Highly active transposase protein for transposon systems and its uses

Abstract

The present invention relates to a highly active transposase protein for a transposon system and its uses. By improving the activity of the transposase, genes can be delivered effectively, which can be useful for developing genome-modified cell lines that express various genes. In addition, when T cells are transformed using the highly active transposase of the present invention, cytotoxic T cells (CD8) that have anti-cancer effects can be generated. + The proportion of T cells increases, and T cells persist in the body. CM (Central memory T cell), T SCM Since the proportion of memory-type T cells (stem cell-like memory T cells) increased, it is expected that TCR-T cells and CAR-T cells with good in vivo persistence can be produced using the transposon system of the present invention.

Description

[Technical Field]

[0001] The present invention relates to a highly active transposase protein of a transposon system and uses thereof.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2023-0014296, filed on February 2, 2023, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Background technology

[0004] CAR (Chimeric Antigen Receptor)-T cells are cell therapy agents in which antibody sequences that bind to tumor antigens (e.g., CD19) are linked to domains required for T cell signaling, such as CD3 / 4-1BB / CD28, and then inserted into T cells. There are various methods for inserting these CAR genes into T cells, but most use a lentivirus delivery system. A unique feature of lentiviruses is that they integrate into the cell's chromosomes, allowing for continuous gene expression. While the high production costs of lentiviruses are a major factor in increasing the price of therapeutic agents, they have the advantage that they can be used on a large number of patients once they are produced.

[0005] On the other hand, therapeutically customized TCR-T cells are produced by identifying TCR (T-cell receptor) sequences that react with each patient's neoantigens and delivering those sequences into T cells via a gene delivery system. However, because this is personalized, the TCR sequences applied to each patient are different, making it nearly impossible to apply this to lentiviruses. Therefore, it is necessary to develop TCR-T cells using transposons, which are non-viral vectors that are easier and less expensive to produce than lentiviruses and can be integrated into chromosomes for continuous gene expression.

[0006] Therefore, in order to meet the above-mentioned needs, the present inventors developed a transposon as a gene carrier capable of integrating an exogenous gene into the genome of a target cell, particularly an immune cell (Korean Patent Registration No. 10-2602485).

[0007] Furthermore, in order to improve the efficiency of gene delivery via the transposon system, the inventors conducted extensive research and confirmed that improving the activity of the transposase in the transposon system resulted in excellent gene delivery efficiency, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] Summary of the Invention

[0009] The present inventors have completed the present invention by confirming that the efficiency of gene delivery via a transposon system is improved when transposase activity is improved by optimizing the nucleic acid sequence of the transposase.

[0010] An object of the present invention is to provide a transposase expression vector comprising a nucleic acid sequence encoding the transposase set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0011] Another object of the present invention is to provide an mRNA encoding the transposase represented by SEQ ID NO:12.

[0012] It is yet another object of the present invention to provide a transposase expressed from the transposase expression vector of the present invention or the mRNA encoding the transposase of the present invention.

[0013] It is yet another object of the present invention to provide a transposon system for target DNA delivery, comprising: (a) a transposon vector into which target DNA has been inserted; and (b) a transposase expression vector containing a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, an mRNA encoding the transposase shown in SEQ ID NO: 12, or a transposase according to the present invention.

[0014] It is yet another object of the present invention to provide a transposon kit for targeted DNA delivery, which includes the transposon system for targeted DNA delivery according to the present invention and an instruction manual.

[0015] Yet another object of the present invention is to provide (a) a transposon vector into which a target DNA has been inserted; and (b) a transposase expression vector comprising a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 or SEQ ID NO: 3, mRNA encoding the transposase represented by SEQ ID NO: 12, or a cell into which the transposase of the present invention has been introduced.

[0016] It is yet another object of the present invention to provide a method for inserting a target DNA sequence into the genome of a cell, comprising the steps of: (a) a transposon vector into which a target DNA has been inserted; and (b) introducing into the cell a transposase expression vector containing a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, mRNA encoding the transposase shown in SEQ ID NO: 12, or a transposase according to the present invention.

[0017] (blank)

[0018] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0019] Means to solve the problem

[0020] To achieve the above object, the present invention provides a transposase expression vector comprising a nucleic acid sequence encoding the transposase represented by SEQ ID NO:2 or SEQ ID NO:3.

[0021] In one embodiment of the present invention, the nucleic acid sequence encoding the transposase may further comprise, but is not limited to, a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0022] In another embodiment of the present invention, the nucleic acid sequence encoding the nuclear localization signal may be, but is not limited to, one or more of the nucleic acid sequences represented by SEQ ID NOs: 4 to 11.

[0023] In yet another embodiment of the present invention, the nucleic acid sequence encoding the nuclear localization signal may be, but is not limited to, included in the 5' to 3' direction at the 5' or 3' end of the nucleic acid sequence encoding the transposase.

[0024] The present invention also provides an mRNA encoding the transposase represented by SEQ ID NO:12.

[0025] In one embodiment of the present invention, the mRNA can be prepared by in vitro transcription using the transposase expression vector of the present invention, but is not limited thereto.

[0026] In another embodiment of the present invention, the transposase expression vector may be, but is not limited to, a transposase expression vector comprising a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3.

[0027] The present invention also provides a transposase expressed from a transposase expression vector according to the present invention or an mRNA encoding a transposase according to the present invention.

[0028] In one embodiment of the present invention, when the transposase expression vector comprises a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3, the transposase can be expressed from mRNA prepared by in vitro transcription of the transposase expression vector, but is not limited thereto.

[0029] In another embodiment of the present invention, the transposase may comprise, but is not limited to, the amino acid sequence set forth in SEQ ID NO:13.

[0030] The present invention also provides a transposon system for target DNA delivery, comprising: (a) a transposon vector into which target DNA has been inserted; and (b) a transposase expression vector containing a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, mRNA encoding the transposase shown in SEQ ID NO: 12, or mRNA encoding the transposase of the present invention; or a transposase of the present invention.

[0031] In one embodiment of the present invention, the transposon vector and transposase expression vector, or the mRNA encoding the transposase, or the transposase may be contained in a mass ratio of 0.1 to 10:1, but is not limited thereto.

[0032] The present invention also provides a transposon kit for targeted DNA delivery, which includes the transposon system for targeted DNA delivery according to the present invention and an instruction manual.

[0033] The present invention also provides (a) a transposon vector into which a target DNA has been inserted; and (b) a transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, mRNA encoding the transposase shown in SEQ ID NO: 12, or mRNA encoding the transposase of the present invention; or a cell into which the transposase of the present invention has been introduced.

[0034] In one embodiment of the present invention, the target DNA may be excised from the transposon-based vector by the transposase in the cell, and the excised target DNA may be inserted into the genome of the cell, but is not limited thereto.

[0035] In other embodiments of the present invention, the cells may be selected from the group consisting of, but not limited to, T cells, NK cells, B cells, dendritic cells, macrophages, and mast cells.

[0036] In yet another embodiment of the present invention, the cells may be co-cultured with feeder cells after the transposon-containing vector is introduced, but is not limited to this.

[0037] In yet another embodiment of the present invention, the feeder cells may be, but are not limited to, irradiated cells.

[0038] In yet another embodiment of the present invention, the cells can express the target DNA for, but are not limited to, 7 days or more after introduction of the transposon-based vector.

[0039] The present invention also provides a method for inserting a target DNA sequence into the genome of a cell, comprising the steps of: (a) a transposon vector into which a target DNA has been inserted; and (b) a transposase expression vector containing a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, mRNA encoding the transposase shown in SEQ ID NO: 12, or mRNA encoding a transposase according to the present invention; or introducing a transposase according to the present invention into the cell.

[0040] In one embodiment of the present invention, the introduction can be performed by, but is not limited to, electroporation.

[0041] In another embodiment of the present invention, the method may further comprise, but is not limited to, a step of co-culturing the cells into which the transposon vector has been inserted with feeder cells after the introduction step.

[0042] In yet another embodiment of the present invention, the step of co-culturing with the feeder cells may be performed immediately after the step of introducing, but is not limited to this. [Effects of the Invention]

[0043] Effect of the invention

[0044] The present invention relates to a highly active transposase protein of a transposon system and its uses. By improving the activity of the transposase, genes can be delivered effectively, and the protein can be usefully used in the development of genome-modified cell lines that express various genes.

[0045] Furthermore, when T cells are transformed using the hyperactive transposase of the present invention, cytotoxic T cells (CD8 + The proportion of T cells increases, and T cells persist in the body. CM (Central memory T cell), T SCM Since the proportion of memory-type T cells (stem cell-like memory T cells) increased, it is expected that TCR-T cells and CAR-T cells with good in vivo persistence can be produced using the transposon system of the present invention.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS [Brief explanation of the drawings]

[0047] [Figure 1] Figures 1 to 3 show the results of confirming 1G4 TCR expression in T cells 7 days (Figure 1), 10 days (Figure 2), and 14 days (Figure 3) after electroporation in PBMCs in the group in which PBMCs were subjected to electroporation (EP) without any plasmid (EP only), the group in which pBat transposon containing the 1G4 TCR gene and the original transposase were introduced (conventional transposase), and the group in which pBat transposon containing the 1G4 TCR gene and the optimized transposase were introduced (optimized transposase). [Figure 2] Same as above. [Figure 3] Same as above.

[0048] [Figure 4] Figure 4 shows the results of comparing the percentage of 1G4 TCR-expressing T cells within T cells on days 7, 10, and 14 after electroporation in the group in which PBMCs were subjected to electroporation (EP) alone without any plasmid (EP only), the group in which pBat transposon containing the 1G4 TCR gene and the original transposase were introduced (conventional transposase), and the group in which pBat transposon containing the 1G4 TCR gene and the optimized transposase were introduced (optimized transposase).

[0049] [Figure 5] Figure 5 shows the results of examining CD4+ and CD8+ cells within 1G4 TCR-expressing T cells 7, 10, and 14 days after electroporation in PBMCs in a group in which PBMCs were subjected to electroporation (EP) without any plasmid (EP only), a group in which pBat transposon containing the 1G4 TCR gene and the original transposase were introduced (conventional transposase), and a group in which pBat transposon containing the 1G4 TCR gene and the optimized transposase were introduced (optimized transposase).

[0050] [Figure 6] Figure 6 shows the results of a comparison of the percentages of CD4+ cells and CD8+ cells within 1G4 TCR-expressing T cells on days 7, 10, and 14 after electroporation between a group in which PBMCs were transfected with the pBat transposon containing the 1G4 TCR gene and the original transposase (conventional transposase) and a group in which PBMCs were transfected with the pBat transposon containing the 1G4 TCR gene and the optimized transposase (optimized transposase).

[0051] [Figure 7] Figure 7 shows the results of confirming memory-type T cells using CD45RA and CCR7 markers in 1G4 TCR-expressing T cells 7, 10, and 14 days after electroporation in a group in which PBMCs were subjected to electroporation (EP) alone without a plasmid (EP only), a group in which pBat transposon containing the 1G4 TCR gene and the original transposase were introduced (conventional transposase), and a group in which pBat transposon containing the 1G4 TCR gene and the optimized transposase were introduced (optimized transposase).

[0052] [Figure 8a]Figures 8a to 10b show the results of electroporation of Jurkat cells with either the pBat transposon containing the GFP gene and the original transposase (P5081 + conventional transposase) or the pBat transposon containing the GFP gene and the optimized transposase (P5081 + optimized transposase). GFP expression levels were confirmed by FACS analysis on days 1 (Figures 8a and 8b), 7 (Figures 9a and 9b), and 14 (Figures 10a and 10b). [Figure 8b] Same as above. [Figure 9a] Same as above. [Figure 9b] Same as above. [Figure 10a] Same as above. [Figure 10b] Same as above.

[0053] [Figure 11] FIG. 11 shows the results of PCR amplification to obtain an optimized transposase gene in mRNA form.

[0054] [Figure 12] FIG. 12 shows the results of confirming the structure through enzyme mapping after infusion cloning between an mRNA template (Vector) and a transposase (Insert) to prepare an mRNA template plasmid.

[0055] [Figure 13] FIG. 13 shows the results of confirming the total number of 1G4 TCR-T cells produced by transposase gene delivery.

[0056] [Figure 14] FIG. 14 shows the results of examining the viability of 1G4 TCR-T cells produced by transposase gene delivery.

[0057] [Figure 15]FIG. 15 shows a FACS analysis method for comparing GFP gene expression according to the delivery form of the transposase gene after electroporation.

[0058] [Figure 16] FIG. 16 shows the results of FACS analysis confirming the expression of the GFP gene depending on the form of transposase after electroporation.

[0059] [Figure 17] FIG. 17 shows the FACS analysis method to compare 1G4 TCR gene expression according to the delivery form of the transposase gene after electroporation.

[0060] [Figure 18] FIG. 18 shows the results of confirming the expression of the 1G4 TCR gene by the form of transposase after electroporation.

[0061] [Figure 19] FIG. 19 shows memory T cells classified by expression of CD45RO / CD62L markers.

[0062] [Figure 20] FIG. 20 shows the results of confirming the percentage of memory T cells within T cells expressing 1G4 TCR after electroporation using transposase gene delivery.

[0063] [Figure 21] FIG. 21 shows the results of confirming the percentage of CD4 and CD8 in T cells expressing 1G4 TCR after electroporation using transposase gene delivery.

[0064] [Figure 22]Figures 22 and 23 show a portion of the sequence of the synthesized template required for cloning to add an NLS (nuclear localization sequence) gene to the 5' end (Figure 22) or 3' end (Figure 23) of the optimized transposase. [Figure 23] Same as above.

[0065] [Figure 24a] Figures 24a to 26 show the results of confirming the GFP gene delivery efficiency 1 day (Figures 24a and 24b), 7 days (Figures 25a and 25b), and 14 days (Figure 26) after transfection (electroporation) of an optimized transposase with an NLS (nuclear localization sequence) gene added to the 5' or 3' end. [Figure 24b] Same as above. [Figure 25a] Same as above. [Figure 25b] Same as above. [Figure 26] Same as above.

[0066] [Figure 27] Figure 27 is a graph showing the results of FACS analysis of GFP gene expression 1 day and 7 days after transfection (electroporation) of optimized transposase with an NLS (nuclear localization sequence) gene added to the 5' or 3' end.

[0067] [Figure 28] Figure 28 is a graph showing the results of FACS analysis of high-intensity GFP gene expression 1 and 7 days after transfection (electroporation) of optimized transposase with an NLS (nuclear localization sequence) gene added to the 5' or 3' end.

[0068] [Figure 29a]Figures 29a and 29b show the results of transfection (electroporation) of an optimized transposase with an NLS (nuclear localization sequence) gene added to the 5' or 3' end. Cells expressing GFP on day 14 were sorted on day 15 and further cultured for 9 days, after which the efficiency of GFP gene delivery was confirmed. [Figure 29b] Same as above.

[0069] [Figure 30a] Figures 30a to 31d show the results of confirming the 1G4 TCR gene delivery efficiency and the proportion of CD4 and CD8 within T cells expressing 1G4 TCR on the 7th (Figures 30a to 30d) and 14th (Figures 31a to 31d) days after transfection (electroporation) of an optimized transposase with an NLS (nuclear localization sequence) gene added to the 5' or 3' end. [Figure 30b] Same as above. [Figure 30c] Same as above. [Figure 30d] Same as above. [Figure 31a] Same as above. [Figure 31b] Same as above. [Figure 31c] Same as above. [Figure 31d] Same as above.

[0070] [Figure 32] Figure 32 shows the transposon and lentiviral CD19 CAR vector structures.

[0071] [Figure 33] FIG. 33 shows co-culture conditions for in vitro killing analysis.

[0072] [Figure 34] Figure 34 shows the results of enzyme mapping to confirm the structure of the pBat CD19 CAR transposon plasmid vector.

[0073] [Figure 35] Figures 35 and 36 show the total cell number (Figure 35) and cell viability (Figure 36) of cultured T cells after gene delivery to LK032 PBMCs and LK053 PBMCs, respectively, using lentivirus and transposon transduction and electroporation methods to produce CD19 CAR-T cells, followed by culture for 7 and 14 days. [Figure 36] Same as above.

[0074] [Figure 37] Figure 37 shows the FACS analysis method to compare the expression of CD19 CAR protein by gene transfer lentivirus and transposon.

[0075] [Figure 38] Figure 38 shows the results of confirming CD19 CAR expression by gene transfer vector lentivirus and transposon in LK032 PBMCs on days 7 and 14.

[0076] [Figure 39] Figure 39 shows the results of confirming CD19 CAR expression by gene transfer lentivirus and transposon in LK053 PBMCs on days 7 and 14.

[0077] [Figure 40] FIG. 40 shows memory T cells were sorted by expression of the CD45RA / CD62L markers.

[0078] [Figure 41] FIG. 41 shows the results of examining the proportion of memory T cells among T cells induced by gene transfer vectors lentivirus and transposon in LK032 PBMC.

[0079] [Figure 42]Figure 42 shows the results of confirming the proportion of memory T cells among CD19 CAR-expressing T cells in LK053 PBMCs using gene transfer lentivirus and transposon.

[0080] [Figure 43] Figure 43 shows the results of confirming the proportion of memory T cells among CD19 CAR-expressing T cells using gene transfer lentivirus and transposon in LK032 PBMC and LK053 PBMC.

[0081] [Figure 44] Figures 44 and 45 show the results of confirming the proportion of CD4+ expressing T cells (Figure 44) and CD8+ expressing T cells (Figure 45) in CD19 CAR-expressing T cells in LK032 PBMCs and LK053 PBMCs using gene transfer lentivirus and transposon. [Figure 45] Same as above.

[0082] [Figure 46] Figures 46 and 47 show the % killing (mean) of FLAG (CD19 CAR) expressing T cells transformed with gene transfer lentivirus (Figure 46) or transposon (Figure 47). [Figure 47] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0083] Best Mode for Carrying Out the Invention

[0084] The present inventors have completed the present invention by confirming that the efficiency of gene delivery via a transposon system is improved when transposase activity is improved by optimizing the nucleic acid sequence of the transposase.

[0085] (blank)

[0086] The present invention will be described in detail below.

[0087] (blank)

[0088] The present invention provides a transposase expression vector comprising a nucleic acid sequence encoding the transposase set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0089] In the present invention, "transposase" refers to an enzyme that recognizes and binds to both ends of a transposon (especially an inverted repeat sequence), cleaves the end, and moves and inserts the gene fragment between the ends (i.e., the site containing the target DNA) to another location within the chromosome.

[0090] The transposase can be introduced into a cell either as the protein itself, or in the form of a vector ("transposase vector," "transposase plasmid," "transposase expression vector," or "transposase expression plasmid") containing a sequence encoding the transposase protein, or a nucleic acid molecule (DNA or RNA molecule) containing a sequence encoding the transposase protein, and then expressed in the cell.

[0091] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. Specifically, the term "vector" refers to any vehicle for introducing and / or transferring bases into a host cell in vitro, ex vivo, or in vivo, and may be a replication unit to which another DNA fragment can be attached and which can cause replication of the attached fragment. The term "replication unit" refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an autonomous unit of DNA replication in vivo, i.e., that can replicate under its own control. Examples of vectors include, but are not limited to, bacteria, plasmids, phages, cosmids, episomes, viruses, and insertable DNA fragments, i.e., fragments that can be inserted into a host cell genome by homologous recombination.

[0092] In the present invention, a "transposase expression vector" refers to a nucleic acid molecule capable of transporting a nucleic acid sequence encoding a transposase or a template nucleic acid sequence for producing mRNA encoding a transposase, and may also be referred to as a "transposase vector," a "transposase plasmid," a "transposase expression plasmid," etc.

[0093] According to one embodiment of the present invention, the transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 may contain the nucleic acid sequence of SEQ ID NO: 14, and the transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3 may contain the nucleic acid sequence of SEQ ID NO: 15, but is not limited thereto.

[0094] The vector according to the present invention may be, but is not limited to, a plasmid DNA, linear DNA, hairpin DNA, or minicircle DNA, and may be composed of double-stranded DNA or a recombinant viral vector. The vector may be any vector that contains a nucleic acid sequence encoding a transposase or a template nucleic acid sequence for producing mRNA encoding a transposase and can deliver it into target cells. Those skilled in the art can select and use various vectors commonly known in the art.

[0095] The vector of the present invention may preferably include a promoter, which is a transcription initiation factor to which RNA polymerase binds, an optional operator sequence for regulating transcription, an enhancer sequence, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, a terminator, etc. More preferably, it may further include a polyhistidine tag (an amino acid motif consisting of at least five histidine residues), a signal peptide gene, an endoplasmic reticulum retention signal peptide, a cloning site, etc. It may also further include a tagging gene, a selectable marker gene such as an antibiotic resistance gene for selecting transformants, etc. In the vector, the sequence of each gene is operably linked to the promoter. As used herein, the term "operably linked" refers to the functional connection between a nucleotide expression regulatory sequence, such as a promoter sequence, and another nucleotide sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleotide sequence.

[0096] The vectors of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vectors of the present invention are expression vectors and prokaryotic cells are used as hosts, they generally contain a strong promoter capable of driving transcription (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. When eukaryotic cells are used as hosts, the replication origin that the vector operates in eukaryotic cells may include, but is not limited to, the f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, and BBV origin. Furthermore, promoters derived from the genomes of mammalian cells (e.g., metallothionine promoter) or mammalian virus promoters (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV tk promoter) can be used, and generally contain a polyadenylation sequence as a transcription termination sequence. Furthermore, the signal sequence may include, but is not limited to, a poly A signal.

[0097] Representative examples of the tagging gene include Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag (polyhistidine tag), Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, etc. Preferably, the vector according to the present invention may contain a myc tag.

[0098] In the present invention, the vector can be delivered into a cell using various techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into a prokaryotic or eukaryotic host cell. For example, the vector according to the present invention can be inserted into a cell by, but is not limited to, calcium phosphate coprecipitation; electroporation; microfluidic gene editing; nucleofection; cell squeezing; sonoporation; optical transfection; impalefection; gene gun; magnetofection; viral transduction; DEAE-dextran transfection; lipofection; or transfection via a dendrimer, liposome, or cationic polymer.

[0099] According to one embodiment of the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 is a sequence optimized for good expression in cells (particularly T cells) by codon optimization of the original transposase DNA sequence (pBat transposase, SEQ ID NO: 1) in order to improve transposase activity, and is characterized by providing the transposase in the form of (plasmid) DNA (a vector containing a sequence encoding the transposase protein).

[0100] In the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3 is a sequence obtained by optimizing the DNA sequence of the original transposase (pBat transposase, SEQ ID NO: 1) to conform to the mRNA form. According to one embodiment of the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3 is mRNA prepared by in vitro transcription using a transposase expression vector containing the nucleic acid sequence, i.e., the transposase is provided in the form of mRNA. Therefore, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3 is preferably a template nucleic acid sequence for preparing mRNA encoding the transposase.

[0101] In the present invention, the nucleic acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 3 is an optimized nucleic acid sequence, and the transposase protein expressed therefrom is identical to the original transposase protein.

[0102] In the present invention, the term "nucleic acid" or "nucleic acid molecule" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic building blocks of nucleic acids, include not only natural nucleotides but also analogs with modified sugars or base moieties. The sequences of the nucleic acids of the present invention may be modified. Such modifications include the addition, deletion, or non-conservative or conservative substitution of nucleotides. The nucleic acids of the present invention also include nucleotide sequences that show substantial identity to the nucleotide sequences. "Substantial identity" refers to a nucleotide sequence that shows at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology, when the nucleotide sequences of the present invention are aligned with any other sequence to maximize correspondence and the aligned sequences are analyzed using algorithms commonly used in the art.

[0103] That is, in the present invention, a polynucleotide consisting of a nucleotide sequence represented by a specific SEQ ID NO is not limited to the nucleotide sequence, and variants of the nucleotide sequence are also included within the scope of the present invention. The nucleic acid molecule consisting of a nucleotide sequence represented by a specific SEQ ID NO of the present invention includes functional equivalents of the nucleic acid molecule that constitutes it, for example, variants in which a portion of the nucleotide sequence of the nucleic acid molecule has been modified by deletion, substitution, or insertion, but which can perform the same function as the nucleic acid molecule. Specifically, the polynucleotide disclosed in the present invention may contain a nucleotide sequence that has 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more sequence identity with the nucleotide sequence represented by a specific SEQ ID NO. For example, polynucleotides having 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity are included. "Percent sequence identity" for polynucleotides is determined by comparing two optimally aligned sequences and a comparison region, and portions of the polynucleotide sequence in the comparison region may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0104] According to one embodiment of the present invention, the nucleic acid sequence encoding the transposase may further comprise a nucleic acid sequence encoding a nuclear localization signal (NLS).

[0105] In the present invention, the term "nuclear localization signal (NLS)" refers to a peptide chain that helps various proteins used in the nucleus to be transported into the nucleus after being synthesized in the cytoplasm. The NLS is well known in the art and can be any known or functional NLS widely described in the literature. According to one embodiment of the present invention, the nuclear localization signal can be SV40 or C-myc.

[0106] In the present invention, the nucleic acid sequence encoding the nuclear localization signal may be one or more of the nucleic acid sequences represented by SEQ ID NOs: 4 to 11. Specifically, SEQ ID NOs: 4 to 9 are sequences encoding SV40, and SEQ ID NOs: 10 to 11 are sequences encoding C-myc. One or more nuclear localization signals may be included, or two or more types of nuclear localization signals may be mixed.

[0107] According to one embodiment of the present invention, the nucleic acid sequence encoding the nuclear localization signal may also be optimized, and the optimized nucleic acid sequence of SV40 may include the nucleic acid sequence of SEQ ID NO: 9, and the optimized nucleic acid sequence of C-myc may include the nucleic acid sequence of SEQ ID NO: 11.

[0108] Since the optimization was performed on the nucleic acid sequence, the SV40 proteins expressed from SEQ ID NOs: 4 to 9 and the C-myc proteins expressed from SEQ ID NOs: 10 to 11 are identical.

[0109] According to one embodiment of the present invention, the nucleic acid sequence encoding the nuclear localization signal may be included in the 5' to 3' direction at the 5' or 3' end of the nucleic acid sequence encoding the transposase. Specifically, when an SV40 or C-myc nucleic acid sequence is added to the 5' end of the transposase nucleic acid sequence to improve transposase activity, a kozak sequence (5'-GCCACC-3') may be further added before the start codon of the transposase, but this is not limitative (see Figure 22).

[0110] In the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 to which the optimized SV40 nucleic acid sequence (SEQ ID NO: 9) has been added at the 5' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 16.

[0111] In the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 to which an optimized C-myc nucleic acid sequence (SEQ ID NO: 11) has been added at the 5' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 18.

[0112] In the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 to which the optimized SV40 nucleic acid sequence (SEQ ID NO: 9) has been added at the 3' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 20.

[0113] In the present invention, the nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 to which the optimized C-myc nucleic acid sequence (SEQ ID NO: 11) has been added at the 3' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 22.

[0114] In the present invention, a transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2, which contains a kozack sequence (5'-GCCACC-3') before the start codon of the transposase and has an optimized SV40 nucleic acid sequence (SEQ ID NO: 9) added to the 5' end, may contain or consist of the nucleic acid sequence of SEQ ID NO: 17 (see Figure 22).

[0115] In the present invention, a transposase expression vector containing a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2, which includes a kozack sequence (5'-GCCACC-3') before the start codon of the transposase and an optimized C-myc nucleic acid sequence (SEQ ID NO: 11) added to the 5' end, may comprise or consist of the nucleic acid sequence of SEQ ID NO: 19 (see Figure 22).

[0116] In the present invention, a transposase expression vector comprising a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 2 to which an optimized SV40 nucleic acid sequence (SEQ ID NO: 9) has been added at the 3' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 21 (see Figure 23).

[0117] In the present invention, a transposase expression vector containing a nucleic acid sequence encoding a transposase represented by SEQ ID NO: 2 to which an optimized C-myc nucleic acid sequence (SEQ ID NO: 11) has been added at the 3' end may comprise or consist of the nucleic acid sequence of SEQ ID NO: 23 (see Figure 23).

[0118] (blank)

[0119] The present invention also provides an mRNA encoding the transposase represented by SEQ ID NO: 12, which may be produced by in vitro transcription using a transposase expression vector according to the present invention. Specifically, the transposase expression vector may be a transposase expression vector comprising a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3.

[0120] (blank)

[0121] The present invention also provides a transposase expressed from the transposase expression vector of the present invention or the mRNA encoding the transposase of the present invention. According to one embodiment of the present invention, the nucleic acid sequence encoding the transposase contained in the transposase expression vector and the template nucleic acid sequence for producing the mRNA encoding the transposase have been optimized using nucleic acid sequences and therefore contain the same amino acid sequence as the original transposase protein, as shown in SEQ ID NO: 13.

[0122] In the present invention, when the transposase expression vector comprises a nucleic acid sequence encoding the transposase represented by SEQ ID NO: 3,

[0123] The transposase may be expressed from mRNA prepared by in vitro transcription using a transposase expression vector, but is not limited thereto.

[0124] (blank)

[0125] The present invention also provides a method for producing a transposon-based vector comprising: (a) a transposon-based vector into which a target DNA has been inserted; and

[0126] (b) A transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, an mRNA encoding the transposase shown in SEQ ID NO: 12, or a transposon system for targeted DNA delivery comprising the transposase of the present invention.

[0127] As used herein, the term "transposon" refers to a polynucleotide that can excise a specific gene from a donor polynucleotide (e.g., a vector), thereby changing its location within a genome and integrating into a target site (e.g., the genome of a cell or extrachromosomal DNA). A transposon is a polynucleotide containing a nucleic acid sequence flanked by cis-acting nucleotide sequences, where at least one cis-acting nucleotide sequence is located at the 5' end of the nucleic acid sequence and at least one cis-acting nucleotide sequence is located at the 3' end of the nucleic acid sequence. The cis-acting nucleotide sequences contain at least one inverted repeat (IR) at each end of the transposon, called an ITR (inverted terminal repeat), to which a transposase binds.

[0128] According to one embodiment of the present invention, the transposon-based vector and transposase expression vector, or the mRNA encoding the transposase, or the transposase may be contained in a mass ratio of, but is not limited to, 0.1 to 10:1, 0.1 to 8:1, 0.1 to 5:1, 0.1 to 3:1, 0.1 to 1:1, 0.5 to 10:1, 0.5 to 8:1, 0.5 to 5:1, 0.5 to 3:1, 0.5 to 1:1, 1 to 10:1, 1 to 8:1, 1 to 5:1, 1 to 4:1, 1 to 3:1, 1 to 2:1, or 1:1.

[0129] The transposon-based vector of the present invention can be constructed by conventional methods generally known in the art, with reference to Korean Patent Registration No. 10-2602485.

[0130] In the present invention, "target DNA" refers to an exogenous DNA molecule to be delivered into a cell using a transposon. It is sufficient for the target DNA to be inserted into a transposon vector and be expressed after being introduced into a target cell. That is, it is clear that the target DNA is not limited to a specific type of DNA, and those skilled in the art can freely select a desired target DNA depending on the purpose.

[0131] In one embodiment of the present invention, the target DNA sequence may encode an antibiotic resistance protein, a therapeutic polypeptide, an siRNA, an miRNA, a reporter protein, a cytokine, a kinase, an antigen, an antigen-specific receptor, a cytokine receptor, a suicide polypeptide, a recombinant antibody, a neutralizing antibody against various viruses or other antigens, or a portion thereof, for example, but not limited to, a CAR (Chimeric Antigen Receptor), a TCR (T cell receptor), or a portion thereof.

[0132] In the present invention, the term "therapeutic polypeptide" refers to a polypeptide or peptide that is effective in preventing, ameliorating, and / or treating any disease, and those skilled in the art can appropriately select a polypeptide that exhibits a therapeutic effect on a specific disease depending on the purpose. The disease is not limited to a specific type, but in one embodiment, the disease may be cancer.

[0133] (blank)

[0134] The present invention also provides a transposon kit for targeted DNA delivery, which includes the transposon system for targeted DNA delivery according to the present invention and an instruction manual.

[0135] The instructions may include pamphlets, recordings, diagrams, or other media (e.g., CDs, VCDs, DVDs, US(B)) that can be used to communicate or inform how to use the transposon system of the present application. The instructions may be attached to the container or may be packaged separately from the container containing the transposon system of the present application.

[0136] The kit may further include a container for containing the transposon system of the present application.

[0137] The kit may further include a buffer solution for stabilizing the transposon system or for cell transfection. The buffer solution may be, for example, but is not limited to, phosphate buffered saline, Tris-based saline, Tris-EDTA buffer, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, or (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer.

[0138] The present invention also provides a method for producing a transposon-based vector comprising: (a) a transposon-based vector into which a target DNA has been inserted; and

[0139] (b) A transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3, an mRNA encoding the transposase shown in SEQ ID NO: 12, or a cell into which the transposase of the present invention has been introduced.

[0140] In the present invention, the cell may be one in which the target DNA has been excised from the transposon-based vector by the transposase within the cell, and the excised target DNA has been integrated into the genome of the cell. That is, the target DNA is inserted into the genome of the target cell by the transposon and transposase of the present invention and can be stably expressed. The target DNA inserted into the genome of the cell can be expressed in the cell for, but is not limited to, 5 days or more, 7 days or more, 10 days or more, 15 days or more, 20 days or more, or 30 days or more after the transposon-based vector and transposase are introduced into the cell.

[0141] That is, the present invention provides genetically engineered cells in which target DNA has been inserted into the genome by the transposon. In this invention, "engineered" refers to any manipulation of a cell that results in a detectable change in the cell, including, but not limited to, inserting heterologous / homologous polynucleotides and / or polypeptides into the cell and mutating polynucleotides and / or polypeptides native to the cell.

[0142] In one embodiment of the present invention, the cells are one or more immune cells selected from the group consisting of myeloid cells such as T cells, B cells, natural killer cells (NK cells), monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes, and dendritic cells; or stem cells derived from bone marrow, adipose tissue, peripheral blood, umbilical cord blood, or dental pulp, but are not limited thereto. The cells may also be derived from insects, plants, fish, or mammals, particularly humans.

[0143] According to one embodiment of the present invention, the T cells can be activated using, but not limited to, CD3 / CD28 beads before the transposon vector is introduced.

[0144] As used herein, the term "immune cell" refers collectively to cells that play a role in the immune response.

[0145] Alternatively, the cells may be co-cultured with feeder cells after transfection with the transposon vector and transposase (protein or nucleic acid molecule). Feeder cells are auxiliary cells that do not proliferate themselves but provide extracellular secretions, including growth factors, to enable the proliferation of cells transfected with target DNA. The feeder cells are not limited to a specific type, and any cells known in the art to function as feeder cells may be used. Non-limiting examples include fibroblasts, human bone marrow-derived mesenchymal cells, human amniotic epithelial cells, adipose-derived mesenchymal stem cells, and melanoma cells (A375 cells). Preferably, the feeder cells may be irradiated before co-culture with the cells transfected with target DNA.

[0146] Co-culture with the support cells can improve gene delivery efficiency, particularly when introducing a CAR or TCR into immune cells using the transposon system of the present invention, and can contribute to increasing the proliferation of the gene-introduced cells and the expression rate of the gene. The method for activating the gene-introduced cells is not limited to co-culture with support cells, and any appropriate cell activation method can be used depending on the type of target cell. For example, when the cells are T cells, they can be activated using TransAct or Dynabeads.

[0147] The co-culture with the feeder cells is preferably carried out immediately after the transposon vector and transposase are introduced into the cells using electroporation or the like, but is not limited to this. The co-culture can be carried out within 1 to 10 days, 1 to 5 days, 1 to 3 days, 1 to 2 days, 1 day, 20 hours, 10 hours, 5 hours, 3 hours, 1 hour, 30 minutes, or 10 minutes after the introduction.

[0148] (blank)

[0149] The present invention also provides a method for producing a transposon-based vector comprising: (a) a transposon-based vector into which a target DNA has been inserted; and

[0150] (b) A method for inserting a target DNA sequence into the genome of a cell, the method comprising introducing into the cell a transposase expression vector comprising a nucleic acid sequence encoding the transposase set forth in SEQ ID NO:2 or SEQ ID NO:3, an mRNA encoding the transposase set forth in SEQ ID NO:12, or a transposase according to the present invention.

[0151] Furthermore, the method may further comprise, after the introducing step, a step of co-culturing the cells into which the transposon vector has been inserted with feeder cells.

[0152] As used herein, the term "introduction" refers to the introduction (delivery) of a polynucleotide (e.g., a transposon vector or a transposase vector) into a cell or organism. The nucleic acid of the polynucleotide may be in the form of naked DNA or RNA, may be associated with various proteins, or may be integrated into a vector. The term "transduction" as used herein is intended to convey the broadest possible meaning and includes, for example, transfection methods (methods by which a polynucleotide is introduced into a eukaryotic cell by physical and / or chemical treatment), transformation methods (methods by which a polynucleotide is introduced into a prokaryotic cell by physical and / or chemical treatment), viral / viral transduction methods (methods by which a polynucleotide is introduced into a eukaryotic and / or prokaryotic cell by a virus or viral vector), conjugation methods (methods by which a polynucleotide is introduced from one cell to another by direct cell-to-cell contact or by a cytoplasmic bridge between cells), and fusion methods (methods by which two cells are fused, including homotypic and heterotypic cell fusion). Preferably, the transduction is carried out by electroporation.

[0153] The present invention also provides compositions for various uses, which contain as an active ingredient cells into whose genome target DNA has been inserted by the transposon-based vector of the present invention, wherein the cells may be autologous or allogeneic.

[0154] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating immune-related diseases, which comprises the cells of the present invention as an active ingredient.

[0155] As used herein, the term "immune-related disease" refers to diseases and / or conditions in which the immune system is involved in the pathogenesis of the disease or in which appropriate stimulation or suppression of the immune system can result in treatment and / or prevention of the disease. Exemplary immune-related diseases that can be treated by the present invention include, but are not limited to, tumors, infectious diseases, allergies, autoimmune diseases, graft-versus-host disease, or inflammatory diseases.

[0156] The present inventors have confirmed, based on specific examples, that CAR T cells produced using the transposon of the present invention differentiate into cytotoxic T cells, memory T cells, etc. in response to antigens. Therefore, those skilled in the art can produce genetically engineered cells with more activated immune functions by using the transposon of the present invention to deliver an appropriate antigen-specific CAR or TCR gene into immune cells, and can use these to prevent or treat immune-related diseases.

[0157] For example, a person skilled in the art can insert a gene encoding a target antigen into a transposon according to the present invention, deliver it to immune cells, and enhance the immune function of the cells against that antigen. Enhancement of immune function can mean, but is not limited to, activating the function of antigen-presenting cells, natural killer cells, T cells (particularly cytotoxic T cells), etc. against the antigen, or modulating the activity of regulatory T cells, myeloid-derived suppressor cells (MDSCs), M2 macrophages, etc.

[0158] MODE FOR CARRYING OUT THE INVENTION

[0159] Hereinafter, preferred examples will be presented to aid in understanding the present invention. However, the following examples are provided merely to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples. [Example]

[0160] (blank)

[0161] [Example]

[0162] Example A. Optimization of pBat Transposase

[0163] To further improve the gene delivery efficiency of the transposon system, the DNA sequence of a conventional transposase was optimized using Genscript's "OptimumGene Codon Optimization" program to ensure good expression in cells (especially T cells).

[0164] Furthermore, to improve the gene delivery efficiency of the transposon system by overcoming the problems that can arise with DNA-type transposases, we constructed an mRNA-type transposase, which has lower endogenous toxicity and a relatively short half-life compared to DNA-type transposases (Example D).

[0165] [Table 1-1]

[0166] [Table 1-2]

[0167] [Table 1-3]

[0168] Example B. Confirmation of gene delivery efficiency of pBat-optimized transposase with optimized DNA sequence

[0169] To further improve the gene delivery efficiency of the pBat transposon system, the transposase DNA sequence was optimized for better expression in cells, and the gene delivery efficiencies using conventional transposase and the optimized transposase were compared.

[0170] (blank)

[0171] [method]

[0172] 1. DNA and cells

[0173] To confirm the gene delivery efficiency of the pBat optimized transposase, whose DNA sequence was optimized, pBat transposon 3M3-5M3-1G4 TCR (1.8 μg / μL) was used as the pBat transposon plasmid vector, and for transposase expression, pBat transposase plasmid (1.5 μg / μL) and pBat optimized transposase plasmid (1.4 μg / μL) were used as the pBat transposase plasmid vector.

[0174] The control cells used were PBMC (LK045) isolated from a healthy human and A375 (ATCC, Cat no. CRL-1619, Lot no. 70032966) irradiated with 50 Gy.

[0175] (blank)

[0176] 2. PBMC electroporation

[0177] Gene delivery into cells was achieved by electroporation, specifically as follows:

[0178] (1) Prepare 1.5 mL tubes for each electroporation condition shown in Table 2 below. Inject the transposase plasmid and transposon plasmid into 5.0 × 10 PBMCs depending on the conditions. 6 I put 5ug into each one.

[0179] [Table 2]

[0180] (blank)

[0181] (2) 5.0 × 10 PBMCs 6 Each was added to a 1.5 mL tube (1).

[0182] (3) 5.0 x 10 cells of the cell suspension (2) in OC100 x 2 assembly 6 Each 50 μL was carefully added to avoid creating bubbles.

[0183] (4) Resting T cells 14-3 protocol was selected with Maxcyte STx for electroporation.

[0184] (5) The OC100x2 assembly from (3) was inserted into STx, and the protocol was followed for electroporation.

[0185] (6) After electroporation, remove the cell suspension (5.0 × 10 cells) from the OC100 × 2 assembly. 6 50 μL / well) was transferred to a T25 flask.

[0186] (7) The OC100x2 assembly from (6) was washed with 50 μL of AlyS medium and added to each T25 flask.

[0187] (8) After allowing the electroporated cells to recover for 20 minutes in a 37°C, 5% CO2 incubator, 2.0 × 10 irradiated A375 cells were added to each flask. 6 Each well was added with 3 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2).

[0188] (9) The cells were cultured in a 37°C, 5% CO2 incubator.

[0189] (blank)

[0190] 3. Culturing and Storage of T Cells After Electroporation

[0191] (1) Three days after electroporation, 5 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2) was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0192] (2) Six days after electroporation, 10 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2) was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0193] (3) Seven days after electroporation, the cultured cells were suspended, and 3 mL of the suspension was collected from each flask and subjected to FACS analysis.

[0194] (4) Eight days after electroporation, the cells in the culture were suspended, and then 8 mL of each flask was transferred to a new T25 flask and divided into two flasks. 8 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2) was added to each flask, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0195] (5) Ten days after electroporation, the cells were suspended in the culture medium, and 2 mL of the medium was collected from each flask and analyzed by FACS. The remaining cells in the T25 flasks were transferred to T75 flasks, and 10 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2) was added to each flask. The cells were then cultured at 37°C in a 5% CO2 incubator.

[0196] (6) 13 days after electroporation, 10 mL of medium (ALyS + 3% HS + 200 IU / mL IL-2) was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0197] (7) 14 days after electroporation, the cultured cells were suspended, and 2 mL of the suspension was collected from each flask and subjected to FACS analysis.

[0198] (8) 14 days after electroporation, the cells remaining in the flasks in 7) were transferred to 50 mL conical tubes.

[0199] (9) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was removed.

[0200] (10) The cell pellet was suspended in 5 mL of ALyS medium and the cells were counted.

[0201] (11) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was removed.

[0202] (12) The cell pellet was suspended in 1 mL of CS10 and frozen according to protocol 6 of the CRF (Controlled Rate Freezer, ThermoFisher, CryoMed TSCM34PV, S / N 300503011, 300503015), and then stored in a nitrogen tank.

[0203] (blank)

[0204] 4.FACS analysis

[0205] FACS analysis was performed 7, 10 and 14 days after electroporation.

[0206] (1) After suspending the cells in each flask, 2 to 3 mL of each was transferred to a FACS tube.

[0207] (2) After centrifugation at 1,500 rpm at 4°C for 5 minutes and removal of the supernatant, the cells were suspended in 1 mL of FACS buffer (PBS + 2% FBS).

[0208] (3)(2) was repeated once more to wash.

[0209] (4) The mixture was centrifuged at 1,500 rpm at 4°C for 5 minutes, and the supernatant was removed.

[0210] (5) 5 μL of human TruStain FcX and 95 μL of FACS buffer were added to each tube, and the mixture was allowed to react at room temperature for 5 minutes.

[0211] (6) 2 uL of each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-mTCRβ) was added to each tube, and the mixture was incubated at room temperature in the dark for 30 minutes.

[0212] (7) 1 mL of FACS buffer (PBS + 2% FBS) was added to each well, and the mixture was centrifuged at 4°C, 1,500 rpm for 5 minutes.

[0213] (8) After removing the supernatant, the cells were suspended in 1 mL of FACS buffer (PBS + 2% FBS).

[0214] (9) The mixture was centrifuged at 4°C and 1,500 rpm for 5 minutes, and the supernatant was removed.

[0215] (10) The cells were suspended in 200 μL of 1× FACS buffer (PBS + 2% FBS) with or without DAPI, and subjected to FACS analysis.

[0216] (blank)

[0217] [result]

[0218] 1. 1G4 TCR Expression Analysis 7 Days After Electroporation

[0219] Since the constant region of 1G4 TCR is composed of the constant region gene sequence of mouse TCR, 1G4 TCR expression was confirmed by expression of mTCRβ using anti-mTCRβ antibody. + The percentage of cells expressing 1G4 TCR in T cells was confirmed. As shown in Figure 1, the percentage was 0% in the "EP only group (negative control group)" in which only electroporation (EP) was performed without a plasmid, while the percentage was 27.7% in the "conventional transposase group" and 56.0% in the "optimized transposase group" for the 3M3-5M3-1G4 TCR transposon. This confirmed that the percentage of mTCRβ-expressing cells was twice as high when the optimized transposase was used.

[0220] (blank)

[0221] 2. 1G4 TCR Expression Analysis 10 Days After Electroporation

[0222] 10 days after electroporation, CD3 +The percentage of T cells expressing 1G4 TCR was confirmed, and as shown in Figure 2, it was 0% in the "EP only group (negative control group)," and it was confirmed that it increased overall in the transposon group from day 7 onwards. Specifically, with the 3M3-5M3-1G4 TCR transposon, it was 62.7% in the "conventional transposase group" and 79.9% in the "optimized transposase group." As with day 7, the percentage of mTCRβ-expressing cells was 1.3 times higher when the optimized transposase was used.

[0223] (blank)

[0224] 3. 1G4 TCR Expression Analysis 14 Days After Electroporation

[0225] 14 days after electroporation, CD3 + The percentage of cells expressing 1G4 TCR in T cells was confirmed to be 0% in the "EP only group (negative control group)," and overall the percentage in the transposon groups was similar to that after 10 days, as shown in Figure 3. Specifically, for the 3M3-5M3-1G4 TCR transposon, the percentage was 59.0% in the "conventional transposase group" and 76.7% in the "optimized transposase group." As with days 7 and 10, the percentage of mTCRβ-expressing cells was 1.3 times higher when the optimized transposase was used.

[0226] Furthermore, when the percentage of 1G4 TCR-expressing T cells was compared 7, 10, and 14 days after electroporation, it was confirmed that the percentage of 1G4 TCR-expressing T cells increased at both 10 and 14 days compared to 7 days, as shown in Figure 4. When comparing transposases, the percentage of 1G4 TCR-expressing cells was higher with the "optimized transposase" than with the "conventional transposase" at all times after 7, 10, and 14 days, indicating that the optimized transposase had better gene delivery efficiency.

[0227] (blank)

[0228] 4.1G4 TCR-expressing T cells expressing CD4 + Cells and CD8 + cell analysis

[0229] In 1G4 TCR-expressing T cells, helper T cells (CD4 + T cells) and cytotoxic T cells (CD8 + In the "EP only" group, there were no 1G4 TCR expressing T cells, and CD3 + CD4 on T cells + Cells and CD8 + As a result, as shown in Figure 5, in the "EP only group," CD4 + Cells and CD8 + After 10 days, the proportion of CD4 + CD8+ cells accounted for 33.0% of the total, and CD8+ cells accounted for 62.5%. + Many cells were present, and even after 14 days, CD4 + 23% CD8 cells + The cells were 72.5% and CD8 + It was confirmed that there were many CD8 cells during the culture period. + As the cells proliferate, CD3 + CD8 in T cells + This was thought to be due to an increase in the proportion of cells.

[0230] In addition, the 3M3-5M3-1G4 TCR transposon + mTCRβ + CD4 on T cells + Cells and CD8 + As a result of examining the cells, as shown in Figures 5 and 6, the CD4 + The percentage of CD8 cells was 40.5% after 7 days, 44.9% after 10 days, and 29.0% after 14 days. + The percentage of CD4 cells was 55.5% after 7 days, 51.8% after 10 days, and 67.2% after 14 days. + Cells and CD8 + Although there was little difference between the cells, after 14 days, CD8 +The number of CD4 cells was 2.3 times higher in the "optimized transposase group." + The percentage of CD8 cells was 24.7% after 7 days, 22.1% after 10 days, and 14.4% after 14 days. + The percentage of CD4 cells was 71.4% after 7 days, 74.8% after 10 days, and 82.2% after 14 days. + CD8 from cells + It was confirmed that the number of cells increased by 2.9 times after 7 days, 3.4 times after 10 days, and 5.7 times after 14 days.

[0231] (blank)

[0232] 5. Analysis of memory T cells in 1G4 TCR-expressing T cells

[0233] The percentage of memory T cells was analyzed using CD45RA and CCR7 markers in 1G4 TCR-expressing T cells. When classifying memory T cells using CD45RA and CCR7 markers, CD45RA + CCR7 - T cells are T EFF , CD45RA - CCR7 - T cells are T EM , CD45RA - CCR7 + T cells are T CM , CD45RA + CCR7 + T cells are T TCM In the "EP only group," there are no 1G4 TCR-expressing T cells, and therefore CD3 + As shown in Figure 7, memory T cells were identified as T cells with increasing culture period. CM and T TCM decreases, and T EM As shown in Figure 7 and Table 3, the proportion of memory-type T cells within 1G4 TCR-expressing T cells increased after 7 days in the "conventional transposase group." EFF 29.7%, T EM 35.3%, T CM 15.1%, TSCM 20.0%, T after 10 days EFF 39.8%, T EM 31.0%, T CM 14.2%, T SCM 15.1%, T after 14 days EFF 38.3%, T EM 38.8%, T CM 8.2%, T SCM 14.8%, and T CM and T SCM gradually decreased, while T EM and T EFF In the "optimized transposase group," T EFF 29.7%, T EM 32.8%, T CM 14.4%, T SCM 23.1%, T after 10 days EFF 42.8%, T EM 19.5%, T CM 14.4%, T SCM 22.5%, T after 14 days EFF 38.7%, T EM 26.1%, T CM 8.4%, T SCM 27.0%, and T CM While T SCM increases, and T CM and T SCM It was confirmed that the total percentage of cells was higher than that of conventional transposase.

[0234] [Table 3]

[0235] As described above, when generating 1G4 TCR-T cells using the pBat transposon system in PBMCs, we investigated whether there was a difference in the delivery and expression efficiency of the 1G4 TCR gene when using a conventional transposase or an optimized transposase. As a result, we confirmed that the optimized transposase had a higher gene delivery efficiency in LK045 PBMCs. In addition, CD4 +or CD8 + The percentage of cells was also significantly higher than that of CD8 cells after 14 days when using conventional transposase. + T cells are CD4 + T cells were 2.3 times more numerous than CD8 T cells, but when using the optimized transposase, the number of CD8 T cells increased from 7 days onwards. + The percentage of T cells was 2.9 times higher, and the difference became increasingly larger thereafter. Analysis of memory types in 1G4 TCR-expressing T cells using CD45RA and CCR7 markers revealed that the optimized transposase resulted in a higher percentage of T cells. CM and T SCM Therefore, when the optimized transposase was used to generate TCR-T cells using the pBat transposon system, a high yield of cytotoxic T cells (CD8 + It was determined that it could produce TCR-T cells with a high proportion of T) cells.

[0236] (blank)

[0237] Example C. Confirmation of gene delivery efficiency of optimized transposase in Jurkat cells

[0238] 1. Method

[0239] Jurkat cells (ATCC, Cat No. TIB-152, Lot No. 70017560) were used as control cells to confirm the gene delivery efficiency of the optimized transposase.

[0240] The transposon plasmid vector used was the pBat transposon 3M3-5M3-EGFP vector containing EGFP, and the pBat transposase plasmid and pBat optimized transposase plasmid were used to compare the transposase delivery efficiency.

[0241] GFP fluorescence expressed in Jurkat cells was observed by FACS analysis on days 1, 7, and 14 after electroporation, in the same manner as in Example B above.

[0242] (blank)

[0243] 2.Results

[0244] On day 1, as shown in Figures 8a and 8b, there was no difference in the percentage of GFP-expressing cells between the two transposases. On day 7, as shown in Figures 9a and 9b, there was no difference in the percentage of total GFP-expressing cells between the two transposases, but the percentage of highly expressing GFP cells was approximately two-fold higher with the optimized transposase. On day 14, as shown in Figures 10a and 10b, the percentage of total GFP-expressing cells and highly expressing GFP cells was approximately two-fold higher with the optimized transposase.

[0245] The results confirmed that the optimized transposase had a GFP expression rate two times higher than that of the conventional transposase.

[0246] (blank)

[0247] Example D. Confirmation of increased gene expression efficiency by the mRNA form of pBat-optimized transposase

[0248] By comparing the gene delivery efficiency of conventional transposase and codon-optimized transposase, we confirmed that the codon-optimized transposase had a higher gene delivery efficiency.

[0249] However, when delivering genes in DNA form, using excessive amounts can induce toxicity in the body due to the action of intracellular mechanisms that recognize foreign DNA. Furthermore, if the transposase gene delivered into cells in DNA form continues to exist in the cell and transposase expression persists for a long period of time, it may remobilize the inserted foreign gene. Therefore, we created an mRNA-type transposase, which has low toxicity in the body and a relatively short half-life, and compared its gene delivery efficiency with that of DNA-type transposase.

[0250] (blank)

[0251] [method]

[0252] 1. Cells and DNA

[0253] To confirm the increased gene expression efficiency of the mRNA form of pBat transposase, Jurkat, Clone E6-1 (ATCC, Cat No. TIB-152, Lot No. 70017560), and PBMC LK053 isolated from a healthy human were used as control cells.

[0254] The transposon plasmid vectors used were the pBat transposon vector containing the GFP gene and the pBat transposon vector containing the 1G4 TCR gene. To confirm the gene delivery efficiency depending on the transposase gene delivery form, we used the optimized transposase in the form of plasmid DNA (pBat optimized transposase plasmid) and the optimized transposase in the form of mRNA.

[0255] (blank)

[0256] 2. Preparation of template DNA for mRNA synthesis

[0257] We requested GenScript Inc. to optimize the original transposase gene sequence (SEQ ID NO: 1) to fit the mRNA form, and requested Bionix Inc. to synthesize the DNA sequence (SEQ ID NO: 3) optimized for the mRNA form in the GenSmart Optimization Report (Tool Version Beta 1.0). TM The gene with the DNA sequence optimized for mRNA format was cloned into the Cloning kit for mRNA Template (Cat. #6143) in the T7 mRNA system kit as follows:

[0258] For infusion cloning of the DNA sequence optimized for mRNA form, PCR was performed for insert amplification using the DNA sequence (SEQ ID NO: 3) optimized for mRNA form synthesized by Bionics Co., Ltd. as a template, and the resulting DNA was loaded onto an agarose gel. After confirming that a band of the desired size appeared as shown in Figure 11, the gel was extracted. TM Infusion cloning was performed between the Cloning kit for mRNA Template (Vector) included in the T7 mRNA Synthesis system and the DNA sequence optimized for mRNA form (Insert, SEQ ID NO: 3). As shown in Figure 12, the structure was confirmed through enzyme mapping to create the pBat optimized transposase mRNA template plasmid required for producing the optimized transposase in mRNA form using an in vitro transcription method.

[0259] (blank)

[0260] 3. IVT (in vitro transcription) process for modifying the mRNA form of pBat-optimized transposase

[0261] To synthesize mRNA using the pBat-optimized transposase mRNA template plasmid prepared in 2., the template plasmid was linearized with Hind III restriction enzyme, and the IVT reaction was performed using this as a template as follows:

[0262] (1) The pBat optimized transposase mRNA template plasmid was linearized by adding the restriction enzyme Hind III and reacting it at 37°C for 3 hours.

[0263] (2) Purify the linearized mRNA template plasmid using EtOH precipitation, as follows:

[0264] (2-1) 20 μL of 3 M sodium acetate was added to the reaction mixture of (1), mixed well, and then left to stand at −20° C. for 15 minutes or more.

[0265] (2-2) Centrifugation was carried out at 12,000 rpm for 15 minutes at 4°C.

[0266] (2-3) The supernatant was carefully removed, and 1 mL of 70% EtOH was added, followed by centrifugation at 12,000 rpm for 15 minutes at 4°C.

[0267] (2-4) After carefully removing the supernatant, the DNA pellet was thoroughly dried.

[0268] (2-5) 200 μL of nuclease-free water was added to the solution to give a final concentration of 0.5 to 1.0 μg / μL, and the solution was stored at −20°C until use.

[0269] (3) Takara IVTpro TM The reagents included in the T7 mRNA Synthesis system were dissolved at room temperature, mixed well, and then spun down. The 10X enzyme mix was placed on ice.

[0270] (4) Each reagent was added to the E-tube in the order shown in Table 4 below, with the total amount being 120 uL.

[0271] [Table 4]

[0272] (5) After mixing well, the mixture was incubated at 37°C for 2 hours. 24 μL of the reaction mixture from (4) was treated with Dnase I in the E-tube and incubated at 37°C for 15 minutes. (6) The optimized transposase in mRNA form was purified through the LiCl precipitation process as follows:

[0273] (6-1) 180 μL of nuclease-free water was added to (5), and 180 μL of LiCl was added.

[0274] (6-2) After thorough mixing, the mixture was reacted at −20° C. for at least 30 minutes and then centrifuged at 12,000 rpm for 15 minutes at 4° C.

[0275] (6-3) The supernatant was carefully removed, 1 mL of 70% EtOH was added, and the mixture was centrifuged at 12,000 rpm for 15 minutes at 4°C.

[0276] (6-4) The supernatant was carefully removed, and the DNA pellet was thoroughly dried. The DNA was then thoroughly dissolved in 200 μL of nuclease-free water.

[0277] (6-5) The solution was aliquoted in 10 μL portions and stored at −80°C until use.

[0278] (blank)

[0279] 4.PBMC activation

[0280] The efficiency of gene delivery using two different transposase forms for two different genes (GFP / 1G4 TCR) in PBMCs was compared using the electroporator devices Neon and Maxyte (see Table 5). When the GFP gene was delivered to PBMCs using the transposon system using the Neon device, the efficiency of gene delivery using the Miltenyi TransAct TM PBMCs were activated for 3 days using the Maxyte device without adding feeder cells during cell culture. On the other hand, when the 1G4 TCR gene was delivered to PBMCs using the Maxyte device with the transposon system, PBMCs were not activated and were added with feeder cells during cell culture.

[0281] [Table 5]

[0282] (1) RPMI medium (RPMI + 10% FBS + 1x P / S) was preheated in a water bath at 37°C. (2) PBMC LK053 was removed from the nitrogen tank and quickly thawed in a water bath at 37°C.

[0283] (3) 30 mL of culture medium was prepared in a 50 mL tube, and the thawed PBMCs were added.

[0284] (4) After centrifugation at 1,500 rpm for 5 minutes and removal of the supernatant, the cells were suspended in 40 mL of RMPI medium and then counted.

[0285] (5) 1.2 × 10 PBMCs in a T175 flask 8 Put 1.2 x 10 pieces 8 RPMI medium was added so that the volume became 120 cells / mL.

[0286] (6) Add IL-2 to a final concentration of 20 IU / mL, and adjust TransAct to 2.0 x 10 7 100 μL of IL-2 was added per cell (1.0 × 10 6 The concentration of IU / mL was diluted 1 / 10 with medium to 1.0 x 10 5 IU / mL and used).

[0287] (7) The cells were cultured for 2 days at 37°C in a 5% CO2 incubator.

[0288] (blank)

[0289] 5. PBMC Electroporation

[0290] 5-1. Neon Device Conditions (PBMC Activation Progression, No Feeder Cells)

[0291] (1) 3 mL of E2 buffer stored at 4°C was dispensed into each of three Neon tubes.

[0292] (2) 1.4 mL of medium (AlyS+3% HS+IL-2 200 IU / mL) was dispensed into each well of a 12-well plate, with four wells per group.

[0293] (3) PBMC cells were collected after activation.

[0294] (4) Centrifuged at room temperature at 1,500 rpm for 5 minutes.

[0295] (5) After removing the supernatant, the cell pellet was suspended in 1.0 mL of Opti-MEM buffer.

[0296] (6) Centrifuged at room temperature at 1,500 rpm for 5 minutes.

[0297] (7) After removing the supernatant, the suspension was diluted with Opti-MEM buffer to 1.0 × 10 6 The cells were suspended at a concentration of 100 μL.

[0298] (8) For each condition, transposon and transposase plasmid were transfected into 1.0 × 10 cells. 6 3 μg of each was added per cell as shown in Table 6 below (no vector was added to the control group). Then, the mixture of cells and plasmid was pipetted and mixed well.

[0299] [Table 6]

[0300] (9) A Neon tube containing the E2 buffer (1) was attached to the Neon device.

[0301] (10) Using a Neon pipette and Neon tip, 100 μL of the cell suspension from (8) was slowly drawn up and then inserted into the Neon device.

[0302] (11) After electroporation under conditions of 1,700 V, 20 ms, and 1 pulse, the completed PBMCs were dispensed into 12-well plates containing culture medium and cultured in an incubator at 37°C and 5% CO2.

[0303] (12) One day after transfection, cells were collected from two wells for each condition (observation plate after one day) and subjected to FACS analysis. 2 mL of culture medium was added to the remaining second well (observation plate after seven days), which was then transferred to a T75 flask and cultured in a 37°C, 5% CO2 incubator.

[0304] (13) Five days after transfection, 2 mL of culture medium was added to each well, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0305] (14) Seven days after transfection, the culture medium from each flask was pipetted to suspend the PBMCs for FACS analysis, and 1 mL of cells was collected from the total 4 mL of culture medium. 5 mL of culture medium was added to the remaining culture medium and cultured.

[0306] (15) Eight, 11, and 13 days after transfection, 5 mL, 10 mL, and 20 mL of fresh culture medium were added, respectively, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0307] (16) 14 days after transfection, the culture medium from each flask was pipetted to suspend the PBMCs, and 200 μL of cells were collected for FACS analysis.

[0308] (blank)

[0309] 5-2. Maxcyte device conditions (no PBMC activation, with support cells)

[0310] (1) PBMC LK053 was removed from the nitrogen tank and quickly thawed in a 37°C water bath. 30 mL of culture medium was then prepared in a 50 mL tube, and the PBMC was placed in the culture medium.

[0311] (2) The mixture was centrifuged at 1,500 rpm for 5 minutes, and the supernatant was removed.

[0312] (3) The cell pellet was suspended in 20 mL of medium (ALYS505N-O + 3% HS), and the cells were counted.

[0313] (4) Centrifuged at room temperature at 1,500 rpm for 5 minutes.

[0314] (5) The supernatant was removed, and the cell pellet was suspended in 20 mL of DPBS.

[0315] (6) After centrifugation at 1,500 rpm for 5 minutes at room temperature and complete removal of the supernatant, the cell pellet was suspended in 5 mL of Opti-MEM medium.

[0316] (7) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was completely removed.

[0317] (8) Cell pellet: 5.0 × 10 6 The cells were suspended in 50uL of warm Opti-MEM medium at a concentration of 1 / 50uL.

[0318] (9) 5 μg of transposon vector and transposase vector were added per well to the tubes in (8) as shown in Table 7 below (no vector was added to the control group).

[0319] [Table 7]

[0320] (10) OC100 x 2 assembly of 5.0 x 10 cells suspension (9) 6 50 μL of each cell was added carefully to avoid creating bubbles. (11) For electroporation, the resting T cell 14-3 protocol was selected using Maxcyte STx.

[0321] (12) The OC100x2 assembly (10) was inserted into the chamber in the STx, and the protocol was followed to perform electroporation.

[0322] (13) After electroporation, the cell suspension from the OC100x2 assembly was transferred to a T25 flask (5.0 x 10 6 pcs / 50uL / well).

[0323] (14) 2 wells of OC100 were washed with 50 μL of Opti-MEM medium and added to each well of the plate in (13).

[0324] (15) The cells were allowed to recover for 20 minutes in an incubator at 37°C and 5% CO2.

[0325] (16) A375 cell stock irradiated at 100 Gy was quickly thawed in a 37°C water bath, slowly added to 10 mL of medium, and then centrifuged at 1,500 rpm for 5 min.

[0326] (17) The supernatant was removed, and the cells were suspended in 10 mL of medium and then counted.

[0327] (18) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was completely removed.

[0328] (19) 2.0 × 10 6 The cells were resuspended in complete medium (ALYS505N-O + 3% HS + 200 IU / mL IL-2) to a concentration of 3 cells / mL.

[0329] (20) After the process (15), 3 mL of the cell suspension (19) was added to each flask of PBMCs, and the flasks were then cultured at 37°C in a 5% CO2 incubator for 2 days.

[0330] (21) Two days after electroporation, 2 mL of medium and IL-2 were added to each flask to a final concentration of 200 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0331] (22) Five days after electroporation, 10 mL of medium and IL-2 were added to a final concentration of 200 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0332] (23) Seven days after electroporation, the cells were suspended in the culture medium. 500 μL of the cell suspension was transferred to a new tube for FACS analysis. The remaining cell suspension was combined to a total of 15 mL, and new medium (ALYS505N-O + 3% HS) and IL-2 were added to a final concentration of 200 IU / mL. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0333] (24) Nine days after electroporation, 5 mL of medium (ALYS505N-O + 3% HS) was added to a total volume of 20 mL, and IL-2 was added to a final concentration of 200 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0334] (25) Twelve days after electroporation, 5 mL of medium (ALYS505N-O + 3% HS) was added to a total volume of 25 mL, and IL-2 was added to a final concentration of 200 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0335] (26) 14 days after electroporation, the cells in the culture were suspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. 2 × 10 cells per group were used for in vitro killing analysis. 7 The cells were collected and cultured in 20 mL of resting medium (ALYS505N-O + 3% HS) for 24 hours. The remaining suspension was used as a stock, and the supernatant was removed. After that, CS10 medium was added and the suspension was stored at -80°C for 1 day before being transferred to a nitrogen tank.

[0336] (blank)

[0337] 6.FACS analysis

[0338] FACS analysis was performed 7 and 14 days after electroporation.

[0339] (1) After suspending the cells in each flask, 500 μL was taken and transferred to a FACS tube.

[0340] (2) After centrifugation at 1,500 rpm at 4°C for 5 minutes and removal of the supernatant, the cells were suspended in 1 mL of washing buffer (PBS + 2% FBS).

[0341] (3)(2) was repeated once more to wash.

[0342] (4) The mixture was centrifuged at 1,500 rpm at 4°C for 5 minutes, and the supernatant was removed.

[0343] (5) 5 μL of human TruStain FcX and 95 μL of FACS buffer were added to each tube, and the mixture was allowed to react at room temperature for 5 minutes.

[0344] (6) 2 μL of each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RO, anti-CD62L) was added per tube, and 0.5 μL of anti-mTCRβ antibody was added per tube, and the mixture was incubated at room temperature in the dark for 30 minutes.

[0345] (7) 1 mL of FACS buffer (PBS + 2% FBS) was added to each well, and the mixture was centrifuged at 4°C, 1,500 rpm for 5 minutes.

[0346] (8) After removing the supernatant, the cells were suspended in 1 mL of FACS buffer (PBS + 2% FBS).

[0347] (9) The mixture was centrifuged at 4°C and 1,500 rpm for 5 minutes, and the supernatant was removed.

[0348] (10) The cells were suspended in 200 μL of FACS buffer (PBS + 2% FBS) containing 1× DAPI, and FACS analysis was performed.

[0349] (blank)

[0350] 7. Peptide Pulsing of T2-Luc Cells

[0351] (1) T2-Luc cells cultured in IMDM medium (IMDM + 10% FBS + 1x P / S) were collected into a 15 mL conical tube.

[0352] (2) Centrifuged at 1,500 rpm at room temperature for 5 minutes.

[0353] (3) The supernatant was removed, the cell pellet was suspended in 5 mL of IMDM medium, and the cells were counted.

[0354] (4) 1.0 × 10 6 IMDM medium was added so that the concentration was 1 / mL, and the mixture was placed in a polypropylene (PP) tube according to the conditions in Table 8 below.

[0355] [Table 8]

[0356] (5) To the T2-Luc cell pulsing group, 100 mg / mL NY-ESO-1 peptide and 0.5 mg / mL β2m were added to achieve the same treatment concentrations as those in the peptide pulsing conditions listed in Table 9. To the non-pulsing group, DMSO was added in the same volume as the peptide added. (6) The reaction was carried out at room temperature for 2 hours, with tapping every 30 minutes.

[0357] (7) After centrifugation at 1,500 rpm at room temperature for 5 minutes, the supernatant was removed.

[0358] (8) The cell pellet was suspended in 10 mL of PBS and centrifuged at 1,500 rpm at room temperature for 5 minutes.

[0359] (9) The supernatant was removed, and the cell pellet was suspended in 1 mL of ALyS medium (ALYS505N-O + 3% HS), followed by cell counting.

[0360] (10) 2.0 × 10 T2-Luc cells 4 ALyS medium (ALYS505N-O+3% HS) was added to the cells / 50 μL to prepare target cells.

[0361] (blank)

[0362] 8. Luciferase-based cytotoxicity assay after co-culture of 1G4 TCR-T cells with pulsed T2-Luc cells

[0363] (1) 5-2. 1G4 TCR-T cells that had been resting for 24 hours under the conditions of the Maxcyte device were collected into 50 mL conical tubes.

[0364] (2) Centrifuged at 1,500 rpm at room temperature for 5 minutes.

[0365] (3) The supernatant was removed, and the cell pellet was suspended in 10 mL of ALyS medium (ALYS505N-O + 3% HS), followed by cell counting.

[0366] (4) 6.0 × 10 1G4 TCR-T cells 5 ALyS medium (ALYS505N-O+3% HS) was added to give a concentration of cells / 50 μL to prepare effector cells.

[0367] (5) To prepare effector cells (E) and target cells (T) in two 96-well white plates at ratios of 30:1, 10:1, 3:1, and 1:1, first, dispense 50 μL of medium into lanes B to D and F to H according to the conditions in the table below. Then, dispense 9.0 × 10 1G4 TCR-T cells as effector cells (E) into lanes A and E. 5 75uL of each sample was added. Serial dilutions were made in 25uL increments from A to D and E to H using a multichannel pipette.

[0368] (6) 2.0 × 10 T2-Luc cells prepared in 7. 4 Each solution (50 μL) was added according to the conditions in the table below and mixed well.

[0369] (7) Only T2 cells were added according to the conditions in Tables 9 and 10 below, and 20 μL of 10% Triton-X was added to some of the only T2 (Lysis) wells and mixed well.

[0370] (8) The total volume of all wells was adjusted to 100 μL with ALyS medium.

[0371] [Table 9]

[0372] (blank)

[0373] [Table 10]

[0374] (9) The cells were co-cultured for 4 hours in a 37°C, 5% CO2 incubator.

[0375] (10) 100 μL of luciferase assay reagent was added to each well, and the wells were covered with aluminum foil and allowed to react at room temperature for 2 minutes.

[0376] (11) The 96-well plate was placed in a spectrophotometer and the luminescence was measured.

[0377] (blank)

[0378] [result]

[0379] 1. mRNA transposase cloning

[0380] PCR was performed to amplify the insert using a DNA sequence (SEQ ID NO: 3) optimized for mRNA synthesis by Bionics Co., Ltd. as a template for infusion cloning. The insert was loaded onto an agarose gel, and after confirming that a band of the desired size appeared, as shown in Figure 11, it was extracted from the gel.

[0381] Then, Takara IVTpro TMInfusion cloning was performed between the Cloning Kit for mRNA Template (Vector) included in the T7 mRNA Synthesis System and a DNA sequence optimized for mRNA form (Insert, SEQ ID NO: 3). After extracting the cloned plasmid DNA, it was digested with Cla I and Nde I restriction enzymes, and final candidate clones were selected through enzyme mapping. The structure is shown in Figure 12.

[0382] (blank)

[0383] 2. Confirmation of total cell number cultured

[0384] After delivering the 1G4 TCR gene to PBMCs using Maxcyte electroporation, the cells were cultured with feeder cells for 14 days, and the total cell number and cell viability of the cultured cells were determined. As shown in Figure 13, the total cell number showed slight differences in proliferation rate depending on the delivery method of the transposase gene. At the time of electroporation, the initial cell number was 5.0 x 10 6 cells, whereas in the group without electroporation after 14 days of culture, the number of cells was 1.3 × 10 7 The group using the optimized transposase DNA grew to 1.4 × 10 cells. 7 cells, and the group using the optimized transposase mRNA form was 1.8 × 10 7 The cells showed the highest proliferation rate. As shown in Figure 14, the cell viability was 89.4% in the group without electroporation, 91.7% in the group using the optimized transposase in DNA form, and 90.3% in the group using the optimized transposase in mRNA form.

[0385] (blank)

[0386] 3. FACS analysis of optimized transposase gene delivery after Neon electroporation (CD3 / CD28 bead activation, no feeder cells)

[0387] To confirm GFP gene expression in the mRNA and DNA forms of the transposase gene in PBMC cells, they were transfected by Neon electroporation and gene expression was confirmed by FACS analysis on days 7 and 14 of culture. In this experiment, PBMCs were activated with CD3 / CD28 beads before electroporation and cultured without feeder cells. As shown in Figure 15, lymphocytes, singlets, live cells, and CD3 + T cells, GFP + Gating was performed in the order of T cells, and CD3 + The efficiency of GFP gene delivery and expression in T cells was confirmed.

[0388] To confirm the expression of the GFP gene in PBMC cells using the optimized transposase in mRNA form and the optimized transposase in DNA form, CD3 + The percentage of GFP-expressing T cells was examined. As shown in Figure 16, after 7 days of culture, no GFP expression was observed in the negative control (electroporation only) group ("EP only"), and the transposon-only group ("pBat 5098 (pBat 3M3-5M3)"), with GFP expression at 0.06% and 0.02%, respectively, confirmed almost no GFP expression. Meanwhile, the GFP expression rates in the DNA-optimized transposase group were 8.5% and 7.4%, respectively, and in the mRNA-optimized transposase group were 18.2% and 18.5%, respectively, demonstrating a GFP expression rate approximately 10% higher in the mRNA-optimized transposase group. After 14 days of culture, the expression rates in the DNA-optimized transposase group were 3.0% and 3.8%, respectively, while those in the mRNA-optimized transposase group were 8.3% and 8.8%, respectively. Consistent with the results from day 7, the expression rate in the mRNA-optimized transposase group was confirmed to be approximately 5% higher.

[0389] (blank)

[0390] 4. FACS analysis of the optimized transposase gene delivery form after Maxcyte electroporation (without activation and containing feeder cells)

[0391] To confirm 1G4 TCR gene expression in PBMC cells in the mRNA and DNA forms of the transposase gene, gene expression was confirmed by FACS analysis on days 7 and 14 of culture after transfection with Maxcyte electroporation. In this experiment, PBMCs were not activated with CD3 / CD28 beads before electroporation, but were cultured with the addition of feeder cells. The analysis method was as shown in Figure 17, using lymphocytes, singlets, live cells, and CD3 + T cells, 1G4 + Gating was performed in the order of T cells, and CD3 + The efficiency of 1G4 TCR gene delivery and expression into T cells was confirmed.

[0392] (blank)

[0393] 4-1. Comparison of 1G4 TCR gene expression by optimized transposase gene delivery method

[0394] To confirm the expression of the 1G4 TCR gene in PBMC cells using the mRNA and DNA forms of the optimized transposase, CD3 +The percentage of T cells expressing 1G4 TCR was examined. As shown in Figure 18, after 7 days of culture, no 1G4 TCR expression was observed in the negative control group (EP only group) that underwent electroporation alone. Meanwhile, the 1G4 TCR expression rates in the DNA-optimized transposase group were 49.2% and 50.8%, respectively, and in the mRNA-optimized transposase group were 79.0% and 79.0%, respectively, confirming that the expression rate in the mRNA-optimized transposase group was approximately 29% higher. After 14 days of culture, the DNA-optimized transposase group was 54.7% and 57.0%, respectively, while the mRNA-optimized transposase group was 77.2% and 73.5%, respectively. Consistent with the results from day 7, the mRNA-optimized transposase group exhibited an approximately 20% higher expression rate.

[0395] (blank)

[0396] 4-2. Confirmation of the percentage of 1G4 TCR-T intracellular memory-type T cells using optimized transposase gene delivery methods

[0397] Optimized transposase gene delivery induces T cells (CD3 + 1G4 + The percentage of memory T cells was analyzed using the CD45RO and CD62L markers. - CD62L - T cells are T EFF (Effector T cell), CD45RO - CD62L + T cells are T SCM (Stem cell like memory T cell), CD45RO + CD62L - T cells are T EM (Effect memory T cell), CD45RO + CD62L+ T cells are T CM (Central memory T cells), and the positions of T cells of each memory type in the quadrant plot are the same as those shown in Figure 19.

[0398] To compare the rate of memory T cells depending on the delivery form of the transposase gene, the rate of memory T cells was determined on days 7 and 14 after transfection of the 1G4 TCR gene with a transposon carrier. As a result, as shown in Figure 20 and Table 11, in the case of the optimized transposase in DNA form, the rate of memory T cells was significantly higher on day 7 after transfection. SCM and T CM The percentage of the optimized transposase in mRNA form was confirmed to be approximately 97.0%, and it was also confirmed that the percentage was approximately 96.0%, similar to that in DNA form. SCM and T CM The proportion of the mRNA-optimized transposase was approximately 75%, and even with the mRNA-optimized transposase, it was 69%, and both forms were T SCM and T CM We confirmed that the proportion of memory T cells was high.

[0399] [Table 11]

[0400] (blank)

[0401] 4-3. Confirmation of the percentage of CD4 and CD8 in 1G4 TCR-expressing T cells

[0402] To confirm the percentage of CD4 and CD8 in T cells expressing 1G4 TCR due to the transposase gene delivery system, the respective percentages were measured on days 7 and 14 after transfection. As a result, as shown in Figure 21 and Table 12, in the case of the optimized transposase in DNA form, CD8 + and CD4 +The percentages of CD8 and CD8C were 86.3% and 20.8%, respectively, and the optimized transposase mRNA was + and CD4 + The percentages of CD8 and CD9 were 82.1% and 20.5%, respectively. + and CD4 + The ratios of the mRNA-optimized transposase were approximately 80.2% and 20.3%, respectively. + and CD4 + The proportions of CD8 were approximately 82.5% and 17.8%, respectively, with no difference between the two forms. + A high percentage of cells was confirmed.

[0403] [Table 12]

[0404] (blank)

[0405] 4-4. Results of luciferase-based cytotoxicity assay

[0406] For the in vitro killing test of 14-day-cultured 1G4 TCR-T cells, they were co-cultured with T2-Luc cells pulsed with 100 μg / mL of peptide at effector cell:target cell ratios of 30:1, 10:1, 3:1, and 1:1. The luciferase detected after target cell death was calculated as a percentage (%). As shown in Figure 22 and Table 13, in the "No E / P (negative control) group" where 1G4 TCR is not expressed, the luciferase assay expression rate (%) was below 7.0% at the 30:1 ratio and was not detected at the 10:1, 3:1, or 1:1 ratios. When 1G4 TCR was introduced using the transposon system, the luciferase expression rates (%) for the group using the optimized transposase in mRNA form were 67.3%, 48.1%, and 13.4% at 30:1, 10:1, and 3:1 ratios, respectively, and were not measured at 1:1. The group using the optimized transposase in DNA form was confirmed to have 54.2% and 32.2% at 30:1 and 10:1 ratios, respectively, but not measured at 3:1 or 1:1. Consequently, cytotoxic activity was confirmed to be more than 10% higher in 1G4 TCR-T cells delivered with the optimized transposase in mRNA form.

[0407] [Table 13]

[0408] As described above, when genes are delivered to PBMC cells via the transposon system, GFP gene expression was confirmed by FACS analysis on days 7 and 14 of culture to confirm whether the transposase delivery form (mRNA or DNA) affected GFP gene expression after transfection with Neon electroporation. On day 7, the mRNA-optimized transposase group showed a GFP expression rate approximately 10% higher than the DNA-optimized transposase group, and on day 14, the mRNA-optimized transposase group showed a GFP expression rate approximately 5% higher. Furthermore, the 1G4 TCR gene was transfected using a different electroporator, the Maxcyte electroporator, and 1G4 TCR gene expression was confirmed by FACS analysis on days 7 and 14 of culture. The negative control group (EP only), which received only electroporation, showed no expression of the 1G4 TCR gene. On the other hand, 7 days after transfection, the expression of the 1G4 TCR gene in the mRNA-optimized transposase group was confirmed to be approximately 29% higher than that in the DNA-optimized transposase group. After 14 days of culture, the expression rate in the mRNA-optimized transposase group was confirmed to be approximately 20% higher than that in the DNA-optimized transposase group. SCM and T CMThe percentage of memory cells and the percentage of CD4 and CD8 T cells were confirmed to be consistent with the gene delivery format. For the in vitro killing test of 1G4 TCR-expressing T cells cultured for 14 days, T2-Luc cells pulsed with 100 μg / mL of peptide were co-cultured at effector cell:target cell ratios of 30:1, 10:1, 3:1, and 1:1. Luciferase activity was detected after target cell death and calculated as a percentage. In the "No E / P" (negative control) group, which does not express the 1G4 TCR, no luciferase activity was detected under any condition. Cytotoxicity analysis results showed that the mRNA-optimized transposase group had a 10% or higher cytotoxicity than the DNA-optimized transposase group under all ratio conditions. These results confirm that the transposase gene delivery format in the transposon system is superior in gene expression and cytotoxicity when delivered in mRNA form compared to DNA form.

[0409] (blank)

[0410] Example E. Confirmation of gene delivery efficiency of pBat transposase with added NLS (nuclear localization sequence) gene

[0411] After being translated into protein, the transposase is transported into the cell nucleus. To increase the efficiency of transposase transport into the nucleus, we added a nuclear localization sequence (NLS) gene to the 5' or 3' end of the optimized transposase, and then confirmed the gene delivery efficiency.

[0412] SV40 and C-myc were selected as NLSs to be added to the 5' or 3' end of the optimized transposase, and the DNA sequences of SV40 and C-myc were also optimized to ensure good expression in T cells.

[0413] [Table 14]

[0414] As shown in Figures 22 and 23, optimized SV40 or C-myc was added to the 5' or 3' end of the optimized transposase (SEQ ID NO: 2) to optimize the optimized transposase.

[0415] (blank)

[0416] 1. Confirmation of GFP gene delivery efficiency ×

[0417] The NLS was added to the 5' or 3' end of the optimized transposase, and the GFP gene delivery efficiency of the optimized transposase was confirmed. To confirm the GFP gene delivery efficiency, Jurkat cells (1 × 10 5 Neon transfection (electroporation) was performed using a 24-well plate (1 well per well) at 1,600 V, 10 ms, and 3 pulses. Plasmids were used, with the transposon and DNA form optimized transposase listed in Table 15 below, at a transposon:transposase ratio of 1 μg:1 μg. One, seven, and 14 days after transfection, the level of GFP expression in the cells was confirmed using a fluorescent microscope and FACS. Jurkat cells cultured in a 24-well plate were transferred to a T25 flask one day after Neon transfection.

[0418] [Table 15]

[0419] As shown in Figures 24a and 24b, one day after electroporation, there was no difference in the type or position (5' or 3') of NLS. As shown in Figures 25a and 25b, seven days after electroporation, high levels of total GFP and high-intensity GFP expression were observed when c-myc was placed at the 5' end. The GFP expression rate 14 days after electroporation was the same as that shown in Figure 26.

[0420] To confirm the efficiency of the NLS transposase in Jurkat cells, FACS analysis was performed on GFP expression 1 and 7 days after electroporation. As shown in Figure 27, 1 day after electroporation, 3'SV showed the highest GFP expression, while 7 days after electroporation, 5'Myc showed the highest GFP expression, followed by 3'Myc and the optimized transposase without an NLS. Furthermore, as shown in Figure 28, the high-intensity GFP expression rate was 8% for the optimized transposase without an NLS, 17% for 5'Myc, and 11% for 3'Myc. The 5'Myc-inserted transposase exhibited the most stable transfection efficiency in Jurkat cells.

[0421] Jurkat cells expressing GFP on day 14 after electroporation were sorted on day 15 and cultured for an additional 9 days. As a result, as shown in Figures 29a and 29b, it was confirmed that the GFP-expressing cells were stably maintained at a GFP expression rate of 95%.

[0422] (blank)

[0423] 2. Confirmation of 1G4 TCR gene delivery efficiency

[0424] The NLS was added to the 5' or 3' end of the transposase, and the optimized transposase was used to confirm the efficiency of 1G4 TCR gene delivery in PBMCs. Specifically, PBMCs (LK048,17011) stored in an LN2 tank were used. Transfection (electroporation) was performed using Maxcyte to OC100 × 2 and 5 × 10 resting T cells using the 14-3 protocol. 6 The transfection was performed using PBMC / 50 μL Opti-MEM buffer. The transposon vector and DNA-optimized transposase shown in Table 16 below were used as plasmids at a transposon:transposase ratio of 8 μg:2 μg. FACS analysis was performed 7 and 14 days after transfection.

[0425] [Table 16]

[0426] The efficiency of 1G4 TCR gene delivery in PBMCs was confirmed using 8 μg / well transposon and 2 μg / well transposase. Seven days after transfection, FACS analysis was performed, confirming a high 1G4 TCR expression rate in the C-myc group, as shown in Figures 30a-30d. Fourteen days after transfection, FACS analysis was performed, confirming a high 1G4 TCR expression rate in the C-myc group, as shown in Figures 31a-31d.

[0427] (blank)

[0428] Example F. Comparison of gene delivery efficiency of pBat transposon and lentivirus using CD19 CAR

[0429] The gene delivery efficiency of pBat transposon as a non-viral vector and lentivirus as a viral vector was compared.

[0430] (blank)

[0431] [method]

[0432] 1. DNA and cells

[0433] To compare the pBat transposon as a non-viral vector and the lentivirus as a viral vector, the pBat transposon plasmid vector and the pBat optimized transposase plasmid vector (SEQ ID NO: 14) were used for the pBat transposon as a non-viral vector, and the lentiviral CD19 CAR transfer plasmid vector and the lentiviral packaging vector were used for lentivirus production.

[0434] The target cells used were PBMC (LK032 / LK053), Lenti-X 293T (Takara, Cat. No. 632180, Lot No. AIY0002S), Jurkat, Clone E6-1 (ATCC, Cat. No. TIB-152, Lot No. 70017560), and NALM6, a GFP-luciferase reporter cell line.

[0435] (blank)

[0436] 2. Construction of pBat CD19 CAR plasmid vector

[0437] To compare gene delivery efficiency with lentiviral transfer vectors during CAR-T production, the EGFP gene portion of the pBat transposon plasmid vector containing EGFP was replaced with CD19 CAR by cloning as follows to create the pBat CD19 CAR plasmid vector:

[0438] (1) The CD19 CAR gene used as an insert was prepared by PCR using primers and a plasmid containing the CD19 CAR gene as a template. The resulting DNA was then loaded onto an agarose gel, and the DNA of the band of the desired size was eluted.

[0439] (2) The pBat transposon plasmid vector containing EGFP was treated with the restriction enzymes BstB I and EcoR I, and the DNA of the band of the desired size was eluted.

[0440] (3) Gibson assembly cloning was performed using the DNA obtained in (1) and (2).

[0441] (4) To confirm the identity of pBat CD19 CAR among the clones for which cloning was completed, we performed enzyme mapping using the restriction enzyme EcoR V, selected candidate clones, and finally confirmed their sequencing.

[0442] (blank)

[0443] 3.PBMC activation

[0444] (1) RPMI medium (RPMI + 10% FBS + 1x P / S) was prepared by pre-warming it in a 37°C water bath.

[0445] (2) PBMC LK032 and LK053 were removed from the nitrogen tank and quickly thawed in a 37°C water bath.

[0446] (3) Two 50 mL tubes were prepared, and 30 mL of culture medium was added to each tube. Then, the thawed cells from the PBMC LK032 and PBMC LK053 vials were placed in each 50 mL tube.

[0447] (4) After centrifugation at 1,500 rpm for 5 minutes and removal of the supernatant, the cells were suspended in 40 mL of RMPI medium and counted.

[0448] (5) 1.2 × 10 PBMCs in a T175 flask 8 Put 1.2 x 10 pieces 8 RPMI medium was added so that the volume became 120 cells / mL.

[0449] (6) IL-2 was added to a final concentration of 20 IU / mL, and the solution was then transacted. TM to 2.0 x 10 7 100 μL of IL-2 was added per cell (1.0 × 10 6 The concentration of IU / mL was diluted 1 / 10 with culture medium, resulting in 1.0 x 10 5 IU / mL was used.)

[0450] (7) Cultured in a 5% CO2 incubator at 37°C for 2 days.

[0451] (blank)

[0452] 4. PBMC electroporation

[0453] A non-viral transposon system containing the CD19 CAR gene was delivered into activated PBMCs as follows:

[0454] (1) Activated PBMCs were collected in a conical tube and centrifuged at 300×g for 10 minutes, and the supernatant was removed.

[0455] (2) The cell pellet was suspended in 20 mL of medium (ALYS505N-O + 3% HS), and the cells were counted.

[0456] (3) After centrifugation at 1,500 rpm for 5 minutes at room temperature, the supernatant was removed and the cell pellet was suspended in 20 mL of DPBS.

[0457] (4) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was completely removed.

[0458] (5) The supernatant was removed, and the cell pellet was suspended in 5 mL of Opti-MEM medium.

[0459] (6) The mixture was centrifuged at room temperature at 1,500 rpm for 5 minutes, and the supernatant was completely removed.

[0460] (7) Cell pellet: 5.0 × 10 6 The cells were suspended in 50uL of warm Opti-MEM medium at a concentration of 1 / 50uL.

[0461] (8) 5 μg of transposon vector and transposase vector were added to the tubes in (7) as shown in Table 17 below. However, no vector was added to the control group.

[0462] [Table 17]

[0463] **N=2 per group. In the case of LK053, the treatment progressed only under the condition of IL-2 200IU / mL.

[0464] (9) 5 x 10 cells of the cell suspension (7) in 2 x OC100 assemblies 6 Each 50 μL was carefully added to avoid creating bubbles.

[0465] (10) Resting T cells 14-3 protocol was selected with Maxcyte STx for electroporation.

[0466] (11) The OC100x2 assembly from (9) was inserted into the chamber in the STx and electroporated according to the protocol.

[0467] (12) After electroporation, the cell suspension from the OC100x2 assembly was transferred to a T25 flask (5x10 6 pcs / 50uL / well).

[0468] (13) 2 wells of OC100 were washed with 50 μL of Opti-MEM medium and added to each well of the plate in (11).

[0469] (14) The cells were allowed to recover for 20 minutes in an incubator at 37°C and 5% CO2.

[0470] (15) 10 mL of complete medium (ALYS505N-O + 3% HS + 200 IU / mL IL-2) was carefully added to each T25 flask containing PBMCs, and the flask was again placed in a 37°C, 5% CO2 incubator and cultured for 2 days.

[0471] (16) Two days after electroporation, 20 mL of medium (ALYS505N-O + 3% HS + 1 × P / S) was added to the control group, and 10 mL of medium was added to the CAR group. IL-2 was then added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0472] (17) Five days after electroporation, IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured at 37°C in a 5% CO2 incubator.

[0473] (18) Seven days after electroporation, the cells in culture were suspended. 500 μL of the cell suspension was transferred to a new tube for FACS analysis. The remaining cell suspension was diluted to 5.0 × 10 6 Fresh medium (ALYS505N-O + 3% HS + 1xP / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0474] (19) Nine days after electroporation, the cells in culture were suspended, and half of the suspension was stored as stock after removing the supernatant and adding CS10 medium. It was then stored in a deep freezer for 1 day and then transferred to a nitrogen tank.

[0475] (20) The remaining half of the suspension was 5.0 × 10 6 Fresh medium (ALYS505N-O + 3% HS + 1x P / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL. The cells were cultured in a 37°C, 5% CO2 incubator.

[0476] (21) 12 days after electroporation, the cells in culture were suspended and then 5.0 × 10 6 Fresh medium (ALYS505N-O + 3% HS + 1xP / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0477] (22) 14 days after electroporation, the cells in culture were suspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. 2.0 × 10 cells per group were used for in vitro killing analysis. 7The cells were collected and cultured in 20 mL of resting medium (RPMI1640 + 10% FBS + 1x P / S). The supernatant was removed from the remaining suspension, which was then stocked with CS10 medium and stored in a deep freezer for 1 day before being transferred to a nitrogen tank.

[0478] (blank)

[0479] 5. Lentiviral Transduction

[0480] Lentivirus containing the CD19 CAR gene was delivered into activated PBMCs as follows:

[0481] (1) Activated PBMCs were collected in a 50 mL conical tube and centrifuged at 300 × g for 10 minutes at room temperature.

[0482] (2) The supernatant was removed, and the cell pellet was suspended in 20 mL of culture medium (RPMI1640 + 10% FBS + 1x P / S) and counted.

[0483] (3) PBMCs 5.0 × 10 6 The cells were suspended in culture medium at a density of 2 cells / 2 mL.

[0484] (4) 5.0 × 10 PBMCs in a 12-well plate 6 Only the required number of wells were seeded at 2 mL / well.

[0485] (5) Each well was treated with 10 μg / mL protamine and 4 MOI lentivirus.

[0486] (6) Spinoculation was performed at 1,200 × g for 90 minutes.

[0487] (7) After centrifugation, the PBMCs in each well were transferred to a T75 flask.

[0488] (8) Add culture medium to a T75 flask so that the total volume becomes 10 mL, and the test groups are as shown in Table 18 below.

[0489] [Table 18]

[0490] (9) IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured at 37°C in a 5% CO2 incubator. (10) Two days after transduction, the cells were suspended in the culture, and the cell suspension was diluted with 5.0 × 10 6 Fresh medium (RPMI1640 + 10% FBS + 1xP / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0491] (11) Five days after transduction, fresh medium (RPMI1640 + 10% FBS + 1x P / S) was added in an amount equal to the culture medium volume, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0492] (12) Seven days after transduction, the cells in culture were suspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. The remaining cell suspension was diluted to 5.0 × 10 6 Fresh medium (RPMI1640 + 10% FBS + 1xP / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0493] (13) Nine days after transfection, the cells were suspended in culture. Half of the suspension was used as a stock, after which the supernatant was removed. The stock was then transferred to a nitrogen tank after adding CS10 medium and storing in a deep freezer for 1 day. The remaining half of the suspension was transferred to a nitrogen tank after adding 5.0 × 10 6Fresh medium (RPMI1640 + 10% FBS + 1x P / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL. The cells were cultured in a 37°C, 5% CO2 incubator.

[0494] (14) 12 days after transfection, the cells in culture were suspended and then 5.0 × 10 6 Fresh medium (RPMI1640 + 10% FBS + 1xP / S) was added to a concentration of 100 cells / mL, and IL-2 was added to a final concentration of 200 IU / mL or 400 IU / mL, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0495] (15) 14 days after transduction, the cells in culture were suspended, and 500 μL of the cell suspension was transferred to a new tube for FACS analysis. 2.0 × 10 cells per group were used for in vitro killing analysis. 7 The cells were collected and cultured in 20 mL of resting medium (RPMI1640 + 10% FBS + 1x P / S). The remaining suspension was stocked after removing the supernatant and adding CS10 medium. It was then stored in a deep freezer for 1 day and then transferred to a nitrogen tank.

[0496] (blank)

[0497] 6.FACS analysis

[0498] FACS analysis was performed 7, 10 and 14 days after electroporation.

[0499] (1) After suspending the cells in each flask, 3 mL of each was transferred to a FACS tube.

[0500] (2) After centrifugation at 1,500 rpm at 4°C for 5 minutes and removal of the supernatant, the cells were suspended in 1 mL of washing buffer (PBS + 2% FBS).

[0501] (3)(2) was repeated once more to wash.

[0502] (4) The mixture was centrifuged at 1,500 rpm at 4°C for 5 minutes, and the supernatant was removed.

[0503] (5) 5 μL of human TruStain FcX and 95 μL of FACS buffer were added to each tube, and the mixture was allowed to react at room temperature for 5 minutes.

[0504] (6) 2uL of each antibody (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, anti-CCR7, anti-CD62L, anti-FLAG TAG) was added to each tube, and the mixture was incubated at room temperature in the dark for 30 minutes.

[0505] (7) 1 mL of FACS buffer (PBS + 2% FBS) was added to each well, and the mixture was centrifuged at 4°C, 1,500 rpm for 5 minutes.

[0506] (8) After removing the supernatant, the cells were suspended in 1 mL of FACS buffer (PBS + 2% FBS).

[0507] (9) The mixture was centrifuged at 4°C and 1,500 rpm for 5 minutes, and the supernatant was removed.

[0508] (10) The cells were suspended in 200 μL of FACS buffer (PBS + 2% FBS) containing 1× DAPI, and FACS analysis was performed.

[0509] (blank)

[0510] 7. Luciferase-based cytotoxicity analysis after co-culture of CD19 CAR-T cells with GFP-luciferase reporter NALM6 cells

[0511] (1) GFP-luciferase reporter NALM6 cells cultured in a T75 flask were collected into a 50 mL tube.

[0512] (2) Centrifuged at 1,500 rpm at room temperature for 5 minutes.

[0513] (3) The supernatant was removed, and the cell pellet was suspended in 5 mL of RPMI medium and counted.

[0514] (4) 2.0 × 10 GFP-luciferase reporter NALM6 cells 4 RPMI medium (10% FBS + 1% P / S) was added to prepare a cell density of 100 cells / 100 μL.

[0515] (5) After counting the two types of resting CD19 CAR-T cells (cells prepared in 6. and cells prepared in 7.), 6.0 × 10 5 The cells were prepared so that the number of cells was 100 μL.

[0516] (6) As shown in Figure 33, 100 μL of RPMI medium (10% FBS + 1% P / S) was pre-filled into lanes B to D and F to H of two white 96-well flat plates, and 150 μL (9.0 × 10) of the CD19 CAR-T cells (5) was added to lanes A and E. 5 100 pieces) and serially diluted the PBMCs in three steps using a multichannel pipette at 50uL aliquots to achieve NALM6 / PBMC ratios of 30:1, 10:1, 3:1, and 1:1 (Plate 1: for the transposon system, Plate 2: for the lentivirus system).

[0517] (7) The NALM6 cells prepared in (4) were placed in lanes A to D, 100 μL each, as shown in Figure 33, and co-cultured at 200 μL / well.

[0518] (8) In the PBMC-only group (lanes E to H), 100 μL of complete RPMI was added to adjust the volume to 200 μL per well.

[0519] (9) As shown in Figure 33, 100 μL of only NALM6 cells from 4) and 100 μL of complete RPMI were added to lanes 11 and 12, and the total volume was adjusted to 200 μL per well.

[0520] (10) For lysis, 20 μL of 10% Triton-X was added to the 12-lane well and mixed well.

[0521] (11) The reaction was allowed to proceed by culturing the cells in a 5% CO2 incubator at 37°C for 4 hours.

[0522] (12) Thaw the required amount of Bright-Glo reagent mixture (100 mL of Bright-Glo luciferase assay buffer + substrate) in a 37°C, 5% CO2 incubator.

[0523] (13) After thoroughly suspending each well in the co-culture wells of a 96-well plate, 100 μL was removed.

[0524] (14) 100 μL of Bright-Glo reagent was added to each well, and the wells were covered with aluminum foil and left at room temperature for 2 minutes.

[0525] (15) After the 96-well plate was placed in a spectrophotometer, luminescence was measured.

[0526] (blank)

[0527] [result]

[0528] 1. pBat CD19 CAR Plasmid Cloning Results

[0529] To clone the pBat CD19 CAR plasmid vector, it was digested with BstBI and EcoR I, and then Gibson assembly cloning was performed with the CD19 CAR gene used as an insert. To confirm successful cloning, candidate clones were selected after enzyme mapping using the restriction enzyme EcoR V as shown in Figure 34, and finally confirmed by sequencing.

[0530] (blank)

[0531] 2. Confirmation of total cell number cultured

[0532] To produce CD19 CAR-T cells, genes were delivered to PBMCs using lentivirus and transposon transduction and electroporation, respectively, and the total cell count and cell viability of the cultured T cells were determined after 7 and 14 days of culture. As shown in Figure 35 and Table 19, the total cell count showed a tendency for viability to decrease after electroporation on day 7 in the transposon-treated group compared to the control group (not treated with a vector). However, the cell proliferation rate increased over time.

[0533] [Table 19]

[0534] As shown in Figure 36 and Table 20, the cell viability was 85.8% to 94.4% for lentivirus and 87.3% to 94.4% for transposon on day 7, and 77.7% to 96.2% for lentivirus and 91.4% to 93.4% for transposon on day 14, confirming that the viability of T cells produced using lentivirus vectors was slightly lower than that of T cells produced using transposon vectors.

[0535] [Table 20]

[0536] 4. Comparison of CD19 CAR protein expression by gene transfer vector

[0537] The expression of CD19 CAR was confirmed by FACS using an anti-FLAG tag antibody against the FLAG tag protein expressed by the FLAG tag gene located between the leader sequence and CD19scFv in the CD19 CAR gene. The analysis method was as shown in Figure 37, using singlets, live cells, lymphocytes, and CD3 + T cells, FLAG + Gating was performed in the order of T cells, and CD3+ The efficiency of CD19 CAR gene delivery and expression into T cells was confirmed.

[0538] (blank)

[0539] 4-1.LK032 PBMC

[0540] After transfection of the CD19-CAR gene into LK032 PBMCs using lentiviral and transposon vectors, CD3 + The percentage of T cells expressing FLAG (CD19-CAR) was determined. As a result, as shown in Figure 38 and Table 21, it was confirmed that FLAG (CD19-CAR) was not expressed in the negative control group (activation only group) in which only activation was performed with each transfectant, and the negative control group (EP only group) in which only electroporation was performed. After transfection with each transfectant, the percentage of cells expressing FLAG (CD19-CAR) in the groups cultured for 7 days with 200 IU / mL IL-2 was 41.0% and 54.6% for the lentivirus and transposon transfectants, respectively, confirming that the percentage was approximately 14% higher in the cells using the transposon transfectant. In the group cultured with 400 IU / mL IL-2, the percentages were 43.4% and 59.6%, respectively, confirming that the percentage was approximately 15% higher in the cells using the transposon transfectant. Furthermore, after 14 days of culture, the percentage of cells expressing FLAG (CD19-CAR) in the group with 200 IU / mL IL-2 added was 67.30% and 61.0%, respectively, indicating a slightly lower level in the transposon-transfected group, but in the group with 400 IU / mL IL-2 added, the percentages were 62.5% and 63.5%, respectively, indicating a similar level. Meanwhile, the percentage of cells expressing FLAG at a high intensity was higher in the transposon-transfected group on day 7, but was higher in the lentiviral-transfected group on day 14.

[0541] [Table 21]

[0542] 4-2.LK053 PBMC

[0543] After transfection of the CD19 CAR gene into LK053 PBMCs using lentiviral and transposon vectors, CD3 + The percentage of T cells expressing FLAG (CD19 CAR) was determined. As shown in Figure 39 and Table 22, the negative control group (activation only group) and the negative control group (EP only group) were confirmed to not express FLAG (CD19 CAR) for each transfectant. After transfection, the percentage of cells expressing FLAG (CD19 CAR) in the groups cultured for 7 days with 200 IU / mL IL-2 was 44.3% and 59.0% for the lentivirus and transposon transfectants, respectively, demonstrating a 15% higher percentage for the transposon transfectant. After 14 days of culture, the percentage of cells expressing FLAG (CD19 CAR) in the group cultured for 14 days with 200 IU / mL IL-2 was 49.1% and 57.3%, respectively, demonstrating a 8% higher percentage for the transposon transfectant. Furthermore, unlike the LK032 case, the proportion of cells expressing FLAG at high intensity among the cells expressing FLAG was confirmed to be high on both days 7 and 14, indicating that the transposon carriers were high.

[0544] [Table 22]

[0545] 5. Confirmation of the proportion of memory T cells by gene transfer

[0546] T cells transformed with two vectors (CD3 +The percentage of memory T cells was analyzed using the CD45RA and CD62L markers. + CD62L - T cells are T EFF (Effector T cell), CD45RA - CD62L + T cells are T EM (Effect memory T cell), CD45RA - CD62L + T cells are T CM (Central memory T cell), CD45RA + CD62L + T cells are T SCM (Stem cell-like memory T cell), and the position of each memory type T cell in the quadrant plot is as shown in Figure 40.

[0547] (blank)

[0548] 5-1.LK032 PBMC

[0549] After transfection of LK032 PBMCs with the CD19 CAR gene using lentiviral and transposon vectors, the percentage of total intracellular memory T cells cultured on days 7 and 14 was examined. As a result, as shown in Figure 41 and Table 23, the negative control group (activation only group) in which only activation was performed and the negative control group (EP only group) in which only electroporation was performed for each vector showed a significant increase in T cells. SCM and T CM The total percentage of T cells was similar, ranging from 56% to 65% on average. In the case of lentiviral vectors, on day 7 after transfection, T cells were observed in all groups cultured with 200 IU / mL and 400 IU / mL IL-2. SCM and T CMThe total percentage of T cells was approximately 61%, while in the case of transposon-transduced cells, the percentage was approximately 80% to 82% in all groups cultured with 200 IU / mL or 400 IU / mL IL-2, which was approximately 20% higher than that of lentiviral-transduced cells. SCM and T CM The total percentage of T was confirmed to be about 43% to 48%, and in the case of transposon carriers, it was confirmed to be slightly higher at 47% to 56%. SCM and T CM We confirmed that the proportion of memory T cells was high.

[0550] [Table 23]

[0551] 5-2.LK053 PBMC

[0552] After transfection of LK053 PBMCs with the CD19 CAR gene using lentiviral and transposon vectors, the percentage of total intracellular memory T cells cultured on days 7 and 14 was examined. As a result, as shown in Figure 42 and Table 24, the negative control group for lentiviral vectors showed significantly higher T cell numbers than the control group. SCM and T CM The total percentage of T was approximately 46% to 57% on day 7, while the negative control group of transposon transmitters was approximately 65%. SCM and T CM It was confirmed that the proportion of memory T cells in the negative control group was high. SCM and T CM The total percentage of T cells was confirmed to be approximately 22% to 33% for lentiviral transducers and 43% for transposon transducers on day 14. SCM and T CMIn the test group, the proportion of memory T cells was confirmed to be high. In the case of lentiviral delivery, the proportion of memory T cells was high in all groups cultured with 200 IU / mL and 400 IU / mL IL-2 on the 7th day. SCM and T CM The percentage of T cells was approximately 57%-62% in the transposon-transduced group, while in the group cultured with 200 IU / mL IL-2, it was approximately 75%, which was approximately 15% higher than in the lentiviral-transduced group. On day 14, the percentage was approximately 35%-42% in the lentiviral-transduced group, while in the transposon-transduced group, it was slightly higher at 46%, showing that the T cells in the transposon-transduced group were generally higher. SCM and T CM We confirmed that the proportion of memory T cells was high.

[0553] [Table 24]

[0554] 6. Identification of memory T cells within T cells expressing FLAG (CD19 CAR)

[0555] The memory type of T cells transduced with the two delivery systems and expressing FLAG (CD19 CAR) was confirmed. As shown in Figure 43 and Table 25, the lentiviral delivery system increased T cells in both LK032 and LK053 PBMCs on day 7. SCM and T CM The total percentage of T cells was approximately 59%-65% for transposon-transduced cells, while it was approximately 78%-85% for transposon-transduced cells, approximately 20% higher than for lentiviral-transduced cells. However, on day 14, the lentiviral-transduced cells showed a T cell count of 100 in both LK053 and LK032. SCM and T CM The total percentage of the two types of carriers was approximately 40% to 54%, while that of the transposon carrier was approximately 48% to 57%, confirming that the two types of carriers were similar.

[0556] [Table 25]

[0557] 7. Confirmation of the proportion of CD4 and CD8 T cells expressing FLAG (CD19 CAR)

[0558] To confirm the percentage of CD4 and CD8 T cells transduced with the two delivery systems and expressing FLAG (CD19-CAR), the respective percentages were measured on days 7 and 14 after transduction. As a result, as shown in Figure 44, Figure 45, and Table 26, in the case of lentiviral delivery, on day 7 after transduction, CD8 T cells were detected in all groups cultured with 200 IU / mL and 400 IU / mL IL-2. + and CD4 + The percentages of CD8 and CD9 were 28% to 38% and 45% to 54%, respectively. + and CD4 + The percentages of CD8 were 56%-59% and 53%-59%, respectively, and the percentage of CD8 was 5%-10% higher than that of lentivirus. + and CD4 + The ratios of CD8 to CD8 were approximately 39% to 51% and 58% to 75%, respectively. + and CD4 + The percentages of CD8 were approximately 64% to 72% and 57% to 61%, respectively, and were higher than those of lentivirus. + The proportion of patients with HIV infection was found to be approximately 20% to 25% higher.

[0559] [Table 26]

[0560] 8. In Vitro Killing Assay Results

[0561] For in vitro killing tests of CAR-T cells cultured for 14 days, they were co-cultured with GFP-luciferase reporter-NALM6 cells at ratios of 30:1, 10:1, 3:1, and 1:1 (effector cells:target cells), and the luciferase detected after target cell death was calculated as a percentage (%). As a result, as shown in Figure 46 and Table 27, in the case of lentiviral transfection, the luciferase assay expression rates (%) in the "No Transduction (negative control) group" that did not express CD19 CAR were measured at ratios of 30:1, 10:1, 3:1, and 1:1, respectively, ranging from 5.3% to 17.2%, 3.4% to 8.9%, 1.0% to 7.4%, and 0% to 9.3%. However, in the lentiviral transfection group, the luciferase expression rates (%) in the lentiviral transfection group were measured at ratios of 30:1, 10:1, 3:1, and 1:1, respectively, ranging from 52.8% to 55.9%, 41.5% to 44.6%, 21.4% to 23.6%, and 0% to 5.2%, confirming a dose-dependent increase in CD19 CAR-T cells.

[0562] [Table 27]

[0563] Furthermore, in the case of transposon carriers, as shown in Figure 47 and Table 28, the luciferase assay expression rates (%) for the "No E / P (negative control) group" not expressing CD19 CAR were measured at ratios of 30:1, 10:1, 3:1, and 1:1, respectively, ranging from 7.8% to 40.8%, 9.9% to 29.9%, 0% to 17.3%, and 0% to 24.3%. However, for the transposon-transformed group, the luciferase expression rates (%) for ratios of 30:1, 10:1, 3:1, and 1:1 were measured at ratios of 63.8% to 78.1%, 41.5% to 64.7%, 4.5% to 42.5%, and 0% to 26.6%, respectively, confirming a dose-dependent increase in CD19 CAR-T cells. In conclusion, we confirmed that CD19 CAR-T cells produced with transposon vectors had approximately 20% higher in vitro killing activity against actual target cells than CD19 CAR-T cells produced with lentivirus under all conditions.

[0564] [Table 28]

[0565] As explained above, the stability of the self-inactivating vector (SIN vector) system has been verified. We attempted to confirm the performance of the pBat transposon vector by analyzing the gene delivery efficiency between the widely used third-generation lentivirus vector as a viral vector and the pBat transposon vector as a non-viral vector, as well as the percentage of CD19 CAR-expressing cells depending on the culture period. As a result, PBMCs transfected with the CD19 CAR gene using lentiviral and transposon vectors were cultured for 14 days, and the cell viability was examined. It was confirmed that T cells produced using lentiviral vectors had a slightly lower viability than T cells produced using transposon vectors. Furthermore, on day 7 of culture, CD3 +The percentage of T cells expressing FLAG (CD19 CAR) was confirmed to be approximately 15% higher in both LK032 and LK053 PBMCs in the transposon-transduced cells. On day 14, the transposon-transduced group showed a similar or slightly higher FLAG (CD19-CAR) expression rate compared to the lentivirus-transduced group. Furthermore, the T cells (CD3 + and FLAG + The proportion of memory T cells was analyzed using CD45RA and CD62L markers. Both LK032 and LK053 PBMCs were found to be T cells in the transposon carriers. SCM and T CM The results showed that the percentage of memory T cells was high, and the percentage of CD4 and CD8 T cells in T cells expressing FLAG (CD19 CAR) was higher than that of lentivirus on day 7. + The percentage of T cells was approximately 5%-10% higher, and by day 14 it was approximately 20%-25% higher. Finally, the in vitro killing activity of CAR-T cells cultured for 14 days was examined, and it was confirmed that T cells produced with transposon vectors had approximately 20% higher in vitro killing activity against actual target cells under all conditions than T cells produced with lentiviral vectors. Based on these results, it was determined that the pBat transposon vector can be used as a gene delivery vehicle that can replace lentiviral delivery vehicles.

[0566] (blank)

[0567] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting.

[0568] (blank)

[0569] In Table 29 below, the bold part of SEQ ID NO: 14 is the sequence of SEQ ID NO: 2, the bold part of SEQ ID NO: 15 is the sequence of SEQ ID NO: 3, the underlined part of SEQ ID NO: 16 is the sequence of SEQ ID NO: 9, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 17 is the sequence of SEQ ID NO: 9, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 18 is the sequence of SEQ ID NO: 11, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 19 is the sequence of SEQ ID NO: 11, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 20 is the sequence of SEQ ID NO: 9, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 21 is the sequence of SEQ ID NO: 9, the bold part is the sequence of SEQ ID NO: 2, the underlined part of SEQ ID NO: 22 is the sequence of SEQ ID NO: 11, the bold part is the sequence of SEQ ID NO: 2, and the underlined part of SEQ ID NO: 23 is the sequence of SEQ ID NO: 11, the bold part is the sequence of SEQ ID NO: 2.

[0570] [Table 29-1]

[0571] [Table 29-2]

[0572] [Table 29-3]

[0573] [Table 29-4]

[0574] [Table 29-5]

[0575] [Table 29-6]

[0576] Table 29-7

[0577] Table 29-8

[0578] Table 29-9

[0579] Table 29-10

[0580] Table 29-11

[0581] Table 29-12

[0582] Table 29-13

[0583] Table 29-14

[0584] Table 29-15

[0585] Table 29-16

[0586] Table 29-17

[0587] Table 29-18

[0588] Table 29-19

[0589] Table 29-20

[0590] Table 29-21

[0591] Table 29-22

[0592] Table 29-23

[0593] Table 29-24

[0594] Table 29-25

[0595] Table 29-26 [Industrial Applicability]

[0596] The present invention relates to a highly active transposase protein of a transposon system and its uses. By improving the activity of the transposase, genes can be delivered effectively, and the protein can be usefully used in the development of genome-modified cell lines that express various genes.

[0597] Furthermore, when T cells are transformed using the hyperactive transposase of the present invention, cytotoxic T cells (CD8 + The proportion of T cells increases, and T cells persist in the body. CM (Central memory T cell), T TCM Since the proportion of memory-type T cells (stem cells like memory T cells) has increased, it is expected that the transposon system of the present invention can be used to produce TCR-T cells and CAR-T cells with good in vivo persistence, which has industrial applicability.

Claims

1. A transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO:2 or SEQ ID NO:

3.

2. The transposase expression vector of claim 1, wherein the nucleic acid sequence encoding the transposase further comprises a nucleic acid sequence encoding a nuclear localization signal (NLS).

3. The transposase expression vector according to claim 2, wherein the nucleic acid sequence encoding the nuclear localization signal is one or more of the nucleic acid sequences shown in SEQ ID NOs: 4 to 11.

4. The transposase expression vector of claim 2, wherein the nucleic acid sequence encoding the nuclear localization signal is contained in the 5' to 3' direction at the 5' or 3' end of the nucleic acid sequence encoding the transposase.

5. mRNA encoding the transposase represented by SEQ ID NO:

12.

6. The mRNA according to claim 5, wherein the mRNA is produced by in vitro transcription using the transposase expression vector according to claim 1.

7. The mRNA according to claim 6, wherein the transposase expression vector is a transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO:

3.

8. A transposase expressed from the transposase expression vector of claim 1 or the mRNA encoding the transposase of claim 5.

9. When the transposase expression vector comprises a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 3, The transposase according to claim 8, wherein the transposase is expressed from mRNA prepared by in vitro transcription using a transposase expression vector.

10. The transposase of claim 8, characterized in that the transposase comprises the amino acid sequence shown in SEQ ID NO:

13.

11. (a) a transposon-based vector into which target DNA has been inserted; and (b) a transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3; An mRNA encoding the transposase set forth in SEQ ID NO: 12, or A transposon system for targeted DNA delivery comprising the transposase of claim 8.

12. The transposon system according to claim 11, wherein the transposon vector and the transposase expression vector, or the mRNA encoding the transposase, or the transposase are contained in a mass ratio of 0.1 to 10:

1.

13. A transposon kit for targeted DNA delivery, comprising the transposon system for targeted DNA delivery according to claim 11 and an instruction manual.

14. (a) a transposon-based vector into which target DNA has been inserted; and (b) a transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3; an mRNA encoding the transposase set forth in SEQ ID NO: 12, or A cell into which the transposase described in claim 8 has been introduced.

15. The cell of claim 14, wherein the target DNA is excised from the transposon-based vector by the transposase in the cell, and the excised target DNA is inserted into the genome of the cell.

16. The cell of claim 14, wherein the cell is selected from the group consisting of a T cell, a NK cell, a B cell, a dendritic cell, a macrophage, and a mast cell.

17. The cell according to claim 14, wherein the cell is co-cultured with feeder cells after the transposon vector is introduced.

18. The cells of claim 17, wherein the feeder cells are irradiated cells.

19. The cell of claim 14, wherein the cell expresses the target DNA for 7 days or more after introduction of the transposon vector.

20. (a) a transposon-based vector into which target DNA has been inserted; and (b) a transposase expression vector comprising a nucleic acid sequence encoding the transposase shown in SEQ ID NO: 2 or SEQ ID NO: 3; An mRNA encoding the transposase set forth in SEQ ID NO: 12, or The transposase according to claim 8.

10. A method for inserting a target DNA sequence into the genome of a cell, comprising the step of introducing into the cell

21. 21. The method of inserting a target DNA sequence into the genome of a cell according to claim 20, wherein the introduction is carried out by electroporation.

22. The method for inserting a target DNA sequence into the genome of a cell according to claim 20, further comprising the step of co-culturing the cells into which the transposon vector has been inserted with support cells after the introduction step.

23. 23. The method for inserting a target DNA sequence into the genome of a cell according to claim 22, wherein the step of co-culturing with the feeder cells is carried out immediately after the step of introducing.

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