB through transfection of a single MICA/MICB gene, and a cell line expressing a single MICA/MICB is antigen specific for a specific MICA/MICB. It is characterized in that the antibody response can be measured.

The H1ME-5 cell line of the present invention has subtypes of HLA-A*02:01/*02:01, HLA-B*07:02/*07:02 and HLA-C*07:02/*07:02 It can be prepared from HEK 293T cells.

The H1ME-5 cell line is a cell line in which HLAA-A, HLA-B, HLA-C and MICA/B genes have been deleted on the genome using the CRISPR-Cas9 system, and may be a HEK 293T cell-based cell line. More specifically, the H1ME-5 cell line is deleted between exons 2 and 3 of each of HLAA-A, HLA-B, HLA-C and MICA/B using the CRISPR-Cas9 system, 293T cell lines deficient in HLA-C and MICA/B genes were completely removed from the genome. The RNA-guided CRISPR (clustered regularly interspaced short palindrome repeats)-associated nuclease Cas9 is a target knock-out, transcriptional activation and single guide RNA (sgRNA) (i.e., crRNA-tracrRNA fusion transcript) provides a breakthrough technology for suppression using , which targets numerous genetic loci.

The CRISPR-Cas9 system can be applied to various HLA subtypes and can simultaneously delete several genes, compared to gene control and editing technologies using shRNA to regulate mRNA levels and ZFN to edit genomic DNA to suppress HLA expression. Artificial expression of HLA molecules has the advantage of being possible.

sgRNAs containing target sequences in exons 2 and 3 of the HLAA-A, HLA-B, HLA-C and MICA/B genes for establishing the HLAA-A, HLA-B, HLA-C and MICA/B deficient 293T cell lines A plasmid expressing; and transfecting 293T cells with a plasmid expressing the Cas9 protein, recognizing the target gene by sgRNA containing the target sequence, forming a complex with the Cas9 protein, and cleavage of the target DNA by the Cas9 protein HLAA-A , between exons 2 and 3 of HLA-B, HLA-C and MICA/B can be deleted.

According to one embodiment of the present invention, in order to induce a large deletion between exon 2 and exon 3 of each HLA class I allele, six gRNA-Cas9 plasmids were simultaneously or sequentially transfected into wild-type 293T cells, and the transfection After 6 days, HLAA-A, HLA-B and HLA-C negative cells are selected using Moflo FACS salter and HLA class I monoclonal antibody, and seeded into single cells in a 96-well plate. Divided into single cells and after 2 to 3 weeks, single cell clones are established and cultured. To analyze the genotype, PCR and electrophoresis are performed using each specific primer set. Then, Sanger sequencing is performed on selected clones to analyze the genome sequence. HLA class I deficient cell lines can be established by confirming HLA class I expression using flow cytometry analysis and HLA class I monoclonal antibodies.

Next, three gRNA-Cas9 plasmids were simultaneously or sequentially transfected to induce a large deletion between exons 2 and 3 of the MICA and MICB genes into the HLA class I deficient cell line, and 6 days after transfection, Moflo FACS MICA/MICB-negative cells are selected using Salter and MICA/MICB monoclonal antibodies, and seeded into single cells in a 96-well plate. Divided into single cells and after 2 to 3 weeks, single cell clones are established and cultured. To analyze the genotype, PCR and electrophoresis are performed using each specific primer set. Then, Sanger sequencing is performed on selected clones to analyze the genome sequence. HLA class I and MICA/MICB deficient cell lines can be established by confirming MICA/MICB expression using flow cytometry analysis and a MICA/MICB monoclonal antibody.

According to one embodiment of the present invention, the sgRNA sequences targeting exons 2 and 3 of HLA class I and MICA/MICB are as follows:

HLA-A exon 2 (SEQ ID NO: 1) and exon 3 (SEQ ID NO: 2);

HLA-B exon 2 (SEQ ID NO: 3) and exon 3 (SEQ ID NO: 4);

HLA-C exon 2 (SEQ ID NO: 5) and exon 3 (SEQ ID NO: 6);

exon 2 of MICA (SEQ ID NO: 7);

exon 2 of MICB (SEQ ID NO: 8); and

Exon 3 of MICA/B (SEQ ID NO: 9)

As used herein, the term "cleavage" refers to breakage of the covalent backbone of a nucleotide molecule.

The guide RNA (sgRNA) contains target sequences of HLA class I and MICA/MICB genes and is expressed through transcription after delivery into cells to recognize HLA class I and MICA/MICB genes and form a complex with Cas9 protein. RNA that brings the Cas9 protein into the HLA class I and MICA/MICB genes. Accordingly, the guide RNA may include target sequences of HLA class I and MICA/MICB genes; and a guide RNA scaffold consisting of a non-variable sequence independent of the target.

The target sequences of the HLA class I and MICA/MICB genes refer to sequences that allow guide RNA to recognize target genes, and appropriate modifications may be applied depending on the type of host cell, type of target gene, or insertion site.

The guide RNA scaffold may be composed of two RNAs, namely CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single-stranded RNA generated by fusion of essential parts of crRNA and tracrRNA. (single-chain guide RNA, sgRNA). The guide RNA may be a dual RNA including crRNA and tracrRNA. Any guide RNA scaffold can be used in the present invention, provided that the guide RNA scaffold includes essential parts of crRNA and tracrRNA and a part complementary to the target. The crRNA may hybridize with target DNA.

Preferably, it may be a single-chain guide RNA.

The terminator is linked to the end of the DNA encoding the guide RNA to terminate transcription of the DNA encoding the guide RNA, and can be selected and used at an appropriate level of selection by those skilled in the art according to the promoter, and is not particularly limited. For example, RNA Polymerase III terminator or -TTTTTT- sequence.

The Cas9 protein refers to an essential protein element in the CRISPR/Cas9 system, and when complexed with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), an active endonuclease form Information on the Cas9 gene and protein can be obtained from GenBank at the national center for biotechnology information (NCBI), but is not limited thereto.

When the Cas9 protein is delivered into cells, it can be linked to a protein transduction domain. The protein transduction domain may be a poly-arginine domain or a TAT protein derived from HIV, but is not limited thereto.

The Cas9 protein may be delivered through transfection in the form of a vector expressing the Cas9 protein, that is, a vector containing a Cas9 protein-encoding nucleic acid. The Cas9 protein-encoding nucleic acid may be in the form of a vector such as a plasmid containing the Cas9 coding sequence under a promoter such as CMV or CAG.

Delivery of the plasmid expressing the sgRNA and the plasmid expressing the Cas9 protein may be performed by microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle- It may be delivered into cells by various methods known in the art, such as, but not limited to, mediated transfection, protein transduction domain-mediated introduction, virus-mediated gene transfer, and PEG-mediated transfection in protozoa.

As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. One type of vector is a “plasmid,” which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in a host cell when introduced into the host cell (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the host cell's genome when introduced into the host cell and replicate together with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are usually in the form of plasmids, and since the plasmid is the most commonly used type of vector, "plasmid" and "vector" may be used interchangeably. However, the present invention provides other forms such as viral vectors (e.g., adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retroviral vectors, lentiviral vectors, baculovirus vectors) that provide equivalent functionality. Expression vectors of are also included. Preferably, a lentiviral vector may be used. Transformation includes any method of introducing nucleic acids into organisms, cells, tissues, or organs, and can be performed by selecting the above-mentioned standard techniques suitable for the host cell as known in the art.

The HLA class I and MICA/MICB gene deficient H1ME-5 cell line of the present invention is a human cell line in which both HLA class I and MICA/MICB have been clearly removed, and was patented at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on July 26, 2018. (Null-293T (H1ME-5), accession number: KCTC 13602BP).

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); And a composition for detecting a MICA/MICB-specific antibody in serum comprising an expression vector into which the nucleic acid sequence of the MICA/MICB gene has been inserted.

The HLA class I and MICA/MICB gene deficient H1ME-5 cell line of the present invention can be transfected with a single MICA/MICB gene to establish a plurality of cell lines expressing a single MICA/MICB, and can react with the patient's serum before transplantation to determine specific The presence or absence of antibodies specific to MICA/MICB can be investigated.

Therefore, the composition of the present invention may be included in the form of an H1ME-5 cell line transfected with a single MICA/MICB gene or an exosome derived from the cell line.

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); and a composition for preparing an immunosuppressive formulation comprising an expression vector into which a nucleic acid sequence of an immunosuppression-related protein is inserted.

The HLA class I and MICA/MICB gene deficient H1ME-5 cell lines of the present invention are transfected with genes encoding immunosuppression-related proteins to minimize immunogenicity through H1ME-5 cell lines expressing immunosuppression-related proteins. By making some, it can be used for immunosuppression.

Accordingly, the immunosuppressive formulation of the composition of the present invention may be an H1ME-5 cell line transfected with a gene encoding an immunosuppression-related protein or an exosome derived from the cell line.

The immunosuppression-related protein may be one or more selected from the group consisting of galectin-3, matrix metalloproteinase (MMP-9), heat shock protein 72 (HSP72), and prolactin-inducible protein (PIP).

The immunosuppressive formulation is suitable for use in graft-versus-host disease, autoimmune disease, hyperproliferative skin disease, chronic obstructive pulmonary disease (COPD), allergic asthma asthma, bronchitis, allergic rhinitis and autoimmune hepatitis. It may be for improvement, prevention or treatment of an immune disease selected from (autoimmune hepatitis).

The present invention is also a Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (Accession Number: KCTC 13602BP); an expression vector into which a nucleic acid sequence of a co-stimulatory molecule is inserted; and an expression vector into which a nucleic acid sequence of an HLA class I gene is inserted.

The HLA class I and MICA/MICB gene deficient H1ME-5 cell lines of the present invention are transfected with co-stimulatory molecules and HLA class I genes to stimulate T cells through the H1ME-5 cell line having antigen presenting ability or exosomes derived from the cell line It can be used for immunotherapy by inducing a CTL response by

Accordingly, the immunotherapeutic formulation of the composition of the present invention may be an H1ME-5 cell line transfected with a co-stimulatory molecule and an HLA class I gene having antigen presenting ability or exosomes derived from the cell line.

The co-stimulatory molecule may be one or more selected from the group consisting of CD80, CD83, CD54, CD32, 4-1BBL and CD70.

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.

<Example 1> Establishment of HLA class I and MICA/MICB gene Null-293T (H1ME-5) cell line

To establish HLA class I and MICA/MICB gene Null-293T (H1ME-5) cell lines, a cell line from which HLA class I molecules were completely removed was established, and MICA/MICB molecules were removed from the HLA class I null-293T cells. .

HEK (human embryonic kidney) 293T cells were used to establish the Null-293T (H1E-25) cell line. HEK 293T cells (CRL-3216; ATCC, Manassas, VA) were cultured in DMEM medium (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin. T2 cells were cultured in RPMI-1640 medium (Lonza) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.

For complete removal of HLA class I molecules, six plasmids encoding Cas9 and gRNA targeting exons 2 and 3 of each HLA-A, -B, and -C gene were used (Fig. 1):

1) gRNA (target: HLA-A*02:01 exon2 (AE2: 5'-GAGCCAGAGGATGGAGCCGCGGG-3', SEQ ID NO: 1), exon3 (AE3: 5'-GAAGGAGACGCTGCAGCGCACGG-3', SEQ ID NO: 2) , HLA-B*07:02 exon2 (BE2: 5'-GCTGTCGAACCTCACGAACTGGG-3', SEQ ID NO: 3), exon3 (BE3: 5'-GAGCATGTACGGCTGCGACGTGG-3', SEQ ID NO: 4), HLA-C* 07:02 exon2 (CE2: 5'-GACACAGAAGTACAAGCGCCAGG-3', SEQ ID NO: 5), exon3 (CE3: 5'-CCAGAGGATGTCTGGCTGCGACC-3', SEQ ID NO: 6))

2) Cas9 protein co-expression all-in-one vector (Genscript, PX458, PX459).

HEK 293T cells were plated at 2×10 ⁶ /10 mL using DMEM (Lonza) without antibiotics. After 24 hours, the 293T cell line was transfected with 6 all-in-one plasmids (3.3 μg each) specific for each of the 6 targets. 48 hours after transfection, cells were subjected to flow cytometry. Six days after transfection, HLA-A, HLA-B and HLA-C negative cells were isolated and clones were established. At 2-3 weeks after sorting, HLA-A, HLA-B and HLA-C negative single cell clones were established and cultured in 6-well plates. Clone genomic DNA was isolated from each clone of 1×10 ⁵ -5×10 ⁶ cells using the TIANamp Genomic DNA Kit (Tiangen, China) according to the manufacturer's instructions. Amplification of each target region was performed using primer sets specific for HLA-A, HLA-B and HLA-C exons 2 and 3 (HLA-A forward primer: 5'-GAA ACS GCC TCT GYG GGG AGA AGC AA-3'; HLA -A reverse primer: 5'-TGT TGG TCC CAA TTG TCT CCC CTC-3'; HLA-B forward primer: 5'-GGG AGG AGC GAG GGG ACC SCA G-3'; HLA-B reverse 50-GGA GGC CAT CCC CGG CGA CCT AT-30; HLA-C forward primer: 5'-CTC MTC CTG CTG CTC TCG GGA-3'; HLA-C reverse primer: 5'-TGG GAG GCC ATS CCG GGA GAT-3') It was performed via PCR. PCR products of the selected clones were analyzed by Sanger sequencing (Cosmo Genetech, Korea). Then, HLA-A, -B, -C null 293T clones (Null-293T (H1E-25)) were selected.

Among the HLA-A, -B, and -C null 293T clones selected above, the Null-293T (H1E-25) cell line (accession number: KCTC 13601BP) was used to establish a MICA/MICB deficient cell line. The H1E-25 cell line was cultured in DMEM medium (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin. T2 cells were cultured in RPMI-1640 medium (Lonza) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin-streptomycin.

For complete removal of MICA/MICB, a CRISPR/Cas9 vector targeting exons 2 and 3 of the MICA and MICB genes was constructed:

1) gRNA (Target: MICA exon2 (MICA_2: 5'-GGCAAAGCCCCAGGGACAGT-3', SEQ ID NO: 7), MICB exon2 (MICB_2: 5'-GCTATGACAGGCAGAAACGC-3', SEQ ID NO: 8), MICAB exon3 (MICAB_3 : 5'-GTCCTCCAGAGCTCAGACCT-3', SEQ ID NO: 9)

2) Cas9 protein co-expression all-in-one vector (Genscript, PX458)

H1E-25 cells were plated at 2×10 ⁶ /10 mL using antibiotic-free DMEM (Lonza). After 24 hours, three all-in-one plasmids (MICA exon2 target vector 5μg, MICB exon2 target vector 5μg, MICAB exon3 target vector 10μg) specific to each of the three targets were transfected using Lipofectamine (Invitrogen). The H1E-25 cell line was transfected. 48 hours after transfection, cells were subjected to flow cytometry. Six days after transfection, MICA/B negative cells were isolated and clones were established. At 2-3 weeks after sorting, MICA/B negative single cell clones were established and cultured in 6-well plates. Clone genomic DNA was isolated from each clone of 1×10 ⁵ -5×10 ⁶ cells using the TIANamp Genomic DNA Kit (Tiangen, China) according to the manufacturer's instructions. Amplification of each target region was performed using a primer set specific for MICA, MICB exons 2 and 3 (MICA forward: 5'-CGTTCTTGTCCCTTTGCCCGTGTGC-3', MICA reverse: 5'-GAATTGGAGGGAGAGGAGAGC-3', MICB forward: 5'-AGCCCCACAGTCTTCGTTAC-3 ', MICB reverse: 5'-CCAGGGTCGGTACCTGTTCT-3'). PCR products of the selected clones were analyzed by Sanger sequencing (Cosmo Genetech, Korea). Thereafter, HLA-A, -B, -C, MICA, and MICB null 293T clones (Null-293T (H1ME-5)) were selected.

<Example 2> Expression of MIC molecules and establishment of negative cell clones

For complete removal of MICA/MICB molecules using HLA-A, -B, -C deficient H1E-25 cells, six gRNAs targeting exons 2 and 3 of each MICA/MICB gene and Cas9 encoding were used. A plasmid was used (FIG. 1). These three plasmids were co-transfected into H1E-25 cells. Six days after transfection, MICA/MICB-specific antibody was stained, and cells expressing the lowest expression were isolated and plated as single cells in a 6-well plate or eight 96-well plates. On day 16 of the cells cultured in the 6-well plate, the MICA/MICB-negative cell population was 21.0% (FIG. 2). After 2-3 weeks after sorting, 48 clones were cultured in a total of 768 wells and MICA/MICB gene mutation and expression were analyzed.

<Example 3> Genetic analysis and flow cytometry analysis of MICA/MICB-negative cell clones

To detect the induced mutations, PCR was performed using specific primer pairs including gRNA target sites in exons 2 and 3 of each MICA/MICB gene (FIG. 3). Shorter (deletion), longer (insertion) or non-amplified products were termed detectable changes (DCP) when the size of the PCR product changed compared to the size of the control cells. When a PCR product was detected as a single band, it was considered homozygous or heterozygous when it was detected as a double band (Fig. 3). The non-amplified product is a homozygous deletion, presumably a larger deletion affecting the PCR. PCR products were considered to contain no mutations, substitutions or small insertions or deletions when they were identical or similar in size. These results demonstrated that mutations in MICA/MICB occur frequently in the order. The mutation frequency of each MICA/MICB locus can indicate the efficiency of gRNA targeting to each MICA/MICB locus.

By screening 48 cultured clones, only 7 homozygous clones (HLA class I null clones, 3, 5, 7, 15, 23, 25, 26, 42) with null deletion of both MICA/MICB were finally obtained ( Fig. 3).

In addition, for the selection of MICA/MICB-deficient cell lines, the nucleotide sequences of three homozygous clones (5, 15, and 26) with predicted deletions in the MICA/MICB gene were identified (FIG. 4). All three clones showed a large deletion between the regions close to the expected two cleavage sites at each targeted MICA/MICB gene location.

<Example 4> Measurement of expression level in HLA_MIC deficient single cells and single MICA expressing cells

MICA*002, MICA*004, MICA*007, MICA*007, MICA*008, MICA*010, MICA*012, MICA*027, MICA*049 transfection into HLA_MIC deficient single cell line (H1ME-5) on day 6 MICA-positive cells were sorted.

As shown in Figure 5, MICA expression was not confirmed in the H1ME-5 cell line.

<Example 5> Detection of MICA-specific antibodies in serum using single MICA-expressing cells

Reaction of 2Х10 ⁵ /20 μl of single MICA-expressing cells (MICA-H1ME-5) and 5 μl of serum (MICA/HLA(+/-): MP, MICA/HLA(+/+): P) for 30 minutes at room temperature and washed twice with 200 μl of PBS. 2 μl of polyclonal anti-human IgG FITC antibody and 50 μl of PBS were added and reacted at room temperature for 30 minutes. After washing three times with 200 μl of PBS and fixing by adding 300 μl of fixative solution, flow cytometry analysis was performed.

As shown in FIG. 6, different MIC-specific antibodies were detected according to each patient, and an MFI of 200 or higher was determined to be positive.

Therefore, a patent was deposited on July 26, 2018 for the human H1ME-5 clone in which both the HLA class I and MICA/MICB were definitely removed (accession number: KCTC 13602BP).

Korea Research Institute of Bioscience and Biotechnology KCTC13602BP 20180726

<110> CATHOLIC UNIVERSITY INDUSTRY ACADEMIC COOPERATION FOUNDATION <120> HLA class I null cell line constructed from multiplex CRISPR-Cas9 system and its use <130> P19U11C0490 <160> 6 <170> KopatentIn 2.0 <210> 1 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-A exon 2 target sequence <400> 1 gagccagagg atggagccgc ggg 23 <210> 2 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-A exon 3 target sequence <400> 2 gaaggagacg ctgcagcgca cgg 23 <210> 3 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-B exon 2 target sequence <400> 3 gctgtcgaac ctcacgaact ggg 23 <210> 4 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-B exon 3 target sequence <400> 4 gagcatgtac ggctgcgacg tgg 23 <210> 5 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-C exon 2 target sequence <400> 5 gacacagaag tacaagcgcc agg 23 <210> 6 <211> 23 <212> DNA <213> artificial sequence <220> <223> HLA-C exon 3 target sequence <400> 6 ccagaggatg tctggctgcg acc 23

Claims (11)

HLA class I and MICA/MICB gene deficient Null-293T (H1ME-5) cell line (accession number: KCTC 13602BP).
According to claim 1,
Null-293T (H1ME-5) cell line has exons 2 and 3 of HLA-A*02:01, exons 2 and 3 of HLA-B*07:02, exons 2 and 3 of HLA-C*07:02, MICA HEK 293T in which HLA-A, -B and -C genes were deleted from the genome by the CRISPR-Cas9 system using the following single guide RNA (sgRNA) targeting exons 2 and 3 of MICB and exons 2 and 3 of MICB Cell line, Null-293T (H1ME-5) cell line:
HLA-A exon 2 (SEQ ID NO: 1) and exon 3 (SEQ ID NO: 2);
HLA-B exon 2 (SEQ ID NO: 3) and exon 3 (SEQ ID NO: 4);
HLA-C exon 2 (SEQ ID NO: 5) and exon 3 (SEQ ID NO: 6);
exon 2 of MICA (SEQ ID NO: 7);
exon 2 of MICB (SEQ ID NO: 8); and
Exon 3 of MICA/B (SEQ ID NO: 9).
Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (accession number: KCTC 13602BP); and
A composition for detecting a MICA/MICB-specific antibody in serum comprising an expression vector into which a nucleic acid sequence of a MICA/MICB gene is inserted.
According to claim 3,
The composition comprises a Null-293T (H1ME-5) cell line transfected with a single MICA/MICB gene or in the form of an exosome derived from the cell line, a composition for detecting MICA/MICB-specific antibodies in serum.
Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (accession number: KCTC 13602BP); and
A composition for preparing an immunosuppressive formulation comprising an expression vector into which a nucleic acid sequence of an immunosuppression-related protein is inserted.
According to claim 5,
The immunosuppressive formulation is a composition for preparing an immunosuppressive formulation comprising a Null-293T (H1ME-5) cell line transfected with a gene encoding an immunosuppression-related protein or an exosome derived from the cell line.
According to claim 5,
The immunosuppressive formulation, wherein the immunosuppressive-related protein is at least one selected from the group consisting of galectin-3, matrix metalloproteinase (MMP-9), heat shock protein 72 (HSP72), and prolactin-inducible protein (PIP). composition for manufacture.
According to claim 5,
Immunosuppressive formulations are used to treat graft-versus-host disease, autoimmune disease, hyperproliferative skin disease, chronic obstructive pulmonary disease (COPD), allergic asthma asthma, bronchitis, allergic rhinitis and autoimmune hepatitis ( A composition for preparing an immunosuppressive formulation for improvement, prevention or treatment of an immune disease selected from autoimmune hepatitis).
Null-293T (H1ME-5) cell line deficient in HLA class I and MICA/MICB genes (accession number: KCTC 13602BP);
an expression vector into which a nucleic acid sequence of a co-stimulatory molecule is inserted; and
A composition for preparing an immunotherapeutic formulation comprising an expression vector into which a nucleic acid sequence of an HLA class I gene is inserted.
According to claim 9,
The immunotherapeutic formulation comprises a Null-293T (H1ME-5) cell line having an antigen presenting ability transfected with a co-stimulatory molecule and an HLA class I gene or an exosome derived from the cell line, a composition for preparing an immunotherapeutic formulation.
According to claim 9,
The co-stimulatory molecule is at least one selected from the group consisting of CD80, CD83, CD54, CD32, 4-1BBL and CD70, a composition for preparing an immunotherapeutic formulation.